Methods and compositions for treating ctnnb1-related disorders

Abstract

The present invention relates to methods and compositions comprising RNAi agents (e.g., double-stranded RNA (dsRNA) agents) that target the beta-catenin (CTNNB1) gene for the treatment of CTNNB1-related disorders, such as cancer, such as hepatocellular carcinoma and colorectal cancer. The invention also relates to RNAi agents (e.g., double-stranded RNA (dsRNA) agents) that target the beta-catenin (CTNNB1) gene in combination with one or more therapeutic and / or therapeutic agents (e.g., immunotherapeutic agents, e.g., one or more immune checkpoint inhibitors, e.g., PD-1 inhibitors (e.g., anti-PD-1 antibodies or antigen binding fragments thereof), PD-L1 inhibitors, CTLA-4 inhibitors; and / or a VEGF inhibitor for use in the treatment of a subject having a CTNNB1-related disorder, such as cancer, such as hepatocellular carcinoma and colorectal cancer.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 530,716, filed August 4, 2023, and U.S. Provisional Application No. 63 / 662,507, filed June 21, 2024. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0003] This application also relates to U.S. Provisional Application No. 63 / 224,901, filed July 23, 2021; U.S. Provisional Application No. 63 / 293,851, filed December 27, 2021; U.S. Patent Application No. 18 / 405,072, filed January 5, 2024; U.S. Patent Application No. 18 / 581,511, filed February 20, 2024; and PCT Application No. PCT / US2022 / 037794, filed July 21, 2022. The entire contents of each of the foregoing applications are incorporated herein by reference. Background of the Invention

[0005] Wnt / β-catenin signaling is an evolutionarily conserved and functionally diverse pathway known to be involved in embryonic development, tissue homeostasis, and various human diseases. Aberrant activation of this pathway leads to the accumulation of β-catenin in the cell nucleus and promotes the transcription of many oncogenes such as c-Myc and Cyclin D-1. Therefore, it is helpful in the carcinogenesis and tumor progression of various cancers, including hepatocellular carcinoma, colon cancer, pancreatic cancer, lung cancer, and ovarian cancer (Khramtsov AI et al., Am J Pathol. 2010;176:2911-2920; Tao J et al., Gastroenterology. 2014; 147:690-701; Kobayashi M et al., Br J Cancer. 2000;82:1689-1693; Damsky WE et al., Cancer Cell. 2011;20:741-754; Gekas C et al., Leukemia. 2016;30:2002-2010).

[0006] β-catenin, encoded by the CTNNB1 gene, is a multifunctional protein that plays a central role in physiological homeostasis. It functions as both a transcriptional co-regulator and an aptamer for intracellular adhesion. Wnt is a major regulator of β-catenin and belongs to a family of 19 glycoproteins that regulate both β-catenin-dependent (classical Wnt) and non-classical Wnt-dependent (non-classical Wnt) signaling pathways (van Ooyen A, Nusse R. Cell. 1984;39:233-240).

[0007] In the classical Wnt pathway, Dsh, β-catenin, glycogen synthase kinase 3β (GSK3β), adenomatous polyposis gene (APC), AXIN, and T-cytokine (TCF) / lymphoenhancing factor (LEF) have been identified as signaling sensors, with β-catenin being the core molecule (Behrens J et al., Nature. 1996;382:638-642; Peifer M et al., Dev Biol. 1994;166:543-556; Rubinfeld B et al., Science. 1996;272:1023-1026; Yost C et al., Genes Dev. 1996;10:1443-1454). In the absence of Wnt ligands, β-catenin is maintained at low levels via the ubiquitin-proteasome system (UPS), leading to ubiquitination and proteasome degradation of β-catenin. Following mutations in genes activating or containing Wnt components, β-catenin accumulates in the cytoplasm and then translocates to the nucleus. Thereafter, it binds to other proteins, such as LEF-1 / TCF4, to promote the transcription of target genes, most of which encode oncoproteins, in a tissue-specific manner. Therefore, abnormally high expression of β-catenin contributes to a variety of diseases, including cancer.

[0008] Furthermore, high levels of cytoplasmic expression and nuclear localization of β-catenin induce tumorigenesis features and promote cancer cell proliferation and survival (Valkenburg KC et al., Cancers (Basel) 2011;3:2050-2079). In addition, β-catenin promotes tumor progression by inhibiting T cell responses (Hong Y et al., Cancer Res. 2015;75:656-665).

[0009] Current treatments for cancer include surgery, radiotherapy, and chemotherapy. However, these methods are not entirely effective and can cause serious side effects. Therefore, there is a need in the field for alternative treatments for subjects with CTNNB1-related conditions such as cancers, such as hepatocellular carcinoma. Summary of the Invention

[0010] This invention provides methods and compositions comprising iRNA that achieves RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript encoding the β-catenin (CTNNB1) gene for the treatment of CTNNB1-related conditions, such as cancers, e.g., hepatocellular carcinoma and colorectal cancer. This invention also provides combination therapies for treating subjects with CTNNB1-related conditions (such as cancers, e.g., hepatocellular carcinoma and colorectal cancer) using an RNAi agent (e.g., a double-stranded RNA (dsRNA) agent) targeting the β-catenin (CTNNB1) gene and one or more therapeutic and / or therapeutic agents, such as immunotherapeutic agents (e.g., one or more immune checkpoint inhibitors, e.g., PD-1 inhibitors (e.g., anti-PD-1 antibodies or their antigen-binding fragments), PD-L1 inhibitors, CTLA-4 inhibitors); and / or VEGF inhibitors.

[0011] Therefore, in one aspect, the present invention provides a method for treating a subject suffering from cancer. The method includes administering to the subject a double-stranded ribonucleic acid (dsRNA) agent at a dose of about 0.01 mg / kg to about 1.5 mg / kg (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or about 1.5 mg / kg) to inhibit the expression of β-catenin (CTNNB1), wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand differs from the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3' by no more than 4 bases (e.g., 4, 3, 2, 1, or 0 bases), and the antisense strand differs from the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc The -3' difference is no more than 4 (e.g., 4, 3, 2, 1, or 0) bases, where a, g, c, and u are 2'-O-methyl(2'-OMe)adenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; Af, Gf, Cf, and Uf are 2'-fluoroadenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; s is a thiophosphate bond; VP is a vinylphosphonate; dT is 2'-deoxythymidine-3'-phosphate; dG is 2'-deoxyguanosine-3'-phosphate; and dA is 2'-deoxyadenosine-3'-phosphate, thereby treating subjects with cancer.

[0012] In another aspect, the present invention provides a method for treating a subject suffering from cancer. The method includes selecting a subject suffering from cancer containing a Wnt-pathway activating mutation and administering to the subject a double-stranded RNA (dsRNA) agent at a dose of about 0.01 mg / kg to about 1.5 mg / kg to inhibit the expression of β-catenin (CTNNB1), wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand differs from the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3' by no more than 4 bases (e.g., 4, 3, 2, 1, or 0 bases), and the antisense strand differs from the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc The -3' difference is no more than 4 bases (e.g., 4, 3, 2, 1, or 0), where a, g, c, and u are 2'-O-methyl(2'-OMe)adenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; Af, Gf, Cf, and Uf are 2'-fluoroadenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; s is a thiophosphate bond; VP is a vinylphosphonate; dT is 2'-deoxythymidine-3'-phosphate; dG is 2'-deoxyguanosine-3'-phosphate; and dA is 2'-deoxyadenosine-3'-phosphate, thereby treating subjects with cancer.

[0013] Cancer can be, for example, hepatocellular carcinoma (e.g., advanced or metastatic hepatocellular carcinoma) or colorectal cancer (e.g., colorectal cancer with liver metastases).

[0014] In one implementation, the subject is a human being.

[0015] In one implementation, the sense strand comprises the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3', and the antisense strand comprises the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc-3'.

[0016] In one implementation, the sense strand consists of the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3', and the antisense strand consists of the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc-3'.

[0017] In one embodiment, the dsRNA agent is present in the pharmaceutical composition.

[0018] In one embodiment, the pharmaceutical composition comprises lipids.

[0019] In one embodiment, the lipid is a cationic lipid.

[0020] In one embodiment, the cationic lipid contains one or more biodegradable groups.

[0021] In one embodiment, the lipid-containing structure

[0022] .

[0023] In one embodiment, the pharmaceutical composition comprises

[0024] (a) ;

[0025] (b) Cholesterol;

[0026] (c) DSPC; and

[0027] (d) PEG-DMG.

[0028] In one embodiment, the pharmaceutical composition comprises

[0029] DSPC, cholesterol and PEG-DMG exist in molar ratios of 50:12:36:2 or 50:10:38.5:1.5, respectively.

[0030] In one implementation, the subject is given a dose of dsRNA of about 0.01 mg / kg to about 1.5 mg / kg approximately every three weeks.

[0031] In one implementation, the subject is given premedication prior to administration of the dsRNA agent, the premedication being selected, for example, from the group consisting of dexamethasone, acetaminophen, diphenhydramine, and ranitidine, and combinations thereof.

[0032] In one implementation, the dsRNA agent is administered intravenously to the subject.

[0033] In one embodiment, the method further includes administering additional treatment (e.g., radiotherapy) and / or a therapeutic agent (e.g., selected from immunotherapeutic agents, chemotherapeutic agents, growth inhibitors, anti-angiogenic agents, antitumor compositions, and any combination of one or more of the foregoing) to a subject for the treatment of cancer.

[0034] In one implementation, additional treatment and / or therapeutic agents are administered intravenously to the subject (e.g., by infusion).

[0035] In one implementation, the additional therapeutic agent is an immunotherapy agent.

[0036] In one embodiment, the method further includes administering to the subject a combination of immunotherapeutic agents (e.g., one or more checkpoint inhibitors, such as one or more anti-programmed death-1 (PD-1) antibodies or antigen-binding fragments thereof, anti-programmed death-ligand 1 (PD-L1) antibodies or antigen-binding fragments thereof, and anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibodies or antigen-binding fragments thereof).

[0037] In one implementation, the immunotherapeutic agent is an anti-programmed death-1 (PD-1) antibody or an antigen-binding fragment thereof.

[0038] In one embodiment, the anti-PD1 antibody or its antigen-binding fragment is a humanized monoclonal antibody or its antigen-binding fragment.

[0039] In one implementation, the humanized monoclonal anti-PD1 antibody or its antigen-binding fragment is pembrolizumab.

[0040] In one implementation, the subject is administered a dose of approximately 200 mg of pembrolizumab.

[0041] In one implementation, subjects are given a 200 mg dose of pembrolizumab approximately every three weeks.

[0042] In one implementation, pembrolizumab is administered intravenously to the subject.

[0043] In one implementation, intravenous administration includes intravenous infusion of pembrolizumab over approximately 30 minutes.

[0044] In one implementation, the dsRNA agent is administered to the subject before, after, or concurrently with the administration of the anti-PD-1 antibody or its antigen-binding fragment.

[0045] In one aspect, the present invention provides a method for treating a subject suffering from cancer. The method comprises administering to the subject a double-stranded ribonucleic acid (dsRNA) agent for inhibiting β-catenin (CTNNB1) expression at a dose of about 0.01 mg / kg to about 1.5 mg / kg (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or about 1.5 mg / kg), and an anti-programmed death-1 (PD-1) antibody or an antigen-binding fragment thereof at a dose of about 200 mg, thereby treating the subject suffering from cancer.

[0046] In another aspect, the present invention provides a method for treating a subject with cancer. The method includes selecting a subject with cancer containing a Wnt-pathway activation mutation and administering to the subject a double-stranded ribonucleic acid (dsRNA) agent for inhibiting β-catenin (CTNNB1) expression at a dose of about 0.01 mg / kg to about 1.5 mg / kg, and an anti-programmed death-1 (PD-1) antibody or its antigen-binding fragment at a dose of about 200 mg, thereby treating the subject with cancer.

[0047] Cancer can be, for example, hepatocellular carcinoma (e.g., advanced or metastatic hepatocellular carcinoma) or colorectal cancer (e.g., colorectal cancer with liver metastases).

[0048] In one implementation, the subject is a human being.

[0049] In one embodiment, the dsRNA agent comprises a sense strand containing at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides, the consecutive nucleotides differing from the nucleotide sequence 5'-UACUGUUGGAUUGAUUCGAAA-3' by no more than 3 (e.g., 3, 2, 1, or 0) nucleotides; and an antisense strand containing at least 15 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23) consecutive nucleotides, the consecutive nucleotides differing from the nucleotide sequence 5'-UTUCGAAUCAATCCAACAGUAGC-3' by no more than 3 (e.g., 3, 2, 1, or 0) nucleotides.

[0050] In one embodiment, the dsRNA agent comprises a sense strand containing the nucleotide sequence 5'-UACUUGUUGGAUUGAUUCGAAA-3' and an antisense strand containing the nucleotide sequence 5'-UTUCGAAUCAATCCAACAGUAGC-3'.

[0051] In one implementation, all nucleotides in the sense strand and all nucleotides in the antisense strand contain nucleotide modifications.

[0052] In one embodiment, at least one nucleotide modification is selected from the group consisting of: deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, baseless nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinonucleotides, phosphoramides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl alcohol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate, nucleotides containing 5'-phosphate mimics, heat-labile nucleotides, ethylene glycol-modified nucleotides (GNA), nucleotides containing 2'-phosphate, and 2-O-(N-methylacetamide)-modified nucleotides; and combinations thereof.

[0053] In another embodiment, at least one nucleotide modification is selected from the group consisting of: LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluorine, 2'-deoxy, 2'-hydroxy and ethylene glycol; and combinations thereof.

[0054] In one embodiment, at least one nucleotide modification is selected from the group consisting of: deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, ethylene glycol modified nucleotides (GNA), nucleotides containing 2' phosphate groups, nucleotides containing thiophosphate groups, and vinyl-phosphonate nucleotides; and combinations thereof.

[0055] In one embodiment, at least one nucleotide modification is a nucleotide modified with a heat-labile nucleotide modification.

[0056] In one embodiment, the thermally unstable nucleotide modification is selected from the group consisting of: debasement modification; mismatch with the relative nucleotide in the duplex; unstable sugar modification, 2'-deoxy modification, acyclic nucleotide, nonlocked nucleic acid (UNA), and glycerol nucleic acid (GNA).

[0057] In one implementation, the length of the double-stranded region is 19-30 nucleotide pairs.

[0058] In one implementation, the length of each chain independently does not exceed 30 nucleotides.

[0059] In another implementation, each chain is independently 19-30 nucleotides long.

[0060] In one implementation, the length of the sense strand is 21 nucleotides, and the length of the antisense strand is 23 nucleotides.

[0061] In one implementation, at least one chain contains a 3' overhang of at least one nucleotide.

[0062] In another embodiment, at least one chain contains a 3' overhang of at least two nucleotides.

[0063] In one embodiment, the dsRNA agent further comprises a ligand.

[0064] In one embodiment, the dsRNA agent further comprises at least one phosphate thioester or methylphosphonate nucleoside linker.

[0065] In one embodiment, the thiophosphate or methylphosphonate nucleoside linkage is located at the 3'-end of one chain (e.g., the antisense chain or the sense chain).

[0066] In another embodiment, the thiophosphate or methylphosphonate nucleoside linkage is located at the 5'-end of one chain (e.g., the antisense chain or the sense chain).

[0067] In one embodiment, the thiophosphate or methylphosphonate nucleoside linkage is located at both the 5'- and 3'-termini of a chain.

[0068] In one implementation, the chain is an antisense chain.

[0069] In one embodiment, the dsRNA agent comprises a positive strand differing from the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3' by no more than 4 bases (e.g., 4, 3, 2, 1, or 0 bases), and a negative strand differing from the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc -3' differs by no more than 4 bases (e.g., 4, 3, 2, 1, or 0 bases) in the antisense strand, where a, g, c, and u are 2'-O-methyl(2'-OMe)adenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; Af, Gf, Cf, and Uf are 2'-fluoroadenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; s is a thiophosphate bond; VP is a vinylphosphonate; dT is 2'-deoxythymidine-3'-phosphate; dG is 2'-deoxyguanosine-3'-phosphate; and dA is 2'-deoxyadenosine-3'-phosphate.

[0070] In one embodiment, the dsRNA agent comprises a sense strand containing the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3' and an antisense strand containing the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc-3', wherein a, g, c, and u are 2'-O-methyl(2'-OMe)adenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; Af, Gf, Cf, and Uf are 2'-fluoroadenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; s is a thiophosphate bond; VP is a vinylphosphonate; dT is 2'-deoxythymidine-3'-phosphate; dG is 2'-deoxyguanosine-3'-phosphate; and dA is 2'-deoxyadenosine-3'-phosphate.

[0071] In one embodiment, the dsRNA agent is present in the pharmaceutical composition.

[0072] In one embodiment, the pharmaceutical composition comprises lipids.

[0073] In one embodiment, the lipid is a cationic lipid.

[0074] In one embodiment, the cationic lipid contains one or more biodegradable groups.

[0075] In one embodiment, the lipid-containing structure

[0076] .

[0077] In one embodiment, the pharmaceutical composition comprises

[0078] (a) ;

[0079] (b) Cholesterol;

[0080] (c) DSPC; and

[0081] (d) PEG-DMG.

[0082] In one embodiment, the pharmaceutical composition comprises

[0083] DSPC, cholesterol and PEG-DMG exist in molar ratios of 50:12:36:2 or 50:10:38.5:1.5, respectively.

[0084] In one implementation, the subject is given a dose of dsRNA of about 0.01 mg / kg to about 1.5 mg / kg approximately every three weeks.

[0085] In one implementation, prior to administration of the dsRNA agent, the subject is given a pre-treatment drug, such as selected from the group consisting of dexamethasone, acetaminophen, diphenhydramine, and ranitidine, and combinations thereof.

[0086] In one implementation, the dsRNA agent is administered intravenously to the subject.

[0087] In one embodiment, the anti-PD1 antibody or its antigen-binding fragment is a humanized monoclonal antibody or its antigen-binding fragment.

[0088] In one embodiment, the humanized monoclonal anti-PD1 antibody or its antigen-binding fragment is pembrolizumab (Keytruda®).

[0089] In one implementation, a 200 mg dose of pembrolizumab was administered to the subject.

[0090] In one implementation, subjects are given a 200 mg dose of pembrolizumab every three weeks.

[0091] In one implementation, pembrolizumab is administered intravenously to the subject.

[0092] In one implementation, intravenous administration includes an intravenous infusion of pembrolizumab over approximately 30 minutes.

[0093] In one implementation, the dsRNA agent is administered to the subject before, after, or concurrently with the administration of the anti-PD-1 antibody or its antigen-binding fragment.

[0094] In one embodiment, the method further includes administering additional treatment (e.g., radiotherapy) and / or a therapeutic agent (e.g., selected from immunotherapeutic agents, chemotherapeutic agents, growth inhibitors, anti-angiogenic agents, antitumor compositions, and any combination of one or more of the foregoing) to a subject for the treatment of cancer.

[0095] In one implementation, the additional therapeutic agent is an immunotherapy agent.

[0096] In one embodiment, the method further includes administering to the subject a combination of immunotherapeutic agents (e.g., one or more immune checkpoint inhibitors, such as one or more anti-programmed death-1 (PD-1) antibodies or antigen-binding fragments thereof, anti-programmed death-ligand-1 (PD-L1) antibodies or antigen-binding fragments thereof, and anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibodies or antigen-binding fragments thereof).

[0097] In one implementation, the Wnt pathway activation mutation is a mutation in a gene selected from the group consisting of: Axin1, Axin2, APC, CTNNB1, RNF43, ZNRF3, RSPO1, RSPO2, RSPO3, and RSPO4, and combinations thereof.

[0098] Brief description of the attached figures

[0099] Figure 1A This is a graph depicting the effects of intravenous administration of a single 0.1 mg / kg or 0.3 mg / kg dose of AD-1548393 on days 5, 15, and 29 post-dose. The percentage of residual CTNNB1 mRNA relative to pre-dose levels is shown.

[0100] Figure 1B This is a graph depicting the effects of intravenous administration of a single 0.1 mg / kg or 0.3 mg / kg dose of AD-1548459 on days 5, 15, and 29 post-dose. The percentage of residual CTNNB1 mRNA relative to pre-dose levels is shown.

[0101] Figure 1C This is a graph depicting the effects of a single intravenous dose of 0.1 mg / kg or 0.3 mg / kg of AD-1548488 on days 5, 15, and 29 post-dose. The percentage of residual CTNNB1 mRNA relative to pre-dose levels is shown.

[0102] Figure 2 This diagram illustrates a phase 1 / 1b study of ALN-BCAT as monotherapy and in combination with pembrolizumab in subjects with advanced or metastatic hepatocellular carcinoma or colorectal cancer with liver metastases. CRC = colorectal cancer; HCC = hepatocellular carcinoma; IV = intravenous; q3w = every 3 weeks; pembro = pembrolizumab; RDFE = recommended extension dose. Recruitment for Part B can begin once at least two dose levels in Part A have been evaluated and determined to be safe, or the MTD or RDFE of Part A has been determined. The starting dose of ALN-BCAT in Part B will be at least one dose level lower than the dose at which Part A was determined to be safe. Invention Details

[0104] This invention provides methods and compositions comprising iRNA that achieves RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript encoding the β-catenin (CTNNB1) gene for the treatment of CTNNB1-related conditions, such as cancers, e.g., hepatocellular carcinoma and colorectal cancer. This invention also provides combination therapies for treating subjects with CTNNB1-related conditions (such as cancers, e.g., hepatocellular carcinoma and colorectal cancer) using an RNAi agent (e.g., a double-stranded RNA (dsRNA) agent) targeting the β-catenin (CTNNB1) gene and one or more therapeutic and / or therapeutic agents, such as immunotherapeutic agents (e.g., one or more checkpoint inhibitors, e.g., PD-1 inhibitors, e.g., anti-PD-1 antibodies or their antigen-binding fragments) and / or VEGF inhibitors.

[0105] The iRNA of the present invention comprises an RNA strand (antisense strand) having a region of length of about 30 nucleotides or less, for example, lengths of 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-24, 19-23, 19-25, 19-26, 19-25, 19-24, 19-23, 19-25, 19-26, 19-27, 19-28, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-25, 19-26, 19-27, 19-28, 19-29 ...9, 19-28, 19-29, 19-29, 19-28, 19-29, 19-29, 19-29, 19-29, 19 2, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 nucleotides, said region being substantially complementary to at least a portion of the mRNA transcript of the CTNNB1 gene.

[0106] In some embodiments, one or both strands of the double-stranded RNAi agent of the present invention are up to 66 nucleotides in length, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides in length, having a region of at least 19 consecutive nucleotides that is substantially complementary to at least a portion of the mRNA transcript of the CTNNB1 gene. In some embodiments, such iRNA agents with a longer antisense strand may, for example, comprise a second RNA strand (sense strand) of 20-60 nucleotides in length, wherein the sense and antisense strands form a doublet of 18-30 consecutive nucleotides.

[0107] The use of iRNA in this invention enables targeted degradation of the mRNA of the corresponding gene (CTNNB1 gene) in mammals. Using in vitro assays, the inventors have demonstrated that iRNA targeting the CTNNB1 gene effectively mediates RNAi, resulting in significant repression of CTNNB1 gene expression. Therefore, methods and compositions comprising these iRNAs can be used to treat subjects suffering from CTNNB1-related conditions, such as cancers, such as hepatocellular carcinoma (HCC), including HCC containing Wnt pathway activating mutations.

[0108] The following detailed description discloses how to prepare and use compositions containing iRNA to inhibit the expression of the CTNNB1 gene, as well as compositions, uses, methods, and combination therapies for treating subjects who would benefit from the inhibition and / or reduction of CTNNB1 gene expression (e.g., subjects who are susceptible to or have been diagnosed with CTNNB1-related conditions, such as cancers, such as hepatocellular carcinoma and colorectal cancer).

[0109] I. Definition

[0110] To facilitate understanding of the invention, certain terms are first defined. Furthermore, it should be noted that whenever a value or range of a parameter is mentioned, it is intended that values ​​and ranges between said values ​​also be included as part of the invention.

[0111] The article “a / an” used in this article refers to the grammatical object of one or more articles (i.e., at least one). For example, “an element” refers to one or more elements, such as multiple elements.

[0112] The term “including / contains / contains” as used in this article refers to the phrase “including / contains / contains but not limited to”, and is used interchangeably with the phrase “including / contains / contains but not limited to”.

[0113] Unless the context clearly indicates otherwise, the term “or” as used herein means “and / or” and is used interchangeably with the term “and / or”. For example, “justice chain or antisense chain” is understood as “justice chain or antisense chain or justice chain and antisense chain”.

[0114] As used herein, the term “about” refers to a tolerance range typical in the art. For example, “about” can be understood as approximately 2 standard deviations from the mean. In some implementations, “about” means ±10%. In some implementations, “about” means ±5%. When “about” appears before a series of numbers or ranges, it is understood that “about” may modify each number in the series or range.

[0115] The terms “at least,” “not less than,” or “more than” preceding a number or series of numbers are understood to include the number immediately preceding the term “at least” as well as all subsequent numbers or integers that are logically implied from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For instance, “at least 19 nucleotides in a nucleic acid molecule of 21 nucleotides” means that 19, 20, or 21 nucleotides have the indicated property. When “at least” precedes a series of numbers or a range, it is understood that “at least” may modify each number in the series or range.

[0116] As used herein, “no more than” or “or less” is understood as the value of the immediately preceding phrase plus a logically lower value or integer, up to zero, depending on the context. For example, a double-stranded strand with a dangling “no more than 2 nucleotides” has a dangling of 2, 1, or 0 nucleotides. When “no more than” appears before a series of numbers or ranges, it is understood that “no more than” can modify each number in the series or range. As used herein, a range includes both the upper and lower limits.

[0117] As used herein, detection methods may include determining the amount of analyte present below the method's detection level.

[0118] In the event of a conflict between the specified target site and the nucleotide sequence of the sense or antisense strand, the specified sequence shall prevail.

[0119] In the event of a conflict between the sequence and its designated site on the transcript or other sequences, the nucleotide sequence described in the instruction manual shall prevail.

[0120] As used in this article, “β-catenin” (which can be used interchangeably with the term “CTNNB1”) refers to a structural protein in the cadherin-mediated cell-cell adhesion system and is also known as a key transcriptional activator in the Wnt signaling pathway. The Wnt / β-catenin signaling pathway, also known as the classical Wnt signaling pathway, is a conserved signaling axis that participates in a variety of physiological processes, such as proliferation, differentiation, apoptosis, migration, invasion and tissue homeostasis (Choi B et al., Cell Rep. 2020;31(5):107540). Dysregulation of the Wnt / β-catenin cascade contributes to the development and progression of certain solid tumors and hematologic malignancies, such as hepatocellular carcinoma (HCC) (Ge X et al., Journal of hematology & oncology. 2010;3:33; He S et al., Biomed Pharmacother. 2020;132:110851; Gajos-Michniewicz A et al., Int J Mol Sci 2020, 21(14); Suzuki T et al., J Gastroenterol Hepatol. 2002;17:994-1000). Indeed, β-catenin plays a crucial role in promoting tumor progression by stimulating tumor cell proliferation and reducing the activity of the cell adhesion system, and is associated with poor prognosis, especially in patients with poorly differentiated HCC (Inagawa S et al., Clin Cancer Res. 2002;8:450-456). CTNNB1 is also known as catalin β, Armadillo, NEDSDV, MRD19, or EVR7.

