HEPATITIS B VIRUS (HBV) iRNA COMPOSITION, AND METHOD FOR USING THE SAME

Abstract

To provide an effective treatment for hepatitis B virus (HBV) infection which inhibits virus replication and can recover immunological control.SOLUTION: The present invention relates to RNAi agent targeting hepatitis B virus (HBV) genome which is double-stranded RNAi agent, for example, a method for using the RNAi agent for inhibiting an expression of one or more HBV genes, and a method for treating a subject having HBV infection and / or HBV relate d disorder which is Type B chronic hepatitis infection, for example.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 077,799, filed November 10, 2014, and U.S. Provisional Patent Application No. 62 / 137,464, filed March 24, 2015. The entire contents of each of the foregoing patent applications are incorporated herein by reference.

[0002] This application also claims priority to U.S. Provisional Patent Application No. 62 / 077,672, filed November 10, 2014, the entire contents of which are hereby incorporated by reference herein.

[0003] This application is related to PCT / US2015 / XXXXX, filed November 10, 2015, entitled "Hepatitis D Viris (HDV) iRNA Compositions and Methods of Use Thereof," the entire contents of which are incorporated herein by reference.

[0004] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 5, 2015, is named 121301-02320_SL.txt and is 385,791 bytes in size. [Background technology]

[0005] More than 400 million people worldwide are chronically infected with HBV and are therefore at high risk of developing serious liver diseases such as chronic hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma (HCC), which cause an estimated 600,000 deaths each year.

[0006] The natural evolution of chronic HBV infection involves four successive stages: (1) an initial "immune tolerance" stage, characterized by high levels of viral replication and minimal hepatitis; (2) an immune response stage, characterized by significant liver inflammation and elevated serum aminotransferases; (3) a "non-replicating" stage, characterized in some patients by seroconversion to anti-HBe, undetectable or low levels of viremia (less than 2000 IU / ml by PCR-based assays), and resolution of liver inflammation; and (4) HBeAg-negative chronic hepatitis B (resulting from the emergence of specific viral mutations that prevent HBeAg production but not viral replication). This form of chronic hepatitis B (CHB) is characterized by fluctuating serum HBV DNA and serum aminotransferase (ALT and AST) levels, as well as progressive liver disease. It is important to note that CHB can exist as either HBeAg-positive or HBeAg-negative CHB. Longitudinal studies of CHB patients have shown that the 5-year cumulative incidence of developing cirrhosis ranges from 8 to 20%. The 5-year cumulative incidence of hepatic decompensation is approximately 20%. The incidence of HCC is increasing worldwide and is now the fifth most common cancer. The annual incidence of HBV-associated HCC is high, ranging from 2 to 5% in those with cirrhosis.

[0007] The primary goals of HBV treatment are to permanently suppress HBV replication and ameliorate liver disease. Clinically important short-term goals are to achieve HBeAg seroconversion, normalization of serum ALT and AST levels, resolution of hepatitis, and prevention of hepatic decompensation. The ultimate goal of treatment is to achieve a durable response to prevent the development of cirrhosis and liver cancer and prolong survival. HBV infection cannot be completely eradicated because a specific form of viral covalently closed circular DNA (ccc HBV DNA) persists in the nuclei of infected hepatocytes. However, treatment-induced serum HBsAg clearance is a marker of the termination of chronic HBV infection and is associated with the best long-term outcomes.

[0008] The current standard of care for HBV treatment involves suppressing viral production through interferon- or thymosin a1-based immunotherapy and inhibition of HBV polymerase. While HBV polymerase inhibitors are effective in reducing viral production, they have little to no effect in rapidly reducing HBsAg levels (as with tenofovir disoproxil fumarate), or can slowly reduce HBsAg levels with long-term treatment in a limited number of patients. Interferon-based immunotherapy can achieve both a reduction in viral production and early elimination of HBsAg from the blood, but only in a small proportion of treated patients. The generally accepted role of HBsAg in the blood is to sequester anti-HBsAg antibodies, allowing infectious viral particles to evade immune detection, which is likely one of the reasons why HBV infection remains chronic. In addition, HBsAg, HBeAg and HBcAg all have immunosuppressive properties, and the persistence of these viral proteins in a patient's blood after administration of any of the currently available HBV treatments is likely to have significant implications in preventing a patient from achieving immunological control of their HBV infection.

[0009] Although all three major HBV proteins (HBsAg, HBeAg, and HBcAg) have immunosuppressive properties, HBsAg accounts for the vast majority of circulating HBV proteins in HBV-infected subjects. Additionally, while elimination of HBeAg (via seroconversion) or reduction of serum viremia is not associated with sustained control of HBV infection upon treatment discontinuation, elimination of serum HBsAg from the blood (and seroconversion) in HBV infection is a well-recognized prognostic indicator of an antiviral response during treatment that can lead to control of HBV infection upon treatment discontinuation (although this occurs in only a small proportion of patients receiving immunotherapy). Therefore, while reduction of all three major HBV proteins (HBsAg, HBeAg, and HBcAg) may result in optimal elimination of the inhibitory effects, elimination of HBsAg alone appears to be sufficient by itself to eliminate the majority of viral suppression of immune function in HBV-infected subjects. Summary of the Invention [Problem to be solved by the invention]

[0010] Therefore, while there are currently no treatment regimens that can restore immunological control of HBV in most patients, there is a need for effective treatments for HBV infection that can both inhibit viral replication and restore immunological control in the majority of patients. Accordingly, there is a need in the art for alternative and combination therapies for subjects infected with HBV and / or with HBV-related diseases. [Means for solving the problem]

[0011] The present invention provides iRNA compositions that cause RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of Hepatitis B virus (HBV) genes. The HBV genes may be located within a cell, e.g., within a cell within the body of a subject, such as a human.

[0012] The present invention also provides methods and therapies for treating subjects with disorders that may benefit from inhibiting or reducing the expression of HBV genes, e.g., HBV infection and / or HBV-related diseases, e.g., chronic hepatitis B infection (CHB), cirrhosis, liver failure, and hepatocellular carcinoma (HCC), using iRNA compositions that cause RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of HBV genes to inhibit the expression of HBV genes.

[0013] The RNAi agents of the present invention are designed to target regions within the HBV genome that are conserved across all eight HBV serotypes. Additionally, the RNAi agents of the present invention are designed to inhibit all stages of the HBV life cycle, such as viral replication, assembly, secretion, and secretion of subviral antigens, by inhibiting the expression of two or more HBV genes. Specifically, because transcription of the HBV genome generates polycistronic overlapping RNAs, RNAi agents of the present invention targeting a single HBV gene will significantly inhibit the expression of most or all HBV transcripts. For example, because the HBV genome is transcribed into a single mRNA, RNAi agents of the present invention targeting the S gene will inhibit not only S gene expression but also the expression of the "downstream" reverse transcriptase gene. Furthermore, the RNAi agents of the present invention are designed to inhibit HBV viral replication by targeting the HBV structural genes and HBV X gene, thereby enabling the subject's immune system to detect and respond to the presence of HBsAg, produce anti-HBV antibodies, and eliminate HBV infection. Without intending to be limited by theory, it is believed that a combination or partial combination of the aforementioned properties and the specific target sites and / or specific modifications of these RNAi agents confers improved efficacy, stability, safety, potency, and durability to the RNAi agents of the present invention.

[0014] Thus, in one aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of hepatitis B virus (HBV) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and the antisense strand comprising at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0015] In one embodiment, one or more of the three nucleotide differences in the nucleotide sequence of the antisense strand are nucleotide mismatches in the antisense strand.

[0016] In another embodiment, one or more of the three nucleotide differences in the nucleotide sequence of the antisense strand are nucleotide mismatches in the sense strand.

[0017] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0018] In one embodiment, the sense and antisense strands comprise a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the sequences listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26.

[0019] In one embodiment, the at least one modified nucleotide is selected from the group consisting of a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic.

[0020] In one embodiment, at least one strand comprises a 3' overhang of at least one nucleotide, hi another embodiment, at least one strand comprises a 3' overhang of at least two nucleotides.

[0021] In one embodiment, the double-stranded region is 15 to 30 nucleotide pairs in length. In another embodiment, the double-stranded region is 17 to 23 nucleotide pairs in length. In yet another embodiment, the double-stranded region is 17 to 25 nucleotide pairs in length. In one embodiment, the double-stranded region is 23 to 27 nucleotide pairs in length. In another embodiment, the double-stranded region is 19 to 21 nucleotide pairs in length. In yet another embodiment, the double-stranded region is 21 to 23 nucleotide pairs in length.

[0022] In one embodiment, each strand has between 15 and 30 nucleotides, hi another embodiment, each strand has between 19 and 30 nucleotides.

[0023] In one embodiment, the ligand is [ka] is.

[0024] In one embodiment, the RNAi agent is shown in the following schematic diagram: [ka] wherein X is O or S.

[0025] In one embodiment, the RNAi agent is selected from the group of RNAi agents listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26.

[0026] In one aspect, the present invention provides a double-stranded RNAi agent for inhibiting expression of hepatitis B virus (HBV) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5) and the antisense strand comprising 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6), substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0027] The present invention also provides RNAi agents comprising sense and antisense nucleotide sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over their entire length to the aforementioned sense and antisense nucleotide sequences.

[0028] In another aspect, the present invention provides a double-stranded RNAi agent for inhibiting expression of hepatitis B virus (HBV) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7) and the antisense strand comprising 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8), substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. The present invention also provides RNAi agents comprising sense and antisense nucleotide sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over their entire length to the aforementioned sense and antisense nucleotide sequences.

[0029] In another aspect, the present invention provides a double-stranded RNAi agent for inhibiting expression of hepatitis B virus (HBV) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising 5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9) and the antisense strand comprising 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10), substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. The present invention also provides RNAi agents comprising sense and antisense nucleotide sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over their entire length to the aforementioned sense and antisense nucleotide sequences.

[0030] In another aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of hepatitis B virus (HBV) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising 5'-CGUGGUGGUCUUCUCUAAAUU-3' (SEQ ID NO: 37) and the antisense strand comprising 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38); Substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. The present invention also provides RNAi agents comprising sense and antisense nucleotide sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over their entire length to the aforementioned sense and antisense nucleotide sequences.

[0031] In another aspect, the present invention provides a double-stranded RNAi agent for inhibiting expression of hepatitis B virus (HBV) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11) and the antisense strand comprising 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. The present invention also provides RNAi agents comprising sense and antisense nucleotide sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over their entire length to the aforementioned sense and antisense nucleotide sequences.

[0032] In another aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of hepatitis B virus (HBV) in cells. The double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) and the antisense strand comprises 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. The present invention also provides an RNAi agent comprising sense and antisense nucleotide sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over their entire length to the aforementioned sense and antisense nucleotide sequences.

[0033] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand comprise modified nucleotides.

[0034] In certain embodiments, the modified nucleotide is selected from the group consisting of a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic.

[0035] In one embodiment, the 5'-phosphate mimic is 5'-vinyl phosphate (5'-VP).

[0036] In one embodiment, the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13), and the antisense strand comprises 5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0037] In another embodiment, the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15) and the antisense strand comprises 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0038] In one embodiment, the sense strand comprises 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17) and the antisense strand comprises 5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0039] In another embodiment, the sense strand comprises 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19) and the antisense strand comprises 5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0040] In one embodiment, the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21), and the antisense strand comprises 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0041] In another embodiment, the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23), and the antisense strand comprises 5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0042] In another embodiment, the sense strand comprises 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) and the antisense strand comprises 5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; dA, dC, dG, and dT are deoxyribose A, C, G, and T; and s is a phosphorothioate linkage.

[0043] In one embodiment, the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) and the antisense strand comprises 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0044] In another embodiment, the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27) and the antisense strand comprises 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0045] In another embodiment, the sense strand comprises 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41), and the antisense strand comprises 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0046] In one embodiment, the ligand is [ka] is.

[0047] In one embodiment, the RNAi agent is shown in the following schematic diagram: [ka] wherein X is O or S.

[0048] In one embodiment, P is a 5'-phosphate mimic. In one embodiment, the 5'-phosphate mimic is 5'-vinyl phosphate (5'-VP).

[0049] In another aspect, the present invention provides a composition comprising two or more double-stranded RNAi agents for inhibiting expression of Hepatitis B virus (HBV) in a cell, each double-stranded RNAi agent independently comprising a sense strand and an antisense strand forming a double-stranded region, each of the sense strands independently comprising at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, and each of the antisense strands independently comprising at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides, substantially all of the nucleotides of each of the sense strands and substantially all of the nucleotides of each of the antisense strands are independently modified nucleotides, each of the sense strands is independently conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0050] In one embodiment, one or more of the three nucleotide differences in the nucleotide sequence of the antisense strand are nucleotide mismatches in the antisense strand. In another embodiment, one or more of the three nucleotide differences in the nucleotide sequence of the antisense strand are nucleotide mismatches in the sense strand.

[0051] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0052] In one embodiment, the sense strand and the antisense strand comprise a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the sequences listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26.

[0053] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxynucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic.

[0054] In another aspect, the present invention provides a composition for inhibiting expression of Hepatitis B virus (HBV) in a cell, the composition comprising: (a) a first double-stranded RNAi agent comprising a first sense strand and a first antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, the first sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. and (b) a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, the second sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; each of the first and second sense strands independently comprises 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5), 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), 5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9), 5'-CGUGGUGGUCUUCUCUAAAUU-3' (SEQ ID NO: 37), 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the aforementioned nucleotide sequence), and the first and second antisense strands each independently comprise: 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6), 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8), 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10), 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38), 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the aforementioned nucleotide sequence).