[0121] The sequences of human CTNNB1 mRNA transcripts can be found, for example, in GenBank accession number GI: 1519314571 (NM_001904.4; SEQ ID NO: 1; reverse complementary sequence, SEQ ID NO: 2). The sequences of mouse CTNNB1 mRNA can be found, for example, in GenBank accession number GI: 260166638 (NM_007614.3; SEQ ID NO: 3; reverse complementary sequence, SEQ ID NO: 4). The sequences of rat CTNNB1 mRNA can be found, for example, in GenBank accession number GI: 46048608 (NM_053357.2; SEQ ID NO: 5; reverse complementary sequence, SEQ ID NO: 6). The sequence of CTNNB1 mRNA from cynomolgus monkeys (Macacafascicularis) can be found, for example, in GenBank accession number GI: 985482040 (NM_001319394.1; SEQ ID NO: 7; reverse complementary sequence, SEQ ID NO: 8). The sequence of CTNNB1 mRNA from rhesus monkeys (Macacamulatta) can be found, for example, in GenBank accession number GI: 383872646 (NM_001257918.1; SEQ ID NO: 9; reverse complementary sequence, SEQ ID NO: 10).

[0122] Additional examples of the CTNNB1 mRNA sequence are readily available from publicly available databases such as GenBank, UniProt, OMIM, and the Macaque Genome Project website.

[0123] Further information about CTNNB1 can be found at, for example, www.ncbi.nlm.nih.gov / gene / ?term=CTNNB1.

[0124] The entire contents of each of the aforementioned GenBank accession numbers and gene database numbers are incorporated herein by reference as of the date of this application.

[0125] As used herein, the term CTNNB1 also refers to variations of the CTNNB1 gene, including variants available in SNP databases. Numerous sequence variations within the CTNNB1 gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, www.ncbi.nlm.nih.gov / snp / ?term=CTNNB1, the entire contents of which are incorporated herein by reference as of the date of this application).

[0126] As used herein, a “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the CTNNB1 gene, including the mRNA (which is the product of RNA processing of the primary transcription product). In one embodiment, the target portion of the sequence is at least long enough to serve as a substrate for iRNA-guided cleavage at or near a portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the CTNNB1 gene.

[0127] The target sequence can be approximately 18-36 nucleotides in length, for example, approximately 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, or 19-30 nucleotides. For example, the target sequence may be about 19-30 nucleotides in length, specifically 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. In some embodiments, the target sequence is 19-23 nucleotides in length, optionally 21-23 nucleotides. The ranges and lengths described above are also encompassed as part of this disclosure.

[0128] As used herein, the term "chain containing a sequence" refers to an oligonucleotide chain containing a nucleotide chain described using the sequence referenced in standard nucleotide nomenclature.

[0129] “G,” “C,” “A,” “T,” and “U” typically refer to nucleotides containing guanine, cytosine, adenine, thymine, and uracil as bases, respectively. However, it should be understood that the terms “ribonucleotide” or “nucleotide” can also refer to modified nucleotides (as further detailed below) or substituted moieties (see, for example, Table 1). Those skilled in the art will readily recognize that guanine, cytosine, adenine, and uracil can be substituted with other moieties without substantially altering the base-pairing properties of the oligonucleotide containing the nucleotide with such substitutions. For example, but not limited to, nucleotides containing inosine as a base can base-pair with nucleotides containing adenine, cytosine, or uracil. Therefore, nucleotides containing uracil, guanine, or adenine in the nucleotide sequence of the dsRNA characterized in this invention can be substituted with nucleotides containing, for example, inosine. In further instances, adenine and cytosine at any position in the oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU-wobble base pairing with the target mRNA. Sequences containing such alternative portions are applicable to the compositions and methods characterized in this invention.

[0130] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent,” which may be used interchangeably herein, refer to an agent containing RNA (as defined herein) that mediates the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates (e.g., inhibits) the expression of the CTNNB1 gene in cells (e.g., hepatocytes) within subjects (such as mammalian subjects).

[0131] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence (e.g., the CTNNB1 target mRNA sequence) to guide the cleavage of the target RNA. Without wishing to be bound by any theory, it is believed that long double-stranded RNA introduced into the cell is cleaved into siRNA by a type III nuclease known as Dicer (Sharp et al., (2001) Genes Dev. 15:485). Dicer (a ribonuclease-III-like enzyme) processes dsRNA into short interfering RNA of 19-23 base pairs, which typically has a two-base 3' overhang (Bernstein et al., (2001) Nature 409:363). The siRNA is then integrated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188). Therefore, in one aspect, the present invention relates to intracellularly generated single-stranded RNA (siRNA) that promotes the formation of the RISC complex, thereby achieving the silencing of a target gene (i.e., the CTNNB1 gene). Therefore, the term "siRNA" is also used herein to refer to the aforementioned iRNA.

[0132] In some embodiments, the RNAi agent may be a single-stranded siRNA (ssRNAi), which is introduced into a cell or organism to inhibit the target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which subsequently cleaves the target mRNA. Single-stranded siRNAs are typically 15-30 nucleotides long and chemically modified. The design and detection of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894 (the entire contents of each of these are incorporated herein by reference). Any antisense nucleotide sequence described herein may be used as a single-stranded siRNA, either as described herein or chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

[0133] In some embodiments, the “iRNA” used in the compositions, uses, and methods of the present invention is double-stranded RNA and is referred herein as a “double-stranded RNA agent,” a “double-stranded RNA (dsRNA) molecule,” a “dsRNA agent,” or “dsRNA.” The term “dsRNA” refers to a ribonucleic acid molecular complex having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands (referred to as having “sense” and “antisense” orientations relative to the target RNA (i.e., the CTNNB1 gene). In some embodiments of the present invention, the double-stranded RNA (dsRNA) triggers the degradation of the target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism referred herein to as RNA interference or RNAi.

[0134] Typically, most nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Furthermore, as used herein, “iRNA” may contain chemically modified ribonucleotides; iRNA may contain substantial modifications at multiple nucleotide sites. As used herein, the term “modified nucleotide” refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide link, or a modified nucleobase, or any combination thereof. Therefore, the term modified nucleotide encompasses substitution, addition, or removal (e.g., functional groups or atoms) of internucleotide links, sugar moieties, or nucleobases. Modifications of agents suitable for use in this invention include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modifications used in siRNA-type molecules are covered by “iRNA” or “RNAi agent”.

[0135] In some embodiments of this disclosure, if the RNAi agent is present, the presence of deoxynucleotides may be considered as constituting modified nucleotides.

[0136] The double-stranded region can have any length that allows for specific degradation of the desired target RNA via the RISC pathway, and the length can range from approximately 19 to 36 base pairs. For example, a length of approximately 19-30 base pairs, such as approximately 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs, such as a length of approximately 19-30... 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. In some embodiments, the length of the double-stranded region is 19-21 base pairs, for example, 21 base pairs. The ranges and lengths described above are also considered part of this disclosure.

[0137] The two strands constituting a double-stranded structure can be different parts of a larger RNA molecule, or they can be independent RNA molecules. When the two strands are part of a larger molecule and thus connected by a continuous nucleotide chain between the 3' end of one strand and the 5' end of the corresponding other strand to form a double-stranded structure, the connecting RNA strands are called a "hairpin loop." A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23, or more unpaired nucleotides. In some embodiments, a hairpin loop may contain 10 or fewer nucleotides. In some embodiments, a hairpin loop may contain 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop may contain 4-10 unpaired nucleotides. In some embodiments, a hairpin loop may contain 4-8 nucleotides.

[0138] When two substantially complementary strands of dsRNA are contained in separate RNA molecules, those molecules do not necessarily, but can, be covalently linked. When the two strands are covalently linked in a manner other than a continuous nucleotide chain between the 3' end of one strand and the 5' end of the corresponding other strand to form a double-stranded structure, the linking structure is called a "linker". The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any dangling overhangs present in the double-stranded structure. In addition to the double-stranded structure, RNAi may contain one or more nucleotide dangling overhangs. In one embodiment of the RNAi agent, at least one strand contains a 3' dangling overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' dangling overhang of at least two nucleotides, such as 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, at least one strand of the RNAi agent contains a 5' dangling overhang of at least one nucleotide. In some embodiments, at least one strand contains a 5' overhang of at least two nucleotides, such as 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, both the 3' and 5' ends of one strand of the RNAi agent contain overhangs of at least one nucleotide.

[0139] In some embodiments, the iRNA agent of the present invention is dsRNA (each strand of which contains 19-23 nucleotides) that interacts with a target RNA sequence (e.g., the CTNNB1 gene) to guide the cleavage of the target RNA.

[0140] In some embodiments, the iRNA of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence (e.g., the CTNNB1 target mRNA sequence) to guide the cleavage of the target RNA.

[0141] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of a double-stranded iRNA. For example, a nucleotide overhang exists when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; alternatively, the overhang may contain at least two, three, four, five, or more nucleotides. The nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). The overhang may be located on the sense strand, antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide may be present at the 5' end, 3' end, or both ends of the antisense strand or sense strand of the dsRNA.

[0142] In one embodiment, the antisense strand of the dsRNA has 1-10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, hanging at the 3' or 5' end. In another embodiment, the sense strand of the dsRNA has 1-10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, hanging at the 3' or 5' end. In yet another embodiment, one or more of the hanging nucleotides are replaced by nucleoside phosphate thioesters.

[0143] In some embodiments, the antisense strand of the dsRNA has 1-10 nucleotides, such as 0-3, 1-3, 2-4, 2-5, 4-10, or 5-10 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides hanging at the 3' or 5' end. In one embodiment, the sense strand of the dsRNA has 1-10 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides hanging at the 3' or 5' end. In another embodiment, one or more of the hanging nucleotides are replaced by nucleoside phosphate thioesters.

[0144] In some embodiments, the antisense strand of the dsRNA has 1-10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, dangling at the 3' or 5' end. In some embodiments, the dangling on the sense or antisense strand (or both) may contain an extension of more than 10 nucleotides, such as 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides. In some embodiments, the extended dangling is located on the sense strand of the duplex. In some embodiments, the extended dangling is present at the 3' end of the sense strand of the duplex. In some embodiments, the extended dangling is present at the 5' end of the sense strand of the duplex. In some embodiments, the extended dangling is located on the antisense strand of the duplex. In some embodiments, the extended dangling is present at the 3' end of the antisense strand of the duplex. In some embodiments, the extended dangling is present at the 5' end of the antisense strand of the duplex. In some embodiments, one or more nucleotides in the extended pendant are replaced by nucleoside thiophosphates. In some embodiments, the pendant includes a self-complementary portion, enabling it to form a stable hairpin structure under physiological conditions.

[0145] "Blunt" or "blunt end" refers to a double-stranded RNA agent that has no unpaired nucleotides at its ends, i.e., no nucleotide dangling. A "blunt-end" double-stranded RNA agent is double-stranded throughout its entire length, meaning there is no nucleotide dangling at either end of the molecule. The RNAi agents of this invention include RNAi agents without nucleotide dangling at one end (i.e., agents with one dangling and a blunt end) or RNAi agents without nucleotide dangling at either end. Most commonly, such molecules will be double-stranded throughout their entire length.

[0146] The term “antisense strand” or “guide strand” refers to the strand of iRNA (e.g., dsRNA) that contains a region that is substantially complementary to the target sequence (e.g., CTNNB1 mRNA).

[0147] As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence, such as the CTNNB1 nucleotide sequence as defined herein). When the complementary region is not perfectly complementary to the target sequence, mismatches can occur in the interior or terminal regions of the molecule. Typically, the most tolerable mismatches are located in terminal regions, such as within 5, 4, or 3 nucleotides of the 5' or 3' end of the iRNA. In some embodiments, the double-stranded RNA agent of the present invention contains nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention contains no more than 4 mismatches with the target mRNA, for example, 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention contains no more than 4 mismatches with the sense strand, for example, 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention contains nucleotide mismatches in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present invention contains no more than four mismatches with the antisense strand; for example, the sense strand contains 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatches are located, for example, within 5, 4, or 3 nucleotides from the 3' end of the iRNA. In other embodiments, the nucleotide mismatches are located, for example, in the 3' terminal nucleotides of the iRNA agent. In some embodiments, the mismatches are not located in the seed region.

[0148] Therefore, the RNAi agents described herein may contain one or more mismatches with the target sequence. In one embodiment, the RNAi agents described herein contain no more than three mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain no more than two mismatches. In one embodiment, the RNAi agents described herein contain no more than one mismatch. In one embodiment, the RNAi agents described herein contain zero mismatches. In some embodiments, if the antisense strand of the RNAi agent contains a mismatch with the target sequence, the mismatch may optionally be limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, in such embodiments, for a 23-nucleotide RNAi agent, the strand complementary to the region of the CTNNB1 gene typically does not contain any mismatches within the middle 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch with the target sequence effectively inhibits the expression of the CTNNB1 gene. It is important to consider the effectiveness of mismatched RNAi agents in suppressing CTNNB1 gene expression, especially if specific complementary regions of the CTNNB1 gene are known to have polymorphic sequence variations in the population.

[0149] As used herein, the term "sense chain" or "passenger chain" refers to the chain of iRNA that contains regions substantially complementary to the regions of the antisense chain (as defined herein).

[0150] As used in this article, “virtually all nucleotides are modified” means that most, but not all, nucleotides are modified and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0151] As used herein, the term "cleavage region" refers to a region immediately adjacent to a cleavage site. A cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases located at any end of the cleavage site and immediately adjacent to it. In some embodiments, the cleavage region comprises two bases located at any end of the cleavage site and immediately adjacent to it. In some embodiments, the cleavage site is specifically located at the site where antisense strand nucleotides 10 and 11 are joined, and the cleavage region comprises nucleotides 11, 12, and 13.

[0152] As used herein, and unless otherwise indicated, the term "complementarity," when used to describe the relationship between a first nucleotide sequence and a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence under specific conditions and form a double-stranded structure, as will be understood by those skilled in the art. Such conditions can be, for example, stringent conditions, which may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12–16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook et al., (1989) Cold SpringHarbor Laboratory Press). Other conditions may also be used, such as physiologically relevant conditions that may be encountered in vivo. Those skilled in the art will be able to determine the set of conditions most suitable for detecting the complementarity of the two sequences based on the final application of the hybridized nucleotides.

[0153] Complementary sequences within iRNAs, such as those within dsRNAs as described herein, include base pairings of an oligonucleotide or polynucleotide containing a first nucleotide sequence with an oligonucleotide or polynucleotide containing a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as “perfectly complementary” to each other. However, when the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may be perfectly complementary, or they may form one or more (but typically no more than 5, 4, 3, or 2) mismatched base pairs when hybridizing into a doublet of up to 30 base pairs, while retaining hybridization ability under conditions most relevant to their final application (e.g., in vitro or in vivo repression of gene expression). However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches in relation to the determination of complementarity. For example, a dsRNA containing one oligonucleotide of length 21 and another oligonucleotide of length 23, wherein the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may still be referred to as “perfectly complementary” for the purposes described herein.

[0154] As used herein, “complementary” sequences may also contain non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, or may consist entirely of non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, provided that the requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairings.

[0155] The terms “complementary,” “fully complementary,” and “substantially complementary” used in this article can refer to base pairing between the sense and antisense strands of dsRNA, or between two oligonucleotides or polynucleotides (such as the antisense strand of a double-stranded RNA agent and its target sequence), as will be understood in the context in which they are used.

[0156] As used herein, a polynucleotide that is “at least partially substantially complementary” to messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding the CTNNB1 gene). For example, a polynucleotide is complementary to at least a portion of the CTNNB1 mRNA if the sequence is substantially complementary to a non-discontinuous portion of the mRNA encoding the CTNNB1 gene.

[0157] Therefore, in some embodiments, the antisense polynucleotide disclosed herein is completely complementary to the target CTNNB1 sequence. In other embodiments, the antisense polynucleotide disclosed herein is substantially complementary to the target CTNNB1 sequence and comprises a continuous nucleotide sequence that is at least 80% complementary over its entire length to an equivalent region or fragment of any of the nucleotide sequences in SEQ ID NO: 1, 3, 5, 7, or 9, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0158] In other embodiments, the antisense polynucleotide disclosed herein is substantially complementary to the target CTNNB1 sequence and comprises a continuous nucleotide sequence that is at least about 80% complementary over its entire length to any of the positive strand nucleotide sequences in any one of Tables 2, 3, 5, or 6, or to a fragment of any of the positive strand nucleotide sequences in any one of Tables 2, 3, 5, or 6, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%.

[0159] In one embodiment, the RNAi agent of this disclosure comprises a positive strand substantially complementary to an antisense polynucleotide, which is identical to the target CTNNB1 sequence, and wherein the positive strand polynucleotide comprises a continuous nucleotide sequence that is at least about 80% complementary over its entire length to an equivalent region of the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8 or 10 or to a fragment of any of SEQ ID NO: 2, 4, 6, 8 or 10, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100%.

[0160] In some embodiments, the iRNA of the present invention comprises a positive strand substantially complementary to an antisense polynucleotide, which in turn is complementary to a target CTNNB1 sequence, and wherein the positive strand polynucleotide comprises a continuous nucleotide sequence that is at least about 80% complementary over its entire length to any fragment of any antisense nucleotide sequence in any of Tables 2, 3, 5 or 6, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100%.

[0161] Typically, "iRNA" comprises chemically modified ribonucleotides. Such modifications may include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modifications used in dsRNA molecules are covered by "iRNA".

[0162] In some embodiments of this disclosure, if the RNAi agent contains deoxynucleotides, then the presence of deoxynucleotides can be considered as constituting modified nucleotides.

[0163] In one aspect of the invention, the agent used in the methods and compositions of the invention is a single-stranded antisense oligonucleotide molecule that inhibits the target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotide can inhibit translation stoichiometrically by pairing with mRNA bases and physically blocking translation mechanisms, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule can be about 14 to 30 nucleotides in length and has a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule may comprise a sequence of at least about 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides derived from any of the antisense sequences described herein.

[0164] As used herein, the phrase “contacting cells with iRNA (such as dsRNA)” includes contacting cells by any possible means. Contacting cells with iRNA includes contacting cells with iRNA in vitro or in vivo. Contact can be direct or indirect. Thus, for example, an individual performing the method may place iRNA in physical contact with cells, or alternatively, may place iRNA in a situation that would allow or enable subsequent contact with cells.

[0165] For example, in vitro cell contact can be achieved by incubating cells with iRNA. In vivo cell contact can be achieved, for example, by injecting iRNA into or near the tissue containing the cells, or by injecting iRNA into another area (e.g., the bloodstream or subcutaneous space) so that the agent subsequently reaches the tissue containing the cells to be contacted. For example, iRNA may contain or be coupled to a ligand (e.g., GalNAc) that guides the iRNA to a site of interest (e.g., the liver). Combinations of in vitro and in vivo contact methods are also possible. For example, cells can also be contacted with iRNA in vitro and subsequently transplanted into a subject.

[0166] In some embodiments, contacting cells with iRNA includes “introducing” or “delivering” iRNA into cells by promoting or achieving uptake or absorption into the cells. The uptake or absorption of iRNA can occur through non-assisted diffusion or active cellular processes, or it can be performed by an adjuvant or device. Introducing iRNA into cells can be in vitro or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and liposome transfection. Further methods are described below or are known in the art.

[0167] The term "cationic lipid" includes lipids having one or two fatty acid or aliphatic chains and an amino acid head group, which can be protonated at physiological pH to form cationic lipids. In some embodiments, cationic lipids are referred to as "amino acid-coupled cationic lipids".

[0168] The term "biodegradable cationic lipid" refers to a cationic lipid having one or more biodegradable groups located in the middle or distal portion of the lipid moiety (e.g., a hydrophobic chain) of the cationic lipid. Incorporating biodegradable groups into cationic lipids results in faster metabolism and removal from the body after the active pharmaceutical ingredient has been delivered to the target region.

[0169] The term "lipid nanoparticle" or "LNP" refers to a vesicle that contains a lipid layer and encapsulates a drug-active molecule, such as a nucleic acid molecule, like iRNA or a plasmid from which iRNA is transcribed. For example, LNPs are described in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069 (the entire contents of which are incorporated herein by reference).

[0170] As used herein, “subject” is an animal that expresses the target gene endogenously or heterologously, such as mammals, including primates (such as humans, non-human primates, such as monkeys and chimpanzees), non-primates (such as cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice), or birds. In embodiments, a subject is a person, such as a person being treated or evaluated for a disease or condition that would benefit from reduced CTNNB1 expression; a person at risk of having a disease or condition that would benefit from reduced CTNNB1 expression; a person having a disease or condition that would benefit from reduced CTNNB1 expression; or a person being treated for a disease or condition that would benefit from reduced CTNNB1 expression, as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In yet another embodiment, the subject is a child subject.

[0171] As used herein, the term "treatment" refers to a beneficial or desired outcome, such as reducing at least one sign or symptom of CTNNB1-related disorder in a subject. Treatment also includes reducing one or more signs or symptoms associated with undesired CTNNB1 expression; reducing the degree of undesired CTNNB1 activation or stabilization; and improving or alleviating undesired CTNNB1 activation or stabilization. "Treatment" can also refer to prolonged survival compared to expected survival without treatment.

[0172] In the context of CTNNB1 levels or disease markers or symptoms in subjects, the term "lower" refers to a statistically significant reduction or decrease in such levels. A reduction can be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher. In some implementations, the reduction is at least 20%. In some implementations, the reduction in disease markers (e.g., protein or gene expression levels) is at least 50%. "Lower" in the context of CTNNB1 levels in subjects means a reduction to a level within the normal range for individuals accepted as not having such a condition. In some implementations, "lower" refers to a reduction in the difference between the level of a marker or symptom in a subject with the disease and the level accepted as within the normal range for an individual. The term "reduced / decreased / fallen" can also be used in connection with normalizing the symptoms of a disease or condition, i.e., reducing the difference between the levels of a subject with CTNNB1-related symptoms and those of a normal subject without CTNNB1-related symptoms, bringing them closer to or to the levels of the normal subject. As used herein, "normal" is considered the upper limit of normal if the disease is associated with elevated symptom values. "Normal" is considered the lower limit of normal if the disease is associated with decreased symptom values.

[0173] As used herein, “prevention” or “avoidance” when referring to a disease, condition, or illness that can be treated or improved by reducing the expression of the CTNNB1 gene means a reduced likelihood that a subject will develop symptoms associated with such a disease, condition, or illness (e.g., CTNNB1-related conditions, such as cancer, such as symptoms of hepatocellular carcinoma). Failure to develop a disease, condition, or illness, or a reduction in the development of symptoms associated with such a disease, condition, or illness (e.g., a reduction of at least approximately 10% on a clinically accepted scale for the disease or condition), or a delay in the development of symptoms (e.g., a delay of days, weeks, months, or years), are considered effective prevention.

[0174] As used herein, the terms "β-catenin-associated disease" or "CTNNB1-associated disease" refer to a disease or condition caused by or associated with CTNNB1 gene expression or CTNNB1 protein production. The term "CTNNB1-associated disease" includes diseases, conditions, or illnesses that benefit from reduced CTNNB1 gene expression, replication, or protein activity. In some embodiments, CTNNB1-associated disease is cancer, such as hepatocellular carcinoma and colorectal cancer.

[0175] As used in this article, the term "cancer" refers to a group of cells exhibiting abnormally high levels of proliferation and growth. Cancer can be benign (also known as a benign tumor), premalignant, or malignant. Cancer cells can be solid cancer cells or leukemia cancer cells. As used in this article, the term "cancer growth" refers to the proliferation or growth of one or more cells that constitute cancer, resulting in a corresponding increase in the size or extent of the cancer.

[0176] Examples of cancer include, but are not limited to, carcinoma, lymphoma, germ cell tumor, sarcoma, myeloma, and leukemia. In some embodiments, cancer includes solid tumor cancers. In other embodiments, cancer includes hematologic cancers such as leukemia, lymphoma, or myeloma. More specific, non-limiting examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous non-small cell lung cancer), lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, renal cell carcinoma, hepatocellular carcinoma, hepatoblastoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, stomach cancer, melanoma, and various types of head and neck cancers (including head and neck squamous cell carcinoma).

[0177] In some implementations, CTNNB1-related disease is hepatocellular carcinoma (HCC). As used herein, the term “hepatocellular carcinoma” refers to a major type of primary liver cancer and is also a rare human tumor associated with viral factors. Chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) has been involved in approximately 80% of cases worldwide (Wang W et al., J Gastroenterol. 2017 Apr; 52(4):419-431). Gene mutations and aberrant activation of signal transduction pathways involved in cell proliferation, apoptosis, metabolism, splicing, and the cell cycle are known to promote the development of HCC. In particular, the Wnt / β-catenin signaling pathway is known to be activated in up to 50% of HCCs (Lee JM et al., Cancer Lett. 2014 Feb 1; 343(1):90-7; Vilchez V et al., World J Gastroenterol. 2016 Jan 14; 22(2):823-32). The Wnt / β-catenin pathway regulates a variety of cellular processes involved in the occurrence, growth, survival, migration, differentiation, and apoptosis of HCCs (Wang Z et al., Mol Clin Oncol. 2015 Jul; 3(4):936-940). Mutations in β-catenin have been identified in these tumors, and β-catenin mutations have been shown to affect the prognosis of HCC (Prange W et al., J Pathol. 2003;201:250-259; Torbenson M et al., Am J Clin Pathol. 2004;122:377-382).

[0178] In some implementations, CTNNB1-related disease is hepatocellular carcinoma (HCC).

[0179] In some implementations, hepatocellular carcinoma is advanced or metastatic hepatocellular carcinoma.

[0180] As used in this article, the term “hepatocellular carcinoma” refers to a major type of primary liver cancer and is also a rare human tumor with a viral etiology. Chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) has been involved in approximately 80% of cases worldwide (Wang W et al., J Gastroenterol. 2017 Apr; 52(4):419-431). Gene mutations and aberrant activation of signaling pathways involved in cell proliferation, apoptosis, metabolism, splicing, and the cell cycle are known to promote the development of HCC. In particular, the Wnt / β-catenin signaling pathway is known to be activated in up to 50% of HCC (Lee JM et al., Cancer Lett. 2014 Feb 1; 343(1):90-7; Vilchez V et al., World J Gastroenterol. 2016 Jan 14; 22(2):823-32). The Wnt / β-catenin pathway regulates multiple cellular processes involved in the occurrence, growth, survival, migration, differentiation, and apoptosis of HCC (Wang Z et al., Mol Clin Oncol. 2015 July; 3(4):936-940). Mutations in β-catenin have been identified in these tumors, and β-catenin mutations have been shown to affect the prognosis of HCC (Prange W et al., J Pathol. 2003;201:250-259; Torbenson M et al., Am J Clin Pathol. 2004;122:377-382).

[0181] In some implementations, the subject with hepatocellular carcinoma has HCC containing Wnt-pathway activation mutations.

[0182] As used in this article, a “Wnt-pathway activation mutation” is a mutation in a gene involved in the classical Wnt signaling pathway that results in an alteration in the normal function of the protein encoded by that gene.

[0183] WNT pathway activation mutations include, for example, mutations in any one or more of the following genes: Axin1, Axin2, APC, CTNNB1, RNF43, ZNRF3, RSPO1, RSPO2, RSPO3, and RSPO4.

[0184] In one implementation, HCC contains a mutation in Axin1.

[0185] The Axin1 gene encodes Axis repressor protein 1 (Axin 1), a cytoplasmic protein containing a G-protein signaling (RGS) domain as well as disheveled and axin (DIX) domains. Axin1 acts as a negative regulator of the wingless MMTV integration site family member 1 (WNT) signaling pathway and induces apoptosis.

[0186] The nucleotide and amino acid sequences of Axin1 are known and can be found in one or more of the following public databases, for example: HGNC: 903, NCBI Gene: 8312, Ensembl: ENSG00000103126, OMIM®: 603816, UniProtKB / Swiss-Prot: O15169.

[0187] Mutations in the Axin1 protein that activate the classical Wnt pathway found in HCC include, for example, D94A, L106R, R146X, F201C, P263T, P345L, G425S, E465X, D495E, A526V, G625X, G651S, R841Q, P849T, G289fs413X, G613fs710X, and D341fs413X.

[0188] Mutations in the Axin1 gene that activate the classical Wnt pathway found in HCC include, for example, A393C, T429G, T704G, C1146T, G1385A, G2063A, C2657A, a 1807-frameshift deletion of 8 bp, a 1076-frameshift deletion of 1 bp, and a 1714-frameshift insertion of 12 bp that leads to QVHH insertion.

[0189] The Axin2 gene encodes Axis repressor protein 2 (Axin 2), also known as conductor. Axin 2 plays an important role in regulating β-catenin stability in the Wnt signaling pathway. Like Axin 1, Axin 2 acts as a scaffold protein to help assemble the β-catenin disruption complex and negatively regulates β-catenin-dependent Wnt signaling.

[0190] The nucleotide and amino acid sequences of Axin2 are known and can be found in one or more of the following public databases, for example: HGNC: 904, NCBI Gene: 8313, Ensembl: ENSG00000168646, OMIM®: 604025, UniProtKB / Swiss-Prot: Q9Y2T1.

[0191] Mutations in the Axin2 protein that activate the classical Wnt pathway found in HCC include, for example, E184D, E198X, R659W, D746N, and deletions of T672-R675.

[0192] Mutations in the Axin2 gene that activate the classical Wnt pathway found in HCC include, for example, C2064T and a 12 bp deletion at nucleotide position 2102.

[0193] The APC gene encodes adenomatous polyposis protein (APC) (also known as the APC regulator of the WNT signaling pathway), a tumor suppressor protein that acts as an antagonist of the Wnt signaling pathway. APC protein is a negative regulator that controls the concentration of β-catenin and interacts with E-cadherin, which is involved in cell adhesion.

[0194] The nucleotide and amino acid sequences of APCs are known and can be found in one or more of the following public databases, for example: HGNC: 583, NCBI Gene: 324, Ensembl: ENSG00000134982, OMIM®: 611731, UniProtKB / Swiss-Prot: P25054.

[0195] Mutations in APC proteins that activate the classical Wnt pathway found in HCC include, for example, V85I, E262X, R1158S, E1573X, and G1635R.

[0196] Mutations in the APC gene that activate the classical Wnt pathway found in HCC include, for example, the change of codon 208 from CAG (encoding glutamine) to TAG (stop codon) and the deletion of a single base pair at codon 568 (AGT to GT).

[0197] Mutations in the CTNNB1 protein that activate the classical Wnt pathway found in HCC include, for example, D32G, S33P, S33F, S33Y, S33C, GTS insertion of amino acid after amino acid position 33, G34E, G34V, G34R, G34V, S37Y, H36P, T41A, S45F, S45P, S45A, S45C (simultaneous deletion of amino acid residues 46 and 47), Q4SfsX3, T3-A126 deletion, L10-N141 deletion, V22-Y64 deletion, V22-D145 deletion, L31-I35 deletion, A5-AC80 deletion, A5-A80 deletion, and W25-I140 deletion.

[0198] CTNNB1 gene mutations that activate the classical Wnt pathway and have been found in HCC include, for example, A95G, T97C, C98T, G101A, G100A, A107C, C110A, A121G, and C134T.

[0199] The RNF43 gene encodes ring finger protein 43 (also known as the ring-type E3 ubiquitin transferase RNF43), which negatively regulates WNT signaling activation. It has been found that RNF43 deficiency causes impaired hepatocyte regeneration, leading to an imbalance in hepatocyte differentiation and proliferation, ultimately resulting in hepatocellular carcinoma.

[0200] The nucleotide and amino acid sequences of APCs are known and can be found in one or more of the following public databases, for example: HGNC: 18505, NCBI Gene: 54894, Ensembl: ENSG00000108375, OMIM®: 612482, UniProtKB / Swiss-Prot: Q68DV7.

[0201] Mutations in the RNF43 protein that activate the classical Wnt pathway found in HCC include, for example, R609L, Q344H, and G24V.

[0202] ZNRF3 is a gene encoding zinc finger and ring finger proteins 3, which participate in cellular protein metabolism and negatively regulate the Wnt signaling pathway. It has been found that ZNRF3 deficiency causes impaired hepatocyte regeneration, leading to an imbalance in hepatocyte differentiation and proliferation, which in turn results in hepatocellular carcinoma.

[0203] The nucleotide and amino acid sequences of APCs are known and can be found in one or more of the following public databases, for example: HGNC: 18126, NCBI Gene: 84133, Ensembl: ENSG00000183579, OMIM®: 612062, UniProtKB / Swiss-Prot: Q9ULT6.

[0204] ZNRF3 protein mutations that activate the classical Wnt pathway and have been found in HCC include, for example, C233S, E304X, C781R, P794R, G647G, R689R, and P793P.

[0205] The RSPO1, RSPO2, RSPO3, and RSPO4 genes encode the proteins R-spondin (RSPO) 1, RSPO2, RSPO3, and RSPO4, respectively. R-spondin (RSPO) proteins are a family of four secreted glycoproteins (RSPO1-4) and are pleiotropic signal transduction ligands. The most well-known molecular function of RSPO is its ability to agonize the Wnt / β-catenin signaling pathway. Tissue microarray analysis revealed increased RSPO2 expression in HCC, and overexpression of RSPO2 led to increased expression of phosphorylated STAT3, β-catenin, and c-Myc.

[0206] The nucleotide and amino acid sequences of RSPO1 are known and can be found in one or more of the following publicly available databases, such as HGNC: 21679, NCBI Gene: 284654, Ensembl: ENSG00000169218, OMIM®: 609595, UniProtKB / Swiss-Prot: Q2MKA7.

[0207] The nucleotide and amino acid sequences of RSPO2 are known and can be found in one or more of the following publicly available databases, such as HGNC: 28583, NCBI Gene: 340419, Ensembl: ENSG00000147655, OMIM®: 610575, UniProtKB / Swiss-Prot: Q6UXX9.

[0208] The nucleotide and amino acid sequences of RSPO3 are known and can be found in one or more of the following publicly available databases, for example: HGNC: 20866, NCBI Gene: 84870, Ensembl: ENSG00000146374, OMIM®: 610574, UniProtKB / Swiss-Prot: Q9BXY4.

[0209] The nucleotide and amino acid sequences of RSPO4 are known and can be found in one or more of the following publicly available databases, for example: HGNC: 16175, NCBI Gene: 343637, Ensembl: ENSG00000101282, OMIM®: 610573, UniProtKB / Swiss-Prot: Q2I0M5.

[0210] Mutations in the RSPO1 protein that activate the classical Wnt pathway found in HCC include, for example, C56S.

[0211] Mutations in the RSPO2 protein that activate the classical Wnt pathway found in HCC include, for example, gene rearrangement caused by a 46.4 kb microdeletion on chromosome 8q23.1, and R158S.

[0212] RSPO3 mutations that activate the classical Wnt pathway and have been found in HCC include, for example, EIF3E-RSPO2 and PTPRK-RSPO3 gene fusions.

[0213] Mutations in the RSPO4 protein that activate the classical Wnt pathway found in HCC include, for example, GLY67ARG.

[0214] In some implementations, CTNNB1-related disease is colorectal cancer.

[0215] In some implementations, colorectal cancer is colorectal cancer with liver metastases.

[0216] Mutations in CTNNB1 / β-catenin in colorectal cancer are typically found in its N-terminal domain, particularly exon 3, which carries multiple phosphorylation sites for CK1 and GSK3β, including amino acids Ser33, Ser37, Thr41, and Ser45 (Cancer Genome Atlas Network, 2012; Dar et al., 2017; Jamieson et al., 2011). Mutations or deletions at these sites (usually in only one allele) prevent phosphorylation of β-catenin, leading to its accumulation and subsequent activation of Wnt pathway target genes.

[0217] As used herein, “therapeutic effective amount” is intended to include an amount sufficient to treat the disease (e.g., by reducing, improving, or maintaining the existing disease or one or more symptoms of the disease) when an RNAi agent is administered to a subject with CTNNB1-related conditions. “Therapeutic effective amount” can vary depending on the RNAi agent, how it is administered, the disease and its severity, and the subject’s medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics.

[0218] As used herein, “preventive effective dose” is intended to include an amount sufficient to prevent or improve the disease or one or more symptoms of the disease when an RNAi agent is administered to a subject with CTNNB1-related disease. Improvement of the disease includes slowing disease progression or reducing the severity of later stages of the disease. “Preventive effective dose” can vary depending on the RNAi agent, how the agent is administered, the level of disease risk, and the subject’s medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics.

[0219] "Therapeutic effective dose" or "preventive effective dose" also includes a certain amount of RNAi agent that produces some desired therapeutic effect with a reasonable benefit / risk ratio in any applicable treatment. The iRNA used in the method of the present invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0220] The phrase “pharmaceutically acceptable” as used in this article refers to compounds, materials, compositions, or dosage forms that, within reasonable medical judgment, are suitable for use in human and animal subjects’ tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and have a reasonable benefit / risk ratio.

[0221] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, zinc stearate, or stearic acid), or solvent encapsulating material, for carrying or delivering the subject compound from one organ or body site to another. Each carrier must be "acceptable" in terms of compatibility with other components of the formulation and must not cause harm to the treated subject. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers for injection administration.

[0222] "Immunotherapy agents" refer to any agent that utilizes a subject's own immune system to recognize and kill cancer cells, thereby eliminating cancer cells. Exemplary immunotherapy agents include immune checkpoint inhibitors, monoclonal antibodies, vaccines, and T-cell transfer therapies, such as tumor-infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor (CAR) T-cell therapy.

[0223] "Immune checkpoint inhibitors" are a type of cancer immunotherapy that enhances the anti-cancer immune response by targeting immune receptors on the surface of T-lymphocytes and works to fight tumor cells by revitalizing the host's immune system. Immune checkpoints maintain a balance between pro-inflammatory and anti-inflammatory signaling under homeostatic conditions. These immune checkpoints are a group of inhibitory and stimulatory pathways that influence the activity of immune cells. Exemplary antibodies targeting immunosuppressive receptors include antibodies targeting CTLA-4, PD-1, and PD-L (e.g., PD-L1). Various antibodies and small molecule compounds targeting a variety of immune checkpoint proteins are in clinical development, including B7H3, CD39, CD73, adenosine A2A receptor, and CD47.

[0224] Exemplary checkpoint inhibitors used in this invention include atezolizumab (Tecentriq®), avelumab (Bavencio®), cimiprizumab (Libtayo®), dotalimumab (Jemperli), durvalumab (Imfinzi™), iplimumab (Yervoy®), nivolumab (Opdivo®), pembrolizumab (Keytruda®), julalimumab, rivanlimumab (Zynyz), and tesimumab (Imjudo®).

[0225] "Vascular endothelial growth factor (VEGF) inhibitors" are agents that block or inhibit tumor angiogenesis. Exemplary VEGF inhibitors used in this invention include, for example, anti-VEGF antibodies and tyrosine kinase inhibitors, such as axitinib, bevacizumab, bevacizumab-awwb, bevacizumab-bvzr, Cyramza, Fotivda, Inlyta, lenvatinib, lenvima, Mvasi, Nexavar, pazopanib, ramucirumab, cepretinib, serpatinib, sorafenib, sunitinib, surufatinib, sunitinib, tevozaniib, Votrient, and Zirabev.

[0226] Chemotherapy agents are chemical compounds used to treat cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide (Cytoxan®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; and acetogenins. (Especially bullatacin and bullatacinone; camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards include chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, and uracil. Mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1 (see Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;In addition to neocarzinostatin chromophores and related chromogenic proteins (ene diyne antibiotic chromophores), aclacinomycins, actinomycins, autramycins, azaserine, bleomycins, cactinomycin C, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin D, daunorubicin, detorubicin, 6-diazol-5-oxo-L-leucine, and Adriamycin® doxorubicin. (Including morpholine doxorubicin, cyanomorpholine doxorubicin, 2-pyrrole doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, and rodorubicin. Streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, and trimetrexate; and purine analogs, such as fludarabine, mercaptopurine, thiamiprine, and thioguanine.Pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenal agents, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as folinic acid; and aceglatone. Aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defosfamide; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan ; Lonidamine; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine;PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, roridin). A and anguidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, such as Taxol® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Abraxane® Cremophor-free, albumin-modified nanoparticle paclitaxel formulations (American Pharmaceutical Partners, Saborg, Illinois), and Taxotere® docetaxel (Rhone-Poulenc). Rorer (Antoine, France); chlorambucil; Gemzar® gemcitabine; thioguanine; mecaptopurine; methotrexate; platinum analogs, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Navelbine® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate;Irinotecan (Camptosar, CPT-11) (including irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; compretastatin; leucovorin (LV); oxaliplatin, including oxaliplatin regimens (FOLFOX); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)), and VEGF-A that reduce cell proliferation; and any pharmaceutically acceptable salt, acid, or derivative thereof.

[0227] Further non-limiting exemplary chemotherapeutic agents include: anti-hormonal agents that act to regulate or inhibit the effects of hormones on cancer, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trivoxifen, koxifen, LY117018, onapril ketone, and Fareston® toremifene; aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megase® medroxyprogesterone acetate, Aromasin® exemestane, formestanie, fatrazole, and Rivisor® vortexilamide. Femara® letrozole and Arimidex® anastrozole; and antiandrogens such as flutamide, nilumethamide, bicalutamide, leuprorelin and goserelin; and trisatabine (a 1,3-dioxolane cytosine analog); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Raf and H-Ras; ribozymes such as VEGF expression inhibitors (e.g., Angiozyme® ribozyme) and HER2 expression inhibitors; vaccines such as gene therapy vaccines, such as Allovectin® vaccine, Leuvectin® vaccine and Vaxid® vaccine; Proleukin® rIL-2; Lurtotecan® topoisomerase 1 inhibitor; Abarelix® rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0228] In some embodiments, the iRNA of the present invention may be further used with gemcitabine-based chemotherapy, wherein one or more chemotherapeutic agents comprising gemcitabine or comprising gemcitabine and nab-paclitaxel are administered. In some such embodiments, the iRNA of the present invention may be administered with at least one chemotherapeutic agent selected from gemcitabine, nab-paclitaxel, leucovorin (folic acid), 5-fluorouracil (5-FU), irinotecan, and oxaliplatin. FOLFIRINOX is a chemotherapy regimen comprising leucovorin, 5-FU, irinotecan (such as liposomal irinotecan injection), and oxaliplatin. In some embodiments, the iRNA of the present invention may be further administered with gemcitabine-based chemotherapy. In some embodiments, the iRNA of the present invention may be further administered with at least one agent selected from: (a) gemcitabine; (b) gemcitabine and nab-paclitaxel; and (c) FOLFIRINOX. In some embodiments, at least one agent is gemcitabine. In some of these implementations, the cancer to be treated is pancreatic cancer.

[0229] "Anti-angiogenic agents" or "angiogenic inhibitors" refer to a low molecular weight substance, polynucleotide (including, for example, repressive RNA (RNAi or siRNA)), polypeptide, isolated protein, recombinant protein, antibody, or conjugate or fusion protein thereof that directly or indirectly inhibits angiogenesis, vascularization, or undesirable vascular permeability. It should be understood that anti-angiogenic agents include those that bind to and block the angiogenic activity of angiogenic factors or their receptors. For example, anti-angiogenic agents are antibodies or other antagonists against angiogenesis, such as antibodies against VEGF-A (e.g., bevacizumab (Avastin®)) or antibodies against VEGF-A receptors (e.g., KDR receptors or Flt-1 receptors), anti-PDGFR inhibitors such as Gleevec® (imatinib mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787 / ZK2284, SU6668, Sutent® / SU11248 (sunitinib malate), AMG706, or, for example, those described in international patent application WO 2004 / 113304). Anti-angiogenic agents also include natural angiogenesis inhibitors such as angiostatin, endostatin, etc. See, for example, Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., Table 3 lists anti-angiogenic therapies in malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table 2 lists known anti-angiogenic factors); and Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1 lists anti-angiogenic agents used in clinical trials).

[0230] As used herein, “growth inhibitor” refers to a compound or composition that inhibits the growth of cells (such as cells expressing VEGF) in vitro or in vivo. Therefore, a growth inhibitor can be an inhibitor that significantly reduces the percentage of cells in the S phase (such as cells expressing VEGF). Examples of growth inhibitors include, but are not limited to, agents that block cell cycle progression (in non-S phase), such as those inducing G1 and M phase arrest. Classic M phase arrestors include vinca alkaloids (vincristine and vincristine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those that arrest G1 phase also cause S phase arrest, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, dichloromethyldiethylamine, cisplatin, methotrexate, 5-fluorouracil, and cytarabine. Further information can be found in Chapter 1 of *The Molecular Basis of Cancer*, edited by Mendelsohn and Israel, entitled “Cellcycle regulation, oncogenes, and antineoplastic drugs,” by Murakami et al. (WB Saunders, Philadelphia, 1995), for example, on page 13. Taxanes (paclitaxel and docetaxel) are two anticancer drugs derived from the yew tree. Docetaxel (Taxotere®, Rhone-Poulenc Rorer), derived from the European yew, is a semi-synthetic analogue of paclitaxel (Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote microtubule assembly from tubulin dimers and stabilize microtubules by preventing depolymerization, thereby inhibiting mitosis in cells.

[0231] The term "antitumor composition" refers to a composition for treating cancer that comprises at least one active therapeutic agent. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, cancer immunotherapy agents, apoptosis inducers, anti-microtubule agents, and other agents for treating cancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (Tarceva®)), platelet-derived growth factor inhibitors (e.g., Gleevec® (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMA, or VEGF receptors, and other biologically active and organic chemical agents. Combinations thereof are also included in this invention.

[0232] As used herein, the term "sample" includes a collection of similar bodily fluids, cells, or tissues isolated from a subject, as well as bodily fluids, cells, or tissues present within the subject. Examples of biological bodily fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, eye discharge, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs, or localized areas. For example, a sample may be derived from a specific organ, a portion of an organ, or bodily fluids or cells within those organs. In some embodiments, a sample may be derived from the liver (e.g., the entire liver or portions of the liver or certain types of cells in the liver, such as, for example, hepatocytes). In some embodiments, "sample derived from a subject" refers to urine obtained from the subject. "Sample derived from a subject" may refer to blood from the subject or blood-derived serum or plasma.

[0233] II. The prevention and treatment methods of the present invention

[0234] This invention provides a method for preventing or treating CTNNB1-related conditions, such as cancers like hepatocellular carcinoma and colorectal cancer, by using the iRNA of this invention or a composition containing the iRNA of this invention (e.g., a pharmaceutical composition). In the method of this invention, cells may be contacted with the siRNA in vitro or in vivo, i.e., the cells may be located within the body of a subject.

[0235] Treatment of subjects who would benefit from reduced and / or suppressed CTNNB1 gene expression includes therapeutic treatment (e.g., subjects with cancer) and preventative treatment (e.g., subjects without cancer or subjects at risk of developing cancer).

[0236] The in vivo method of the present invention may include administering to a subject a composition comprising iRNA, wherein the iRNA comprises a nucleotide sequence complementary to at least a portion of the RNA transcript of the CTNNB1 gene of the mammal to which the RNAi agent is to be administered. The composition may be administered in any manner known in the art, including but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, intraocular (e.g., periocular, conjunctival, subfascial, anterior chamber, vitreous, intraocular, anterior or posterior juxtascleral, subretinal, subconjunctival, retrobulbar, or tubular injection), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), and local (including buccal and sublingual) administration.

[0237] In some embodiments, the composition is administered via intravenous infusion or injection. In some embodiments, the composition is administered via subcutaneous injection. In some embodiments, the composition is administered via intramuscular injection.

[0238] In one implementation, the iRNA is administered subcutaneously, i.e., by subcutaneous injection. The desired dose of iRNA can be delivered to the subject using one or more injections. Injections can be repeated over a period of time.

[0239] Application can be repeated periodically. In some embodiments, treatment can be administered at a lower frequency basal level after the initial treatment regimen. Repeated dosing regimens may include periodic administration of therapeutic doses of iRNA, such as once monthly to once a year. In some embodiments, iRNA is administered approximately once monthly to approximately once every three months, or approximately once every three months to approximately once every six months.

[0240] The method of application can be selected based on whether local or systemic treatment is required, and based on the area to be treated. The route and site of application can be chosen to enhance targeting.

[0241] In some implementations, the RNAi agent is administered to the subject at an amount that effectively inhibits CTNNB1 expression in the subject's cells. The amount of CTNNB1 expression effectively inhibited in the subject's cells can be assessed using the methods discussed above, including methods involving assessing inhibition of CTNNB1 mRNA, CTNNB1 protein, or related variants (such as tumorigenesis).

[0242] The iRNA of this invention can be administered as "free iRNA". Free iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA can be present in a suitable buffer solution. The buffer solution may contain acetate, citrate, proline, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and osmotic pressure of the iRNA-containing buffer solution can be adjusted to suit its administration to the subject.

[0243] Alternatively, the iRNA of the present invention can be administered as a pharmaceutical composition, such as a dsRNA liposome formulation.

[0244] Subjects who benefit from inhibiting CTNNB1 gene expression are those who are susceptible to or have been diagnosed with CTNNB1-related conditions, such as cancers, like hepatocellular carcinoma. In one embodiment, the method includes administering the composition particularly described herein to reduce the expression of the target CTNNB1 gene, such as for each dose for approximately 1, 2, 3, 4, 5, 6, 1-6, 1-3, or 3-6 months. In some embodiments, the composition is administered every 3-6 months.

[0245] In one embodiment, the iRNA used in the methods and compositions particularly described herein specifically targets the target RNA (primary or processed RNA) of the CTNNB1 gene. Compositions and methods for inhibiting the expression of these genes using iRNA can be prepared and implemented as described herein.

[0246] Therefore, in one aspect, the present invention provides a method for treating a subject suffering from cancer. The method comprises administering to the subject a double-stranded ribonucleic acid (dsRNA) agent at a dose of about 0.01 mg / kg to about 1.5 mg / kg to inhibit the expression of β-catenin (CTNNB1), wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand differs from the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3' by no more than 4 bases, and the antisense strand differs from the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc-3' by no more than 4 bases. The bases, wherein a, g, c, and u are 2'-O-methyl(2'-OMe)adenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; Af, Gf, Cf, and Uf are 2'-fluoroadenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; s is a thiophosphate bond; VP is a vinylphosphonate; dT is 2'-deoxythymidine-3'-phosphate; dG is 2'-deoxyguanosine-3'-phosphate; and dA is 2'-deoxyadenosine-3'-phosphate, are used to treat subjects with cancer.

[0247] In another aspect, the present invention provides a method for treating a subject suffering from cancer. The method includes selecting a subject suffering from cancer containing a Wnt-pathway activating mutation and administering to the subject a double-stranded RNA (dsRNA) agent at a dose of about 0.01 mg / kg to about 1.5 mg / kg to inhibit the expression of β-catenin (CTNNB1), wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand differs from the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3' by no more than 4 bases, and the antisense strand differs from the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc The -3' difference is no more than 4 bases, where a, g, c, and u are 2'-O-methyl(2'-OMe)adenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; Af, Gf, Cf, and Uf are 2'-fluoroadenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; s is a thiophosphate bond; VP is a vinylphosphonate; dT is 2'-deoxythymidine-3'-phosphate; dG is 2'-deoxyguanosine-3'-phosphate; and dA is 2'-deoxyadenosine-3'-phosphate, thereby treating subjects with cancer.

[0248] In some implementations, CTNNB1-related conditions are cancer.

[0249] Examples of cancer include, but are not limited to, carcinoma, lymphoma, germ cell tumor, sarcoma, myeloma, and leukemia. In some embodiments, cancer includes solid tumor cancers. In other embodiments, cancer includes hematologic cancers such as leukemia, lymphoma, or myeloma. More specific, non-limiting examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous non-small cell lung cancer), lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, renal cell carcinoma, hepatocellular carcinoma, hepatoblastoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, stomach cancer, melanoma, and various types of head and neck cancers (including head and neck squamous cell carcinoma).

[0250] In some implementations, the cancer is hepatocellular carcinoma.

[0251] In one embodiment, the hepatocellular carcinoma comprises Wnt pathway activation mutations, for example, mutations in genes selected from the group consisting of Axin1, Axin2, APC, CTNNB1, RNF43, ZNRF3, RSPO1, RSPO2, RSPO3, and RSPO4, and combinations thereof. In some embodiments, the hepatocellular carcinoma is advanced or metastatic hepatocellular carcinoma.

[0252] In some implementations, the cancer is colorectal cancer.

[0253] In some embodiments, the colorectal cancer is colorectal cancer with liver metastases.

[0254] Subjects with cancer (such as hepatocellular carcinoma (HCC) containing Wnt-pathway activating mutations) can be identified and selected using any method known in the art, including those described in the Examples section of this document. For example, such subjects can be selected using nucleotide sequencing, such as next-generation sequencing (NGS), of Wnt-pathway genes (e.g., Axin1, Axin2, APC, CTNNB1, RNF43, ZNRF3, RSPO1, RSPO2, RSPO3, and RSPO4) present in samples obtained from the subject (e.g., tissue or biological fluid samples). Examples of suitable samples for this purpose include biopsy samples obtained from the subject, such as core needle biopsy samples of tumors (e.g., HCC), or biological fluid samples obtained from the subject (e.g., blood samples, liver fluid samples, urine samples containing circulating tumor DNA (ctDNA)).

[0255] The present invention further provides methods and uses of iRNA agents or pharmaceutical compositions thereof for treating subjects who would benefit from reduced and / or suppressed CTNNB1 gene expression (e.g., subjects with CTNNB1-related conditions), wherein the iRNA agents or pharmaceutical compositions thereof are used in combination with other drugs and / or other treatments (e.g., known drugs and / or known treatments, such as methods currently used to treat these conditions).

[0256] Therefore, in some aspects of the invention, the method of administering the iRNA agent of the invention further includes administering one or more treatments and / or one or more additional therapeutic agents to the subject.

[0257] For example, in some embodiments, the iRNA targeting CTNNB1 is administered in combination with an agent, for example, for treating CTNNB1-related conditions. Exemplary adjunctive therapies and treatments for treating CTNNB1-related conditions (e.g., cancer) may include surgery, immunotherapy, chemotherapy, radiation therapy, or administration of one or more adjunctive anticancer agents, such as immunotherapeutic agents and / or VEGF inhibitors, chemotherapeutic agents, growth inhibitors, antiangiogenic agents, and / or antitumor compositions. Non-limiting examples of anticancer agents, immunotherapeutic agents, VEGF inhibitors, chemotherapeutic agents, growth inhibitors, antiangiogenic agents, and antitumor compositions that may be used in combination with the iRNA of the present invention are as follows.

[0258] In one implementation, the iRNA targeting CTNNB1 is administered in combination with an immunotherapeutic agent.

[0259] In one embodiment, the method further includes administering to the subject an immunotherapeutic agent (e.g., one or more checkpoint inhibitors, such as one or more anti-programmed death-1 (PD-1) antibodies or antigen-binding fragments thereof, anti-programmed death-ligand 1 (PD-L1) antibodies or antigen-binding fragments thereof, and anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibodies or antigen-binding fragments thereof), and / or a combination of VEGF inhibitors.

[0260] In some implementations, the iRNA targeting CTNNB1 is administered in combination with an anti-programmed death-1 (PD-1) antibody or an antigen-binding fragment thereof. The anti-PD-1 antibody may be a humanized monoclonal antibody or an antigen-binding fragment thereof, such as pembrolizumab.