[0055] In one embodiment, all of the nucleotides of the first and second sense strands and / or the first and second antisense strands comprise the modification.

[0056] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxynucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic.

[0057] In one embodiment, the first and second RNAi agents are: 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13) 5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14); 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15) 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16); 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17) 5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18); 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19) 5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20); 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22); 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23) 5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24); 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) 5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36); 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26); 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27) 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf are 2'-fluoro A, G, C, or U; dA, dC, dG, and dT are deoxyribose A, C, G, and T; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimetic.

[0058] In one embodiment, the first and second RNAi agents are: 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15) 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16); 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0059] In another embodiment, the first and second RNAi agents are: 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0060] In one aspect, the invention provides a double-stranded RNAi agent comprising an RNAi agent listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26.

[0061] The present invention also provides vectors and cells comprising the double-stranded RNAi agents of the present invention.

[0062] In another aspect, the present invention provides a pharmaceutical composition comprising the double-stranded RNAi agent of the present invention, or the composition of the present invention, or the vector of the present invention.

[0063] In one embodiment, the double-stranded RNAi agent is administered in an unbuffered solution, hi one embodiment, the unbuffered solution is saline or water.

[0064] In another embodiment, the double-stranded RNAi agent is administered with a buffer. In one embodiment, the buffer comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In another embodiment, the buffer is phosphate-buffered saline (PBS).

[0065] In one aspect, the present invention provides a method for inhibiting hepatitis B virus (HBV) gene expression in a cell, comprising the steps of contacting the cell with a double-stranded RNAi agent of the present invention, or a composition of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention, and maintaining the resulting cell for a period of time sufficient to result in degradation of the mRNA transcript of the HBV gene, thereby inhibiting the expression of the HBV gene in the cell.

[0066] In one embodiment, the HBV gene is selected from the group consisting of C, X, P, S, and combinations thereof.

[0067] In one aspect, the present invention provides a method for inhibiting hepatitis B virus (HBV) replication in a cell, the method comprising the steps of contacting the cell with a double-stranded RNAi agent of the present invention, or a composition of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention, and maintaining the resulting cell for a period of time sufficient to result in degradation of mRNA transcripts of the HBV gene, thereby inhibiting HBV replication in the cell.

[0068] In one embodiment, the cell is in a subject. In one embodiment, the subject is a human.

[0069] In one embodiment, the subject is suffering from an HBV-related disease.

[0070] In one embodiment, HBV gene expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

[0071] In one embodiment, replication of HBV in the cells is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

[0072] In one aspect, the present invention provides a method for reducing hepatitis B virus (HBV) DNA levels in a subject infected with HBV, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of the present invention, or a composition of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention, thereby reducing the subject's HBV ccc DNA levels.

[0073] In another aspect, the present invention provides a method for reducing hepatitis B virus (HBV) antigen levels in a subject infected with HBV, comprising the step of administering a therapeutically effective amount of the double-stranded RNAi agent of the present invention, or the composition of the present invention, or the vector of the present invention, or the pharmaceutical composition of the present invention to the subject, thereby reducing the HBV antigen levels in the subject.

[0074] In one embodiment, the HBV antigen is HBsAg, hi another embodiment, the HBV antigen is HBeAg.

[0075] In another aspect, the present invention provides a method for reducing the viral load of hepatitis B virus (HBV) in a subject infected with HBV, comprising the step of administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of the present invention, or a composition of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention, thereby reducing the HBV viral load in the subject.

[0076] In yet another aspect, the present invention provides a method for reducing alanine aminotransferase (ALT) levels in a subject infected with HBV, comprising administering a therapeutically effective amount of a double-stranded RNAi agent of the present invention, or a composition of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention to the subject, thereby reducing the ALT level in the subject.

[0077] In another aspect, the present invention provides a method for reducing aspartate aminotransferase (AST) levels in a subject infected with HBV, comprising administering a therapeutically effective amount of a double-stranded RNAi agent of the present invention, or a composition of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention to the subject, thereby reducing the AST level in the subject.

[0078] In another aspect, the present invention provides a method for increasing anti-hepatitis B virus (HBV) antibody levels in a subject infected with HBV, comprising the step of administering a therapeutically effective amount of the double-stranded RNAi agent of the present invention, or the composition of the present invention, or the vector of the present invention, or the pharmaceutical composition of the present invention to the subject, thereby increasing the anti-HBV antibody level in the subject.

[0079] In one aspect, the present invention provides a method for treating a subject with hepatitis B virus (HBV) infection, comprising the step of administering a therapeutically effective amount of a double-stranded RNAi agent of the present invention, or a composition of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention to the subject, thereby treating the subject.

[0080] In another aspect, the present invention provides a method for treating a subject with a hepatitis B virus (HBV)-associated disorder, comprising the step of administering a therapeutically effective amount of a double-stranded RNAi agent of the present invention, or a composition of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention to the subject, thereby treating the subject.

[0081] In one embodiment, the HBV-related disorder is selected from the group consisting of hepatitis D virus infection, hepatitis delta, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0082] In one embodiment, the HBV-related disorder is chronic hepatitis and the subject is HBeAg positive. In another embodiment, the HBV-related disorder is chronic hepatitis and the subject is HBeAg negative.

[0083] In one aspect, the present invention provides a method for treating a subject with a hepatitis B virus (HBV) infection, the method comprising administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length), and the antisense strand comprises 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' terminus, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, thereby treating the subject.

[0084] In another aspect, the present invention provides a method for treating a subject with a hepatitis B virus (HBV)-associated disorder, comprising administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the aforementioned nucleotide sequence), and the antisense strand comprising 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6) (or the aforementioned nucleotide sequence). and a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the sequence of the present invention, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' terminus, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, thereby treating the subject.

[0085] In one aspect, the present invention provides a method for treating a subject with a hepatitis B virus (HBV) infection, the method comprising administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; The sense strand comprises 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length), and the antisense strand comprises 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length). 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, thereby treating the subject.

[0086] In another aspect, the present invention provides a method of treating a subject having a hepatitis B virus (HBV)-associated disorder, the method comprising administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; The sense strand comprises 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length), and the antisense strand comprises 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length). 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, thereby treating the subject.

[0087] In one aspect, the present invention provides a method for treating a subject with a hepatitis B virus (HBV) infection, the method comprising administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length). 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, thereby treating the subject.

[0088] In another aspect, the present invention provides a method of treating a subject having a hepatitis B virus (HBV)-associated disorder, the method comprising administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length). 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, thereby treating the subject.

[0089] In one aspect, the present invention provides a method for treating a subject with a hepatitis B virus (HBV) infection, the method comprising administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; The sense strand comprises 5'-CGUGGUGGUCUUCUCUAAAUU-3' (SEQ ID NO: 37) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length). 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, thereby treating the subject.

[0090] In another aspect, the present invention provides a method for treating a subject having a hepatitis B virus (HBV)-associated disorder, comprising administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises 5'-CGUGGUGGUCUUCUCUAAAUU-3' (SEQ ID NO: 37) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the aforementioned nucleotide sequence), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the aforementioned nucleotide sequence). the sense strand comprises a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the sense strand, wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, thereby treating the subject.

[0091] In one aspect, the present invention provides a method for treating a subject with a hepatitis B virus (HBV) infection, the method comprising administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length), and the antisense strand comprises 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length). 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, thereby treating the subject.

[0092] In another aspect, the present invention provides a method of treating a subject having a hepatitis B virus (HBV)-associated disorder, the method comprising administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length), and the antisense strand comprises 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length). 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, thereby treating the subject.

[0093] In one aspect, the present invention provides a method for treating a subject with a hepatitis B virus (HBV) infection, the method comprising administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; The sense strand comprises 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length), and the antisense strand comprises 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length). 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, thereby treating the subject.

[0094] In another aspect, the present invention provides a method of treating a subject having a hepatitis B virus (HBV)-associated disorder, the method comprising administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; The sense strand comprises 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length), and the antisense strand comprises 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned nucleotide sequence over its entire length). 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, thereby treating the subject.

[0095] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand comprise a modification.

[0096] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxynucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic.

[0097] In one embodiment, the 5'-phosphate mimic is 5'-vinyl phosphate (5'-VP).

[0098] In one embodiment, the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13), and the antisense strand comprises 5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0099] In another embodiment, the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15) and the antisense strand comprises 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0100] In one embodiment, the sense strand comprises 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17) and the antisense strand comprises 5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0101] In another embodiment, the sense strand comprises 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19) and the antisense strand comprises 5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0102] In one embodiment, the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21), and the antisense strand comprises 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0103] In another embodiment, the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23), and the antisense strand comprises 5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0104] In another embodiment, the sense strand comprises 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) and the antisense strand comprises 5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; dA, dC, dG, and dT are deoxyribose A, C, G, and T; and s is a phosphorothioate linkage.

[0105] In one embodiment, the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) and the antisense strand comprises 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0106] In another embodiment, the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27) and the antisense strand comprises 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0107] In another embodiment, the sense strand comprises 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41), and the antisense strand comprises 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0108] In one embodiment, the ligand is [ka] is.

[0109] In one embodiment, the RNAi agent is shown in the following schematic diagram: [ka] wherein X is O or S.

[0110] In one embodiment, the HBV-related disorder is selected from the group consisting of hepatitis D virus infection, hepatitis delta, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0111] In one embodiment, the HBV-related disorder is chronic hepatitis and the subject is HBeAg positive. In another embodiment, the HBV-related disorder is chronic hepatitis and the subject is HBeAg negative.

[0112] In one aspect, the present invention provides a method for treating a subject with hepatitis B virus (HBV) infection. The method comprises administering to the subject a therapeutically effective amount of a composition for inhibiting expression of hepatitis B virus (HBV) in a cell. The composition comprises: (a) a first double-stranded RNAi agent comprising a first sense strand and a first antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, the first sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; and (b) a first double-stranded RNAi agent comprising a first sense strand and a first antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, the first sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a region, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, the second sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5), 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), 5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9), 5'-CGUGGUGGUCUUCUCUAAAUU-3' (SEQ ID NO: 37), 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to any of the foregoing nucleotide sequences), and the first and second antisense strands each independently comprise: 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6); 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8); 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10); 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38), 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to any of the foregoing nucleotide sequences), and treating a subject therewith.

[0113] In another aspect, the present invention provides a method for treating a subject with a hepatitis B virus (HBV)-associated disorder. The method comprises administering to the subject a therapeutically effective amount of a composition for inhibiting expression of hepatitis B virus (HBV) in a cell. The composition comprises: (a) a first double-stranded RNAi agent comprising a first sense strand and a first antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, the first sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; and (b) a first double-stranded RNAi agent comprising a first sense strand and a first antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, the first sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a region, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, the second sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5), 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), 5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9), 5'-CGUGGUGGUCUUCUCUAAAUU-3' (SEQ ID NO: 37), 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to any of the foregoing nucleotide sequences), and the first and second antisense strands each independently comprise: 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6); 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8); 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10); 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38), 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to any of the foregoing nucleotide sequences), and treating a subject therewith.

[0114] In one embodiment, all of the nucleotides of the first and second sense strands and all of the nucleotides of the first and second antisense strands comprise a modification.

[0115] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxynucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic.

[0116] In one embodiment, the first and second RNAi agents are: 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13) 5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14); 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15) 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16); 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17) 5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18); 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19) 5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20); 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22); 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23) 5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24); 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) 5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36); 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26); 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27) 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf are 2'-fluoro A, G, C, or U; dA, dC, dG, and dT are deoxyribose A, C, G, and T; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimetic.

[0117] In one embodiment, the first and second RNAi agents are: 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15) 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16); and 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0118] In another embodiment, the first and second RNAi agents are: 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0119] In one embodiment, the ligand is [ka] is.

[0120] In one embodiment, the RNAi agent is shown in the following schematic diagram: [ka] wherein X is O or S.

[0121] In one embodiment, the subject is a human.

[0122] In one embodiment, the HBV-related disorder is selected from the group consisting of hepatitis D virus infection, hepatitis delta, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0123] In one embodiment, the HBV-related disorder is chronic hepatitis and the subject is HBeAg positive. In another embodiment, the HBV-related disorder is chronic hepatitis and the subject is HBeAg negative.

[0124] In one embodiment, the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg.

[0125] In one embodiment, the double-stranded RNAi agent is administered at a dose of about 10 mg / kg to about 30 mg / kg. In another embodiment, the double-stranded RNAi agent is administered at a dose of about 3 mg / kg. In one embodiment, the double-stranded RNAi agent is administered at a dose of about 10 mg / kg.

[0126] In one embodiment, the double-stranded RNAi agent is administered at a dose of about 0.5 mg / kg twice a week.

[0127] In one embodiment, the double-stranded RNAi agent is administered at a fixed dose of about 50 mg to 200 mg.

[0128] In one embodiment, the double-stranded RNAi agent is administered subcutaneously. In another embodiment, the double-stranded RNAi agent is administered intravenously.

[0129] In one embodiment, the RNAi agent is administered in two or more doses.

[0130] In one embodiment, the RNAi agent is administered at an interval selected from the group consisting of about once every 12 hours, about once every 24 hours, about once every 48 hours, about once every 72 hours, and about once every 96 hours.

[0131] In one embodiment, the RNAi agent is administered twice weekly. In another embodiment, the RNAi agent is administered every other week.

[0132] In one embodiment, the method of the invention further comprises the step of administering to the subject an additional therapeutic agent.