[0261] Pembrolizumab (Keytruda®) is a humanized IgG4 monoclonal antibody targeting programmed death receptor-1 (PD-1). Upon administration, pembrolizumab binds to PD-1 (an inhibitory signaling receptor expressed on the surface of activated T cells) and blocks the binding and activation of its ligands, leading to an activated T cell-mediated immune response against tumor cells. PD-1 ligands include programmed cell death ligand 1 (PD-L1) (overexpressed on some cancer cells) and programmed cell death ligand 2 (PD-L2) (primarily expressed on antigen-presenting cells). Activated PD-1 negatively regulates T cell activation and plays a crucial role in tumor evasion of the host immune system.

[0262] Pembrolizumab has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, urothelial carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) cancer, colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma, endometrial cancer, tumor mutation burden-high (TMB-H) cancer, squamous cell carcinoma of the skin, and triple-negative breast cancer.

[0263] In some embodiments, pembrolizumab is administered at doses of about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, or about 400 mg.

[0264] In some embodiments, pembrolizumab is administered at a dose of about 100 mg. In some embodiments, pembrolizumab is administered at a dose of about 200 mg. In some embodiments, pembrolizumab is administered at a dose of about 300 mg. In some embodiments, pembrolizumab is administered at a dose of about 400 mg.

[0265] In some implementations, pembrolizumab is administered every three weeks.

[0266] In some implementations, pembrolizumab is administered at a dose of approximately 100 mg every three weeks.

[0267] In some implementations, pembrolizumab is administered at a dose of approximately 200 mg every three weeks.

[0268] In some implementations, pembrolizumab is administered at a dose of approximately 400 mg every six weeks.

[0269] In some embodiments of the method of the present invention, pembrolizumab is administered to the subject approximately every three weeks for 12 weeks or longer. In other embodiments, pembrolizumab is administered to the patient approximately every three weeks for 18 weeks or longer, 24 weeks or longer, 30 weeks or longer, 36 weeks or longer, 42 weeks or longer, 48 weeks or longer, 54 weeks or longer, 60 weeks or longer, 66 weeks or longer, 72 weeks or longer, 78 weeks or longer, 84 weeks or longer, 90 weeks or longer, 96 weeks or longer, or 102 weeks or longer.

[0270] In some embodiments, pembrolizumab is administered to the subject intravenously. In some embodiments, pembrolizumab is administered via intravenous infusion. In one embodiment, pembrolizumab is administered via intravenous infusion for a duration between 25 and 40 minutes, or approximately 30 minutes.

[0271] The heavy chain amino acid sequence of pembrolizumab is: QVQLVQSGVE VKKPGASVKV SCKASGYTFTNYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRDYRFDMGFDYW GQGTTVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK.

[0272] The light chain amino acid sequence of pembrolizumab is: EIVLTQSPAT LSLSPGERAT LSCRASKGVSTSGYSYLHWY QQKPGQAPRL LIYLASYLES GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPLTFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQDSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC.

[0273] Therefore, in one aspect, the present invention provides a method for treating a subject suffering from cancer. The method includes administering to the subject a double-stranded ribonucleic acid (dsRNA) agent at a dose of about 0.01 mg / kg to about 1.5 mg / kg to inhibit the expression of β-catenin (CTNNB1), and an anti-programmed death-1 (PD-1) antibody at a dose of about 200 mg, thereby treating the subject suffering from cancer.

[0274] In another aspect, the present invention provides a method for treating a subject with cancer. The method includes selecting a subject with cancer containing a Wnt-pathway activating mutation and administering to the subject a double-stranded ribonucleic acid (dsRNA) agent at a dose of about 0.01 mg / kg to about 1.5 mg / kg to inhibit the expression of β-catenin (CTNNB1), and an anti-programmed death-1 (PD-1) antibody or its antigen-binding fragment at a dose of about 200 mg, thereby treating the subject with cancer. In some embodiments, the cancer is hepatocellular carcinoma.

[0275] In one embodiment, hepatocellular carcinoma includes Wnt pathway activation mutations, for example, mutations in genes selected from the group consisting of Axin1, Axin2, APC, CTNNB1, RNF43, ZNRF3, RSPO1, RSPO2, RSPO3, and RSPO4 and combinations thereof.

[0276] Subjects with cancer, such as hepatocellular carcinoma (HCC) containing Wnt-pathway activating mutations, can be identified and selected as described above.

[0277] In some implementations, hepatocellular carcinoma is advanced or metastatic hepatocellular carcinoma.

[0278] In some implementations, the cancer is colorectal cancer.

[0279] In some implementations, colorectal cancer is colorectal cancer with liver metastases.

[0280] In one embodiment, the method further includes administering additional treatment (e.g., radiotherapy) and / or a therapeutic agent to the subject, such as a therapeutic agent selected from the group consisting of: immunotherapeutic agents, VEGF inhibitors, chemotherapeutic agents, growth inhibitors, anti-angiogenic agents, antitumor compositions, and any one or more combinations thereof, for the treatment of cancer.

[0281] In one implementation, the additional therapeutic agent is an immunotherapy agent.

[0282] In one embodiment, the method further includes administering to the subject a combination of immunotherapeutic agents (e.g., one or more checkpoint inhibitors, such as one or more anti-programmed death-1 (PD-1) antibodies or antigen-binding fragments thereof, anti-programmed death-ligand 1 (PD-L1) antibodies or antigen-binding fragments thereof, and anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibodies or antigen-binding fragments thereof) and / or VEGF inhibitors.

[0283] iRNA and the adjunctive therapeutic agent may be administered simultaneously and / or in the same combination, such as parenteral administration, or the adjunctive therapeutic agent may be administered as part of a separate composition, or at different times and / or by other methods known in the art or described herein.

[0284] iRNA agents and adjunctive therapeutic agents and / or treatments may be administered simultaneously and / or in the same combination, such as parenteral administration, or the adjunctive therapeutic agents may be administered as part of a separate composition, or at different times and / or by other methods known in the art or described herein.

[0285] III. Methods to Inhibit CTNNB1 Expression

[0286] The present invention also provides a method for inhibiting the expression of the CTNNB1 gene in cells. The method comprises contacting cells with an RNAi agent (e.g., a double-stranded RNA agent) in an amount that effectively inhibits CTNNB1 expression in cells, thereby inhibiting CTNNB1 expression in the cells. In some embodiments of this disclosure, the expression of the CTNNB1 gene is preferentially inhibited in the liver (e.g., hepatocytes).

[0287] Cell contact with iRNA (e.g., a double-stranded RNA agent) can be performed in vitro or in vivo. In vivo contact with iRNA includes contacting cells or cell populations within a subject (e.g., a human subject) with the iRNA. Combinations of in vitro and in vivo cell contact methods are also possible. As discussed above, cell contact can be direct or indirect. Furthermore, cell contact can be achieved via targeting ligands, including any ligands described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, such as the GalNAc3 ligand, or any other ligand that directs the RNAi agent to the site of interest.

[0288] As used herein, the term “inhibition” is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing,” and other similar terms, and includes any level of inhibition.

[0289] The phrase "inhibit CTNNB1 expression" refers to the suppression of the expression of any CTNNB1 gene (such as the mouse CTNNB13 gene, rat CTNNB1 gene, monkey CTNNB1 gene, or human CTNNB1 gene) and variants or mutants of the CTNNB1 gene. Therefore, in the context of gene-manipulated cells, cell populations, or organisms, the CTNNB1 gene can be the wild-type CTNNB1 gene, the mutant CTNNB1 gene, or the transgenic CTNNB1 gene.

[0290] "Inhibition of CTNNB1 gene expression" includes any level of CTNNB1 gene repression, such as at least partial inhibition of CTNNB1 gene expression. CTNNB1 gene expression can be assessed based on the level or changes in the level of any variable associated with CTNNB1 gene expression, such as CTNNB1 mRNA levels or CTNNB1 protein levels. It should be understood that CTNNB1 is primarily expressed in the liver.

[0291] CTNNB1 expression can also be indirectly assessed based on other variables associated with CTNNB1 gene expression, such as the level of β-catenin expression in the cytoplasm, the nuclear localization of β-catenin, or the expression of certain target genes (such as Jun, c-Myc, and Cyclin D-1) or other oncogenes controlled by β-catenin transcription.

[0292] Inhibition can be assessed by a reduction in the absolute or relative levels of one or more variables associated with CTNNB1 expression compared to control levels. Control levels can be any type of control level used in the art, such as baseline levels before administration, or levels measured from similar subjects, cells, or samples that are untreated or treated with controls (such as buffer-only controls or non-active agent controls).

[0293] In some embodiments of the method of the present invention, the expression of the CTNNB1 gene is suppressed by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or suppressed to below the assayed level. In some embodiments, the expression of the CTNNB1 gene is suppressed by at least 70%. Furthermore, it should be understood that it may be desirable to suppress CTNNB1 expression in some tissues (e.g., the liver) without significantly suppressing expression in other tissues (e.g., the brain). In some embodiments, the expression level is determined using the assay method provided in Example 2, in a suitable species-matched cell line, using a 10 nM siRNA concentration.

[0294] In some embodiments, the inhibition of expression in vivo is determined by knocking down the human gene in rodents expressing the human gene (e.g., mice infected with AAV that express the human target gene (i.e., CTNNB1), for example, when administered as a single dose of 3 mg / kg, for example, at the point of nadir of RNA expression. Knockdown of endogenous gene expression in model animal systems can also be determined, for example, after a single dose of 3 mg / kg, at the point of nadir of RNA expression. Such systems are useful when the nucleic acid sequences of the human gene and the model animal gene are sufficiently similar that human iRNA provides effective knockdown of the model animal gene. RNA expression in the liver was determined using the PCR method provided in Example 2.

[0295] Inhibition of CTNNB1 gene expression can be manifested by a reduction in the amount of mRNA expressed in a first cell or cell population (which may be present, for example, in a sample derived from a subject) that is substantially identical to the first cell or cell population but not so treated (control cells, untreated with iRNA, or untreated with iRNA targeting the gene of interest), where the CTNNB1 gene is transcribed and has been treated (e.g., by contacting the cells with the iRNA of the present invention, or by applying the iRNA of the present invention to a subject in which the cells are located or were present). In some embodiments, inhibition is assessed by using a concentration of 10 nMsiRNA in a species-matched cell line, expressed as a percentage of the mRNA level in the treated cells, using the following formula:

[0296]

[0297] In other embodiments, inhibition of CTNNB1 gene expression can be assessed by reducing parameters related to CTNNB1 gene expression function (e.g., CTNNB1 protein levels in the blood or serum of a subject). CTNNB1 gene silencing can be performed in any cells that express CTNNB1 endogenously or heterologously from an expression vector, and can be determined by any assay known in the art.

[0298] Inhibition of CTNNB1 protein expression can be manifested as a decrease in the level of CTNNB1 protein expressed in cells or cell populations or in subject samples (e.g., protein levels in blood samples from subjects). As explained above, for the assessment of mRNA inhibition, inhibition of protein expression levels in treated cells or cell populations can similarly be expressed as a percentage of protein levels in control cells or cell populations, or a change in protein levels in subject samples (e.g., blood or serum derived therefrom).

[0299] Control cells, cell populations, or subject samples that can be used to evaluate CTNNB1 gene expression inhibition include cells, cell populations, or subject samples that have not yet been exposed to the RNAi agent of the present invention. For example, control cells, cell populations, or subject samples may be derived from individual subjects (e.g., human or animal subjects) prior to treatment with the RNAi agent or from appropriately matched population controls.

[0300] The level of CTNNB1 mRNA expressed in cells or cell populations can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the expression level of CTNNB1 in a sample is determined by detecting the transcribed polynucleotide or a portion thereof (e.g., the mRNA of the CTNNB1 gene). RNA can be extracted from cells using RNA extraction techniques, including, for example, extraction using acid phenol / guanidine isothiocyanate (RNAzol B; Biogenesis), RNeasy... TM RNA Preparation Kit (Qiagen®) or PAXgene TM (PreAnalytix TM (Switzerland). Typical assays using ribonucleic acid hybridization include nuclear ligation assays, RT-PCR, ribonuclease protection assays, Northern blotting, in situ hybridization, and microarray analysis.

[0301] In some implementations, the expression level of CTNNB1 is determined using a nucleic acid probe. As used herein, the term "probe" refers to any molecule capable of selectively binding to a specific CTNNB1. Probes may be synthesized by those skilled in the art or may be derived from suitable biological products. Probes may be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0302] The isolated mRNA can be used for hybridization or amplification assays, including but not limited to Southern or Northern blotting, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) capable of hybridizing to CTNNB1 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and then transferring the mRNA from the gel to a membrane, such as a nitrocellulose membrane. In another embodiment, the probe is immobilized on a solid surface and the mRNA is contacted with the probe, for example, in an Affymetrix® gene chip array. Those skilled in the art can readily adapt known mRNA detection methods for determining CTNNB1 mRNA levels.

[0303] Alternative methods for determining CTNNB1 expression levels in samples involve processes such as nucleic acid amplification or reverse transcription (for preparing cDNA) of mRNA in the sample, for example by RT-PCR (experimental implementation described by Mullis, 1987, US Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-persistent sequence replication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al., (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-β replicase (Lizardi et al., (1988) Bio / Technology). The expression level of CTNNB1 can be determined by a method such as rolling circle replication (Lizardi et al., U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by detection of the amplified molecule using techniques well known to those skilled in the art. These detection protocols are particularly useful for detecting nucleic acid molecules if such molecules are present in very low amounts. In a particular aspect of the invention, the expression level of CTNNB1 is determined by quantitative fluorescent RT-PCR (i.e., the TaqMan™ system). In some embodiments, the expression level is determined by the method provided in Example 2, for example, using a 10 nM siRNA concentration in species-matched cells.

[0304] The expression level of CTNNB1 mRNA can be monitored using membrane blotting (such as Northern, Southern, dot hybridization, etc. for hybridization analysis) or microplates, sample tubes, gels, beads, or fibers (or any solid carrier containing bound nucleic acids). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. The determination of CTNNB1 expression levels may also involve the use of nucleic acid probes in solution.

[0305] In some embodiments, branched DNA (bDNA) assays or real-time PCR (qPCR) are used to assess mRNA expression levels. The use of these methods is described and illustrated in the examples provided herein. In some embodiments, expression levels are determined in species-matched cell lines using a 10 nM siRNA concentration, as described in Example 2.

[0306] The expression level of CTNNB1 protein can be determined using any method known in the art for measuring protein levels. Such methods include, but are not limited to, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), ultradiffusion chromatography, liquid or gel precipitation reaction, absorption spectroscopy, colorimetric assay, spectrophotometry, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc.

[0307] In some embodiments, the efficacy of the method of the present invention is assessed by a reduction in CTNNB1 mRNA or protein levels (e.g., in liver biopsy).

[0308] In some embodiments, the efficacy of the method of the present invention can be monitored by detecting or monitoring a reduction in tumor formation. As used herein, “reduction in tumor” includes any reduction in tumor size, number, or severity, or prevention or reduction of tumor formation in the subject’s tissues, which can be assessed in vitro or in vivo using any method known in the art.

[0309] In some embodiments of the method of the present invention, iRNA is administered to a subject, such that the iRNA is delivered to a specific site within the subject's body. Inhibition of CTNNB1 expression can be assessed using measurements of CTNNB1 mRNA or CTNNB1 protein levels or changes in samples of bodily fluids or tissues from a specific site within the subject's body (e.g., liver or blood).

[0310] As used herein, the terms "detecting" or "determining" an analyte level are understood to refer to the steps of performing a determination of the presence of a substance (e.g., protein, RNA). As used herein, a detection or determination method includes detecting or determining analyte levels below the detection level of the method used.

[0311] V. The iRNA of the present invention

[0312] This invention provides an iRNA for inhibiting CTNNB1 gene expression. In some embodiments, the iRNA comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the CTNNB1 gene in cells such as subjects, mammals, or cells in humans prone to developing CTNNB1-related diseases (e.g., cancers, such as hepatocellular carcinoma). The dsRNA iRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during CTNNB1 gene expression. The complementary region is about 19-30 nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length).

[0313] When contacted with cells expressing the CTNNB1 gene, the iRNA inhibits the expression of the CTNNB1 gene (e.g., human, primate, non-primate, or rat CTNNB1 gene) by at least about 50%, as determined by, for example, PCR or branched DNA (bDNA)-based methods, or by protein-based methods (such as immunofluorescence analysis using, for example, Western blotting or flow cytometry). In some embodiments, inhibition of expression is determined by the qPCR method provided in the embodiments herein, using, for example, a concentration of 10 nM siRNA in a suitable somatic cell line provided therein. In some embodiments, inhibition of in vivo expression is determined by knocking down the human gene in rodents expressing human genes (e.g., mice expressing human target genes or mice infected with AAV), for example, when administered at a single dose, for example, 3 mg / kg, at the point of lowest RNA expression.

[0314] dsRNA comprises two complementary RNA strands that hybridize to form a duplex structure under the conditions in which the dsRNA will be used. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially complementary to, and usually perfectly complementary to, the target sequence. The target sequence may be derived from the mRNA sequence formed during the expression of the CTNNB1 gene. The other strand (the sense strand) contains a region complementary to the antisense strand, such that, under suitable conditions, the two strands hybridize to form a duplex structure. As described elsewhere in this document and as is known in the art, the complementary sequence of the dsRNA may also be present in the self-complementary region of a single nucleic acid molecule, rather than in a single oligonucleotide.

[0315] Typically, the length of a double-stranded structure is 15 to 30 base pairs, for example, lengths of 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30. 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. In some embodiments, the length of the double-stranded structure is 18 to 25 base pairs, for example, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, or 24-25 base pairs, for example, 19-21 base pairs. Intermediate ranges and lengths between the above ranges and lengths are also considered part of this disclosure.

[0316] Similarly, the region complementary to the target sequence is 15 to 30 nucleotides in length, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28. 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides, for example, a length of 19-23 nucleotides or a length of 21-23 nucleotides. Intermediate ranges and lengths between the above ranges and lengths are also considered part of this disclosure.

[0317] In some implementations, the length of the double-stranded structure is 19 to 30 base pairs. Similarly, the region complementary to the target sequence is 19 to 30 nucleotides in length.

[0318] In some embodiments, the dsRNA is about 19 to about 23 nucleotides long, or about 25 to about 30 nucleotides long. Typically, the dsRNA is long enough to be used as a substrate for the Dicer enzyme. For example, as is well known to those skilled in the art, dsRNA longer than about 21-23 nucleotides can be used as a substrate for Dicer. As will be recognized by those skilled in the art, the region of RNA targeted for cleavage is most often a larger RNA molecule, typically a portion of an mRNA molecule. In relevant cases, a “portion” of the mRNA target is a contiguous sequence of the mRNA target that is long enough to allow it to be a substrate for RNAi-guided cleavage (i.e., cleavage via the RISC pathway).

[0319] Those skilled in the art will also recognize that the double-stranded region is the major functional part of the dsRNA, for example, the double-stranded region is about 19 to about 30 base pairs, such as about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 base pairs. Therefore, in one embodiment, where the double-stranded region is processed into a functional double-stranded structure (e.g., 15-30 base pairs) and targeted for cleavage of the desired RNA, the RNA molecule or RNA molecule complex having a double-stranded region greater than 30 base pairs is dsRNA. Thus, those skilled in the art will recognize that in one embodiment, the miRNA is dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In yet another embodiment, the iRNA agent used to target CTNNB1 gene expression is not generated in the target cell by cleaving a large dsRNA.

[0320] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, such as 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs with at least one nucleotide overhang exhibit superior repressive properties relative to their blunt-terminal counterparts. The nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). The overhang may be located on the sense strand, antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide may be present at the 5' end, 3' end, or both ends of the antisense strand or sense strand of the dsRNA.

[0321] dsRNA can be synthesized using standard methods known in the art. The double-stranded RNAi compounds of the present invention can be prepared using a two-step method. First, single strands of the double-stranded RNA molecule are prepared separately. Then, the constituent strands are annealed. The single strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis, or both. Organic synthesis offers the advantage of readily preparing oligonucleotide chains containing non-natural or modified nucleotides. Similarly, the single-stranded oligonucleotides of the present invention can also be prepared using solution-phase or solid-phase organic synthesis, or both.

[0322] In one aspect, the dsRNA of the present invention comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand is selected from the group consisting of sequences provided in any of Tables 2, 3, 5, and 6, and the corresponding antisense strand of the sense strand is selected from the group consisting of sequences in any of Tables 2, 3, 5, and 6. In this aspect, one of the two sequences is complementary to the other of the two sequences, and one sequence is substantially complementary to the mRNA sequence generated during CTNNB1 gene expression. Therefore, in this aspect, the dsRNA will comprise two oligonucleotides, one of which is described as the sense strand in any of Tables 2, 3, 5, or 6, and the other oligonucleotide is described as the corresponding antisense strand of the sense strand in any of Tables 2, 3, 5, or 6.

[0323] In some embodiments, the substantially complementary sequence of the dsRNA is contained on different oligonucleotides. In other embodiments, the substantially complementary sequence of the dsRNA is contained on a single oligonucleotide.

[0324] It will be understood that, although the sequences in Table 2, for example, are not described as modified or coupled sequences, the RNA of the iRNA of the present invention (e.g., the dsRNA of the present invention) may comprise any of the sequences listed in any of Tables 2, 3, 5, or 6, which are unmodified, uncoupled, or modified or coupled in a manner different from that described herein. In other words, the present invention covers unmodified, uncoupled, modified, or coupled dsRNAs of Tables 2, 3, 5, or 6, as described herein. For example, although the positive strand of the agents of the present invention shown in Table 5 is coupled to the L96 ligand, these agents may also be uncoupled, as described herein.

[0325] As is well known to those skilled in the art, dsRNAs with a double-stranded structure of about 20 to 23 base pairs (e.g., 21 base pairs) have been shown to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA double-stranded structures can also be effective (Chu and Rana (2007) RNA14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, given the nature of the oligonucleotide sequences provided in any of Tables 2, 3, 5, or 6, the dsRNAs described herein may comprise at least one chain of at least 21 nucleotides in length. It is reasonable to expect that shorter double-stranded structures having any of the sequences in any of Tables 2, 3, 5, or 6, with only a few nucleotides reduced at one or both ends, will also be similarly effective compared to the dsRNAs described above. Therefore, the scope of this invention covers dsRNAs having a sequence of at least 19, 20 or more consecutive nucleotides, said sequence being derived from any of the sequences in any of Tables 2, 3, 5 or 6, and whose ability to inhibit CTNNB1 gene expression differs from that of dsRNAs containing the complete sequence by no more than about 5, 10, 15, 20, 25 or 30%.

[0326] Furthermore, the RNAs provided in Tables 2, 3, 5, or 6 identify sites in the CTNNB1 transcript susceptible to RISC-mediated cleavage. Therefore, this invention further describes iRNAs targeting one of these sites. As used herein, an iRNA is considered to target a specific site within the RNA transcript if it promotes cleavage of the transcript at any location within that specific site. Such iRNAs typically comprise at least about 19 consecutive nucleotides from any of the sequences provided in any of Tables 2, 3, 5, or 6, coupled with other nucleotide sequences derived from the CTNNB1 gene that are adjacent to the selected sequence.

[0327] VI. Modified iRNA of the present invention

[0328] In some embodiments, the RNA (e.g., dsRNA) of the iRNA of the present invention is unmodified and does not contain, for example, chemical modifications or conjugates known in the art and described herein. In other embodiments, the RNA (e.g., dsRNA) of the iRNA of the present invention is chemically modified to enhance stability or other beneficial properties. In some embodiments of the present invention, substantially all nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all or substantially all nucleotides of the iRNA are modified, i.e., no more than 5, 4, 3, 2, or 1 unmodified nucleotides are present in the iRNA chain.

[0329] The nucleic acids described in this invention can be synthesized or modified using methods recognized in the art, such as those described in "Currentprotocols in nucleic acid chemistry," Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, USA (which are incorporated herein by reference). Modifications include, for example, terminal modifications, such as 5' end modifications (phosphorylation, coupling, reverse linking) or 3' end modifications (coupling, DNA nucleotides, reverse linking, etc.); base modifications, such as base substitution with stable bases, unstable bases, or bases paired with a broad repertoire of partners, base removal (base-free nucleotides), or base coupling; sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; or backbone modifications, including modification or substitution of phosphodiester bonds. Specific examples of useful iRNA compounds in the embodiments described herein include, but are not limited to, RNAs containing a modified backbone or lacking natural nucleoside inter-linking. RNAs with a modified backbone particularly include RNAs whose backbone does not contain phosphorus atoms. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs whose internucleotide backbones do not contain phosphorus atoms may also be considered oligonucleotides. In some embodiments, the modified iRNA has phosphorus atoms in its internucleotide backbone.

[0330] Modified RNA backbones include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), hypophosphonates, phosphoramides (including 3'-aminophosphonamides and aminoalkylphosphonamides), thiophosphonamides, thioalkylphosphonates, thioalkyl phosphate triesters, and boron phosphates having normal 3'-5' linkages, their 2'-5' linkage analogs, and those with reverse polarity, wherein adjacent nucleoside unit pairs are linked by 3'-5' to 5'-3' or 2'-5'-2' linkages. Various salt, mixed salt, and free acid forms are also included. In some embodiments of the invention, the dsRNA agent of the invention is in free acid form. In other embodiments of the invention, the dsRNA agent of the invention is in salt form. In one embodiment, the dsRNA agent of the invention is in sodium salt form. In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ions are present in the agent as counterions to substantially all phosphodiester and / or thiophosphate groups present in the agent. Agents in which substantially all phosphodiester and / or thiophosphate bonds have sodium counterions include no more than 5, 4, 3, 2, or 1 phosphodiester and / or thiophosphate bonds without sodium counterions. In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ions are present in the agent as counterions to all phosphodiester and / or thiophosphate groups present in the agent.

[0331] Representative U.S. patents teaching the preparation of the aforementioned phosphorus-containing bonds include, but are not limited to, U.S. patent numbers 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5 ,321,131;5,399,676;5,405,939;5,453,496;5,455,233;5,466,677;5,476,925;5,519,126;5,536,821;5,541,316;5,550,111;5,563,253;5,571,799;5, 587,361;5,625,050;6,028,188;6,124,445;6,160,109;6,169,170;6,172,209;6,239,265;6,277,603;6,326,199;6,346,614;6,444,423;6,531,590;6,5 U.S. Patent RE39464, the entire contents of each of these patents are incorporated herein by reference.

[0332] Modified RNA backbones that do not contain phosphorus atoms have a backbone formed by short-chain alkyl or cycloalkyl nucleosides linked together, mixed heteroatoms linked together with alkyl or cycloalkyl nucleosides, or one or more short-chain heteroatoms or heterocyclic nucleosides linked together. These include backbones with morpholino groups (partially formed from the sugar moieties of nucleosides); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formyl and thioformyl backbones; methyleneformyl and thioformyl backbones; olefin-containing backbones; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and others with mixed N, O, S, and CH2 components.

[0333] Representative U.S. patents teaching the preparation of the aforementioned oligonucleotides include, but are not limited to, U.S. patent numbers 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5 The entire contents of each of the following articles are incorporated herein by reference: ,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

[0334] The iRNAs presented herein consider the use of suitable RNA mimics in which the sugar and nucleoside links (i.e., the backbone) of the nucleotide units are replaced by novel groups. The base units are retained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, which has shown excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and directly or indirectly linked to the nitrogen atom of the amide moiety of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are incorporated herein by reference. Other PNA compounds suitable for use in the iRNAs of this invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0335] Some embodiments described in this invention include RNAs having a thiophosphate backbone and oligonucleotides having a heteroatom backbone, and particularly the --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [referred to as a methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2— and --N(CH3)--CH2--CH2— of U.S. Patent No. 5,489,677, and the amide backbone of U.S. Patent No. 5,602,240, mentioned above. In some embodiments, the RNA described herein has the morpholino backbone structure of U.S. Patent No. 5,034,506, mentioned above. The native phosphodiester backbone can be represented as OP(O)(OH)-OCH2-.