[0133] In one embodiment, the additional therapeutic agent is selected from the group consisting of antiviral agents, reverse transcriptase inhibitors, immunostimulants, therapeutic vaccines, viral entry inhibitors, oligonucleotides that inhibit the secretion or release of HbsAg, capsid inhibitors, cccDNA inhibitors, and combinations of any of the foregoing.

[0134] In another embodiment, the method of the invention further comprises administering to the subject a reverse transcriptase inhibitor, hi yet another embodiment, the method of the invention further comprises administering to the subject a reverse transcriptase inhibitor and an immunostimulant.

[0135] In one embodiment, the reverse transcriptase inhibitor is selected from the group consisting of tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, and AGX-1009.

[0136] In some embodiments, the methods of the present invention further include treating the subject for hepatitis D virus (HDV). Treatment methods can include any treatment method known in the art. In certain embodiments, HDV is treated in the subject using one or more iRNA agents that target HBV as described herein.

[0137] In some embodiments, the methods of the invention further include modulating, e.g., reducing, the expression of PD-L1. Compositions and methods for reducing PD-L1 expression are provided, for example, in WO 2011 / 127180, the entire contents of which are hereby incorporated by reference.

[0138] In one embodiment, the immunostimulant is selected from the group consisting of pegylated interferon alpha 2a (PEG-IFN-α2a), interferon alpha-2b, recombinant human interleukin-7, and a Toll-like receptor 7 (TLR7) agonist.

[0139] In a further aspect, the present invention provides a method of treating a subject having a hepatitis B virus (HBV)-associated disorder, the method comprising administering a therapeutically effective amount of a double-stranded RNAi agent. [The double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 29, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 30; substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, to a subject, thereby treating the subject.

[0140] In another aspect, the present invention also provides a method of treating a subject having a hepatitis B virus (HBV) infection, the method comprising: (a) a first double-stranded RNAi agent comprising a first strand and a first antisense strand forming a double-stranded region, substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides; a first double-stranded RNAi agent, wherein the first sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; and (b) a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a double-stranded region, substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides; the second sense strand is conjugated to a ligand attached at the 3' end, and a second double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; wherein the first sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and the first antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides; the second sense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of SEQ ID NO: 29, and the second antisense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of SEQ ID NO: 30, thereby treating the subject.

[0141] In some embodiments, the first sense strand is 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5), 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), 5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9), 5'-CGUGGUGGUCUUCUCUAAAUU-3' (SEQ ID NO: 37) 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the aforementioned nucleotide sequence), and the second antisense strand comprises: 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6); 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8); 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10); 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38); 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12); and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the aforementioned nucleotide sequence).

[0142] In some embodiments, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand comprise a modification.

[0143] In certain embodiments, at least one of the modified nucleotides is selected from the group consisting of a deoxynucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic.

[0144] In some embodiments, the ligand is [ka] is.

[0145] In a specific embodiment, the RNAi agent is shown in the following schematic diagram: [ka] wherein X is O or S.

[0146] In certain embodiments, the double-stranded RNAi agents and compositions provided herein are used to treat HDV infection and / or HDV-associated disorders.

[0147] Thus, the present invention provides a method for inhibiting replication of hepatitis D virus (HDV) in a cell, comprising the steps of: (a) contacting the cell with a double-stranded RNAi agent, composition, vector, or pharmaceutical composition provided herein; and (b) maintaining the cell resulting from step (a) for a time sufficient to achieve degradation of mRNA transcripts of an HBV gene, thereby inhibiting replication of HDV in the cell.

[0148] In certain embodiments, the cell is in a subject. In certain embodiments, the subject is a human.

[0149] The present invention further provides a method for reducing hepatitis D virus (HDV) antigen levels in a subject infected with HDV, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, composition, vector, or pharmaceutical composition provided herein, thereby reducing the level of an HDV antigen, e.g., S-HDAg or L-HDAg, in the subject.

[0150] The present invention also provides a method for reducing hepatitis D virus (HDV) viral load in a subject infected with HDV, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, composition, vector, or pharmaceutical composition provided herein, thereby reducing the subject's HDV viral load.

[0151] The present invention also provides a method for treating a subject having a hepatitis D virus (HDV) infection, the method comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, composition, vector, or pharmaceutical composition provided herein, thereby treating the subject.

[0152] In certain embodiments, the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region. The sense and antisense strands can be selected from the following RNAi agents, wherein the sense strand comprises 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5) and the antisense strand comprises 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6); the sense strand comprises 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7) and the antisense strand comprises 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8); the sense strand comprises 5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9) and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10); or the sense strand comprises 5'-CGUGGUGGUCUUCUCUAAAUU-3' (SEQ ID NO: 37) and the antisense strand comprises 5'-AAU UGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38); wherein the sense strand comprises 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11) and the antisense strand comprises 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12); or wherein the sense strand comprises 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) and the antisense strand comprises 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, thereby treating a subject.

[0153] In certain embodiments, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand comprise a modification. In certain embodiments, at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl (hydroxly) modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics. In certain embodiments, the 5'-phosphate mimic is 5'-vinyl phosphate (5'-VP).

[0154] In certain embodiments, the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13), and the antisense strand comprises 5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0155] In certain embodiments, the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15), and the antisense strand comprises 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0156] In certain embodiments, the sense strand comprises 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17), and the antisense strand comprises 5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0157] In certain embodiments, the sense strand comprises 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19), and the antisense strand comprises 5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0158] In certain embodiments, the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21), and the antisense strand comprises 5'-AfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0159] In certain embodiments, the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23), and the antisense strand comprises 5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0160] In certain embodiments, the sense strand comprises 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) and the antisense strand comprises 5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; dA, dC, dG, and dT are deoxyribose A, C, G, and T; and s is a phosphorothioate linkage.

[0161] In certain embodiments, the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25), and the antisense strand comprises 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0162] In certain embodiments, the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27), and the antisense strand comprises 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0163] In certain embodiments, the sense strand comprises 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41), and the antisense strand comprises 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

[0164] In certain embodiments, the ligand is [ka] is.

[0165] In certain embodiments, the RNAi agent is represented by the following schematic diagram: [ka] wherein X is O or S.

[0166] The present invention provides a method for treating a subject with hepatitis D virus (HDV) infection. The method includes a composition for inhibiting expression of hepatitis B virus (HBV) in a cell, the composition comprising: (a) a first double-stranded RNAi agent comprising a first sense strand and a first antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, the first sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; and (b) a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, the second sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, wherein the first and second sense strands are each independently 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5), 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), 5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9), 5'-CGUGGUGGUCUUCUCUAAAUU-3' (SEQ ID NO: 37), 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and the first and second antisense strands each independently comprise a sequence selected from the group consisting of: 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6); 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8); 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10); 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38), 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and The method includes administering to a subject a therapeutically effective amount of a composition comprising a sequence selected from the group consisting of: 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40), thereby treating the subject.

[0167] In certain embodiments, all of the nucleotides of the first and second sense strands and all of the nucleotides of the first and second antisense strands comprise a modification. In certain embodiments, at least one of the modified nucleotides is selected from the group consisting of a deoxynucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic.

[0168] In certain embodiments, the first and second RNAi agents are selected from the group: 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13) 5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14); 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15) 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16); 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17) 5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18); 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19) 5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20); 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22); 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23) 5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24); 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) 5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36); 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26); 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27) 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf are 2'-fluoro A, G, C, or U; dA, dC, dG, and dT are deoxyribose A, C, G, and T; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0169] In certain embodiments, the first and second RNAi agents are: 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15) 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16); and 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0170] In certain embodiments, the first and second RNAi agents are: 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42), where A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

[0171] In certain embodiments, the ligand is [ka] is.

[0172] In certain embodiments, the RNAi agent is represented by the following schematic diagram: [ka] wherein X is O or S.

[0173] In certain embodiments, the subject is a human.

[0174] In certain embodiments, the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg. In certain embodiments, the double-stranded RNAi agent is administered at a dose of about 10 mg / kg to about 30 mg / kg. In certain embodiments, the double-stranded RNAi agent is administered at a dose of about 3 mg / kg. In certain embodiments, the double-stranded RNAi agent is administered at a dose of about 10 mg / kg. In certain embodiments, the double-stranded RNAi agent is administered at a dose of about 0.5 mg / kg twice weekly. In certain embodiments, the double-stranded RNAi agent is administered at a fixed dose of about 50 mg to 200 mg.

[0175] In certain embodiments, the double-stranded RNAi agent is administered subcutaneously.

[0176] In certain embodiments, the double-stranded RNAi agent is administered intravenously.

[0177] In certain embodiments, the RNAi agent is administered in two or more doses. In certain embodiments, the RNAi agent is administered at intervals selected from the group consisting of about once every 12 hours, about once every 24 hours, about once every 48 hours, about once every 72 hours, and about once every 96 hours. In certain embodiments, the RNAi agent is administered twice a week. In certain embodiments, the RNAi agent is administered every other week. In certain embodiments, the RNAi agent is administered once a month. In certain embodiments, the RNAi agent is administered once every other month. In certain embodiments, the RNAi agent is administered once every three months.

[0178] In certain embodiments, the RNAi agent is administered to the subject in conjunction with an additional therapeutic agent, such as, for example, an antiviral agent, a reverse transcriptase inhibitor, an immunostimulant, a therapeutic vaccine, a viral entry inhibitor, an oligonucleotide that inhibits the secretion or release of HbsAg, a capsid inhibitor, a covalently closed circular (ccc) HBV DNA inhibitor, and any combination of the foregoing.

[0179] In certain embodiments, the additional agent is a reverse transcriptase inhibitor. In certain embodiments, the additional agent is a reverse transcriptase inhibitor and an immunostimulant. Exemplary reverse transcriptase inhibitors include tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, and AGX-1009. Exemplary immunostimulants include pegylated interferon alpha 2a (PEG-IFN-α2a), interferon alpha 2b, recombinant human interleukin-7, and Toll-like receptor 7 (TLR7) agonists.

[0180] The present invention further provides a method of treating a subject having a hepatitis D virus (HDV) infection, the method comprising a composition for inhibiting expression of hepatitis B virus (HBV) in a cell, the composition comprising: (a) a first double-stranded RNAi agent comprising a first strand and a first antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, the first sense strand is conjugated to a ligand attached at its 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; and (b) a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides. the second sense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the first antisense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of SEQ ID NO: 2, the second sense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of SEQ ID NO: 29, and the second antisense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of SEQ ID NO: 30, thereby treating the subject.

[0181] In certain embodiments, the first sense strand comprises: 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5), 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), 5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9), 5'-CGUGGUGGUCUUCUCUAAAUU-3' (SEQ ID NO: 37), 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and The second antisense strand is 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6); 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8); 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10); 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38), 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40).

[0182] In certain embodiments, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand comprise a modification. In certain embodiments, the additional agent is at least one modified nucleotide selected from the group consisting of deoxynucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxly modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

[0183] In certain embodiments, the ligand is [ka] is.

[0184] In certain embodiments, the RNAi agent is represented by the following schematic diagram: [ka] wherein X is O or S.

[0185] The present invention is further illustrated by the following detailed description and drawings. [Brief explanation of the drawings]

[0186] [Figure 1]The structure of the approximately 3.2 kb double-stranded HBV genome is shown schematically. Replication of the HBV genome occurs through an RNA intermediate, producing four overlapping viral transcripts (approximately 3.5 kb, 2.4 kb, 2.1 kb, and 0.7 kb transcripts) that encode seven viral proteins (pre-S1, pre-S2, S, P, X, pre-C, and C) that are translated across three reading frames. [Figure 2] 1 is a graph depicting the log reduction in HBsAg serum levels normalized to pre-dose HBsAg serum levels following administration of a single 3 mg / kg dose of the indicated iRNA agents. [Figure 3] 1 is a graph depicting the log reduction in HBsAg serum levels normalized to pre-dose HBsAg serum levels following administration of a single 3 mg / kg dose of the indicated iRNA agents. [Figure 4] 4 is a graph showing the percent pre-dose HBsAg remaining at days 5 and 10 after administration of a single 3 mg / kg dose of the indicated iRNA agents. Figure 4 also shows the percent HBsAG remaining at day 10 after administration compared to the percent HBsAG remaining at day 10 after administration in animals administered 3 mg / kg of a control dsRNA targeting mouse / rat transthyretin (mrTTR). [Figure 5] 1 is a graph showing the log reduction in HBsAg serum levels normalized to pre-dose HBsAg serum levels following administration of a single 3 mg / kg dose of AD-65403. [Figure 6A] 1 is a graph showing the reduction in HBsAg serum levels normalized to pre-dose HBsAg serum levels on a standard linear scale following administration of a single subcutaneous 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 9 mg / kg dose of AD-66810. [Figure 6B] 1 is a graph showing the reduction in HBsAg serum levels normalized to pre-dose HBsAg serum levels on a log10 scale following administration of a single subcutaneous 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 9 mg / kg dose of AD-66810. [Figure 7]1 is a graph showing the reduction in HBsAg plasma levels normalized to pre-dose HBsAg plasma levels on a log10 scale after three weekly subcutaneous 3 mg / kg doses of AD-66810. DETAILED DESCRIPTION OF THE INVENTION

[0187] The present invention provides iRNA compositions that induce RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of Hepatitis B virus (HBV) genes. The genes may be present in cells, for example, cells within the body of a subject, such as a human. These iRNAs can be used to target and degrade the mRNA of the corresponding gene (HBV gene) in mammals.