[0336] Modified RNA may also contain one or more substituted sugar moieties. The iRNAs described herein (e.g., dsRNAs) may contain one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and ynyl groups. Exemplary suitable modifications include O[(CH2)] n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, where n and m are 1 to approximately 10. In other embodiments, the dsRNA contains one of the following at the 2' position: C1 to C 10 Lower alkyl groups, substituted lower alkyl groups, alkylaryl groups, aryl alkyl groups, O-alkylaryl or O-aryl alkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalating agents or groups used to improve the pharmacokinetic properties of iRNA, or groups used to improve the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy groups. Other exemplary modifications include 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the examples below; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH3)2. Further exemplary modifications include: 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (R and S isomers of these three classes); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0337] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluorine (2'-F). Similar modifications can also be made at other positions on the iRNA, particularly at the 3' position of the sugar at the 3' terminal nucleotide, or in the 2'-5' linked dsRNA and at the 5' position of the 5' terminal nucleotide. The iRNA can also use sugar mimics such as the cyclobutyl moiety to replace the pentofuranose. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. patent numbers 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which are common to this application. The entire contents of each of the foregoing are incorporated herein by reference.

[0338] iRNA may also contain nucleobase modifications or substitutions (often simply referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as deoxythymidine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halogenated uracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, Cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated (especially 5-bromine), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deadenine and 7-deadenine, and 3-deadenine and 3-deadenine. Further nucleobases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in modified nucleosides in *Biochemistry, Biotechnology and Medicine*, edited by Herdewijn, P., Wiley-VCH, 2008; those disclosed in *The Concise Encyclopedia Of Polymer Science and Engineering*, pp. 858-859, edited by Kroschwitz, J. L., John Wiley & Sons, 1990; those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed in Sanghvi, Y. S., Chapter 15, dsRNA Research and Applications, pp. 289-302, edited by Crooke, ST. and Lebleu, B., CRC Press, 1993. Some of these nucleobases are particularly useful for enhancing the binding affinity of the oligomeric compounds described in this invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-Methylcytosine substitution has been shown to improve the stability of nucleic acid duplexes by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B. eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278), and is a typical base substitution, even more so when combined with 2'-O-methoxyethyl sugar modification.

[0339] Representative U.S. patents teaching the preparation of certain modified nucleobases and other modified nucleobases include, but are not limited to, U.S. patent numbers 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,5 The entire contents of each of the following articles are incorporated herein by reference: 94,121; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088.

[0340] In some embodiments, the RNAi agents of this disclosure may also be modified to include one or more bicyclic sugar groups. A “bicyclic sugar” is a furanose ring modified by a ring formed by bridging two carbon atoms (whether adjacent or non-adjacent). A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a ring formed by bridging two carbon atoms (whether adjacent or non-adjacent) of the sugar ring, thereby forming a bicyclic ring system. In some embodiments, the bridging optionally connects the 4'-carbon and 2'-carbon of the sugar ring via a 2'-acyclic oxygen atom. Therefore, in some embodiments, the agents of the present invention may comprise one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety containing an additional bridging linking the 2' and 4' carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety containing a 4'-CH2-O-2' bridging. This structure effectively “locks” the ribose in a 3'-inner configuration. It has been shown that adding locked nucleic acids to siRNA improves the stability of siRNA in serum and reduces off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides used in the polynucleotides of this invention include, but are not limited to, nucleosides containing bridging between the 4' and 2' ribose ring atoms. In some embodiments, the antisense polynucleotides of this invention comprise one or more bicyclic nucleosides containing 4' to 2' bridging.

[0341] The structure of nucleosides can be represented as (stereochemical structure omitted):

[0342]

[0343] Where B is a nucleobase or modified nucleobase, and L is a linking group connecting the 2'-carbon and 4'-carbon of the ribose ring. Examples of such 4' to 2'-bridged bicyclic nucleosides include, but are not limited to: 4'-(CH2)—O-2' (LNA); 4'-(CH2)—S-2'; 4'-(CH2)2—O-2' (ENA); 4'-CH(CH3)—O-2' (also known as "restricted ethyl" or "cEt") and 4'-CH(CH2OCH3)—O-2' (and its analogues; see, for example, U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)—O-2' (and its analogues; see, for example, U.S. Patent No. 8,278,283); 4'-CH2—N(OCH3)-2' (and its analogues; see, for example, U.S. Patent No. 8,278,425); 4'-CH2—O—N(CH3)-2' (See, for example, U.S. Patent Publication No. 2004 / 0171570); 4'-CH2—N(R)—O-2', wherein R is H, C1-C12 alkyl, or a nitrogen-protecting group (see, for example, U.S. Patent No. 7,427,672); 4'-CH2—C(H)(CH3)-2' (see, for example, Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2—C(═CH2)-2' (and its analogues; see, for example, U.S. Patent No. 8,278,426). The entire contents of each of the foregoing are incorporated herein by reference.

[0344] Other representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; U.S. 2008 / 0039618; and U.S. 2009 / 0012281, the entire contents of which are incorporated herein by reference.

[0345] Any of the aforementioned bicyclic nucleosides can be prepared to have one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0346] The RNA of iRNA can also be modified to include one or more restricted ethyl nucleotides. As used herein, "restricted ethyl nucleotide" or "cEt" refers to a locked nucleic acid containing a bicyclic sugar moiety comprising a 4'-CH(CH3)-O-2' bridging (i.e., the L in the aforementioned structure). In one embodiment, the restricted ethyl nucleotide is in the S configuration, referred to herein as "S-cEt".

[0347] The iRNA of this invention may also comprise one or more "conformation-restricted nucleotides" ("CRNs"). A CRN is a nucleotide analog having a linker connecting the C2' and C4' carbons of the ribose or the C3 and -C5' carbons of the ribose. The CRN locks the ribose ring in a stable conformation and increases hybridization affinity with mRNA. The linker is of sufficient length to position the oxygen in the optimal position for stability and affinity, resulting in reduced ribose ring puckering.

[0348] Representative publications teaching certain CRN preparations mentioned above include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383 and PCT Publication No. WO 2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0349] In some embodiments, the iRNA of the present invention comprises one or more monomers that are UNA (non-locked nucleic acid) nucleotides. A UNA is a non-locked acyclic nucleic acid in which any bonds of the sugar have been removed, forming non-locked “sugar” residues. In one instance, the UNA also encompasses monomers in which the bond between C1' and C4' has been removed (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another instance, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, incorporated by reference).

[0350] Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publications Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0351] Potential stabilizing modifications to the ends of RNA molecules may include N-(acetaminohexanoyl)-4-hydroxyproline (Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyproline (Hyp-C6), N-(acetyl-4-hydroxyproline (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminohexanoyl)-4-hydroxyproline (Hyp-C6-amino), 2-eicosanoyl-uridine-3"-phosphate, and the reverse base dT (idT), etc. Public information on such modifications can be found in PCT Publication No. WO2011 / 005861.

[0352] Other nucleotide modifications to the iRNA of this invention include 5' phosphate or 5' phosphate mimics, such as the 5'-terminal phosphate or phosphate mimics on the antisense strand of the iRNA. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511 (the entire contents of which are incorporated herein by reference).

[0353] A. Modified iRNA containing the motif of this invention

[0354] In certain aspects of the invention, the double-stranded RNA agents of the present invention comprise chemically modified agents, for example, as disclosed in WO2013 / 075035 (the entire contents of which are incorporated herein by way of representation). As shown herein and in WO2013 / 075035, one or more motifs of three identical modifications on three consecutive nucleotides may be introduced into the sense or antisense strand of the dsRNAi agent, particularly at or near a cleavage site. In some embodiments, the sense and antisense strands of the dsRNAi agent may otherwise be completely modified. The introduction of these motifs disrupts the modification pattern of the sense or antisense strand (if present). The dsRNAi agent may optionally be coupled to a GalNAc derivative ligand, for example, on the sense strand.

[0355] More specifically, gene silencing activity of dsRNAi agents is observed when the sense and antisense strands of a double-stranded RNA agent are fully modified such that one or more motifs with three identical modifications on three consecutive nucleotides are present at or near the cleavage site of at least one strand of the dsRNAi agent.

[0356] Therefore, this invention provides a double-stranded RNA agent capable of inhibiting the expression of a target gene (i.e., the CTNNB1 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can be, for example, 17-30 nucleotides long, 25-30 nucleotides long, 27-30 nucleotides long, 19-25 nucleotides long, 19-23 nucleotides long, 19-21 nucleotides long, 21-25 nucleotides long, or 21-23 nucleotides long.

[0357] The sense and antisense strands typically form a double-stranded RNA (“dsRNA”), also referred to herein as a “dsRNAi agent.” The double-stranded region of a dsRNAi agent can be, for example, 27–30 nucleotide pairs, 19–25 nucleotide pairs, 19–23 nucleotide pairs, 19–21 nucleotide pairs, 21–25 nucleotide pairs, or 21–23 nucleotide pairs in length. In other instances, the length of the double-stranded region is selected from 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides.

[0358] In some embodiments, the dsRNAi agent may include one or more overhanging regions or capping groups at the 3' end, 5' end, or both ends of one or both strands. The length of the overhang may be independently 1-6 nucleotides, for example, 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, or 1-2 nucleotides. In some embodiments, the overhanging region may include extended overhanging regions as described above. The overhang may be due to one strand being longer than the other, or due to the misalignment of two strands of the same length. The overhang may form a mismatch with the target mRNA, or it may be complementary to the targeted gene sequence, or it may be other sequences. The first and second strands may also be linked, for example, by forming a hairpin with additional bases, or by other non-base linkers.

[0359] In some embodiments, the nucleotides in the dsRNAi agent hangover region may be independently modified or unmodified nucleotides, including but not limited to 2'-sugar modified nucleotides such as 2'-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof.

[0360] For example, TT can be a dangling sequence at either end of any strand. The dangling sequence may form a mismatch with the target mRNA, or it may be complementary to the targeted gene sequence, or it may be other sequences.

[0361] The 5' or 3' overhang of the sense strand, antisense strand, or both strands of the dsRNAi agent can be phosphorylated. In some embodiments, the overhang region comprises two nucleotides with a phosphate thioester between them, wherein the two nucleotides may be the same or different. In some embodiments, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In some embodiments, the 3' overhang is located in the antisense strand. In some embodiments, the 3' overhang is located in the sense strand.

[0362] dsRNAi agents may contain only a single overhang, which can enhance the interfering activity of RNAi without affecting its overall stability. For example, the single-stranded overhang can be located at the 3' end of the sense strand, or alternatively at the 3' end of the antisense strand. RNAi can also have a blunt end located at the 5' end of the antisense strand (i.e., the 3' end of the sense strand), or vice versa. Typically, the antisense strand of a dsRNAi agent has a nucleotide overhang at the 3' end and a blunt end at the 5' end. While it is not desirable to be bound by theory, the asymmetric blunt end at the 5' end of the antisense strand and the 3' overhang of the antisense strand are beneficial for guiding strand loading into the RISC process.

[0363] In some embodiments, the dsRNAi agent is a double-stranded blunt-ended strand of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0364] In other embodiments, the dsRNAi agent is a double-stranded blunt-ended strand of 20 nucleotides in length, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0365] In another embodiment, the dsRNAi agent is a double-stranded blunt-ended strand of 21 nucleotides in length, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0366] In some embodiments, the dsRNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, wherein one end of the RNAi agent is blunt, and the other end comprises a 2-nucleotide overhang. In one embodiment, the 2-nucleotide overhang is located at the 3' end of the antisense strand.

[0367] When two nucleotides are dangling at the 3' end of the antisense strand, two phosphate-thionucleotide inter-linkages may exist between the three terminal nucleotides, where two of the three nucleotides are dangling nucleotides and the third nucleotide is a pairing nucleotide adjacent to the dangling nucleotide. In one embodiment, the RNAi agent has additional two phosphate-thionucleotide inter-linkages between the three terminal nucleotides at the 5' end of the sense strand and the 5' end of the antisense strand. In some embodiments, each nucleotide (including nucleotides that are part of a motif) in the sense and antisense strands of the dsRNAi agent is a modified nucleotide. In some embodiments, each residue is independently modified with a 2'-O-methyl or 3'-fluorine, for example, in an alternating motif manner. Optionally, the dsRNAi agent further comprises a ligand (such as GalNAc3).

[0368] In some embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotide residues long, starting from the 5' terminal nucleotide (position 1), and positions 1 to 23 of the first strand contain at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues long, and starting from the 3' terminal nucleotide, contains at least 8 ribonucleotides at positions 1-23 of the sense strand to form a double strand; wherein at least the 3' terminal nucleotide of the antisense strand is unpaired from the sense strand, and up to 6 consecutive 3' terminal nucleotides are unpaired from the sense strand, thereby forming a 1-6 nucleotide 3' single-stranded overhang; wherein the 5' end of the antisense strand contains 10- The device comprises 30 consecutive nucleotides, which are unpaired with the sense strand to form a single-stranded 5' overhang of 10-30 nucleotides; wherein, when the sense and antisense strands are aligned for maximum complementarity, at least the 5' and 3' terminal nucleotides of the sense strand are paired with the nucleotide bases of the antisense strand, thereby forming a substantially double-stranded region between the sense and antisense strands; and the antisense strand is sufficiently complementary to the target RNA for at least 19 ribonucleotides along its length to reduce target gene expression when the double-stranded nucleic acid is introduced into mammalian cells; and wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, wherein at least one motif is located at or near a cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near a cleavage site.

[0369] In some embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, wherein the dsRNAi agent comprises a first strand having a length of at least 25 nucleotides and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides, having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form blunt ends, and the second strand is 1-4 nucleotides longer than the first strand at the 3' end; wherein the length of the double-stranded region is at least 25 nucleotides, and at least 19 nucleotides along the length of the second strand are sufficiently complementary to the target mRNA, thereby reducing target gene expression when the RNAi agent is introduced into mammalian cells; and wherein Dicer cleaves the dsRNAi agent to produce siRNA containing the 3' end of the second strand, thereby reducing target gene expression in mammals. Optionally, the dsRNAi agent further comprises a ligand.

[0370] In some implementations, the positive strand of the dsRNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of which is located at a cleavage site on the positive strand.

[0371] In some embodiments, the antisense strand of the dsRNAi agent may also contain at least one motif of three identical modifications on three consecutive nucleotides, one of which is located at or near the cleavage site of the antisense strand.

[0372] For dsRNAi agents with a duplex region length of 19-23 nucleotides, the cleavage site of the antisense strand is typically located at positions approximately 10, 11, and 12 from the 5' end. Therefore, three identically modified motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15, with counting starting from the first nucleotide at the 5' end of the antisense strand, or from the first paired nucleotide within the duplex region at the 5' end of the antisense strand. The cleavage site of the antisense strand can also vary depending on the length of the duplex region from the 5' end of the dsRNAi agent.

[0373] The sense strand of a dsRNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides, the motif located at a cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides, the motif located at or near a cleavage site of the strand. When the sense and antisense strands form a dsRNA duplex, the sense and antisense strands may be arranged such that a motif of three nucleotides on the sense strand overlaps with a motif of three nucleotides on the antisense strand by at least one nucleotide, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

[0374] In some embodiments, the positive strand of the dsRNAi agent may contain more than one motif with three identical modifications on three consecutive nucleotides. The first motif may be located at or near a cleavage site on the strand, and the other motifs may be wing modifications. The term "wing modification" as used herein refers to a motif located on another portion of the strand and separated from motifs located at or near a cleavage site on the same strand. Wing modifications may be adjacent to the first motif or separated by one or more nucleotides. When the motifs are adjacent to each other, their chemical properties are different; and when the motifs are separated by one or more nucleotides, their chemical properties may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may be located at one end relative to the first motif (which is located at or near a cleavage site) or on either side of the main motif.

[0375] Similar to the sense strand, the antisense strand of a dsRNAi agent may also contain more than one motif of three identical modifications on three consecutive nucleotides, with at least one motif located at or near a strand cleavage site. The antisense strand may also contain one or more wing modifications arranged similarly to those present on the sense strand.

[0376] In some implementations, the wing modifications on the sense or antisense strand of the dsRNAi agent typically do not include the first one or two terminal nucleotides at the 3', 5', or both ends of the strand.

[0377] In other embodiments, the wing modifications on the sense or antisense strand of the dsRNAi agent typically do not include the preceding one or two paired nucleotides at the 3', 5', or both ends of the double-stranded region.

[0378] When the sense and antisense strands of a dsRNAi agent each contain at least one wing modification, the wing modification may be located at the same end of the duplex region and have an overlap of one, two or three nucleotides.

[0379] When the sense and antisense strands of a dsRNAi agent each contain at least two wing modifications, the sense and antisense strands can be arranged as follows: two modifications from one strand are located at one end of the double-stranded region, with an overlap of one, two, or three nucleotides; two modifications from one strand are located at the other end of the double-stranded region, with an overlap of one, two, or three nucleotides; and two modifications from one strand are located on each side of the guide motif, with an overlap of one, two, or three nucleotides within the double-stranded region.

[0380] In some embodiments, each nucleotide (including nucleotides that are part of a motif) in the sense and antisense strands of the dsRNAi agent can be modified. Each nucleotide can be modified with the same or different modifications, which may include one or more changes to one or two non-linked phosphate oxygen atoms or one or more linked phosphate oxygen atoms; changes to the ribose sugar composition, such as changes to the 2'-hydroxyl group on the ribose; complete replacement of the phosphate moiety with a "dephosphorylated" linker; modification or substitution of naturally occurring bases; and substitution or modification of the ribose-phosphate backbone.

[0381] Because nucleic acids are polymers of subunits, many modifications occur at repetitive sites within the nucleic acid, such as modifications of bases or phosphate groups or non-linked Os of the phosphate group. In some cases, modifications occur at all subject sites of the nucleic acid, but in many cases, they do not. For example, modifications may occur only at the 3'- or 5' end positions, or only in terminal regions, such as at the position on the terminal nucleotide or at the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in double-stranded regions of RNA or only in single-stranded regions of RNA. For example, phosphate thioester modifications at non-linked O positions may occur only at one or both ends, or only in terminal regions, such as at the position on the terminal nucleotide or at the last 2, 3, 4, 5, or 10 nucleotides of the strand, or may occur in both double-stranded and single-stranded regions, particularly at the ends. The 5' end can be phosphorylated.

[0382] For example, to enhance stability, specific bases may be included in the pendant, or modified nucleotides or nucleotide substitutes may be included in the single-stranded pendant (e.g., a 5' or 3' pendant, or both). For example, the inclusion of purine nucleotides in the pendant is desirable. In some embodiments, all or part of the bases in the 3'- or 5'- pendant may be modified, for example, using modifications described herein. Modifications may include, for example, modifications known in the art at the 2' position of the ribose, such as using a deoxyribonucleotide, a 2'-deoxy-2'-fluoro(2'-F), or a 2'-O-methyl modified ribose instead of the nucleobase, and modifications of the phosphate group, such as thiophosphate modifications. The pendant need not be homologous to the target sequence.

[0383] In some embodiments, each residue of the sense and antisense strands may be independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxy, or 2'-fluorine. The strand may contain more than one modification. In one embodiment, each residue of the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluorine.

[0384] There are usually at least two different modifications on the sense and antisense strands. These two modifications can be 2'-O-methyl or 2'-fluoro, or other modifications.

[0385] In some implementations, N a or N b Modifications containing alternating patterns. As used herein, the term "alternating motif" refers to a motif having one or more modifications, each appearing on alternating nucleotides on one strand. Alternating nucleotides can refer to nucleotides appearing every other nucleotide, every three nucleotides, or similar patterns. For example, if A, B, and C each represent a type of nucleotide modification, then the alternating motif could be "ABABABABABAB…", "AABBAABBAABB…", "AABAABAABAAB…", "AAABAAABAAAB…", "AAABBBAAABBB…", or "ABCABCABCABC…", etc.

[0386] Alternating motifs can contain the same or different types of modifications. For example, if A, B, C, and D are each a type of nucleotide modification, the alternation pattern (i.e., the modification every other nucleotide) can be the same, but each strand of the sense or antisense strand can be selected from several possible modifications in the alternation motif, such as “ABABAB…”, “ACACAC…”, “BDBDBD…”, or “CDCDCD…”.

[0387] In some embodiments, the modification pattern of alternating motifs on the sense strand of the dsRNAi agent of the present invention is offset relative to the modification pattern of alternating motifs on the antisense strand. This offset can cause the modified nucleotide set of the sense strand to correspond to a different modified nucleotide set of the antisense strand, and vice versa. For example, when the sense and antisense strands pair in the dsRNA duplex, within the duplex region, the alternating motif in the sense strand may begin with “ABABAB” from the 5' to 3' of the strand, and the alternating motif in the antisense strand may begin with “BABABA” from the 5' to 3' of the strand. As another example, within the duplex region, the alternating motif in the sense strand may begin with “AABBAABB” from the 5' to 3' of the strand, and the alternating motif in the antisense strand may begin with “BBAABBAA” from the 5' to 3' of the strand, thereby causing a complete or partial offset of the modification patterns between the sense and antisense strands.

[0388] In some embodiments, the dsRNAi agent comprises an initial alternating motif of 2'-O-methyl and 2'-F modifications on the sense strand, the alternating motif initially offset relative to the alternating motif of 2'-O-methyl and 2'-F modifications on the antisense strand, i.e., the nucleotides with 2'-O-methyl modifications on the sense strand pair with the nucleotides with 2'-F modifications on the antisense strand, and vice versa. The first position of the sense strand may begin with a 2'-F modification, and the first position of the antisense strand may begin with a 2'-O-methyl modification.

[0389] Introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense or antisense strand disrupts the initial modification pattern present in the sense or antisense strand. This disruption of the modification pattern in the sense or antisense strand enhances gene silencing activity against the target gene.

[0390] In some implementations, when a motif consisting of three identical modifications on three consecutive nucleotides is introduced into any chain, the modifications of the nucleotides adjacent to the motif are different from the modifications of the motif itself. For example, the sequence portion containing the motif is “…N”. a YYYN b …”, where “Y” represents a modification in a motif with three identical modifications on three consecutive nucleotides, and “N” a " and "N b " represents a modification of the nucleotide adjacent to the motif "YYY", the modification being different from the modification of Y, and wherein N a and N b These can be the same or different modifiers. Alternatively, when a wing modifier exists, N a or N b It may or may not exist.

[0391] The iRNA may further comprise at least one phosphate thioester or methylphosphonate nucleoside linker. Phosphothioester or methylphosphonate nucleoside linker modification may occur on any nucleotide at any position in the sense strand, antisense strand, or both. For example, the nucleoside linker modification may occur on each nucleotide in the sense strand or antisense strand; each nucleoside linker modification may occur in an alternating pattern in the sense strand or antisense strand; or the sense strand or antisense strand may contain two nucleoside linker modifications present in an alternating pattern. The alternating pattern of nucleoside linker modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of nucleoside linker modifications on the sense strand may be offset relative to the alternating pattern of nucleoside linker modifications on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6-8 phosphate thioester nucleoside links. In some embodiments, the antisense strand comprises a two-thiophosphate nucleoside link at the 5' end and a two-thiophosphate nucleoside link at the 3' end, and the sense strand comprises at least two-thiophosphate nucleoside links at the 5' end or the 3' end.

[0392] In some embodiments, the dsRNAi agent in the dangling region refers to a modification containing a phosphate thioester or methylphosphonate nucleoside linker. For example, the dangling region may contain two nucleotides linked by a phosphate thioester or methylphosphonate nucleoside linker. The nucleoside linker may also be modified to connect the dangling nucleotide to a terminal pairing nucleotide within the double-stranded region. For example, at least two, three, four, or all of the dangling nucleotides may be linked by a phosphate thioester or methylphosphonate nucleoside linker, and optionally, additional phosphate thioester or methylphosphonate nucleoside linkers may be present to link the dangling nucleotide to a pairing nucleotide immediately adjacent to the dangling nucleotide. For example, at least two phosphate thioester nucleoside linkers may be present between three terminal nucleotides, wherein two of the three nucleotides are dangling nucleotides, and the third is a pairing nucleotide immediately adjacent to the dangling nucleotide. These three terminal nucleotides may be located at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, or the 5' end of the sense strand.

[0393] In some embodiments, the 2-nucleotide dangling is located at the 3' end of the antisense strand, and there are two phosphate thioester nucleoside links between the terminal three nucleotides, wherein two of the three nucleotides are dangling nucleotides, and the third nucleotide is a pairing nucleotide immediately adjacent to the dangling nucleotide. Optionally, the dsRNAi agent may additionally have two phosphate thioester nucleoside links between the terminal three nucleotides at the 5' end of the sense strand and the 5' end of the antisense strand.

[0394] In one implementation, the dsRNAi agent contains mismatches with the target, the mismatches being located within the double strand, or combinations thereof. Mismatches can occur in overhanging regions or double-stranded regions. Base pairs can be sorted according to their tendency to promote dissociation or dissociation (e.g., based on the association or dissociation free energy of a particular pair; the simplest approach is to examine base pairs on a single-pair basis, although nearest-neighbor analysis or similar analyses can also be used). Regarding promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C, and I:C is preferred over G:C (I = inosine). Mismatches, such as non-canonical base pairs or base pairs different from canonical base pairs (as described elsewhere herein), are preferred over canonical base pairs (A:T, A:U, G:C); and base pairs containing universal bases are preferred over canonical base pairs.

[0395] In some embodiments, the dsRNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs from the 5' end of the antisense strand within the duplex region, said base pairs being independently selected from the group consisting of A:U, G:U, I:C, and mismatched base pairs (e.g., non-canonical base pairs or base pairs different from canonical base pairs, base pairs containing universal bases) to promote dissociation of the antisense strand at the 5' end of the duplex.

[0396] In some embodiments, the nucleotide at the first position of the 5' end of the antisense double-stranded region is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs at the 5' end of the antisense double-stranded region is an AU base pair. For example, the first base pair at the 5' end of the antisense double-stranded region is an AU base pair.

[0397] In other embodiments, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT), or the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). For example, there are short deoxythymidine nucleotide sequences, such as two dT nucleotides at the 3' ends of the sense strand, antisense strand, or both strands.

[0398] In some embodiments, the RNAi agent of the present invention may comprise a small number of nucleotides with 2'-fluorine modification, for example, 10 or fewer nucleotides with 2'-fluorine modification. For example, the RNAi agent may comprise 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides with 2'-fluorine modification. In a specific embodiment, the RNAi agent of the present invention comprises 10 nucleotides with 2'-fluorine modification, for example, 4 nucleotides in the sense strand have 2'-fluorine modification and 6 nucleotides in the antisense strand have 2'-fluorine modification. In another specific embodiment, the RNAi agent of the present invention comprises 6 nucleotides with 2'-fluorine modification, for example, 4 nucleotides in the sense strand have 2'-fluorine modification and 2 nucleotides in the antisense strand have 2'-fluorine modification.

[0399] In other embodiments, the RNAi agent of the present invention may contain a very small amount of nucleotides with 2'-fluorine modification, for example, two or fewer nucleotides with 2'-fluorine modification. For example, the RNAi agent may contain 2, 1, or 0 nucleotides with 2'-fluorine modification. In a specific embodiment, the RNAi agent may contain 2 nucleotides with 2'-fluorine modification, for example, 0 nucleotides with 2'-fluorine modification in the sense strand and 2 nucleotides with 2'-fluorine modification in the antisense strand.

[0400] Various publications describe multimeric iRNAs that can be used in the methods of the present invention. These publications include WO2007 / 091269, U.S. Patent No. 7,858,769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, the entire contents of each of which are incorporated herein by reference.