[0188] The RNAi agents of the present invention are designed to target regions within the HBV genome that are conserved across all eight HBV serotypes. Additionally, the RNAi agents of the present invention are designed to inhibit all stages of the HBV life cycle, such as viral replication, assembly, secretion, and secretion of subviral antigens, by inhibiting the expression of two or more HBV genes. Specifically, because transcription of the HBV genome generates polycistronic overlapping RNAs, RNAi agents of the present invention targeting a single HBV gene will significantly inhibit the expression of most or all HBV transcripts. For example, because the HBV genome is transcribed into a single mRNA, RNAi agents of the present invention targeting the S gene will inhibit not only S gene expression but also the expression of the "downstream" polymerase gene. Furthermore, the RNAi agents of the present invention are designed to inhibit HBV viral replication by targeting the HBV structural genes and HBV X gene, thereby enabling the subject's immune system to detect and respond to the presence of HBsAg, produce anti-HBV antibodies, and eliminate HBV infection. Without intending to be limited by theory, it is believed that a combination or partial combination of the aforementioned properties and the specific target sites and / or specific modifications of these RNAi agents confers improved efficacy, stability, safety, potency, and durability to the RNAi agents of the present invention.

[0189] Using in vitro and in vivo assays, the present inventors have demonstrated that iRNAs targeting HBV genes can potently mediate RNAi, resulting in significant inhibition of the expression of two or more HBV genes. The present inventors have also demonstrated that the RNAi agents of the present invention are highly stable in the cytoplasm and lysosomes. Therefore, methods and compositions comprising these iRNAs are useful for treating subjects with HBV infection and / or HBV-related diseases, such as chronic hepatitis B (CHB).

[0190] Thus, the present invention also provides methods of treating subjects with disorders that may benefit from inhibiting or reducing the expression of HBV genes, e.g., HBV-related diseases such as chronic hepatitis B virus infection (CHB), using iRNA compositions that cause RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of HBV genes.

[0191] In particular, extremely low dosages of the iRNAs of the present invention can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of the expression of the corresponding genes (HBV genes).

[0192] The iRNA of the present invention may be about 30 nucleotides in length or less, for example, 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, The RNA strand (antisense strand) comprises a region of 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 length, which is substantially complementary to at least a portion of an mRNA transcript of the HBV gene.

[0193] The detailed description below discloses how to make and use compositions containing iRNA to inhibit the expression of HBV genes, as well as compositions, uses, and methods for treating subjects with diseases and disorders that may benefit from the inhibition and / or reduction of HBV gene expression.

[0194] I. Definition So that the present invention may be more readily understood, several terms are first defined. Furthermore, it should be noted that whenever a value or range of values ​​for a variable is recited, all values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0195] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0196] The term "including" is used herein to mean, and is used synonymously with, the phrase "including but not limited to."

[0197] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0198] As used herein, the term "hepatitis B virus," used interchangeably with "HBV," refers to a well-known, non-cytopathic, hepatotropic DNA virus belonging to the Hepadnaviridae family. The HBV genome is a partially double-stranded, circular DNA with overlapping reading frames (see, e.g., Figure 1).

[0199] There are four known genes encoded by the HBc genome, designated C, X, P, and S. The core protein is encoded by gene C (HBcAg). Hepatitis B antigen (HBeAg) is produced by proteolytic processing of the precore (preC) protein. DNA polymerase is encoded by gene P. Gene S encodes the surface antigen (HBsAg). The HBsAg gene is a single long open reading frame, but contains three in-frame "start" (ATG) codons that divide the gene into three sections: preS1, preS2, and S. Because of the multiple start codons, three different sized polypeptides, designated large, middle, and small (preS1 + preS2 + S, preS2 + S, or S), are produced. The function of the nonstructural protein encoded by gene X is not fully understood, but it has been associated with the development of liver cancer and encodes a decoy protein that allows circulating HBsAg to sequester anti-HBsAg antibodies, allowing infectious viral particles to evade immune detection.

[0200] Proteins encoded by the HBV genome include envelope proteins—i) small, hepatitis B surface antigen (HBsAg); ii) middle, pre-S2+HBsAg; iii) large, pre-S1+pre-S2+HBsAg; nucleocapsid protein, hepatitis B core antigen (HBcAg). Hepatitis B e antigen (HBeAg) is a nonstructural protein produced during HBV replication that shares 90% amino acids with nucleocapsid HBcAg; and the X protein, a nonstructural protein (HBx) that functions in the cytoplasm to activate various signaling pathways, many of which are controlled by modulation of cytoplasmic calcium, and in the nucleus to regulate transcription by direct interaction with various transcription factors, in some cases enhancing their binding to specific transcriptional elements.

[0201] HBV is one of the few DNA viruses that utilizes reverse transcriptase in a replication process that involves multiple steps, including entry, uncoating, and transport of the viral genome to the nucleus. Initially, replication of the HBV genome involves the production of an RNA intermediate, which is then reverse transcribed to produce the DNA viral genome.

[0202] Upon cell infection with HBV, the viral genome relaxed circular DNA (rcDNA) is transported to the cell nucleus and converted into episomal covalently closed circular DNA (cccDNA), which serves as a transcription template for viral mRNA. After transcription and nuclear export, the cytoplasmic viral pregenomic RNA (pgRNA) assembles with HBV polymerase and capsid proteins to form nucleocapsids, within which polymerase-catalyzed reverse transcription generates minus-strand DNA, which is subsequently copied into plus-strand DNA to form progeny rcDNA genomes. The mature nucleocapsids are then packaged with viral envelope proteins to emerge as virion particles, or shuttled to the nucleus to amplify the cccDNA reservoir via the intracellular cccDNA amplification pathway. cccDNA is an essential component of the HBV replication cycle and is involved in the establishment of infection and viral persistence.

[0203] HBV infection results in the production of two distinct particles: 1) the HBV virus itself (or Dane particle), which contains a viral capsid assembled from HBcAg, is coated with HBsAg, and has the ability to reinfect cells; and 2) subviral particles (or SVPs), which are high-density lipoprotein-like particles composed of lipids, cholesterol, cholesterol esters, and noninfectious small and medium forms of the hepatitis B surface antigen HBsAg. For each viral particle produced, 1,000 to 10,000 SVPs are released into the blood. Thus, SVPs (and the HBsAg protein they carry) represent the overwhelming majority of viral proteins in the blood. HBV-infected cells also secrete a soluble proteolytic product of the precore protein called HBV e antigen (HBeAg).

[0204] Eight genotypes of HBV, designated A through H, have been characterized, each with a distinct geographic distribution. The virus is noncytopathic, and virus-specific cell-mediated immunity is the primary factor determining the outcome of HBV exposure: acute infection with resolution of liver disease within 6 months, or chronic HBV infection with frequent progressive liver damage.

[0205] The term "HBV" includes any of these eight HBV genotypes (A to H). The amino acid and complete coding sequence of the reference sequence of the HBV genome can be found, for example, in GenBank Accession Nos. GI:21326584 (SEQ ID NO:1) and GI:3582357 (SEQ ID NO:3).

[0206] Further examples of HBV mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, and OMIM.

[0207] The term "HBV," as used herein, also refers to naturally occurring DNA sequence variants of the HBV genome.

[0208] As used herein, "hepatitis D virus," which is used interchangeably with the term "HDV," refers to a well-known non-cytopathic hepatotropic DNA virus belonging to the Hepadnaviridae family. See, for example, Ciancio and Rizzetto, Nat.Rev.11:68-71, 2014; Le Gal et al., Emerg.Infect.Dis.12:1447-1450, 2006; and Abbas and afzal, World J.Hep.,5:666-675, 2013 (all of which are incorporated by reference). Unless otherwise indicated, HDV refers to all clades and variants of HDV.

[0209] HDV produces one protein, HDAg, which exists in two forms: the 27 kDa large HDAg (also referred to herein as lHD, L-HDAg, and large HDV antigen) and the 24 kDa small HDAg (also referred to herein as sHD, S-HDAg, and small HDV antigen). These two forms have the same N-terminus, while the C-terminus of the large HDAg differs by 19 amino acids. Both isoforms are produced from the same reading frame, containing a UAG stop codon at codon 196, and normally produce only the small HDAg. However, editing by the cellular enzyme adenosine deaminase-1 to change the stop codon to UCG allows the production of the large HDAg. Despite sharing 90% sequence identity, these two proteins play different roles during infection. HDAg-S is produced early in infection and enters the nucleus to support viral replication. In contrast, HDAg-L is produced late in infection, acts as an inhibitor of viral replication, and is required for viral particle assembly.

[0210] Further examples of HDV mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, and OMIM.

[0211] The term "HDV," as used herein, also refers to naturally occurring DNA sequence variants of the HDV genome.

[0212] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed upon transcription of an HBV gene, including mRNAs that are the product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed upon transcription of an HBV gene.

[0213] The target sequence can be about 9 to 36 nucleotides in length, for example, about 15 to 30 nucleotides in length. For example, the target sequence can be about 15 to 30 nucleotides, 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, 1 It can be 9-28, 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 length. Ranges and lengths intermediate to the above-listed ranges and lengths are also considered part of the invention.

[0214] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides represented by a sequence given using standard nucleotide nomenclature.

[0215] "G," "C," "A," and "U" generally represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides or surrogate replacement moieties, as described in more detail below (see, for example, Table 2). Those skilled in the art will appreciate that guanine, cytosine, adenine, and uracil may be substituted by other moieties without significantly changing the base pairing properties of oligonucleotides containing nucleotides with such replacement moieties. For example, but not limited to, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine may be substituted, for example, by a nucleotide containing inosine in the nucleotide sequence of a dsRNA characterized in the present invention. In another example, adenine and cytosine anywhere in an oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU wobble base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured in the present invention.

[0216] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interfering agent," as used interchangeably herein, refer to an agent that contains RNA and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, as those terms are defined herein. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates (e.g., inhibits) expression of an HBV gene (e.g., one or more HBV genes) in a cell, e.g., a cell in a subject, such as a mammalian subject.

[0217] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, such as an HBV target mRNA sequence, and induces cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into 19-23 base pair short interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the 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 the appropriate target mRNA, one or more endonucleases in the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Here, in one aspect, the present invention relates to single-stranded siRNA (ssRNA) that is produced in cells and promotes the formation of a RISC complex that leads to the silencing of target genes, i.e., HBV genes. Therefore, the term "siRNA" is also used herein to refer to the above-mentioned RNAi.

[0218] In another embodiment, the RNAi agent can be a single-stranded siRNA introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing 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 which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as single-stranded siRNAs chemically modified as described herein or by the methods described in Lima et al., (2012) Cell 150;:883-894.

[0219] In another embodiment, an "iRNA" for use in the compositions, uses, and methods of the invention is double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, which are shown to have "sense" and "antisense" orientations relative to a target RNA, i.e., an HBV gene. In certain embodiments of the invention, the double-stranded RNA (dsRNA) causes degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0220] Generally, the majority of the nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands can also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides.Furthermore, as used herein, "RNAi agent" can also include ribonucleotides with chemical modifications; RNAi agent can contain substantial modifications in multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide bond, and / or a modified nucleobase. Thus, the term modified nucleotide encompasses, for example, the substitution, addition, or removal of a functional group or atom to an internucleoside bond, a sugar moiety, or a nucleobase. Modifications suitable for use in the agents of the present invention include any type of modification disclosed herein or known in the art. Any such modifications when used in siRNA-type molecules are encompassed by "RNAi agent" for the purposes of this specification and claims.

[0221] The double-stranded region can be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and can range from about 9 to 36 base pairs in length, e.g., about 15 to 30 base pairs in length, e.g., about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 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 The nucleic acid sequence may be about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the invention.

[0222] The two strands forming the double-stranded structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of one larger molecule and are therefore connected by a continuous chain of nucleotides between the 3' end of one strand and the 5' end of the other strand that form the double-stranded structure, the connected RNA strands are called "hairpin loops." A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 20, at least 23, or more unpaired nucleotides.

[0223] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules can be covalently linked, but they do not have to be.When the two strands are covalently linked by means other than a continuous chain of nucleotides between the 3'-end of one strand and the 5'-end of the other strand that form a double-stranded structure, the linked structure is called a "linker".The RNA strands can have the same or different number of nucleotides.The maximum number of base pairs is the number of nucleotides of the shortest strand of dsRNA minus the overhang that exists in the double strand.In addition to the double-stranded structure, the RNAi agent can include one or more nucleotide overhangs.

[0224] In one embodiment, the RNAi agent of the present invention is a dsRNA, each strand of which contains 24-30 nucleotides that interact with a target RNA sequence, e.g., an HBV target mRNA sequence, leading to cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Processing of dsRNA by the RNase III-like enzyme Dicer results in 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA then integrates into the 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 the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0225] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, e.g., a dsRNA. 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 can contain an overhang of at least one nucleotide; alternatively, the overhang can contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang can be in the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide can be present at the 5'-end, the 3'-end, or both ends of either the antisense strand or the sense strand of a dsRNA.

[0226] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3'-end and / or 5'-end. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3'-end and / or 5'-end. In another embodiment, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0227] "Blunt" or "blunt-ended" means that there are no unpaired nucleotides at the relevant end of a double-stranded RNAi agent, i.e., there are no nucleotide overhangs. A "blunt-ended" RNAi agent is a dsRNA that is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. The RNAi agents of the present invention include RNAi agents that have a nucleotide overhang at one end (i.e., an agent with one overhang and one blunt end) or RNAi agents that have nucleotide overhangs at both ends.