[0401] In some embodiments, the compositions and methods of this disclosure include vinylphosphonate (VP) modification of the RNAi agents described herein. In an exemplary embodiment, the 5' vinylphosphonate-modified nucleotide of this disclosure has the following structure:

[0402]

[0403] Where X is O or S;

[0404] R is hydrogen, hydroxyl, fluorine, or C1- 20 Alkyl groups (e.g., methoxy or n-hexadecyloxy);

[0405] R5' is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is E- or Z-oriented (e.g., E-oriented); and

[0406] B is a nucleobase or a modified nucleobase, optionally wherein B is adenine, guanine, cytosine, thymine, or uracil.

[0407] The vinylphosphonate of this disclosure may be attached to the antisense or sense strand of the dsRNA of this disclosure. In some embodiments, the vinylphosphonate of this disclosure is attached to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

[0408] The compositions and methods disclosed herein also contemplate vinylphosphonate modification. Exemplary vinylphosphonate structures include the aforementioned structures, wherein R5' is =C(H)-OP(O)(OH)2, and the double bond between the C5' carbon and R5' is E-oriented or Z-oriented (e.g., E-oriented).

[0409] As described in more detail below, iRNAs containing one or more carbohydrate moieties coupled to the iRNA can optimize one or more properties of the iRNA. In many cases, the carbohydrate moieties will be attached to the modifying subunits of the iRNA. For example, the ribose of one or more ribonucleotide subunits of the iRNA can be replaced by another moieties, such as non-carbohydrate (e.g., cyclic) carriers with attached carbohydrate ligands. Ribonucleotide subunits in which the ribose of the subunits has been so replaced are referred to herein as ribose substitution modification subunits (RRMS). Circular carriers can be carbocyclic systems (i.e., all ring atoms are carbon atoms) or heterocyclic systems (i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, or sulfur). Circular carriers can be monocyclic systems or can contain two or more rings, such as fused rings. Circular carriers can be fully saturated cyclic systems or they can contain one or more double bonds.

[0410] Ligands can be attached to polynucleotides via a carrier. The carrier includes (i) at least one “backbone attachment site,” such as two “backbone attachment sites,” and (ii) at least one “linkage attachment site.” As used herein, a “backbone attachment site” refers to a functional group, such as a hydroxyl group, or generally refers to a bond that can be used and is suitable for introducing the carrier into the backbone, such as a phosphate or modified phosphate (e.g., sulfur-containing) backbone of ribonucleic acid. In some embodiments, a “linkage attachment site” (TAP) refers to a constitutive ring atom of the cyclic carrier, such as a carbon atom or heteroatom (different from the atom providing the backbone attachment site) linking a selected portion. The portion may be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected portion is linked to the cyclic carrier via an intermediate linker. Thus, cyclic carriers typically contain functional groups, such as amino groups, or generally provide bonds suitable for incorporating or linking another chemical entity (e.g., a ligand) into or to the constitutive ring.

[0411] iRNA can be coupled to a ligand via a vector, wherein the vector can be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolinyl, isoxazolinyl, morpholinyl, thiazolinyl, isothiazolinyl, quinoxolinyl, pyridazinone, tetrahydrofuranyl, and decahydronaphthyl. In one embodiment, the acyclic group is a serinel backbone or a diethanolamine backbone, PCT / US12 / 068491.

[0412] i. Thermally unstable modification

[0413] In some implementations, dsRNA molecules can be optimized for RNA interference by introducing thermally unstable modifications into the seed region of the antisense strand. As used herein, "seed region" means positions 2-9 or 2-8 of the 5' end of the reference strand. For example, thermally unstable modifications can be introduced into the seed region of the antisense strand to reduce or suppress off-target gene silencing.

[0414] The term "thermally unstable modification" includes modifications that lead to the overall melting temperature (T0) of dsRNA. m The T value of dsRNAs without such modifications is lower than that of T-cell RNAs. m Modifications. For example, thermally unstable modifications can alter the T-type of dsRNA. m Lower the temperature by 1-4°C, such as one, two, three, or four degrees Celsius. Furthermore, the term "thermally unstable nucleotide" refers to a nucleotide containing one or more thermostable modifications.

[0415] It has been found that dsRNAs containing at least one double-stranded thermally unstable modification within the first nine nucleotide positions of the antisense strand, counting from the 5' end of the antisense strand, exhibit reduced off-target gene silencing activity. Therefore, in some embodiments, the antisense strand contains at least one (e.g., one, two, three, four, five, or more) double-stranded thermally unstable modification within the first nine nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more double-stranded thermally unstable modifications are located at positions 2-9, such as positions 4-8, of the 5' end of the antisense strand. In some further embodiments, the double-stranded thermally unstable modification is located at position 6, 7, or 8 of the 5' end of the antisense strand. In still some further embodiments, the double-stranded thermally unstable modification is located at position 7 of the 5' end of the antisense strand. In some embodiments, the double-stranded thermally unstable modification is located at positions 2, 3, 4, 5, or 9 of the 5' end of the antisense strand.

[0416] RNAi agents may include a phosphorus-containing group at the 5' end of either the sense or antisense strand. The 5'-terminal phosphorus-containing group may be a 5'-phosphate (5'-P), a 5'-thiophosphate (5'-PS), a 5'-dithiophosphate (5'-PS2), a 5'-vinylphosphonate (5'-VP), a 5'-methylphosphonate (MePhos), or a 5'-deoxy-5'-C-malonyl group (…). When the 5'-terminal phosphorus-containing group is a 5'-terminal vinylphosphonate (5'-VP), 5'-VP can be a 5'-E-VP isomer (i.e., trans-vinylphosphonate). ), 5'-Z-VP isomer (i.e., cis-vinylphosphonate, (or mixtures thereof).

[0417] In one embodiment, the RNAi agent contains a phosphorus-containing group at the 5' end of the sense strand. In another embodiment, the RNAi agent contains a phosphorus-containing group at the 5' end of the antisense strand.

[0418] In one embodiment, the RNAi agent contains 5'-P. In another embodiment, the RNAi agent contains 5'-P in the antisense strand.

[0419] In one embodiment, the RNAi agent contains 5'-PS. In another embodiment, the RNAi agent contains 5'-PS in the antisense strand.

[0420] In one embodiment, the RNAi agent contains 5'-VP. In one embodiment, the RNAi agent contains 5'-VP in the antisense strand. In one embodiment, the RNAi agent contains 5'-E-VP in the antisense strand. In one embodiment, the RNAi agent contains 5'-Z-VP in the antisense strand.

[0421] In one embodiment, the RNAi agent comprises 5'-PS2. In another embodiment, the RNAi agent comprises 5'-PS2 in the antisense strand.

[0422] In one embodiment, the RNAi agent comprises 5'-PS2. In another embodiment, the RNAi agent comprises a 5'-deoxy-5'-C-malonyl group in the antisense strand.

[0423] In some embodiments, the iRNA used in the method of the present invention is an agent selected from those listed in any of Tables 2, 3, 5, or 6. These agents may further comprise ligands.

[0424] VII. Ligand-coupled iRNA

[0425] Further modifications to the iRNA of this invention involve chemically linking one or more ligands, portions, or conjugates that enhance the activity, cellular distribution, or (e.g., cellular uptake) of the iRNA. Such portions include, but are not limited to, lipid portions, such as cholesterol portions (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556). In other embodiments, the ligand is a cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether such as beryl-S-triphenylmethylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), or an aliphatic chain such as dodecyl glycol or undecyl residue (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990). 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as di-hexadecyl-racemic-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmitic moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237) or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0426] In some embodiments, the ligand alters the distribution, targeting, or lifetime of the iRNA agent into which it is incorporated. In some embodiments, such ligands provide enhanced affinity for selected targets (e.g., molecules, cells or cell types, compartments (e.g., cellular or organ compartments), tissues, organs, or body regions), for example, compared to species that do not contain such ligands. In some embodiments, the ligand does not participate in double-strand pairing in double-stranded nucleic acids.

[0427] Ligands may include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylglucosamine, or hyaluronic acid); or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, for example, synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, diethylene ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphonazines. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide polyamine, pseudopeptide polyamine, dendritic polyamine, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary ammonium salts of polyamines, or α-helical peptides.

[0428] The ligand may also contain a targeting group, such as a cell or tissue target, like a lectin, glycoprotein, lipid, or protein (e.g., an antibody), which binds to a specific cell type, such as kidney cells. The targeting group can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant A, mucin carbohydrates, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimics. In some embodiments, the ligand is a polygalactose, such as N-acetylgalactosamine.

[0429] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralene, mitomycin C), porphyrins (TPPC4, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, bile acids, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O (hexadecyl)glycerol, geranyyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3- (oleoyl) Lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytriphenylmethyl or phenoxazine and peptide conjugates (e.g., antennal peptide, Tat peptide), alkylating agents, phosphates, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled substances, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraza macrocyclic Eu3+ complexes), dinitrophenyl, HRP or AP.

[0430] Ligands can be proteins, such as glycoproteins, or peptides, such as molecules with a specific affinity for a coligand, or antibodies, such as antibodies that bind to a specific cell type (e.g., hepatocytes). Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, or polyfucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0431] The ligand can be a substance, such as a drug, that increases the uptake of iRNA agents into cells, for example, by disrupting the cytoskeleton, such as by disrupting microtubules, microfilaments, or intermediate filaments. Drugs can be, for example, paclitaxel, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0432] In some embodiments, ligands attached to iRNA, as described herein, can serve as pharmacokinetic modulators (PK modulators). PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, bile acids, lithocholic acid, dialkyl glycerides, diacylglycerols, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Oligonucleotides containing multiple thiophosphate bonds are also known to bind to serum proteins; therefore, short oligonucleotides with a backbone containing multiple thiophosphate bonds (e.g., oligonucleotides of about 5, 10, 15, or 20 bases) are also suitable as ligands in this invention (e.g., as PK-regulating ligands). Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable as PK-regulating ligands in the embodiments described herein.

[0433] The ligand-coupled iRNA of this invention can be synthesized using oligonucleotides with dangling reactive functional groups, such as those derived from attaching linker molecules to the oligonucleotides (as described below). The reactive oligonucleotides can react directly with commercially available ligands, ligands with any variety of protecting groups during synthesis, or ligands with attached linker portions.

[0434] The oligonucleotides used in the conjugates of this invention can be conveniently and routinely prepared using well-known solid-phase synthesis techniques. Equipment for such synthesis is sold by several vendors, such as Applied Biosystems® (Foster City, California). Any other such synthetic methods known in the art may also be used, either additionally or alternately. Similar techniques are also known for the preparation of other oligonucleotides, such as phosphate thioesters and alkylated derivatives.

[0435] In the ligand-coupled iRNA and ligand molecules with sequence-specific linker nucleosides of the present invention, oligonucleotides and oligonucleotides can be assembled on a suitable DNA synthesizer using standard nucleotides or nucleoside precursors, nucleotides or nucleoside-coupled precursors with linker portions, ligand-nucleotide or nucleoside-coupled precursors with ligand molecules, or building blocks with non-nucleoside ligands.

[0436] When using nucleotide-conjugated precursors with pre-existing linker moieties, the synthesis of sequence-specific linked nucleosides is routinely performed, and then the ligand molecule is reacted with the linker moieties to form ligand-conjugated oligonucleotides. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized using automated synthesizers, employing not only commercially available standard and non-standard phosphorus amides conventionally used in oligonucleotide synthesis, but also phosphorus amides derived from ligand-nucleoside conjugates.

[0437] A. Lipid conjugates

[0438] In some implementations, the ligand or conjugate is a lipid or lipid-based molecule.

[0439] In one implementation, such lipids or lipid-based molecules bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands allow the conjugate to distribute to target tissues, such as non-renal target tissues of the body. For example, a target tissue could be the liver, including the parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipids or lipid-based ligands can (a) enhance the conjugate's resistance to degradation, (b) improve targeting or transport to target cells or cell membranes, or (c) modulate binding to serum proteins (e.g., HSA).

[0440] Lipid-based ligands can be used to inhibit (e.g., control) the binding of conjugates to target tissues. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to target the kidneys and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target conjugates to the kidneys.

[0441] In some embodiments, lipid-based ligands bind to HSA. In one embodiment, it binds to HSA with sufficient affinity to allow the conjugate to distribute to non-renal tissues. However, it is preferable that the affinity is not so strong as to cause irreversible HSA-ligand binding.

[0442] In other embodiments, the lipid-based ligand binds weakly or not at all to the HSA. In one embodiment, the conjugate is distributed to the kidney. Other renal cell-targeting portions may also be used instead of lipid-based ligands, or other renal cell-targeting portions may be used in addition to lipid-based ligands.

[0443] On the other hand, ligands are components, such as vitamins, that can be taken up by target cells (e.g., proliferating cells). These are particularly useful for treating conditions characterized by undesirable cell proliferation (e.g., malignant or non-malignant types, such as cancer cells). Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by target cells (such as liver cells). Also included are HSA and low-density lipoprotein (LDL).

[0444] B. Cell permeabilizer

[0445] On the other hand, the ligand is a cell permeabilizer, such as a helical cell permeabilizer. In one embodiment, the agent is amphiphilic. Exemplary agents are peptides, such as tat or antennal peptides. If the agent is a peptide, it is modified, including peptide mimics, inverted isomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. In one embodiment, the helical permeabilizer is an α-helical permeabilizer having a lipophilic phase and a lipophobic phase.

[0446] The ligand can be a peptide or a peptide mimic. A peptide mimic (also referred to herein as an oligopeptide mimic) is a molecule capable of folding into a specific three-dimensional structure similar to that of a natural peptide. Attaching peptides and peptide mimics to iRNA agents can affect the pharmacokinetic distribution of iRNA, such as by enhancing cellular recognition and uptake. The peptide or peptide mimic moiety can be about 5–50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0447] The peptide or peptide-like substance can be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., primarily composed of Tyr, Trp, or Phe). The peptide moiety can be a dendritic peptide, a bound peptide, or a cross-linked peptide. In another embodiment, the peptide moiety can contain a hydrophobic membrane transport sequence (MTS). An exemplary peptide containing a hydrophobic MTS is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 14). RFGF analogs (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 15) containing a hydrophobic MTS) can also be targeting moieties. The peptide moiety can be a "delivery" peptide that can carry large polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. For example, sequences from HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 16)) and Drosophila antennal foot protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 17)) have been found to function as delivery peptides. Peptides or peptide-like compounds can be encoded by random DNA sequences, such as those identified from phage display libraries or single-bead single-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Examples of peptides or peptide-like compounds linked to dsRNA agents via an incorporated monomeric unit for cell-targeting purposes are arginine-glycine-aspartic (RGD)-peptides or RGD mimics. The length of the peptide moiety can range from about 5 amino acids to about 40 amino acids. The peptide moiety can have structural modifications, such as to improve stability or direct conformational properties. Any structural modifications described below can be used.

[0448] RGD peptides used in the compositions and methods of the present invention can be linear or cyclic and can be modified, for example, by glycosylation or methylation, to facilitate targeting of specific tissues. RGD-containing peptides and peptide mimics may contain D-amino acids and synthetic RGD mimics. In addition to RGD, other portions targeting integrin ligands, such as PECAM-1 or VEGF, may also be used.

[0449] "Cell-permeable peptides" are capable of penetrating cells, such as microbial cells, like bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-permeable peptides can be, for example, α-helical linear peptides (e.g., LL-37 or CeropinP1), disulfide-containing peptides (e.g., α-defensins, β-defensins, or bactericides), or peptides containing only one or two major amino acids (e.g., PR-39 or indolicidin). Cell-permeable peptides may also contain nuclear localization signals (NLS). For example, cell-permeable peptides can be bipartite amphiphilic peptides, such as MPG, which derives its NLS from the fusion peptide domain of HIV-1 gp41 and the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0450] C. Carbohydrate conjugates

[0451] In some embodiments of the compositions and methods of the present invention, the iRNA further comprises carbohydrates. As described herein, carbohydrate-coupled iRNAs facilitate the in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use. As used herein, “carbohydrate” means a compound that is itself a carbohydrate, consisting of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom attached to each carbon atom; or it means a compound having a carbohydrate moiety (consisting of one or more monosaccharide units each having at least six carbon atoms) as part thereof (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), as well as polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars with C5 or more (e.g., C5, C6, C7, or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0452] In some embodiments, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide.

[0453] In some embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). For example, US 8,106,022 (the entire contents of which are incorporated herein by reference) describes GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives. In some embodiments, GalNAc conjugates are used as ligands to target iRNA to specific cells. In some embodiments, GalNAc conjugates target iRNA to hepatocytes, for example, by acting as ligands for desialylate glycoprotein receptors of liver cells (e.g., hepatocytes).

[0454] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be attached via a linker, such as a divalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is coupled to the 3' end of the positive sense strand. In some embodiments, the GalNAc conjugate is coupled to an iRNA agent (e.g., coupled to the 3' end of the positive sense strand) via a linker (e.g., a linker described herein). In some embodiments, the GalNAc conjugate is coupled to the 5' end of the positive sense strand. In some embodiments, the GalNAc conjugate is coupled to an iRNA agent (e.g., coupled to the 5' end of the positive sense strand) via a linker (e.g., a linker described herein).

[0455] In some embodiments of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a trivalent linker. In still other embodiments of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a tetravalent linker.

[0456] In some embodiments, the double-stranded RNAi agent of the present invention comprises a GalNAc or GalNAc derivative attached to the iRNA agent. In some embodiments, the double-stranded RNAi agent of the present invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each GalNAc or GalNAc derivative being independently attached to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0457] In some embodiments, for example, when the two chains of the iRNA agent of the present invention are part of a larger molecule, and the two chains are connected by an unbroken nucleotide chain between the 3' end of one chain and the 5' end of the corresponding other chain to form a hairpin loop containing a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently contain a GalNAc or a GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extension overhang of one chain in the duplex.

[0458] In some embodiments, for example, when the two chains of the iRNA agent of the present invention are part of a larger molecule, and the two chains are connected by an unbroken nucleotide chain between the 3' end of one chain and the 5' end of the corresponding other chain to form a hairpin loop containing a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently contain a GalNAc or a GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extension overhang of one chain in the duplex.

[0459] In one embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is selected from the group consisting of:

[0460] Formula II,

[0461] Formula III,

[0462] Formula IV,

[0463] Formula V,

[0464] Formula VI,

[0465] Equation VII,

[0466] Formula VIII,

[0467] Formula IX,

[0468] Formula X,

[0469] Formula XI,

[0470] Formula XII,

[0471] Formula XIII,

[0472] Formula XIV,

[0473] Formula XV,

[0474] Formula XVI,

[0475] Formula XVII,

[0476] Formula XVIII,

[0477] Formula XIX,

[0478] Formula XX,

[0479] Formula XXI,

[0480] Formula XXII,

[0481] Formula XXIII,

[0482] Where Y is O or S, and n is 3 - 6 (Equation XXIV),

[0483] Where Y is O or S, and n is 3-6 (Equation XXV),

[0484] Formula XXVI,

[0485] Where X is O or S (Equation XXVII),

[0486] Formula XXVII,

[0487] Formula XXIX,

[0488] Formula XXX,

[0489] Formula XXXI,

[0490] Formula XXXII, and

[0491] Formula XXXIII,

[0492] Formula XXXIV.

[0493] In another embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide. In one embodiment, the monosaccharide is N-acetylgalactosamine, such as...

[0494] Formula II.

[0495] In some implementations, the RNAi agent is attached to the carbohydrate conjugate via a linker, as illustrated in the following schematic diagram, where X is O or S.

[0496] .

[0497] In some implementations, the RNAi agent is coupled with L96 as defined in Table 1 and shown below:

[0498] .

[0499] Another representative carbohydrate conjugate used in the embodiments described herein includes, but is not limited to:

[0500]

[0501] (Formula XXXVI), where one of X or Y is an oligonucleotide and the other is hydrogen.

[0502] In some embodiments, suitable ligands are those disclosed in WO 2019 / 055633 (the entire contents of which are incorporated herein by reference). In one embodiment, the ligand comprises the following structure:

[0503]

[0504] In some embodiments of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a divalent linker. In still other embodiments of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a trivalent linker.

[0505] In one embodiment, the double-stranded RNAi agent of the present invention comprises one or more GalNAc or GalNAc derivatives attached to the iRNA agent. GalNAc can be attached to any nucleotide on the sense or antisense strand via a linker. GalNAc can be attached to the 5' end, 3' end, 5' end, or 3' end of the antisense strand. In one embodiment, GalNAc is attached to the 3' end of the sense strand, for example, via a trivalent linker.

[0506] In other embodiments, the double-stranded RNAi agent of the present invention comprises a plurality of (e.g., 2, 3, 4, 5 or 6) GalNAc or GalNAc derivatives, each GalNAc or GalNAc derivative being independently attached to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of linkers (e.g., monovalent linkers).

[0507] In some embodiments, for example, when the two chains of the iRNA agent of the present invention are part of a larger molecule, the two chains are connected by an unbroken nucleotide chain between the 3' end of one chain and the 5' end of the corresponding other chain to form a hairpin loop containing a plurality of unpaired nucleotides, each unpaired nucleotide in the hairpin loop may independently contain a GalNAc or a GalNAc derivative attached via a monovalent linker.

[0508] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, PK regulators or cell-permeable peptides.

[0509] The additional carbohydrate conjugates and linkers applicable in this invention include those described in PCT Publications WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0510] D. Connector

[0511] In some embodiments, the conjugates or ligands described herein can be attached to iRNA oligonucleotides via various cleavable or non-cleavable linkers.

[0512] The term "linker" or "linking group" refers to the organic part that connects two parts of a compound, for example, covalently connecting two parts of a compound. Linkers typically include direct bonds or atoms such as oxygen or sulfur, or units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or atomic chains such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, aralkyl, areneyl, arynyl, heteroarylalkyl, heteroaryleneyl, heteroarylynyl, heterocyclic alkyl, heterocyclic alkenyl, heterocyclic alkenyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, alkylaralkyl, alkylareneyl, alkylarynyl, alkenylaralkyl, alkenylareneyl, alkenylarynyl, alkynylaralkyl, alkynylareneyl, alkynylarynyl, alkylheteroalkyl, alkylheteroalkenyl, alkylheteroalynyl, alkenylheteroalkyl Alkyl, alkenyl heteroaryl, alkenyl heteroarylynyl, ynyl heteroarylalkyl, ynyl heteroaryl, ynyl heteroarylynyl, alkyl heterocyclic alkyl, alkyl heterocyclic alkenyl, alkyl heterocyclic ynyl, alkenyl heterocyclic alkenyl, alkenyl heterocyclic alkenyl, alkenyl heterocyclic ynyl, ynyl heterocyclic alkenyl, ynyl heterocyclic ynyl, alkyl aryl, alkenyl aryl, ynyl aryl, alkyl heteroaryl, alkenyl heteroaryl, ynyl heterocyclic aryl, alkyl aryl, alkenyl aryl, alkenyl heteroaryl, alkyl heteroaryl, ynyl heteroaryl, alkyl heteroaryl, ynyl heteroaryl, one or more methylene groups may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or substituted or unsubstituted heterocyclic group; wherein R8 is hydrogen, acyl, aliphatic group or substituted aliphatic group. In one embodiment, the linker is about 1-24 atoms, 2-24 atoms, 3-24 atoms, 4-24 atoms, 5-24 atoms, 6-24 atoms, 6-18 atoms, 7-18 atoms, 8-18 atoms, 7-17 atoms, 8-17 atoms, 6-16 atoms, 7-17 atoms, or 8-16 atoms.

[0513] A cleavable linker is a group that is sufficiently stable outside the cell but is cleaved upon entry into a target cell, thereby releasing the two parts linked together by the linker. In an exemplary embodiment, the cleavable linker is cleaved in the target cell or under a first reference condition (e.g., which may be selected to simulate or represent the intracellular environment) at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or more times, or at least 100 times, faster than in the subject's blood or under a second reference condition (e.g., which may be selected to simulate or represent conditions found in blood or serum).

[0514] Cleavable linker groups are susceptible to the influence of cleavage agents, such as pH, redox potential, or the presence of degrading molecules. Typically, cleavage agents are more prevalent or found at higher levels or with higher activity within cells than in serum or blood. Examples of such degrading agents include: redox agents that are selective or non-substrate-specific to a particular substrate, including, for example, oxidases or reductases or reducing agents (such as thiols) present in cells that can degrade redox-cleavable linker groups by reduction; esterases; endosomes or agents that can create an acidic environment, such as those resulting in a pH of 5 or below; and enzymes that can hydrolyze or degrade acid-cleavable linker groups by acting as universal acids, peptidases (which can be substrate-specific), and phosphatases.

[0515] Cleavable linker groups, such as disulfide bonds, can be pH-sensitive. Human serum has a pH of 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomals have a more acidic pH ranging from 5.5 to 6.0, and lysosomes have a pH of about 5.0 or even more. Some linkers have cleavable linker groups that cleave at a selected pH, thereby releasing cationic lipids from the ligand within the cell or into the desired compartment of the cell.

[0516] Linkers may contain cleavable linker groups that can be cleaved by specific enzymes. The type of cleavable linker group incorporated into the linker may depend on the cell type to be targeted. For example, liver-targeting ligands can be linked to cationic lipids via linkers containing ester groups. Hepatocytes are rich in esterases, and therefore linkers are cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0517] When targeting cell types rich in peptidase (such as hepatocytes and synovial cells), linkers containing peptide bonds can be used.

[0518] Typically, the suitability of a candidate cleavable linker can be assessed by detecting its ability to cleave the candidate linker group by a degrading agent (or condition). It is also necessary to assess the ability of the candidate cleavable linker group to resist cleavage in blood or upon contact with other non-target tissues. Thus, the relative sensitivity to cleavage can be determined between first and second conditions, where the first condition is selected to indicate cleavage in target cells, and the second condition is selected to indicate cleavage in other tissues or biological fluids (e.g., blood or serum). This assessment can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. Preliminary assessment under cell-free or culture conditions, followed by confirmation through further assessment in whole animals, can be beneficial. In some embodiments, the useful candidate compound cleaves at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in cells (or under in vitro conditions selected to simulate intracellular conditions) than in blood or serum (or under in vitro conditions selected to simulate extracellular conditions).

[0519] i. Redox-cleavable linker groups

[0520] In some embodiments, the cleavable linker is a redox-cleavable linker that cleaves upon reduction or oxidation. An example of a redox-cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable "redox-cleavable linker," or, for example, whether it is suitable for use with a specific iRNA moiety and a specific target, one may refer to the methods described herein. For example, incubation with dithiothreitol (DTT) or other reducing agents may be used to evaluate the candidate using reagents known in the art to simulate the cleavage rate observed in cells (e.g., target cells). The candidate may also be evaluated under selected conditions simulating blood or serum. In one embodiment, the candidate compound is cleaved in blood at a rate of up to about 10%. In other embodiments, the useful candidate compound is degraded in cells (or under selected in vitro conditions simulating intracellular conditions) at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster than in blood (or under selected in vitro conditions simulating extracellular conditions). The cleavage rate of candidate compounds can be determined using standard enzyme kinetic assays under selected simulated intracellular media conditions and compared with those under selected simulated extracellular media conditions.

[0521] ii. Phosphoric acid-based cleavable linker groups

[0522] In other embodiments, the cleavable linker comprises a phosphate-based cleavable linker group. The phosphate-based cleavable linker group is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents that cleave phosphate groups in cells are cellular enzymes such as phosphatases. Examples of phosphoric acid-based linking groups include -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-, wherein each Rk can independently be a C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl. Exemplary embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. In some embodiments, the phosphoric acid-based linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0523] iii. Acids can cleave linker groups.