[0228] The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., an HBV mRNA. As used herein, the term "region of complementarity," as defined herein, refers to a region of the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., an HBV nucleotide sequence. If the region of complementarity is not perfectly complementary to the target sequence, mismatches can exist in internal or terminal regions of the molecule. Generally, most tolerated mismatches occur in terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotide at the 5' and / or 3' ends of the iRNA. In one embodiment, a double-stranded RNAi agent of the present invention comprises a nucleotide mismatch in the antisense strand. In another embodiment, a double-stranded RNAi agent of the present invention comprises a nucleotide mismatch in the sense strand. In one embodiment, the nucleotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucleotide of the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, at the 3'-terminal nucleotide of the iRNA.

[0229] The term "sense strand," or "passenger strand," as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0230] As used herein, the term "cleavage region" refers to a region located immediately adjacent to the cleavage site. The cleavage site is the site in the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases immediately adjacent to the cleavage site on either side of the cleavage site. In some embodiments, the cleavage region comprises two bases immediately adjacent to the cleavage site on either side of the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0231] As used herein, unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a double-stranded structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under defined conditions, as understood by those of skill in the art. Such conditions may be, for example, stringent conditions, where stringent conditions 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 Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may occur inside an organism, may be applied. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0232] A complementary sequence in an iRNA, e.g., a dsRNA, described herein includes base pairing across the entire length of one or both nucleotide sequences of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be perfectly complementary, or they may form one or more, but generally no more than five, four, three, or two mismatched base pairs upon hybridization to a duplex of up to 30 base pairs while retaining the ability to hybridize under conditions optimal for their ultimate application, e.g., inhibiting gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs shall not be considered mismatches in determining complementarity. For example, for purposes described herein, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length may be referred to as "fully complementary" if the longer oligonucleotide comprises a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide.

[0233] As used herein, "complementary" sequences can also include, or be formed entirely of, non-Watson-Crick base pairs and / or base pairs formed from unnatural and modified nucleotides, so long as the above requirements related to their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairs.

[0234] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in reference to matching bases between the sense and antisense strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as understood in the context of their use.

[0235] As used herein, a polynucleotide "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding an HBV gene), including the 5' UTR, open reading frame (ORF), or 3' UTR. For example, a polynucleotide is complementary to at least a portion of an HBV mRNA if its sequence is substantially complementary to a contiguous portion of an mRNA encoding an HBV gene.

[0236] Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to a target HBV sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to a target HBV sequence, and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the nucleotide sequence of SEQ ID NO: 1, or an equivalent region of a fragment of SEQ ID NO: 1, over its entire length.

[0237] In one embodiment, an RNAi agent of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide, which in turn is complementary to a target HBV sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the nucleotide sequence of any one of SEQ ID NOs: 6, 8, 10, 12, 38, and 40, or an equivalent region of a fragment of any one of SEQ ID NOs: 6, 8, 10, 12, 38, and 40, over its entire length. In another embodiment, the RNAi agent of the present invention comprises an antisense strand that is substantially complementary to a target HBV sequence and comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the nucleotide sequence of any one of SEQ ID NOs: 5, 7, 9, 11, 37, and 39, or an equivalent region of a fragment of any one of SEQ ID NOs: 5, 7, 9, 11, 37, and 39, over its entire length.

[0238] In some embodiments, generally, the majority of the nucleotides in each strand are ribonucleotides, although, as described in detail herein, each or both strands may also include one or more non-ribonucleotides, e.g., deoxyribonucleotides and / or modified nucleotides. Furthermore, an "iRNA" may include ribonucleotides with chemical modifications. Such modifications may include any type of modification disclosed herein or known in the art. Any such modifications when used in an iRNA molecule are encompassed by "iRNA" for purposes of this specification and claims.

[0239] In one embodiment of the present invention, the agent for use in the methods and compositions of the present invention is a single-stranded antisense nucleic acid molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence in the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation stoichiometrically by base pairing to the mRNA and physically interfering with the translation machinery (see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355). The single-stranded antisense RNA molecule can be about 15 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule can contain a sequence that is at least about 15, 16, 17, 18, 19, 20, or more consecutive nucleotides from any one of the antisense sequences described herein.

[0240] As used herein, a "subject" refers to an animal such as a mammal, including a primate (such as a human or a non-human primate, e.g., a monkey or chimpanzee), a non-primate (such as a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, and whale), or a bird (e.g., a duck or goose). In certain embodiments, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition that may benefit from reduced HBV gene expression and / or replication, as described herein; a human being at risk for a disease, disorder, or condition that may benefit from reduced HBV gene expression and / or replication; a human having a disease, disorder, or condition that may benefit from reduced HBV gene expression and / or replication; and / or a human being being treated for a disease, disorder, or condition that may benefit from reduced HBV gene expression and / or replication. In another embodiment, the subject has a hepatitis B virus (HBV) infection. In another embodiment, the subject has both a hepatitis B virus (HBV) infection and a hepatitis D virus (HDV) infection.

[0241] As used herein, the term "treat" or "treatment" refers to, but is not limited to, one or more symptoms associated with unwanted HBV gene expression and / or HBV replication, such as, for example, the presence of serum and / or liver HBV ccc DNA, the presence of serum and / or liver HBV antigens, such as HBsAg and / or HBeAg, elevated ALT, elevated AST, absent or low levels of anti-HBV antibodies, liver damage; cirrhosis; delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma; serum sickness-like syndrome; anorexia; nausea; vomiting, mild fever; muscle pain; fatigue; impaired taste and smell (eating disorders) and tobacco avoidance); and / or right upper quadrant abdominal and epigastric pain (intermittent, mild to moderate); hepatic encephalopathy; somnolence; disturbed sleep patterns; mental confusion; coma; ascites; gastrointestinal bleeding; coagulation abnormalities; jaundice; hepatomegaly (mildly enlarged, soft liver); splenomegaly; palmar erythema; spider nevi; muscle wasting; spider angiomas; vasculitis; variceal bleeding; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (caput medullaris) medusa); elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in the range of 1000-2000 IU / mL, but 100 times higher than the upper limit of normal (ULN), may also be identified; ALT levels higher than AST levels; elevated gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) levels (e.g., ≤3 times the ULN); moderately low albumin levels; elevated serum iron levels; leukopenia (i.e., granulocytopenia); lymphocytosis; increased erythrocyte sedimentation rate (ESR); shortened red blood cell survival; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); presence of serum and / or liver HBsAg, HBeAg, hepatitis B core antibody (anti-HBc), immunoglobulin M (IgM); hepatitis B surface antibody (anti-HBs), hepatitis B e antibody (anti-HBe), and / or HBV DNA; elevated aminotransferases (≤5x ULN); ALT levels higher than AST levels; increased bilirubin levels, prolonged prothrombin time (PT); hyperglobulinemia; the presence of tissue-nonspecific antibodies such as anti-smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA) (10-20%); the presence of tissue-specific antibodies such as antibodies against the thyroid gland (10-20%); elevated levels of rheumatoid factor (RF);"Treatment" refers to beneficial or desired results, including hyperbilirubinemia, prolonged PT, decreased platelet and white blood cell counts, AST levels higher than ALT levels; elevated alkaline phosphatase (ALP) and GGT levels; inflammation with and accompanying lobular, degenerative and reparative hepatocellular changes; and reduction or amelioration of primarily centrilobular necrosis, whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0242] The term "lower" in the context of a subject's level of HBV gene expression and / or HBV replication or disease marker or symptom refers to a statistically significant reduction in such level. The reduction may be, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more, preferably to a level accepted as being within the normal range for individuals without such disorder. In certain embodiments, target expression is normalized, i.e., reduced to a level accepted as being within the normal range for individuals without such disorder, e.g., the level of a disease marker such as ALT or AST is reduced to a level accepted as being within the normal range for individuals without such disorder.

[0243] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition that would benefit from reduced HBV gene expression and / or replication, refers to the prevention or treatment of a disease, disorder, or condition that would benefit from the reduction of HBV gene expression and / or replication, and ... and tobacco avoidance); and / or right upper quadrant abdominal and epigastric pain (intermittent, mild to moderate); hepatic encephalopathy; somnolence; disturbed sleep patterns; mental confusion; coma; ascites; gastrointestinal bleeding; coagulation abnormalities; jaundice; hepatomegaly (mildly enlarged, soft liver); splenomegaly; palmar erythema; spider nevi; muscle wasting; spider angiomas; vasculitis; variceal bleeding; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (caput medullaris) medusa); elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in the range of 1000-2000 IU / mL, but 100 times higher than the upper limit of normal (ULN), may also be identified; ALT levels higher than AST levels; elevated gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) levels (e.g., ≤3 times the ULN); moderately low albumin levels; elevated serum iron levels; leukopenia (i.e., granulocytopenia); lymphocytosis; increased erythrocyte sedimentation rate (ESR); shortened red blood cell survival; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); presence of serum and / or liver HBsAg, HBeAg, hepatitis B core antibody (anti-HBc), immunoglobulin M (IgM); hepatitis B surface antibody (anti-HBs), hepatitis B e antibody (anti-HBe), and / or HBV DNA; elevated aminotransferases (≤5x ULN); ALT levels higher than AST levels; increased bilirubin levels, prolonged prothrombin time (PT); hyperglobulinemia; the presence of tissue-nonspecific antibodies such as anti-smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA) (10-20%);"It refers to a reduced likelihood of developing undesirable symptoms of HBV infection, such as the presence of tissue-specific antibodies (10-20%), such as antibodies against the thyroid gland; elevated levels of rheumatoid factor (RF); hyperbilirubinemia, prolonged PT, decreased platelet and white blood cell counts, AST levels higher than ALT levels; elevated alkaline phosphatase (ALP) and GGT levels; inflammation accompanied by and associated with lobular, degenerative, and reparative hepatocyte changes; and primarily centrilobular necrosis, whether detectable or undetectable. For example, the likelihood of developing liver fibrosis is reduced when, for example, an individual with one or more risk factors for liver fibrosis, e.g., chronic hepatitis B infection, does not develop liver fibrosis or develops liver fibrosis with less severity than a population with the same risk factors but not receiving the treatment described herein. Effective prevention is considered to be the absence of a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition (e.g., by at least about 10% on a clinically accepted measure for the disease or disorder), or a delay in symptoms (e.g., by days, weeks, months, or years);

[0244] As used herein, the term "hepatitis B virus-related disease" or "HBV-related disease" refers to a disease or disorder caused by or associated with HBV infection and / or replication. The term "HBV-related disease" includes diseases, disorders, or conditions that may benefit from reduced HBV gene expression and / or replication. Non-limiting examples of HBV-related diseases include, for example, hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0245] In one embodiment, the HBV-related disease is hepatitis D virus infection. Hepatitis D virus or hepatitis delta virus (HDV) is a human pathogen. However, this virus is defective, and transmission depends on essential helper functions provided by hepatitis B virus (HBV); in fact, HDV requires concurrent or pre-existing HBV infection, specifically a viral envelope containing hepatitis B surface antigen, to become infectious and actively replicate. HDV can lead to severe acute and chronic liver disease associated with HBV. Hepatitis D infection and / or hepatitis delta are highly endemic in several African countries, the Amazon region, and the Middle East, while their prevalence is low in industrialized countries, except in the Mediterranean region.

[0246] HDV transmission can occur through coinfection with HBV (coinfection) or in conjunction with chronic hepatitis B or hepatitis B colonization (superinfection). Both superinfection and coinfection with HDV result in more severe complications than infection with HBV alone. These complications include an increased likelihood of liver failure in acute infection, and rapid progression to cirrhosis and an increased likelihood of developing liver cancer in chronic infection. In combination with hepatitis B virus, hepatitis D has the highest case fatality rate of all hepatitis infections, at 20%.

[0247] In one embodiment, the HBV-related disease is acute hepatitis B. Acute hepatitis B includes inflammation of the liver lasting less than six months. Typical symptoms of acute hepatitis B include fatigue, anorexia, nausea, and vomiting. Extremely elevated aminotransferase levels (>1000 U / L) and hyperbilirubinemia are often observed. Severe cases of acute hepatitis B can rapidly progress to acute liver failure, characterized by liver synthetic dysfunction. This is often defined as a prothrombin time (PT) of 16 seconds or an international normalized ratio (INR) of 1.5 in the absence of preceding liver disease. Acute hepatitis B can progress to chronic hepatitis B.

[0248] In one embodiment, the HBV-related disease is chronic hepatitis. Chronic hepatitis B (CHB) includes liver inflammation lasting for more than six months. Subjects with chronic hepatitis B disease exhibit immune tolerance or have an inactive chronic infection without evidence of active disease, and these subjects may also be asymptomatic. Patients with chronic active hepatitis may have symptoms similar to acute hepatitis, particularly during the replicative state. Persistence of HBV infection in CHB subjects is due to ccc HBV DNA. In one embodiment, subjects with CHB are HBeAg positive. In another embodiment, subjects with CHB are HBeAg negative. Subjects with CHB have a serum HBV DNA level of less than about 105 and persistently elevated transaminases, such as ALT, AST, and γ-glutamyltransferase. Subjects with CHB may have a liver biopsy score (e.g., a necroinflammatory score) of less than about 4. In addition, subjects with CHB may have:

[0249] In one embodiment, the HBV-related disease is acute fulminant hepatitis B. A subject with acute fulminant hepatitis B has symptoms of acute hepatitis, plus confusion or coma (due to the liver's inability to detoxify chemicals) and purpura or bleeding (due to a lack of blood clotting factors).

[0250] Subjects with HBV infection, such as CHB, may develop liver fibrosis.Therefore, in one embodiment, the HBV-related disease is liver fibrosis.Liver fibrosis, or liver cirrhosis, is histologically defined as diffuse liver lesions characterized by fibrosis (excessive fibrous connective tissue) and the transformation of normal liver structure into structurally abnormal nodules.

[0251] The subject with HBV infection, for example, CHB, can develop end-stage liver disease.Therefore, in one embodiment, HBV-related disease is end-stage liver disease.For example, liver fibrosis can progress to the point that, as a result of liver fibrosis, body can no longer compensate for, for example, the decline of liver function, and for example, mental and neurological symptoms and liver failure occur.

[0252] Subjects with HBV infection, such as CHB, may develop hepatocellular carcinoma (HCC), also known as malignant liver cancer. Thus, in one embodiment, the HBV-related disease is HCC. HCC often develops in subjects with CHB and can be of the fibrolamellar type, pseudoductal type (adenoid type), pleomorphic type (giant cell type), or clear cell type.

[0253] An "HDV-associated disorder" or "hepatitis D virus-associated disorder" is a disease or disorder associated with the development of HDV. Exemplary HDV-associated disorders include hepatitis B virus infection, acute hepatitis B, acute hepatitis D; acute fulminant hepatitis D; chronic hepatitis D; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0254] A "therapeutically effective amount," as used herein, is intended to include an amount of an RNAi agent that, when administered to a patient for treating a subject with HBV infection and / or an HBV-related disease, is sufficient to result in treatment of the disease (e.g., by attenuating, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity and medical history, age, weight, family history, genetic makeup, the stage of the pathological process mediated by HBV gene expression, the type of prior or concomitant treatment, if any, and other individual characteristics of the patient to be treated.

[0255] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent sufficient to prevent or ameliorate disease or one or more symptoms of disease when administered to a subject who has not yet developed or exhibited symptoms of HBV infection and / or HBV-related disease, but who may be predisposed to the condition. Ameliorating disease includes slowing the progression of the disease or reducing the severity of subsequent disease development. A "prophylactically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the degree of risk of disease, and the patient's medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, if any, and other individual characteristics of the patient being treated.

[0256] A "therapeutically effective amount" or "prophylactically effective amount" also includes that amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agents used in the methods of the invention can be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0257] The term "sample," as used herein, includes similar fluids, cells, or tissues collected from a subject, as well as collections of fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and the like. Tissue samples can include samples from tissues, organs, or localized regions. For example, samples can be derived from specific organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples can be derived from the liver (e.g., the whole liver or specific parts of the liver, or specific types of cells within the liver, such as hepatocytes), the retina or parts of the retina (e.g., the retinal pigment epithelium), the central nervous system or parts of the central nervous system (e.g., the ventricles or choroid plexus), or the pancreas or specific cells or parts of the pancreas. In some embodiments, a "sample derived from a subject" refers to cerebrospinal fluid obtained from a subject. In preferred embodiments, a "sample derived from a subject" refers to blood or plasma collected from a subject. In a further embodiment, "a sample derived from a subject" refers to liver tissue (or a partial component thereof) or retinal tissue (or a partial component thereof) derived from a subject.

[0258] II. iRNAs of the Invention The present invention provides iRNAs that inhibit the expression of one or more HBV genes. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of an HBV gene in a cell, such as a cell in a subject, e.g., a mammal, such as a human, suffering from an HBV-related disease or disorder, including, but not limited to, chronic hepatitis B. The dsRNA comprises an antisense strand having a region of complementarity that is complementary to at least a portion of an mRNA formed during expression of the HBV gene. The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing an HBV gene, the iRNA inhibits the expression of the HBV gene by at least about 10%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or protein-based methods, such as, for example, by immunofluorescence analysis using Western blot or flow cytometry techniques.

[0259] dsRNA comprises two RNA strands, which are complementary and hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (antisense strand) comprises a complementary region that is substantially complementary to the target sequence, generally completely complementary. The target sequence can be derived from the sequence of mRNA formed during the expression of the HBV gene. The other strand (sense strand) comprises a region complementary to the antisense strand, so that the two strands hybridize to form a double-stranded structure when combined under suitable conditions. As described elsewhere herein and known in the art, the complementary sequence of the dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, rather than being on separate oligonucleotides.

[0260] Generally, the double-stranded structure is 15-30 base pairs in length, e.g., 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, 19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 19-31, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38, 19-39, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61, 19-62, 19-63, 19-64, 19-65, 1 21-23, 21-22, or 21-22 base pairs in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the present invention.

[0261] Similarly, the region of complementarity to the target sequence may be 15-30 nucleotides in length, e.g., 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, 19-40, 19-50, 19-60, 19-70, 19-80, 19-90, 19-100, 19-110, 19-120, 19-130, 19-140, 19-150, 19-210, 19-220, 19-230, 19-240, 19-250, 19-260, 19-270, 19-280, 19-290, 19-300, 19-310, 19-320, 19-330, 19-340, 19-350, 19-360, 19-370, 19-380, 19-410, 19-420, 19-430, 19-440, 19-450, 19-460, 19-470, 19-480, 19-510, 21-23, 21-22, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the present invention.

[0262] In certain embodiments, the dsRNA is about 15 to about 20 nucleotides in length, or about 25 to about 30 nucleotides in length. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21 to 23 nucleotides in length can serve as substrates for Dicer. As those skilled in the art will also recognize, the region of RNA targeted for cleavage is most often a portion of a larger RNA molecule (often an mRNA molecule). Where relevant, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0263] The double-stranded region may be a primary functional portion of the dsRNA, e.g., about 9-36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-3 3, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-2 4, 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, 1 Those skilled in the art will also recognize that a double-stranded region of 9-22, 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 is also a dsRNA. In one embodiment, an RNA molecule or a complex of RNA molecules having a double-stranded region of more than 30 base pairs is a dsRNA, to the extent that it is processed into a functional double-strand of, for example, 15-30 base pairs that targets a desired RNA for cleavage. Thus, those skilled in the art will recognize that, in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting HBV gene expression is not generated in the target cell by cleavage of a larger dsRNA.

[0264] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, for example, 1, 2, 3, or 4 nucleotides. dsRNAs with at least one nucleotide overhang may have unexpectedly superior inhibitory properties compared to their blunt-ended counterparts. The nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may be present on the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0265] dsRNA can be synthesized by standard methods known in the art, for example, by use of an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, Inc.), as further described below.

[0266] The iRNA compounds of the present invention can be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compounds can be prepared using solution phase or solid phase organic synthesis, or both. Organic synthesis offers the advantage that oligonucleotide strands containing unnatural or modified nucleotides can be easily prepared. The single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis, or both.

[0267] In one embodiment, 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 of sequences shown in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26, and the corresponding antisense strand of the sense strand is selected from the group of sequences shown in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of mRNA produced during expression of the HBV gene. Therefore, in this embodiment, dsRNA will comprise two oligonucleotides, wherein one oligonucleotide is represented as the sense strand in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25 and 26, and the second oligonucleotide is represented as the corresponding antisense strand of the sense strand in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25 and 26. In one embodiment, the substantially complementary sequences of dsRNA are comprised in separate oligonucleotides. In another embodiment, the substantially complementary sequences of dsRNA are comprised in one oligonucleotide.

[0268] Although some of the sequences in Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26 are described as modified and / or conjugated sequences, it will be understood that the RNA of the iRNA of the invention, e.g., the dsRNA of the invention, can comprise any one of the sequences set forth in Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26 unmodified, unconjugated, and / or modified and / or conjugated in a manner different from that described in these tables.

[0269] Those skilled in the art are well aware that dsRNAs with a double-stranded structure of about 20-23 base pairs, for example, 21 base pairs, have been found to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others skilled in the art have found that shorter or longer RNA double-stranded structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above embodiment, depending on the nature of the oligonucleotide sequence shown in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26, the dsRNAs described herein may comprise at least one strand with a minimum length of 21 nucleotides. It can reasonably be expected that shorter duplexes having one of the sequences in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26, minus only a few nucleotides at one or both ends, may be similarly effective compared to the above-described dsRNAs. Thus, dsRNAs having at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotide sequences from one of the sequences in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26, whose ability to inhibit HBV gene expression differs from that of a dsRNA containing the complete sequence by no more than about 5, 10, 15, 20, 25, or 30% inhibition, are considered to be within the scope of the present invention.

[0270] Additionally, RNAs set forth in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26 identify sites in the HBV transcript that are susceptible to RISC-mediated cleavage. Accordingly, the present invention also features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a specific site in an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that specific site. Such iRNAs will generally comprise at least about 15 contiguous nucleotides from one of the sequences set forth in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26, linked to additional nucleotide sequences taken from regions adjacent to the selected sequence in the HBV gene.

[0271] Target sequences are generally approximately 15-30 nucleotides in length, although the suitability of specific sequences within this range for directing cleavage of any given target RNA varies. While the various software packages and guidelines described herein provide guidance for identifying optimal target sequences for any given gene target, an empirical approach can also be taken in which a "window" or "mask" of a given size (21 nucleotides, as a non-limiting example) is placed literally or figuratively (including, for example, in silico) over the target RNA sequence to identify sequences within a size range that could serve as target sequences. The next potential target sequence can be identified by gradually shifting the sequence "window" one nucleotide upstream or downstream of the initial target sequence position until a complete set of possible sequences has been identified for any given target size selected. This process, along with systematic synthesis and testing of identified sequences (using assays described herein or known in the art) to identify optimally functioning sequences, can identify RNA sequences that mediate the best inhibition of target gene expression when targeted with an iRNA agent. Thus, for example, while the sequences identified in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26 represent effective target sequences, it is believed that further optimization of inhibitory efficiency may be achieved by gradually "moving the window" one nucleotide upstream or downstream of the given sequence to identify sequences with equivalent or better inhibitory properties.

[0272] Furthermore, for any sequence identified in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26, further optimization could be achieved by systematically adding or removing nucleotides to generate longer or shorter sequences, and testing the resulting sequences by shifting the window of longer or shorter size from that point up or down the target RNA. Furthermore, coupling this approach to generating novel candidate targets with testing the efficacy of iRNAs based on those target sequences in inhibition assays known in the art and / or described herein could further improve the efficiency of inhibition. Furthermore, such optimized sequences could be adjusted, for example, by introducing modified nucleotides described herein or known in the art, adding or altering overhangs, or other modifications known in the art and / or described herein to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermostability, improving transmembrane delivery, targeting to specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes).

[0273] The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. If the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the region of mismatch is not located in the center of the region of complementarity. If the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the mismatches be limited to the last five nucleotides from either the 5' or 3' end of the region of complementarity. For example, for a 23-nucleotide iRNA agent, the strand complementary to a region of the HBV gene generally does not contain any mismatches within the central 13 nucleotides. Using methods described herein or known in the art, it can be determined whether an iRNA containing mismatches with the target sequence is effective in inhibiting HBV gene expression. Considering the effectiveness of iRNAs with mismatches in inhibiting HBV gene expression is important, especially when a particular region of complementarity in the HBV gene is known to have polymorphic sequence variation within the population.

[0274] III. Modified iRNAs of the Invention In one embodiment, the RNA, e.g., dsRNA, of an iRNA of the invention is unmodified, e.g., does not contain chemical modifications and / or conjugates known in the art and described herein. In another embodiment, the RNA, e.g., dsRNA, of an iRNA of the invention is chemically modified to improve stability or other beneficial properties. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified. An iRNA of the invention in which "substantially all of the nucleotides are modified" is mostly, but not completely, modified and may contain no more than five, no more than four, no more than three, no more than two, or no more than one unmodified nucleotide.

[0275] Nucleic acids featured in the present invention can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkage) or 3'-end modifications (conjugation, DNA nucleotide, inverted linkage, etc.); base modifications, such as substitution with a stable base, an unstable base, or a base that base-pairs with a wide range of partners, base removal (abasic nucleotide), or conjugated base; sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; and / or backbone modifications, including modification or substitution of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or that do not contain natural internucleoside linkages. The RNA with modified backbone particularly includes those that do not have a phosphorus atom in the backbone.For the purpose of this specification and as sometimes referred to in the art, the modified RNA that does not have a phosphorus atom in the internucleoside backbone can also be considered as oligonucleoside.In some embodiments, the modified iRNA has a phosphorus atom in its internucleoside backbone.

[0276] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates; phosphinates; phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates; thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters; and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs thereof, and those with reversed polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0277] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 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; 5,32 Specification No. 1,131; Specification No. 5,399,676; Specification No. 5,405,939; Specification No. 5,453,496; Specification No. 5,455,233; Specification No. 5,466,677; Specification No. 5,476,925 Specification No. 5,519,126; Specification No. 5,536,821; Specification No. 5,541,316; Specification No. 5,550,111; Specification No. 5,563,253; Specification No. 5,571,799; Specification No. 5,587 ,361 Specification; 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; 6,534, Nos. 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Reissue Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0278] Modified RNA backbones that do not contain internal phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0279] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 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; Nos. 5,489,677; 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, the entire contents of each of which are incorporated herein by reference.

[0280] In other embodiments, suitable RNA mimics are contemplated for use in iRNA, in which both the sugar and internucleoside linkages, i.e., the backbone of the nucleotide units, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic, that has been shown to have excellent hybridization properties is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are incorporated herein by reference. Additional PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0281] Certain embodiments featured in the present invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly those described in U.S. Pat. No. 5,489,677, such as --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (also known as the methylene (methylimino) or MMI backbone), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (where the natural phosphodiester backbone is represented as --O--P--O--CH2--) and those described in U.S. Pat. No. 5,602,240, such as the amide backbones described in U.S. Pat. In certain embodiments, RNAs featured herein have the morpholino backbone structures described in U.S. Pat. No. 5,034,506, such as the morpholino backbone structures described in U.S. Pat.