[0524] In other embodiments, the cleavable linker comprises an acid-cleavable linker group. An acid-cleavable linker group is a linker group that cleaves under acidic conditions. In some embodiments, the acid-cleavable linker group is cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower) or by an agent that can act as a universal acid (such as an enzyme). In cells, specific low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linker group. Examples of acid-cleavable linker groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid-cleavable linker group may have the general formula -C=NN-, C(O)O, or -OC(O). An exemplary embodiment is that the carbon atom (alkoxy group) attached to the oxygen atom of the ester is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or tert-butyl. These candidates can be evaluated using methods similar to those described above.

[0525] iv. Ester-based linking groups

[0526] In other embodiments, the cleavable linker comprises an ester-based cleavable linker group. Ester-based cleavable linker groups are cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linker groups include, but are not limited to, esters with alkylene, alkenyl, and ynylene groups. Ester-based cleavable linker groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0527] V. Peptide-based cleavage groups

[0528] In other embodiments, the cleavable linker comprises a peptide-based cleavable linker group. The peptide-based cleavable linker group is cleaved by cellular enzymes such as peptidases and proteases. The peptide-based cleavable linker group is a peptide bond formed between amino acids to generate oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable linker group does not include an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenyl, or alkyne groups. A peptide bond is a special type of amide bond formed between amino acids to generate peptides and proteins. Peptide-based cleavable linkers are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to generate peptides and proteins and do not include the entire amide functional group. The peptide-based cleavable linker group has the general formula -NHCHHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0529] In some embodiments, the iRNA of the present invention is coupled to a carbohydrate via a linker. Non-limiting examples of iRNA-carbohydrate conjugates having linkers in the compositions and methods of the present invention include, but are not limited to:

[0530] (Formula XXXVII),

[0531] (Formula XXXVIII),

[0532] (Formula XXXIX),

[0533]

[0534] (XL style)

[0535]

[0536] (Formula XLI),

[0537]

[0538] (Formula XLII)

[0539]

[0540] (Formula XLIII), and

[0541]

[0542] (Formula XLIV), where one of X or Y is an oligonucleotide and the other is hydrogen.

[0543] In some embodiments of the compositions and methods of the present invention, the ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives linked by divalent or trivalent branched linkers.

[0544] In one embodiment, the dsRNA of the present invention is coupled to a bivalent or trivalent branched linker selected from the group consisting of structures of any of formulas (XLV)-(XLVI).

[0545] Style XXXXV Style XLVI

[0546] ,

[0547] or ;

[0548] Type XLVII Type XLVIII

[0549] in:

[0550] q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C are each represented independently as 0-20, and the repeating units can be the same or different;

[0551] P 2A P 2B P 3A P 3B P 4A P 4B P 5A P 5B P 5C T 2A T 2B T 3A T 3B T 4A T 4B T 4A T 5B T 5C Each occurrence is independently represented as non-existent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O;

[0552] Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C Each occurrence is independently represented as absent, alkylene, or substituted alkylene, wherein one or more methylene groups can be O, S, S(O), SO2, or N(R). N One or more interruptions or terminations of C(R') = C(R''), C≡C, or C(O);

[0553] R 2A R 2B R 3A R 3B R 4A R 4B R 5A R 5B R 5C Each occurrence is independently represented as non-existent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R) a -C(O), -C(O)-CH(R) a )-NH-, CO, CH=NO, , , , , Or heterocyclic group;

[0554] L 2A L 2B L 3A L 3B L 4A L 4B L 5A L 5B and L 5C The term "ligand" indicates that each instance of the ligand is independently represented as a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R a The side chain is H or an amino acid. Trivalent conjugated GalNAc derivatives are particularly suitable for use with RNAi agents to inhibit the expression of target genes, such as those shown in formula (XLIX):

[0555] ,

[0556] Where L 5A L 5B and L 5C This refers to monosaccharides, such as GalNAc derivatives.

[0557] Examples of suitable divalent and trivalent branched linking groups for coupling GalNAc derivatives include, but are not limited to, the structures described above as formulas II, VII, XI, X, and XIII.

[0558] Representative U.S. patents teaching the preparation of RNA conjugates include, but are not limited to, U.S. patent numbers 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; and 5,485. 6,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;5,214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5,371,241;5,391,723;5,416,203;5,451,463;5,510,475;5,512,667;5,514,785;5,565,552;5,567 ,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are incorporated herein by reference.

[0559] Not all positions in a given compound need to be uniformly modified, and in fact, more than one of the above modifications can be incorporated into a single nucleotide in a single compound or even within an iRNA. This invention also includes iRNA compounds that are chimeric compounds.

[0560] In the context of this invention, a "chimeric" iRNA compound or "chimera" is an iRNA compound comprising two or more chemically distinct regions, such as dsRNAi agents, each region consisting of at least one monomeric unit, i.e., in the case of dsRNA compounds, each region consisting of at least one nucleotide. These iRNAs typically contain at least one region in which the RNA is modified to confer enhanced resistance to nuclease degradation, enhanced cellular uptake, or enhanced binding affinity to target nucleic acids. Additional regions of the iRNA can serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, ribonuclease H is an endonuclease that cleaves the RNA strand in an RNA:DNA duplex. Therefore, activation of ribonuclease H leads to cleavage of the RNA target, thereby significantly enhancing the efficiency of the iRNA in repressing gene expression. Thus, when using chimeric dsRNAs, shorter iRNAs can generally achieve comparable results compared to phosphate-thioester deoxy dsRNAs hybridized to the same target region. Cleavage of the RNA target can be detected by gel electrophoresis and, if necessary, by routine detection using relevant nucleic acid hybridization techniques known in the art.

[0561] In some cases, the RNA of iRNA can be modified with non-ligand groups. Many non-ligand molecules have been coupled to iRNA to enhance its activity, cellular distribution, or cellular uptake, and methods for such coupling are provided in the scientific literature. These non-ligand moieties include lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), bile acids (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-triphenylmethylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), and thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 1993, 1994, 1995). 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-racemic-glycerol or triethylammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates have been listed above.A typical conjugation protocol involves synthesizing RNA with amino linkers at one or more positions in the sequence. The amino groups are then reacted with the molecules to be conjugated using a suitable conjugating agent or activator. The conjugation reaction can be carried out while the RNA is still bound to a solid support, or in solution after RNA lysis. Typically, the RNA conjugate is purified by HPLC to obtain a pure conjugate.

[0562] VIII. Delivery of the iRNA of the present invention

[0563] The iRNA of this invention can be delivered to cells in a variety of different ways, such as cells in a subject's body, like human subjects (e.g., subjects with a need for it, such as subjects susceptible to or diagnosed with CTNNB1-related conditions (e.g., cancer, such as hepatocellular carcinoma). For example, delivery can be performed by contacting cells with the iRNA of this invention in vitro or in vivo. In vivo delivery can also be performed by directly administering a composition containing iRNA (e.g., dsRNA) to the subject. Alternatively, in vivo delivery can also be performed indirectly by administering one or more vectors that encode and direct iRNA expression. These alternatives are discussed further below.

[0564] Generally, any method of delivering nucleic acid molecules (in vitro or in vivo) is suitable for use with the iRNA of this invention (see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02595, which are incorporated herein by reference in their entirety). For in vivo delivery, factors to be considered for delivering iRNA molecules include, for example, the biostability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. RNA interference has also been successfully delivered locally to the CNS via direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al., (2005) Gene Ther. 12:59-66; Makimura, H. et al., (2002) BMC Neurosci. 3:18; Shishkina, GT. et al., (2004) Neuroscience 129:521-528; Thacker, ER. et al., (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y. et al., (2005) J. Neurophysiol. 93:594-602). Modification of RNA or drug carriers can also allow iRNA to target tissues and avoid undesirable off-target effects. iRNA molecules can be modified by chemically coupling to lipophilic groups (such as cholesterol) to enhance cellular uptake and prevent degradation. For example, systemic injection of an ApoB-targeting iRNA coupled to a lipophilic cholesterol moiety into mice resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178).

[0565] In alternative embodiments, drug delivery systems such as nanoparticles, dendritic molecules, polymers, liposomes, or cationic delivery systems can be used to deliver iRNA. Positively charged cationic delivery systems facilitate the binding of the negatively charged iRNA molecules and also enhance interactions at negatively charged cell membranes, thereby allowing for efficient cellular uptake of iRNA. Cationic lipids, dendritic molecules, or polymers can bind to iRNA or be induced to form vesicles or micelles encapsulating iRNA (see, for example, Kim SH et al., (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents the degradation of iRNA during systemic administration. The methods for preparing and applying the cationic-iRNA complex are entirely within the capabilities of those skilled in the art (see, for example, Sorensen, DR et al., (2003) J. Mol. Biol 327:761-766; Verma, UN et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference). Some examples of non-restricted drug delivery systems that can be used for systemic delivery of iRNA include DOTAP (Sorensen, DR. et al., (2003), ibid.; Verma, UN et al., (2003), ibid.), “solid nucleic acid lipid particles” (Zimmermann, TS et al., (2006) Nature 441:111-114), cardiolipin (Chien, PY et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME et al., (2008) Pharm. Res. Aug 16 Epub preprint; Aigner, A. (2006) J. Biomed. Biotechnol.71659), and Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487) and polyamidoamine (Tomalia, DA et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, iRNA forms a complex with cyclodextrin for systemic administration.Methods of administration of iRNA and cyclodextrin, and pharmaceutical compositions thereof, can be found in U.S. Patent No. 7,427,605 (which is incorporated herein by reference in its entirety). Certain aspects of this disclosure relate to a method for reducing CTNNB1 gene expression in cells, the method comprising contacting the cells with a double-stranded RNAi agent of this disclosure. In one embodiment, the cells are hepatocytes, optionally hepatocyte parenchymal cells. In another embodiment, the cells are extrahepatic cells.

[0566] A. The iRNA encoded by the vector of this invention

[0567] iRNAs targeting the CTNNB1 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture, A et al., TIG. (1996), 12:5-10; Skillern, A et al., International PCT Publication No. WO 00 / 22113, Conrad, International PCT Publication No. WO 00 / 22114, and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (on the order of hours to weeks) or persistent (on the order of weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced in the form of linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative. Transgenes can also be constructed to allow their inheritance as extrachromosomal plasmids (Gassmann et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0568] Viral vector systems that can be used in the methods and compositions described herein include, but are not limited to: (a) adenovirus vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) piconemavirus vectors; (i) poxvirus vectors, such as orthopoxviruses, e.g., vaccinia virus vectors or fowlpoxviruses, e.g., canarypox virus or fowlpox virus; and (j) helper-dependent or enterovirus-free adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors may or may not integrate into the cellular genome. If desired, the construct may contain viral sequences for transfection. Alternatively, the construct may be integrated into vectors capable of free replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA typically require regulatory elements, such as promoters, enhancers, etc., to ensure iRNA expression in target cells. Other aspects of the carrier and the construct are known in the art.

[0569] IX. Pharmaceutical compositions of the present invention

[0570] This invention also includes pharmaceutical compositions and formulations comprising the iRNA of the present invention. In one embodiment, pharmaceutical compositions comprising the iRNA as described herein and a pharmaceutically acceptable carrier are provided herein. Pharmaceutical compositions comprising the iRNA can be used for the prevention or treatment of CTNNB1-related conditions, such as cancers, such as hepatocellular carcinoma.

[0571] Such pharmaceutical compositions are formulated according to the delivery method. One example is that the composition is formulated for systemic administration via parenteral delivery, such as subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. The pharmaceutical compositions of the present invention can be administered at a dose sufficient to inhibit CTNNB1 gene expression.

[0572] In some embodiments, the pharmaceutical composition of the present invention is sterile. In other embodiments, the pharmaceutical composition of the present invention is pyrogen-free.

[0573] The pharmaceutical compositions of the present invention can be administered at doses sufficient to inhibit CTNNB1 gene expression. Typically, suitable doses of the iRNA of the present invention range from about 0.001 to about 200.0 mg per kilogram of recipient body weight per day, typically ranging from about 1 to 50 mg per kilogram of body weight per day. Typically, suitable doses of the iRNA of the present invention range from about 0.1 mg / kg to about 5.0 mg / kg, such as about 0.3 mg / kg and about 3.0 mg / kg. Repeated dosing regimens may include periodic administration of therapeutic doses of iRNA, such as once monthly, every 3-6 months, or annually. In some embodiments, the iRNA is administered about once monthly to every six months.

[0574] Following the initial treatment regimen, treatment can be administered at a lower frequency. The duration of treatment can be determined based on the severity of the disease.

[0575] In other embodiments, a single dose of the pharmaceutical composition may have a long-lasting effect, allowing for administration at intervals not exceeding 1, 2, 3, or 4 months. In some embodiments of the invention, a single dose of the pharmaceutical composition is administered approximately once a month. In other embodiments of the invention, a single dose of the pharmaceutical composition is administered once a quarter (i.e., approximately every three months). In other embodiments of the invention, a single dose of the pharmaceutical composition is administered twice a year (i.e., approximately every six months).

[0576] Those skilled in the art will understand that certain factors can influence the dosage and duration required for effective treatment of a subject, including but not limited to mutations present in the subject, prior treatment, the subject's overall health or age, and any other pre-existing conditions. Furthermore, depending on the circumstances, treatment of a subject with a preventative or therapeutically effective amount of the composition may comprise a single treatment or a series of treatments.

[0577] The pharmaceutical compositions disclosed herein can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be local (including ocular, vaginal, rectal, nasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, for example via an implanted device; or intracranial, for example via intraparenchymal, intrathecal, or intraventricular administration.

[0578] iRNA can be delivered in a way that targets specific tissues, such as the liver.

[0579] Pharmaceutical compositions and formulations for topical application may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous, powder, or oily matrices, thickeners, etc., may be necessary or desirable. Coated condoms, gloves, etc., may also be useful. Suitable topical formulations include those that blend the RNAi agents described in this disclosure with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine DOPE, dimyristoylphosphatidylcholine DMPC, distearylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The RNAi agents described in this disclosure may be encapsulated in liposomes or may form complexes with them, particularly with cationic liposomes. Alternatively, RNAi agents may form complexes with lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, arachidic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, didecanoate, tridecanoate, monooleate, dilaurate, 1-monodecanoate, 1-dodecylazine-2-one, acylcarnitine, acylcholine, or C1-20 alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. For a detailed description of topical formulations, see US 6,747,014, which is incorporated herein by reference.

[0580] In one embodiment, the siRNA (double-stranded RNA agent) of the present invention is administered to cells in the form of a pharmaceutical composition via a local administration route.

[0581] In one embodiment, the pharmaceutical composition may comprise an siRNA compound mixed with a local delivery agent. The local delivery agent may be a variety of microvesicles. The microvesicles may be liposomes. In some embodiments, the liposomes are cationic liposomes.

[0582] In another embodiment, the dsRNA agent is blended with a local penetration enhancer. In one embodiment, the local penetration enhancer is a fatty acid. The fatty acid may be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, didecanoate, tridecanoate, monooleate, dilaurate, 1-monodecanoate, 1-dodecylazine-2-one, acylcarnitine, acylcholine, or C1-10 alkyl esters, monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof.

[0583] In another embodiment, the local penetration enhancer is a bile salt. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidic acid, sodium glycodeoxyfusidic acid, polyoxyethylene-9-lauryl ether, or a pharmaceutically acceptable salt thereof.

[0584] In another embodiment, the penetration enhancer is a chelating agent. The chelating agent may be EDTA, citric acid, salicylate, N-acyl derivatives of collagen, lauryl ether-9, N-aminoacyl derivatives of β-diketone, or mixtures thereof.

[0585] In another embodiment, the penetration enhancer is a surfactant, such as an ionic or nonionic surfactant. The surfactant may be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, perfluorinated chemical emulsions, or mixtures thereof.

[0586] In another embodiment, the penetration enhancer may be selected from the group consisting of: unsaturated cyclic ureas, 1-alkyl-alkane ketones, 1-alkenylazine-alkane ketones, steroidal anti-inflammatory agents, and mixtures thereof. In yet another embodiment, the penetration enhancer may be a diol, pyrrole, azone, or terpene.

[0587] In one aspect, the present invention describes injectable dosage forms of pharmaceutical compositions comprising siRNA compounds, such as double-stranded siRNA compounds or ssiRNA compounds (e.g., precursors, such as larger siRNA compounds that can be processed into ssiRNA compounds, or DNA encoding siRNA compounds (e.g., double-stranded siRNA compounds or ssiRNA compounds or their precursors)). In one embodiment, the injectable dosage form of the pharmaceutical composition comprises a sterile aqueous solution or dispersion and a sterile powder. In some embodiments, the sterile solution may contain a diluent, such as water, physiological saline, fixative oil, polyethylene glycol, glycerol, or propylene glycol.

[0588] The iRNA molecules of this invention can be incorporated into pharmaceutical compositions. Such compositions typically comprise one or more iRNAs and a pharmaceutically acceptable carrier. As used herein, the phrase "pharmaceutically acceptable carrier" refers to any solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic agent, and absorption delay agent compatible with drugs applied to cells (e.g., hepatocytes). The use of such media and agents for pharmaceutically active substances is well known in the art. Within this scope, the use of any conventional media or agent in the composition is also contemplated unless any conventional media or agent is incompatible with the active compound. Additional active compounds may also be incorporated into the composition.

[0589] The pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components, including, but not limited to, pre-formulated liquids, self-emulsifying solids, and self-emulsifying semi-solids. Formulations include those targeting the liver.

[0590] The pharmaceutical formulations of the present invention, readily available in unit dosage forms, can be prepared according to conventional techniques well-known in the pharmaceutical industry. These techniques include the step of combining the active ingredient with a pharmaceutical carrier or excipient. Typically, formulations are prepared by uniformly and tightly mixing the active ingredient with a liquid carrier.

[0591] The iRNA described in this invention can be encapsulated in liposomes or can form complexes with them, particularly with cationic liposomes. Alternatively, the iRNA can form complexes with lipids, particularly with cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, arachidic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, didecanoate, tridecanoate, monooleate, dilaurate, 1-monodecanoate, 1-dodecylazine-2-one, acylcarnitine, acylcholine, or C1-20 alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. A detailed description of the topical formulation is given in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0592] A. iRNA formulations containing membrane-like molecular assemblies

[0593] iRNA used in the compositions and methods of the present invention can be formulated for delivery in the form of membrane-like molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, such as one or more bilayers. Liposomes include monolayer and multilayer vesicles having a membrane formed of a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not contain the iRNA composition, although in some instances it may contain the iRNA composition. Liposomes can be used to transfer and deliver active ingredients to the site of action. Because the structure of the liposome membrane is similar to that of a biological membrane, when liposomes are applied to tissues, the liposome bilayer fuses with the cell membrane bilayer. As liposome-cell fusion proceeds, the aqueous contents containing the iRNA are delivered into the cell, where the iRNA can specifically bind to the target RNA and mediate RNAi. In some cases, liposomes are also specifically targeted, for example, to guide the iRNA to a specific cell type.

[0594] Liposomes containing RNAi agents can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent, causing the lipid component to form micelles. For example, the lipid component can be an amphoteric cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include bile salts, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent formulation is then added to the micelles containing the lipid component. The cationic groups on the lipids interact with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed by, for example, dialysis, thereby obtaining the liposomal formulation of the RNAi agent.

[0595] If necessary, a carrier compound that facilitates polycondensation can be added during the polycondensation reaction, for example, through controlled addition. For instance, the carrier compound can be a polymer other than nucleic acids (e.g., spermine or spermidine). pH can also be adjusted to promote polycondensation.

[0596] Methods for generating stable polynucleotide delivery media (which use polynucleotide / cationic lipid complexes as structural components of the delivery media) are further described, for example, in WO 96 / 37194 (the entire contents of which are incorporated herein by reference). Liposome formulations may also include one or more aspects of the exemplary methods described below: Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Patent No. 4,897,355; U.S. Patent No. 5,171,678; Bangham et al., M. Mol. Biol. 23:238, 1965; Olson et al., Biochim. Biophys. Acta 557:9, 1979; Szoka et al., Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew et al., Biochim. Biophys. Acta 775:169, 1984; Kim et al., Biochim. Biophys. Acta 728:339, 1983; and Fukunaga et al., Endocrinol. 115:757, 1984. Common techniques for preparing appropriately sized lipid aggregates as delivery media include sonication and freeze-thaw extrusion (see, e.g., Mayer et al., Biochim. Biophys. Acta 858:161, 1986). Microfluidics can be used when consistently small (50 to 200 nm) and relatively homogeneous aggregates are desired (Mayhew et al., Biochim. Biophys. Acta 775:169, 1984). These methods are readily adaptable for packaging RNAi agent formulations into liposomes.

[0597] Liposomes are divided into two main categories. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are internalized into the endosomes. Due to the acidic pH in the endosomes, the liposomes rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0598] pH-sensitive or negatively charged liposomes encapsulate nucleic acids instead of forming complexes with them. Because nucleic acids and lipids carry similar charges, repulsion occurs instead of complex formation. Nevertheless, some nucleic acids are encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene into cultured cell monolayers. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0599] A major type of liposome composition includes phospholipids other than those of naturally derived phosphatidylcholine. For example, neutral liposome compositions may be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are typically formed from dimyristoyl phosphatidylglycerol, while anionic fused liposomes are primarily formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC) such as soybean PC and egg PC. Yet another type is formed from phospholipids and / or mixtures of phosphatidylcholine and / or cholesterol.

[0600] Examples of other methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent Nos. 5,283,185; 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0601] Nonionic liposome systems were also investigated to determine their efficacy in drug delivery to the skin, particularly systems containing nonionic surfactants and cholesterol. (The text also mentions the use of Novasome-containing liposomes.) TM I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome TM A nonionic liposome formulation of II (distearate / cholesterol / polyoxyethylene-10-stearyl ether) delivered cyclosporine-A to the dermis of mouse skin. The results showed that this type of nonionic liposome system effectively promoted the deposition of cyclosporine A in different layers of the skin (Hu et al., STP Pharma. Sci., 1994, 4(6) 466).

[0602] Liposomes also include “sterically stable” liposomes, as used herein; this term refers to liposomes containing one or more specific lipids, the incorporation of which results in an extended cycle life relative to liposomes lacking such specific lipids. Examples of sterically stable liposomes include those in which a portion (A) of the lipid portion forming the liposome vesicle contains one or more glycolipids, such as monosialotetrahexosylganglioside G. M1 (A) or (B) those derivatized with one or more hydrophilic polymers (such as polyethylene glycol (PEG) moiety). While not wishing to be bound by any particular theory, it is believed in the art that, at least for spatially stable liposomes containing gangliosides, sphingomyelins, or PEG-derived lipids, the prolongation of the circulating half-life of these spatially stable liposomes is due to reduced uptake by reticuloendothelial system (RES) cells (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0603] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) reported monosialotetrahexosylganglioside G M1 Galactocerebroside sulfate and phosphatidylinositol can prolong the blood half-life of liposomes. Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949) elaborated on these findings. US Patent Nos. 4,837,028 and WO 88 / 04924 (both belonging to Allen et al.) disclose a compound containing (1) sphingomyelin and (2) ganglioside G. M1 Liposomes containing galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. WO 97 / 13499 (Lim et al.) discloses liposomes containing 1,2-sn-dimyristoylphosphatidylcholine.

[0604] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes, while not effectively fusing with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.

[0605] Further advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can be incorporated into a wide range of water-soluble and lipid-soluble drugs; and liposomes can protect the RNAi agents encapsulated in their internal chambers from metabolism and degradation (Rosoff in "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger, and Banker (eds.), 1988, Vol. 1, p. 245). Important factors to consider when preparing liposomal formulations include lipid surface charge, vesicle size, and the aqueous phase volume of the liposome.

[0606] A positively charged synthetic cationic lipid, N-[1-(2,3-dioleoxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes. These complexes are capable of fusing with negatively charged lipids in the cell membranes of tissue cultured cells to deliver RNAi agents (see, for example, Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Patent No. 4,897,355, which describes DOTMA and its use with DNA).

[0607] The DOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonium)propane (DOTAP) can be used in combination with phospholipids to form DNA-complex vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an agent for the efficient delivery of highly anionic nucleic acids into living tissue culture cells. It comprises positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. When a sufficient amount of positively charged liposomes is used, the resulting complex also has a positive net charge. The positively charged complex prepared in this way spontaneously attaches to the negatively charged cell surface, fuses with the plasma membrane, and efficiently delivers functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonium)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moiety is linked by an ester bond rather than an ether bond.

[0608] Other reported cationic lipid compounds include compounds coupled with various moieties, including, for example, carboxysemine, which has been coupled with one of two types of lipids, and compounds such as 5-carboxysemine-glycine dioctyl oleamide (“DOGS”) (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxysemine-amine (“DPPES”) (see, for example, U.S. Patent No. 5,171,678).

[0609] Other cationic lipid conjugates include cholesterol-derived lipids (“DC-Chol”), which have been conjugated with DOPE to form liposomes (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine conjugated with DOPE has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids are claimed to exhibit lower toxicity and provide more efficient transfection than compositions containing DOTMA. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, CA) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, MD). Other cationic lipids suitable for delivering oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0610] Liposome formulations are particularly suitable for topical application, and liposomes offer several advantages compared to other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target site, and the ability to deliver RNAi agents into the skin. In some embodiments, liposomes are used to deliver RNAi agents to epidermal cells and also enhance the penetration of RNAi agents into dermal tissues (e.g., skin). For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, for example, Weiner et al., Journal of Drug Targeting, 1992, Vol. 2, 405-410; and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, RJ and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al., Gene 56:267-276, 1987; Nicolau, C. et al., Meth. Enz. 149:157-176, 1987; Straubinger, RM and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, CY and Huang, L., Proc. Natl. Acad.). Sci. USA 84:7851-7855, 1987)).

[0611] Nonionic liposome systems were also investigated to determine their efficacy in drug delivery to the skin, particularly systems containing nonionic surfactants and cholesterol. Drug delivery to the dermis of mouse skin was performed using nonionic liposome formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether). Such formulations with RNAi agents could be used to treat skin conditions.

[0612] Liposomes containing iRNA can be made highly deformable. This deformability allows the liposomes to pass through pores smaller than the average radius of a liposome. For example, transfersomes are a type of deformable liposome. Transfersomes can be prepared by adding a surface edge activator (typically a surfactant) to a standard liposome composition. Transfersomes containing RNAi agents can be delivered, for example, via subcutaneous injection, thereby delivering the RNAi agent to keratinocytes in the skin. In order to penetrate intact mammalian skin, lipid vesicles must pass through a series of fine pores (each less than 50 nm in diameter) under the influence of a suitable transdermal gradient. Furthermore, due to their lipid properties, these transfersomes are self-optimizing (adapting to the shape of the pores, such as those in the skin), self-repairing, and can frequently reach their targets without breaking, and are often self-loading.

[0613] Other formulations applicable to this invention are described in U.S. Provisional Application Serial Nos. 61 / 018,616 (filed January 2, 2008), 61 / 018,611 (filed January 2, 2008), 61 / 039,748 (filed March 26, 2008), 61 / 047,087 (filed April 22, 2008), and 61 / 051,528 (filed May 8, 2008). Formulations applicable to this invention are also described in PCT Application No. PCT / US2007 / 080331 (filed October 3, 2007).

[0614] Transfer bodies are another type of liposome and are highly deformable lipid aggregates, making them attractive candidates for drug delivery media. Transfer bodies can be described as lipid droplets whose high deformability allows them to easily pass through pores smaller than droplets. Transfer bodies are adaptable to the environment in which they are used; for example, they are self-optimizing (adapting to the shape of skin pores), self-healing, typically reaching their targets without breaking, and often self-loading. To prepare transfer bodies, surface edge activators, typically surfactants, can be added to standard liposome compositions. Transfer bodies have been used to deliver serum albumin into the skin. It has been shown that transfer body-mediated serum albumin delivery is as effective as subcutaneous injection of a solution containing serum albumin.