[0282] Modified RNAs may also contain one or more substituted sugar moieties. iRNAs, e.g., dsRNAs, featured herein, can include 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, where the alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA includes one of the following substituents at the 2'-position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group that improves the pharmacodynamic properties of iRNA, or group that improves the pharmacokinetic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, as described in the Examples herein below.

[0283] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in the RNA of an iRNA, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA and the 5' position of the 5'-terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 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; 5,567,81 Nos. 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, several of which are co-owned with the present application, the entire contents of each of which are incorporated herein by reference.

[0284] iRNAs can also include nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include deoxythymine (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-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), and 5-uracil (pseudouracil). Other synthetic and natural nucleobases include uracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine, and 3-deazaguanine and 3-deazaadenine.Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present 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 substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T., and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), making them exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0285] Representative United States patents that teach the preparation of some of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-mentioned U.S. Patent Nos. 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; 5,594,1 Nos. 21, 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, the entire contents of each of which are incorporated herein by reference.

[0286] The RNA of an iRNA can also be modified to contain one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a two-atom bridge. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in certain embodiments, the agent of the present invention can contain one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with a modified ribose moiety, where the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. In other words, LNAs are nucleotides containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in a 3'-endo structural configuration. The addition of a locking nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce 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 for use in the polynucleotides of the present invention include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention includes one or more bicyclic nucleosides containing a 4'-2' bridge.Examples of such 4'-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 "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,282). 83); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' (where R is H, C1-C12 alkyl), or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of these are incorporated herein by reference.

[0287] Further representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, 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; ,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. Patent Application Publication No. 2008 / 0039618; and U.S. Patent Application Publication No. 2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0288] For example, any of the bicyclic nucleosides described above can be prepared with one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0289] The RNA of an iRNA can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-0-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S configuration, referred to herein as an "S-cEt."

[0290] The iRNA of the present invention may also contain one or more "conformationally restricted nucleotides" ("CRNs"). CRNs are nucleotide analogs with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRNs lock the ribose ring into a stable conformation, increasing hybridization affinity for mRNA. The linker is long enough to position the oxygen in an optimal position for stability and affinity, reducing puckering of the ribose ring.

[0291] Representative publications that teach the preparation of some of the above-described CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383; and PCT Publication No. WO 2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0292] One or more of the nucleotides of the iRNA of the invention can also comprise a hydroxymethyl-substituted nucleotide. A "hydroxymethyl-substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide, also known as an "unlocked nucleic acid" ("UNA") modification.

[0293] Representative U.S. patent publications that teach the preparation of UNAs include, but are not limited to, U.S. Pat. No. 8,314,227; and U.S. Patent Application Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0294] Potentially stabilizing modifications to the termini of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl 4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and the like. Disclosure of this modification can be found in PCT Publication No. WO 2011 / 005861.

[0295] Other modifications of the nucleotides of the iRNAs of the invention include 5' phosphates or 5' phosphate mimics, such as 5' terminal phosphates or phosphate mimics on the antisense strand of an RNAi agent. Suitable phosphate mimics are described, for example, in U.S. Patent Application Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0296] A. Modified iRNAs Containing Motifs of the Invention In certain aspects of the present invention, double-stranded RNAi agents of the present invention include agents having chemical modifications, for example, those disclosed in International Publication No. WO 2013 / 075035, filed November 16, 2012, the entire contents of each of which are incorporated herein by reference. As shown herein and in PCT Application No. WO 2013 / 075035, better results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense and / or antisense strands of the RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the RNAi agent can be completely modified. The introduction of these motifs, if present, disrupts the modification pattern of the sense and / or antisense strands. The RNAi agent can optionally be conjugated with a GalNAc derivative ligand, for example, on the sense strand. The resulting RNAi agent exhibits better gene silencing activity.

[0297] More specifically, it has been surprisingly discovered that when the sense and antisense strands of a double-stranded RNAi agent are fully modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the RNAi agent, the gene silencing activity of the RNAi agent is significantly improved.

[0298] Thus, the present invention provides double-stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., an HBV gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can be in the range of 12 to 30 nucleotides in length. For example, each strand can be 14 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length.

[0299] The sense and antisense strands typically form double-stranded RNA ("dsRNA"), also referred to herein as an "iRNA agent." The double-stranded region of an iRNA agent can be 12 to 30 nucleotide pairs in length. For example, the double-stranded region can be 14 to 30 nucleotide pairs in length, 17 to 30 nucleotide pairs in length, 27 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 21 nucleotide pairs in length, 17 to 19 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, 21 to 25 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0300] In one embodiment, an RNAi agent can include one or more overhang regions and / or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs can be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhangs can form mismatches with the target mRNA, or the overhangs can be complementary to the targeted gene sequence or can be another sequence. The first and second strands can also be joined by additional bases or other non-basic linkers, e.g., to form a hairpin.

[0301] In one embodiment, each nucleotide in the overhang region of an RNAi agent can independently be a modified or unmodified nucleotide, including, but not limited to, 2'-sugar modifications 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. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA, or the overhang can be complementary to the targeted gene sequence, or it can be another sequence.

[0302] The 5'- or 3'-overhang on the sense strand, antisense strand, or both strands of an RNAi agent can be phosphorylated. In some embodiments, the overhang region comprises two nucleotides with a phosphorothioate between them, wherein the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, the antisense strand, or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

[0303] RNAi agent can have only one overhang, which can enhance the interference 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 the 3'-end of the antisense strand.RNAi can also have a blunt end located at the 5'-end of the antisense strand (or the 3'-end of the sense strand) or vice versa.Generally, the antisense strand of RNAi has a nucleotide overhang at the 3'-end, and the 5'-end is blunt.Without wishing to be bound by theory, the asymmetric blunt ends at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand favor the introduction of the guide strand into the RISC process.

[0304] In one embodiment, the RNAi agent is a 19-nucleotide double-ended bluntmer, wherein the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides, positions 7, 8, and 9, from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end.

[0305] In another embodiment, the RNAi agent is a 20-nucleotide long blunt-ended duplex, wherein the sense strand comprises at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0306] In yet another embodiment, the RNAi agent is a blunt-ended duplex 21 nucleotides in length, wherein the sense strand comprises at least one motif of three 2'-F modifications at three consecutive nucleotides, positions 9, 10, and 11, from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end.

[0307] In one embodiment, the RNAi 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 in 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 in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the RNAi agent is blunt while the other end comprises two nucleotide overhangs. Preferably, the two nucleotide overhangs are at the 3' end of the antisense strand.

[0308] When two nucleotide overhangs are at the 3'-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand. In one embodiment, all nucleotides in the sense and antisense strands of the RNAi agent, including nucleotides that are part of a motif, are modified nucleotides. In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in alternating motifs. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).

[0309] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length, and starting from the 5'-terminal nucleotide (position 1), positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, and starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions paired with positions 1 to 23 of the sense strand to form a duplex; at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; and the 5' end of the antisense strand is 10 to 30 consecutive nucleotides that are not paired with the sense strand. The antisense strand comprises at least one ribonucleotide at the 5'-end of the sense strand, thereby forming a 10-30 nucleotide single-stranded 5' overhang; at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides in the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands; the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length so as to reduce expression of the target gene when the double-stranded nucleic acid is introduced into a mammalian cell; the sense strand comprises at least one motif of three 2'-F modifications in three consecutive nucleotides, where at least one of the motifs is located at or near the cleavage site; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at or near the cleavage site.

[0310] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the RNAi agent comprising a first strand having a length of at least 25 and no more than 29 nucleotides, and a second strand having a length of no more than 30 nucleotides, the second strand comprising at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end; the 3' end of the first strand and the 5' end of the second strand form a blunt end, the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, the double-stranded region is at least 25 nucleotides long, the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the second strand length such that the RNAi agent reduces expression of the target gene when introduced into a mammalian cell, and dicer cleavage of the RNAi agent preferentially yields siRNA comprising the 3' end of the second strand, thereby reducing expression of the target gene in a mammal. Optionally, the RNAi agent further comprises a ligand.

[0311] In one embodiment, the sense strand of the iRNA agent contains at least one motif of three identical modifications in three consecutive nucleotides, one of the motifs occurring at the cleavage site of the sense strand.

[0312] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications in three consecutive nucleotides, one of the motifs being at or near the cleavage site on the antisense strand.

[0313] In RNAi agents having a double-stranded region 17-23 nucleotides in length, the cleavage sites in the antisense strand are typically near positions 10, 11, and 12 from the 5' end. Thus, the three identical modification motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end of the antisense strand, or from the first paired nucleotide in the double-stranded region from the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0314] The sense strand of RNAi agent can comprise at least one motif of three identical modifications in three consecutive nucleotides at the break site of strand; antisense strand can have at least one motif of three identical modifications in three consecutive nucleotides at or near the break site of strand.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be aligned such that one motif of three nucleotides in sense strand and one motif of three nucleotides in antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairs.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0315] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications in three consecutive nucleotides. The first motif may be located at or near the cleavage site of the strand, and the other motif may be a wing modification. The term "wing modification" herein refers to a motif located in another part of the strand, away from a motif located at or near the cleavage site of the same strand. The wing modification may be adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are directly adjacent to each other, the chemical structures of the motifs are different from each other; when the motifs are separated by one or more nucleotides, the chemical structures 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 or on either side of the lead motif relative to the first motif at or near the cleavage site.

[0316] Like the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications in three consecutive nucleotides, with at least one of the motifs occurring at or near the site of strand cleavage. The antisense strand may also contain one or more wing modifications in the same sequence as the wing modifications that may be present in the sense strand.

[0317] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0318] In another embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two paired nucleotides within the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0319] When the sense and antisense strands of an RNAi agent each include at least one wing modification, the wing modifications may be located at the same end of the double-stranded region and may have an overlap of 1, 2, or 3 nucleotides.

[0320] When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands can be arranged such that two modifications from one strand are each located at one end of the double-stranded region and have an overlap of one, two, or three nucleotides; two modifications from one strand are each located at the other end of the double-stranded region and have an overlap of one, two, or three nucleotides; or two modifications from one strand are located on either side of the lead motif and have an overlap of one, two, or three nucleotides in the double-stranded region.

[0321] In one embodiment, all nucleotides in the sense and antisense strands of an RNAi agent, including nucleotides that are part of a motif, can be modified. Each nucleotide can be modified with the same or different modifications, and these modifications can include one or more changes to one or both of the non-linked phosphate oxygen and / or one or more linking phosphate oxygens; changes to components of the ribose sugar, such as the 2' hydroxyl of the ribose sugar; large-scale replacement of the phosphate moiety with a "dephosphorylation" linker; modifications or replacement of natural bases; and replacement or modification of the ribose-phosphate backbone.

[0322] Because nucleic acids are polymers of subunits, many modifications, such as modifications of bases, phosphate moieties, or non-linked Os in phosphate moieties, occur at repeated positions within the nucleic acid. In some cases, modifications can occur at all of the intended positions in the nucleic acid, but often this is not the case. For example, modifications can occur only at the 3' or 5' terminal positions, or only in terminal regions, such as at the terminal nucleotide position or the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only in double-stranded regions of RNA, or only in single-stranded regions of RNA. For example, phosphorothioate modifications at non-linked O positions can occur only at one or both ends, or only in terminal regions, such as at the terminal nucleotide position or the last 2, 3, 4, 5, or 10 nucleotides of the chain, or in double-stranded and single-stranded regions, especially at the ends. The 5' or both ends can be phosphorylated.

[0323] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide surrogates in the single-stranded overhang, e.g., the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In certain embodiments, all or some of the bases in the 3' or 5' overhang may be modified, e.g., with the modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar, e.g., deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications in place of the ribosugar of the nucleobase, according to modifications known in the art, and modifications of the phosphate group, e.g., phosphorothioate modifications. The overhang need not be homologous to the target sequence.

[0324] In one embodiment, each residue in the sense strand and the antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. A strand may contain two or more modifications. In one embodiment, each residue in the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0325] At least two different modifications are typically present in the sense and antisense strands, and the two modifications may be 2'-O-methyl or 2'-fluoro modifications, or others.

[0326] In one embodiment, Na and / or Nb comprise an alternating pattern of modifications. As used herein, the term "alternating motif" refers to a motif having one or more modifications, each modification occurring at alternating nucleotides in a strand. The alternating nucleotides may refer to every other nucleotide or every third nucleotide, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif may be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0327] The types of modifications included in the alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternation pattern, i.e., the modifications at every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possibilities for modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0328] In one embodiment, the RNAi agent of the present invention comprises an alternating motif modification pattern in the sense strand that is shifted relative to the alternating motif modification pattern in the antisense strand. This shift can be such that the modification group of the nucleotide of the sense strand corresponds to a different modification group of the nucleotide of the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can start with "ABABAB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can start with "BABABA" from 5' to 3' of the strand in the double-stranded region. As another example, the alternating motif in the sense strand can start with "AABBAABB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can start with "BBAABBAA" from 5' to 3' of the strand in the double-stranded region, thereby resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0329] In one embodiment, the RNAi agent comprises a pattern of alternating motifs of 2'-O-methyl and 2'-F modifications in the sense strand, and this pattern has a first shift with respect to the pattern of alternating motifs of 2'-O-methyl and 2'-F modifications in the antisense strand, i.e., the 2'-O-methyl modified nucleotides in the sense strand form base pairs with the 2'-F modified nucleotides in the antisense strand, and vice versa. Position 1 of the sense strand may start with a 2'-F modification, and position 1 of the antisense strand may start with a 2'-O-methyl modification.