[0615] Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common method for classifying and ranking the properties of many different types of natural and synthetic surfactants is by using the hydrophilic-lipophilic balance (HLB) value. The properties of the hydrophilic group (also known as the “head”) are the most useful method for distinguishing the different surfactants used in formulations (Rieger in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York City, NY, 1988, p. 285).

[0616] If a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceuticals and cosmetics and can be used across a wide pH range. Generally, their HLB values ​​range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters, such as ethylene glycol esters, propylene glycol esters, glycerol esters, polyglycerol esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this category. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant category.

[0617] Surfactants are classified as anionic surfactants if their molecules carry a negative charge when dissolved or dispersed in water. Anionic surfactants include carboxylic acid esters (such as soaps, acyl lactates, and amino acid amides), sulfate esters (such as alkyl sulfates and ethoxylated alkyl sulfates), sulfonates (such as alkylbenzene sulfonates, acyl isothiosulfates, acyl taurate, and sulfosuccinates), and phosphate esters. The most important members of the anionic surfactant category are alkyl sulfates and soaps.

[0618] Surfactants are classified as cationic surfactants if their molecules carry a positive charge when dissolved or dispersed in water. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most widely used members in this category.

[0619] Surfactants are classified as amphoteric surfactants if their molecules can carry either a positive or negative charge. Amphoteric surfactants include acrylic acid derivatives, substituted alkyl amides, N-alkyl betaines, and phospholipids.

[0620] The use of surfactants in pharmaceutical products, formulations, and emulsions has been reviewed (in Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0621] The iRNA used in the method of this invention can also be provided in the form of micelle formulations. "Micelles" are defined herein as a special type of molecular aggregate in which amphiphilic molecules are arranged in a spherical structure such that the hydrophobic portions of all molecules face inwards, leaving the hydrophilic portions in contact with the surrounding aqueous phase. If the environment is hydrophobic, the arrangement is reversed.

[0622] An aqueous solution of the siRNA composition can be mixed with C8 to C8. 22Alkyl sulfate alkali metal salts and micellar-forming compounds are used to prepare mixed micelle formulations suitable for transdermal membrane delivery. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, glyceryl monooleate, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxycholic acid alkyl glycine and its pharmaceutically acceptable salts, glycerol, polyglycerol, lysine, polylysine, trioleic acid glycerol, polyoxyethylene ethers and their analogs, polydocalyl ethers and their analogs, chenodeoxycholic acid, deoxycholic acid, and mixtures thereof. The micelle-forming compounds may be added simultaneously with or after the alkyl sulfate alkali metal salt. Essentially, any type of mixing of the components will form mixed micelles, but vigorous mixing is required to provide micelles of smaller size.

[0623] In one method, a first micelle composition is prepared comprising an siRNA composition and at least an alkyl sulfate alkali metal salt. The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, a micelle composition is prepared by mixing an siRNA composition, an alkyl sulfate alkali metal salt, and at least one micelle-forming compound, followed by the addition of any remaining micelle-forming compound and vigorous mixing.

[0624] Phenol and / or m-cresol can be added to the mixed micelle composition to stabilize the formulation and prevent bacterial growth. Alternatively, phenol and / or m-cresol can be added together with the micelle-forming component. After the mixed micelle composition is formed, an isotonic agent, such as glycerol, may also be added.

[0625] To deliver a micelle formulation as a spray, the formulation is loaded into an aerosol dispenser, and a propellant is added to the dispenser. The propellant, under pressure, is in liquid form within the dispenser. The proportions of the components are adjusted so that the aqueous phase and propellant are combined, forming a single phase. If two phases are present, the dispenser needs to be shaken before dispensing a portion of the contents (e.g., through a metering valve). The dispensed dose of the agent is expelled from the metering valve as a fine mist.

[0626] The propellant may include hydrochlorofluorocarbons, hydrofluorocarbons, dimethyl ether, and diethyl ether. In some embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) may be used.

[0627] The specific concentration of key components can be determined through relatively simple experiments. For oral absorption, the dosage usually needs to be increased, for example, by at least two or three times, the dosage used for injection or administration via the gastrointestinal tract.

[0628] B. Lipid particles

[0629] The iRNA, such as dsRNA, in this invention can be completely encapsulated in a lipid formulation (e.g., LNP) to form, for example, SPLP, pSPLP, SNALP, or other nucleic acid-lipid particles.

[0630] As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles, including SPLP. As used herein, the term "SPLP" refers to nucleic acid-lipid particles containing plasmid DNA encapsulated within lipid vesicles. SNALPs and SPLPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALPs and SPLPs are useful for systemic application because they exhibit prolonged circulation life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the application site). SPLPs include "pSPLPs," which contain condenser-nucleic acid complexes encapsulated as described in PCT Publication No. WO 00 / 03683. The particles of the present invention typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially non-toxic. Furthermore, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010 / 0324120 and PCT Publication No. WO 96 / 40964.

[0631] In one embodiment, the ratio (mass / mass ratio) of lipids to the drug (e.g., the ratio of lipids to dsRNA) ranges from about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Intermediate ranges between these ranges are also considered part of the invention.

[0632] Cationic lipids can be, for example, N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearate-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinyloxy-N,N-dimethylaminopropane (DLenDMA), 1, 2-Dilinoleoylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleoyl-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleothio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleoyloxy-3-trimethylaminopropane hydrochloride (DLin-TMA.Cl), 1, 2-Dilinoleoyl-3-trimethylaminopropane hydrochloride (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ) or 3-(N,N-dilinoleoylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleoylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DL... (in-K-DMA) or its analogues, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopentadieno[d][1,3]dioxacyclopenten-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazidine)docosahexadecyl-2-ol (Tech G1), or mixtures thereof.Cationic lipids may account for approximately 20 mol% to approximately 50 mol% or approximately 40 mol% of the total lipids present in the particles.

[0633] In another embodiment, compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008 (which is incorporated herein by reference).

[0634] In one embodiment, the lipid-siRNA particles comprise 40% 2,2-dilinole-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (molar percentage), have a particle size of 63.0 ±20 nm, and have an siRNA / lipid ratio of 0.027.

[0635] Ionizable / non-cationic lipids can be anionic or neutral lipids, including but not limited to distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. Non-cationic lipids may comprise approximately 5 mol% to approximately 90 mol%, approximately 10 mol%, or approximately 58 mol% of the total lipids present in the particles (if cholesterol is included).

[0636] The coupling lipids that inhibit particle aggregation can be, for example, polyethylene glycol (PEG) lipids, including but not limited to PEG-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. PEG-DAA conjugates can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristoxypropyl (Ci4), PEG-dispalmitoxypropyl (Ci6), or PEG-distearateoxypropyl (C)8. The coupling lipids that prevent particle aggregation can comprise from 0 mol% to about 20 mol% or about 2 mol% of the total lipids present in the particles.

[0637] In some embodiments, the nucleic acid-lipid particles further contain cholesterol, which accounts for, for example, about 10 mol% to about 60 mol% or about 48 mol% of the total lipids present in the particles.

[0638] In one embodiment, lipid-dsRNA nanoparticles (i.e., LNP01 particles) can be prepared using liposomes ND98∙4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, which is incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids).

[0639] In some embodiments of the invention, suitable cationic lipids for use in the compositions of the invention are those described in U.S. Patent No. 9,061,063 and PCT Publication No. WO 2013 / 086354 (the entire contents of which are incorporated herein by reference). In some embodiments, suitable cationic lipids comprise one or more biodegradable groups. A biodegradable group comprises one or more bonds that can undergo bond-breaking reactions in a biological environment (e.g., in an organism, organ, tissue, cell, or organelle). Functional groups comprising biodegradable bonds include, for example, esters, dithiols, and oximes. Biodegradation can be a factor affecting the clearance of the compound from the body upon administration to a subject. Biodegradation can be measured by a cell-based assay in which the formulation comprising the cationic lipid is exposed to cells and sampled at different time points. The lipid fraction can be extracted from the cells and separated and analyzed by LC-MS. The biodegradation rate (e.g., expressed as a t1 / 2 value) can be measured based on the LC-MS data. The cationic lipid comprises a biodegradable group.

[0640] In one embodiment, the cationic lipids in any of the embodiments described herein have an in vivo half-life (t1 / 2) (e.g., in the liver, spleen, or plasma) of less than about 3 hours, such as less than about 2.5 hours, less than about 2 hours, less than about 1.5 hours, less than about 1 hour, less than about 0.5 hours, or less than about 0.25 hours. The cationic lipids preferably remain intact or have a half-life sufficient to form stable lipid nanoparticles that effectively deliver the desired active pharmaceutical ingredient (e.g., nucleic acid) to its target, but subsequently degrade rapidly to minimize any side effects on the subject. For example, in mice, the t1 / 2 of the cationic lipids in the spleen is preferably from about 1 to about 7 hours.

[0641] In another embodiment, the cationic lipid containing one or more biodegradable groups in any of the embodiments described herein has an in vivo half-life (t1 / 2) (e.g., in the liver, spleen, or plasma) that is less than about 10% of the in vivo half-life of the same cationic lipid not containing one or more biodegradable groups (e.g., less than about 7.5%, less than about 5%, less than about 2.5%).

[0642] Representative cationic lipids include, but are not limited to:

[0643] r = 0-2, n = 1-5 and m = 1-5 r = 0, 1 or 2 z r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2 m = 0-5, n = 0, 1 or 2 m = 0-5, n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 n = 0, 1 or 2 r = 0, 1 or 2 r = 0, 1 or 2

[0644] In a preferred embodiment, cationic lipids are

[0645] .

[0646] In some embodiments, the dsRNA agent of the present invention is formulated together with a cationic lipid, such as... Distearate phosphatidylcholine (DSPC), cholesterol (Chol), and 1,2-myristoyl-trans-glycerol-3-methoxy polyethylene glycol (PEG-DMG). In one embodiment, The ratios of DSPC, Chol, and PEG-DMG are 50:12:36:2.

[0647] This invention includes the free form of the cationic lipids described herein, their pharmaceutically acceptable salts, and their stereoisomers. The cationic lipids may be protonated salts of amine cationic lipids. The term "free form" refers to the non-salt form of the amine cationic lipid. The free form can be regenerated by treating the salt with a suitable dilute aqueous solution of an alkaline solution (such as dilute NaOH, potassium carbonate, ammonia, and sodium bicarbonate).

[0648] Pharmaceutically acceptable salts of the cationic lipids of this invention can be synthesized by conventional chemical methods from cationic lipids of this invention containing basic or acidic moieties. Typically, salts of basic cationic lipids can be prepared by ion-exchange chromatography or by reacting a free base with a stoichiometric or excess amount of the desired salting inorganic or organic acid in a suitable solvent or various combinations of solvents. Similarly, salts of acidic compounds are formed by reacting with a suitable inorganic or organic base.

[0649] Therefore, pharmaceutically acceptable salts of the cationic lipids of the present invention include non-toxic salts of the cationic lipids of the present invention formed by reacting the basic cationic lipids with inorganic or organic acids. For example, non-toxic salts include salts derived from inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, etc.) and salts prepared from organic acids (such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, dihydroxynaphthyl acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, hydroxyethanesulfonic acid, and trifluoroacetic acid (TFA)).

[0650] When the cationic lipids of the present invention are acidic, a suitable "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic alkali (including inorganic and organic alkalis). Salts derived from inorganic alkalis include aluminum salts, ammonium salts, calcium salts, copper salts, ferric salts, ferrous salts, lithium salts, magnesium salts, ferric salts, ferrous salts, potassium salts, sodium salts, and zinc salts. In one embodiment, the alkali is selected from ammonium salts, calcium salts, magnesium salts, potassium salts, and sodium salts. Salts derived from pharmaceutically acceptable, non-toxic organic bases include the following: primary, secondary, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, histidine, ammonium hydrobromide, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, and tromethamine.

[0651] It should also be noted that the cationic lipids of the present invention can be internal salts or zwitterions, because under physiological conditions, the deprotonated acidic portion (such as a carboxyl group) in the compound can be anionic, and this charge can then be internally balanced with the cationic charge of the protonated or alkylated basic portion (such as a quaternary nitrogen atom).

[0652] C. Additives

[0653] i. Emulsion

[0654] The compositions of the present invention can be prepared and formulated into emulsions. Emulsions are typically heterogeneous systems in which one liquid is dispersed in droplets in another liquid, the droplets typically having a dia...

Claims

1. A method of treating a subject with cancer, the method comprising administering to the subject a double-stranded ribonucleic acid (dsRNA) agent at a dose of about 0.01 mg / kg to about 1.5 mg / kg to inhibit the expression of β-catenin (CTNNB1), thereby treating the subject with cancer. The dsRNA agent contains a sense strand and an antisense strand. The difference between the sense strand and the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3' is no more than 4 bases, and the difference between the antisense strand and the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc-3' is no more than 4 bases. Where a, g, c, and u are 2'-O-methyl(2'-OMe)adenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; Af, Gf, Cf, and Uf are 2'-fluoroadenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; s is a thiophosphate bond; VP is a vinylphosphonate; dT is 2'-deoxythymidine-3'-phosphate; dG is 2'-deoxyguanosine-3'-phosphate; and dA is 2'-deoxyadenosine-3'-phosphate.

2. A method of treating a subject with cancer, the method comprising selecting a subject with cancer containing a Wnt-pathway activating mutation, and Subjects were administered a double-stranded RNA (dsRNA) agent at doses of approximately 0.01 mg / kg to approximately 1.5 mg / kg to inhibit the expression of β-catenin (CTNNB1), thereby treating subjects with cancer. The dsRNA agent contains a sense strand and an antisense strand. The difference between the sense strand and the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3' is no more than 4 bases, and the difference between the antisense strand and the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc-3' is no more than 4 bases. Where a, g, c, and u are 2'-O-methyl(2'-OMe)adenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; Af, Gf, Cf, and Uf are 2'-fluoroadenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; s is a thiophosphate bond; VP is a vinylphosphonate; dT is 2'-deoxythymidine-3'-phosphate; dG is 2'-deoxyguanosine-3'-phosphate; and dA is 2'-deoxyadenosine-3'-phosphate.

3. The method according to claim 1 or 2, wherein the cancer is hepatocellular carcinoma.

4. The method according to claim 3, wherein the hepatocellular carcinoma is advanced or metastatic hepatocellular carcinoma.

5. The method according to claim 1 or 2, wherein the cancer is colorectal cancer.

6. The method of claim 5, wherein the cancer is colorectal cancer with liver metastases.

7. The method according to any one of claims 1-6, wherein the subject is a human.

8. The method according to any one of claims 1-7, wherein the sense strand comprises the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3', and the antisense strand comprises the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc-3'.

9. The method according to any one of claims 1-7, wherein the sense strand consists of the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3', and the antisense strand consists of the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc-3'.

10. The method according to any one of claims 1-9, wherein the dsRNA agent is present in the pharmaceutical composition.

11. The method of claim 10, wherein the pharmaceutical composition comprises lipids.

12. The method of claim 11, wherein the lipid is a cationic lipid.

13. The method of claim 12, wherein the cationic lipid comprises one or more biodegradable groups.

14. The method of claim 13, wherein the lipid comprises a structure 。 15. The method of claim 14, wherein the pharmaceutical composition comprises: (a) ； (b) Cholesterol; (c) DSPC; and (d) PEG-DMG.

16. The method of claim 15, wherein the pharmaceutical composition comprises substances present in a molar ratio of 50:12:36:2 or 50:10:38.5:1.

5. DSPC, cholesterol, and PEG-DMG.

17. The method according to any one of claims 1-16, wherein the subject is administered a dose of about 0.01 mg / kg to about 1.5 mg / kg of dsRNA about every three weeks.

18. The method according to any one of claims 1-17, wherein the subject is given a pre-treatment drug before the administration of the dsRNA agent.

19. The method of claim 18, wherein the pre-treatment administered to the subject prior to administration of the dsRNA agent is selected from the group consisting of dexamethasone, acetaminophen, diphenhydramine, and ranitidine, and combinations thereof.

20. The method according to any one of claims 1-19, wherein the dsRNA agent is administered intravenously to the subject.

21. The method according to any one of claims 1-20, further comprising administering to the subject additional treatment and / or therapeutic agent for treating cancer.

22. The method of claim 21, wherein the additional therapeutic agent is selected from the group consisting of: immunotherapeutic agents, VEGF inhibitors, chemotherapeutic agents, growth inhibitors, anti-angiogenic agents, antitumor compositions, and any combination thereof.

23. The method of claim 22, wherein the additional therapeutic agent is an immunotherapeutic agent.

24. The method of claim 23, wherein the combination of immunotherapeutic agents is administered to the subject.

25. The method of claim 23 or 24, wherein the immunotherapeutic agent is an immune checkpoint inhibitor.

26. The method of claim 25, wherein the immune checkpoint inhibitor is an anti-programmed death-1 (PD-1) antibody or its antigen-binding fragment, an anti-programmed death-ligand 1 (PD-L1) antibody or its antigen-binding fragment, and / or an anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibody or its antigen-binding fragment.

27. The method of claim 25 or 26, wherein the immunotherapeutic agent is an anti-programmed death-1 (PD-1) antibody or an antigen-binding fragment thereof.

28. The method of claim 27, wherein the anti-PD1 antibody is a humanized monoclonal antibody or an antigen-binding fragment thereof.

29. The method of claim 28, wherein the humanized monoclonal anti-PD-1 antibody or its antigen-binding fragment is pembrolizumab.

30. The method of claim 29, wherein the subject is administered a dose of about 200 mg of pembrolizumab.

31. The method of claim 30, wherein a dose of 200 mg of pembrolizumab is administered to the subject approximately every three weeks.

32. The method according to any one of claims 29-31, wherein pembrolizumab is administered intravenously to the subject.

33. The method of claim 32, wherein intravenous administration comprises intravenous infusion of pembrolizumab over approximately 30 minutes.

34. The method according to any one of claims 27-33, wherein the dsRNA agent is administered to the subject before, after, or simultaneously with the administration of the anti-PD-1 antibody or its antigen-binding fragment.

35. A method of treating a subject with cancer, the method comprising administering to the subject a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of β-catenin (CTNNB1) at a dose of about 0.01 mg / kg to about 1.5 mg / kg, and an anti-programmed death-1 (PD-1) antibody or an antigen-binding fragment thereof at a dose of about 200 mg, thereby treating the subject with cancer.

36. A method of treating a subject with cancer, the method comprising selecting a subject with cancer containing a Wnt-pathway activating mutation, and Subjects with cancer were treated by administering a double-stranded RNA (dsRNA) agent for inhibiting the expression of β-catenin (CTNNB1) at a dose of about 0.01 mg / kg to about 1.5 mg / kg, and an anti-programmed death-1 (PD-1) antibody or its antigen-binding fragment at a dose of about 200 mg.

37. The method according to claim 35 or 36, wherein the cancer is hepatocellular carcinoma.

38. The method of claim 37, wherein the hepatocellular carcinoma is advanced or metastatic hepatocellular carcinoma.

39. The method according to claim 35 or 36, wherein the cancer is colorectal cancer.

40. The method of claim 39, wherein the cancer is colorectal cancer with liver metastases.

41. The method according to any one of claims 36-40, wherein the subject is a human being.

42. The method according to any one of claims 36-41, wherein the dsRNA agent comprises a sense strand comprising at least 15 consecutive nucleotides, the sense strand differing from the nucleotide sequence 5'-UACUGUUGGAUUGAUUCGAAA-3' by no more than 3 nucleotides; and an antisense strand comprising at least 15 consecutive nucleotides, the antisense strand differing from the nucleotide sequence 5'-UTUCGAAUCAATCCAACAGUAGC-3' by no more than 3 nucleotides.

43. The method of claim 42, wherein the dsRNA agent comprises a sense strand containing the nucleotide sequence 5'-UACUGUUGGAUUGAUUCGAAA-3' and an antisense strand containing the nucleotide sequence 5'-UTUCGAAUCAATCCAACAGUAGC-3'.

44. The method according to claim 42 or 43, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand contain nucleotide modifications.

45. The method of claim 44, wherein at least one nucleotide modification is selected from the group consisting of: deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, baseless nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl... 2'-O-alkyl-modified nucleotides, morpholinonucleotides, phosphoramide, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate, nucleotides containing 5'-phosphate mimics, heat-labile nucleotides, ethylene glycol-modified nucleotides (GNA), nucleotides containing 2'-phosphate, and 2-O-(N-methylacetamide)-modified nucleotides; and combinations thereof.

46. ​​The method of claim 44, wherein at least one nucleotide modification is selected from the group consisting of: LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluorine, 2'-deoxy, 2'-hydroxy and ethylene glycol; and combinations thereof.

47. The method of claim 44, wherein at least one nucleotide modification is selected from the group consisting of: deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, ethylene glycol modified nucleotides (GNA), nucleotides containing 2' phosphate groups, nucleotides containing thiophosphate groups, and vinyl-phosphonate nucleotides; and combinations thereof.

48. The method of claim 44, wherein at least one nucleotide modification is a nucleotide modified with a heat-labile nucleotide modification.

49. The dsRNA agent according to claim 48, wherein the heat-labile nucleotide modification is selected from the group consisting of: no base modification, mismatch with the relative nucleotide in the duplex, unstable sugar modification, 2'-deoxy modification, acyclic nucleotide, nonlocked nucleic acid (UNA), and glycerol nucleic acid (GNA).

50. The method according to any one of claims 36-49, wherein the length of the double-stranded region is 19-30 nucleotide pairs.

51. The method according to any one of claims 36-50, wherein the length of each chain independently does not exceed 30 nucleotides.

52. The method according to any one of claims 36-51, wherein the length of each chain is independently 19-30 nucleotides.

53. The method according to any one of claims 36-52, wherein the length of the sense strand is 21 nucleotides and the length of the antisense strand is 23 nucleotides.

54. The method according to any one of claims 36-53, wherein at least one chain comprises a 3' overhang of at least one nucleotide.

55. The method according to any one of claims 36-54, wherein at least one chain comprises a 3' overhang of at least two nucleotides.

56. The method according to any one of claims 36-55, wherein the dsRNA agent further comprises a ligand.

57. The method according to any one of claims 33-56, wherein the dsRNA agent further comprises at least one phosphate thioester or methylphosphonate nucleoside linker.

58. The method of claim 58, wherein the internucleotide link between the thiophosphate or methylphosphonate is located at the 3' end of one chain.

59. The method of claim 58, wherein the chain is an antisense chain.

60. The method of claim 58, wherein the chain is a justice chain.

61. The method of claim 57, wherein the internucleotide link between the thiophosphate or methylphosphonate is located at the 5' end of one chain.

62. The method of claim 61, wherein the chain is an antisense chain.

63. The method of claim 61, wherein the chain is a justice chain.

64. The method of claim 57, wherein the internucleotide link between the thiophosphate or methylphosphonate is located at both the 5' and 3' ends of a chain.

65. The method of claim 64, wherein the chain is an antisense chain.

66. The method according to any one of claims 36-65, wherein the dsRNA agent comprises a sense strand differing from the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3' by no more than 4 bases, and an antisense strand differing from the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc-3' by no more than 4 bases. Where a, g, c, and u are 2'-O-methyl(2'-OMe)adenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; Af, Gf, Cf, and Uf are 2'-fluoroadenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; s is a thiophosphate bond; VP is a vinylphosphonate; dT is 2'-deoxythymidine-3'-phosphate; dG is 2'-deoxyguanosine-3'-phosphate; and dA is 2'-deoxyadenosine-3'-phosphate.

67. The method according to any one of claims 36-66, wherein the dsRNA agent comprises a sense strand containing the nucleotide sequence 5'-usascuguugGfAfUfugauucgasasa-3', and an antisense strand containing the nucleotide sequence 5'-VPudTucdGadAucaadTcCfaacaguasgsc-3'. Where a, g, c, and u are 2'-O-methyl(2'-OMe)adenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; Af, Gf, Cf, and Uf are 2'-fluoroadenosine-, guanosine-, cytidine-, and uridine-3'-phosphate, respectively; s is a thiophosphate bond; VP is a vinylphosphonate; dT is 2'-deoxythymidine-3'-phosphate; dG is 2'-deoxyguanosine-3'-phosphate; and dA is 2'-deoxyadenosine-3'-phosphate.

68. The method according to any one of claims 36-67, wherein the dsRNA agent is present in the pharmaceutical composition.

69. The method of claim 68, wherein the pharmaceutical composition comprises lipids.

70. The method of claim 69, wherein the lipid is a cationic lipid.

71. The method of claim 70, wherein the cationic lipid comprises one or more biodegradable groups.

72. The method of claim 71, wherein the lipid comprises a structure 。 73. The method of claim 72, wherein the pharmaceutical composition comprises (a) ； (b) Cholesterol; (c) DSPC; and (d) PEG-DMG.

74. The method of claim 73, wherein the pharmaceutical composition comprises substances present in a molar ratio of 50:12:36:2 or 50:10:38.5:1.

5. DSPC, cholesterol, and PEG-DMG.

75. The method according to any one of claims 36-74, wherein the subject is administered a dose of about 0.01 mg / kg to about 1.5 mg / kg of dsRNA about every three weeks.

76. The method according to any one of claims 36-75, wherein the subject is given a pre-treatment drug before the administration of the dsRNA agent.

77. The method of claim 76, wherein the pre-treatment administered to the subject prior to administration of the dsRNA agent is selected from the group consisting of dexamethasone, acetaminophen, diphenhydramine, and ranitidine, and combinations thereof.

78. The method according to any one of claims 36-77, wherein the dsRNA agent is administered intravenously to the subject.

79. The method according to any one of claims 36-78, wherein the anti-PD1 antibody or its antigen-binding fragment is a humanized monoclonal antibody or its antigen-binding fragment.

80. The method of claim 79, wherein the humanized monoclonal anti-PD1 antibody or its antigen-binding fragment is pembrolizumab.

81. The method of claim 80, wherein a dose of 200 mg of pembrolizumab is administered to the subject.

82. The method of claim 81, wherein a dose of 200 mg of pembrolizumab is administered to the subject every three weeks.

83. The method according to any one of claims 80-82, wherein pembrolizumab is administered intravenously to the subject.

84. The method of claim 83, wherein intravenous administration comprises intravenous infusion of pembrolizumab over approximately 30 minutes.

85. The method according to any one of claims 36-84, wherein the dsRNA agent is administered to the subject before, after, or simultaneously with the administration of the anti-PD-1 antibody or its antigen-binding fragment.

86. The method according to any one of claims 36-85, further comprising administering to the subject additional treatment and / or therapeutic agent for treating cancer.

87. The method of claim 86, wherein the additional therapeutic agent is selected from the group consisting of: immunotherapeutic agents, VEGF inhibitors, chemotherapeutic agents, growth inhibitors, anti-angiogenic agents, antitumor compositions, and any combination thereof.

88. The method of claim 87, wherein the additional therapeutic agent is an immunotherapeutic agent.

89. The method of claim 88, wherein the combination of immunotherapeutic agents is administered to the subject.

90. The method of claim 88 or 89, wherein the immunotherapeutic agent is an immune checkpoint inhibitor.

91. The method of claim 90, wherein the immune checkpoint inhibitor is an anti-programmed death-1 (PD-1) antibody or its antigen-binding fragment, an anti-programmed death-ligand 1 (PD-L1) antibody or its antigen-binding fragment, and / or an anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibody or its antigen-binding fragment.

92. The method according to any one of claims 2-35 and 37-91, wherein the Wnt-pathway activation mutation is a mutation in a gene selected from the group consisting of: Axin1, Axin2, APC, CTNNB1, RNF43, ZNRF3, RSPO1, RSPO2, RSPO3 and RSPO4 and combinations thereof.

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