[0330] The introduction of one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand interrupts the original modification pattern present in the sense strand and / or antisense strand. This interruption of the modification pattern of the sense strand and / or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand unexpectedly enhances the gene silencing activity against the target gene.

[0331] In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modification of the nucleotides adjacent to the motif is a different modification than the modification of the motif. For example, a portion of a sequence containing the motif is "NaYYYNb," where "Y" represents a modification of the motif of three identical modifications on three consecutive nucleotides, "Na" and "Nb" represent modifications of the nucleotides adjacent to the motif "YYY" that are different from the modification of Y, and Na and Nb can be the same or different modifications. Alternatively, Na and / or Nb may be present or absent when wing modifications are present.

[0332] The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may be present at any nucleotide in the sense strand, the antisense strand, or both strands, at any position in the strand. For example, the internucleotide linkage modification may be present at every nucleotide in the sense strand and / or the antisense strand; each internucleotide linkage modification may be present in an alternating pattern in the sense strand and / or the antisense strand; or the sense strand or the antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications in the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of internucleotide linkage modifications in the sense strand may have a shift relative to the alternating pattern of internucleotide linkage modifications in the antisense strand. In one embodiment, a double-stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In one embodiment, the antisense strand contains two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand contains at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

[0333] In one embodiment, the RNAi comprises a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region can comprise two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. The internucleotide bond modification can also be formed to link the overhang nucleotide with the terminal paired nucleotide in the double-stranded region. For example, at least 2, 3, 4, or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide bonds, and optionally, there can be additional phosphorothioate or methylphosphonate internucleotide bonds that link the overhang nucleotide with the paired nucleotide adjacent to the overhang nucleotide. For example, there can be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of the three nucleotides being overhang nucleotides, and the third nucleotide being the paired nucleotide adjacent to the overhang nucleotide. These terminal three nucleotides can be at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and / or the 5' end of the antisense strand.

[0334] In one embodiment, the two nucleotide overhangs are at the 3'-end of the antisense strand, and there are two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of which are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. Optionally, the RNAi agent can further have two phosphorothioate internucleotide bonds between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.

[0335] In one embodiment, the RNAi agent contains mismatches with the target, mismatches within the duplex, or a combination thereof. Mismatches can occur in overhang regions or duplex regions. Base pairs can be evaluated based on their tendency to promote dissociation or melting (e.g., for the free energy of binding or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but similar or equivalent analyses can also be used). With regard to 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 or non-canonical pairings (described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred over canonical pairings.

[0336] In one embodiment, the RNAi agent includes at least one of the first one, two, three, four, or five base pairs within the double-stranded region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and a mismatch pair, e.g., a non-canonical or non-canonical pairing or a pairing containing a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0337] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0338] In another embodiment, the 3'-terminal nucleotide of the sense strand is deoxythymine (dT). In another embodiment, the 3'-terminal nucleotide of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, e.g., two dT nucleotides, at the 3'-end of the sense strand and / or antisense strand.

[0339] In one embodiment, the sense strand sequence has formula (I): 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3'(I) (In the formula: i and j are each independently 0 or 1; p and q are each independently 0 to 6; each Na independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each Nb independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each np and nq independently represents an overhanging nucleotide; wherein Nb and Y do not have the same modification; XXX, YYY and ZZZ each independently represent one motif of three identical modifications in three consecutive nucleotides. Preferably, all of YYY are 2'-F modified nucleotides.

[0340] In one embodiment, Na and / or Nb comprise an alternating pattern of modifications.

[0341] In one embodiment, the YYY motif is located at or near the cleavage site of the sense strand. For example, if the RNAi agent has a double-stranded region 17 to 23 nucleotides in length, the YYY motif can be located at or near the cleavage site of the sense strand, counting from the first nucleotide from the 5' end; or optionally, counting from the first paired nucleotide in the double-stranded region from the 5' end (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13).

[0342] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand may be represented by the following formula: 5'np-Na-YYY-Nb-ZZZ-Na-nq3'(Ib); 5'np-Na-XXX-Nb-YYY-Na-nq3'(Ic); or 5'np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3'(Id).

[0343] When the sense strand is represented by Formula (Ib), Nb represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0344] When the sense strand is represented by Formula (Ic), Nb represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0345] When the sense strand is represented by formula (Id), each Nb independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5, or 6. Each Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0346] Each of X, Y and Z can be the same or different from each other.

[0347] In other embodiments, i is 0, j is 0, and the sense strand may be represented by the formula: 5'np-Na-YYY-Na-nq3'(Ia).

[0348] When the sense strand is represented by Formula (Ia), each Na can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0349] In one embodiment, the antisense strand sequence of the RNAi has formula (II): 5'nq'-Na'-(Z'Z'Z')k-Nb'-Y'Y'Y'-Nb'-(X'X'X')l-N'a-np'3'(II) (In the formula: k and l are each independently 0 or 1; p' and q' are each independently 0 to 6; each Na' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each Nb' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each np' and nq' independently represents an overhanging nucleotide; wherein Nb' and Y' do not have the same modification; X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications in three consecutive nucleotides. It can be represented by:

[0350] In one embodiment, Na' and / or Nb' comprise an alternating pattern of modifications.

[0351] The Y'Y'Y' motif is present at or near the cleavage site of the antisense strand. For example, if the RNAi agent has a double-stranded region 17 to 23 nucleotides in length, the Y'Y'Y' motif can be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end; or optionally, counting from the first paired nucleotide in the double-stranded region from the 5' end. Preferably, the Y'Y'Y' motif is present at positions 11, 12, or 13.

[0352] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0353] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0354] Thus, the antisense strand can be represented by the following formula: 5'nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y'-Na'-np'3'(IIb); 5'nq'-Na'-Y'Y'Y'-Nb'-X'X'X'-np'3'(IIc); or 5'nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y'-Nb'-X'X'X'-Na'-np'3'(IId).

[0355] When the antisense strand is represented by formula (IIb), Nb' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0356] When the antisense strand is represented by Formula (IIc), Nb' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0357] When the antisense strand is represented by formula (IId), each Nb' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5, or 6.

[0358] In other embodiments, k is 0, l is 0, and the antisense strand may be represented by the formula: 5'np'-Na'-Y'Y'Y'-Na'-nq'3'(Ia).

[0359] When the antisense strand is represented as Formula (IIa), each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0360] Each of X', Y' and Z' can be the same or different from each other.

[0361] Each nucleotide in the sense strand and the antisense strand can be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense strand and the antisense strand can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' can specifically represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0362] In one embodiment, the sense strand of the RNAi agent may include a YYY motif at positions 9, 10, and 11 of the strand, counting from the first nucleotide from the 5' end if the double-stranded region is 21 nucleotides; or optionally, counting from the first paired nucleotide in the double-stranded region from the 5' end; Y represents a 2'-F modification. The sense strand may further include a XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region; XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0363] In one embodiment, the antisense strand may include a Y'Y'Y' motif at positions 11, 12, and 13 of the strand, counting from the first nucleotide from the 5'-end; or optionally, counting from the first paired nucleotide in the double-stranded region from the 5'-end; Y' represents a 2'-O-methyl modification. The antisense strand may further include an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region; X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0364] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId).

[0365] Thus, an RNAi agent for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex has the formula (III): Sense: 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3' Antisense: 3'np'-Na'-(X'X'X')k-Nb'-Y'Y'Y'-Nb'-(Z'Z'Z')l-Na'-nq'5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each Na and Na' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each Nb and Nb' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; where: each np', np, nq', and nq, which may or may not be present, independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. is expressed by

[0366] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; or k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0367] Exemplary combinations of sense and antisense strands that form RNAi duplexes include the following formulas: 5'np-Na-YYY-Na-nq3' 3'np'-Na'-Y'Y'Y'-Na'nq'5' (IIIa) 5'np-Na-YYY-Nb-ZZZ-Na-nq3' 3'np'-Na'-Y'Y'Y'-Nb'-Z'Z'Z'-Na'nq'5' (IIIb) 5'np-Na-XXX-Nb-YYY-Na-nq3' 3'np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Na'-nq'5' (IIIc) 5'np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3' 3'np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Nb'-Z'Z'Z'-Na-nq'5' (IIId)

[0368] When an RNAi agent is represented by formula (IIIb), each Nb independently represents an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides, and each Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0369] When an RNAi agent is represented by formula (IIIc), each Nb, Nb' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0370] When an RNAi agent is represented by Formula (IIId), each Nb, Nb' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na, Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of Na, Na', Nb, and Nb' independently comprises an alternating pattern of modifications.

[0371] When an iRNA agent is represented as formula (IIId), each Nb, Nb' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na, Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of Na, Na', Nb, and Nb' independently includes an alternating pattern of modifications. Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) can be the same or different from one another.

[0372] When an RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can be base-paired with one of the Y' nucleotides, alternatively, at least two of the Y nucleotides are base-paired with the corresponding Y' nucleotide; or all three of the Y nucleotides are base-paired with the corresponding Y' nucleotide.

[0373] When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can be base-paired with one of the Z' nucleotides, or at least two of the Z nucleotides can be base-paired with a corresponding Z' nucleotide; or all three of the Z nucleotides can be base-paired with a corresponding Z' nucleotide.

[0374] When an RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides can be base-paired with one of the X' nucleotides, or at least two of the X nucleotides can be base-paired with a corresponding X' nucleotide; or all three of the X nucleotides can be base-paired with a corresponding X' nucleotide.

[0375] In one embodiment, the modification on a Y nucleotide is different from the modification on a Y' nucleotide, the modification on a Z nucleotide is different from the modification on a Z' nucleotide, and / or the modification on an X nucleotide is different from the modification on an X' nucleotide.

[0376] In one embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0377] In one embodiment, when the RNAi agent is represented by formula (IIIa), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0378] In one embodiment, the RNAi agent is a multimer comprising at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target the same gene at two different target sites.

[0379] In one embodiment, the RNAi agent is a multimer comprising three, four, five, six or more duplexes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the duplexes being linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target the same gene at two different target sites.

[0380] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at one or both of the 5' and 3' ends, and are optionally conjugated to a ligand. Each of the RNAi agents can target the same gene or two different genes; or each of the RNAi agents can target the same gene at two different target sites.

[0381] Various publications describe the multimeric RNAi agent that can be used in the method of the present invention.Such publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, each of which is incorporated herein by reference in its entirety.

[0382] As described in more detail below, RNAi agents that include one or more carbohydrate moieties conjugated to the RNAi agent can optimize one or more properties of the RNAi agent. Often, the carbohydrate moiety is attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been replaced in this manner is referred to herein as a ribose-replacement modified subunit (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms can be a heteroatom, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, such as fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0383] The ligand can be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tether attachment point." As used herein, a "backbone attachment point" refers to a bond available and suitable for incorporation of the carrier into the backbone of a ribonucleic acid, typically containing a functional group, e.g., a hydroxyl group, or a backbone, e.g., a phosphate, or a modified phosphate, e.g., sulfur. A "tether attachment point" (TAP) refers, in certain embodiments, to a ring atom, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. This moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is attached to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain functional groups, such as amino groups, or generally provide bonds suitable for the incorporation or tethering of another chemical moiety, such as a ligand, to the constituent ring.

[0384] An iRNA agent may be conjugated to a ligand via a carrier, which can be a cyclic group or a cyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the cyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0385] In certain embodiments, an iRNA agent for use in the methods of the invention is an agent selected from the group of agents listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26. These agents can further comprise a ligand.

[0386] IV. Ligand-Conjugated iRNA Another modification of the RNA of the iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the iRNA.Such moieties include, but are not limited to, cholesterol moieties (Letsinger et al., Proc. Natl. Acids Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Bior. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (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-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or lipid moieties such as octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0387] In one embodiment, a ligand alters the distribution, targeting, or lifespan of an iRNA agent into which it is incorporated. In preferred embodiments, a ligand provides improved affinity for a selected target (e.g., a molecule, cell, or cell type), compartment (e.g., a cell or organ compartment), tissue, organ, or region of the body, e.g., compared to a species lacking such a ligand. Preferred ligands do not participate in pairing of the two strands in a double-stranded nucleic acid.

[0388] Ligands can 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-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids are polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0389] Ligands can also include targeting groups, e.g., cell or tissue targeting agents, e.g., lectins, glycoproteins, lipids, or proteins, e.g., antibodies that bind to specific cell types such as kidney cells. The targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide, or an RGD peptidomimetic or aptamer.

[0390] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelating agents (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid (cholenic acid), and the like. acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu3+ tetraazamacrocycle conjugate), dinitrophenyl, HRP, or AP.

[0391] A ligand can be a molecule with specific affinity for a protein, e.g., a glycoprotein, or a peptide, e.g., a co-ligand, or an antibody, e.g., an antibody that binds to a particular cell type, such as a hepatocyte. Ligands can also include hormones and hormone receptors. Ligands can also include lipids, lectins, carbohydrates, vitamins, cofactors, non-peptide species such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, or multivalent mannose, multivalent fucose, or aptamers. Ligands can be, for example, lipopolysaccharide, an activator of MAP kinase, or an activator of NF-κB.

[0392] The ligand can be a substance, e.g., a drug, that can enhance uptake of the iRNA agent into the cell, e.g., by disrupting the cellular microtubules, microfilaments, and / or intermediate filaments, e.g., by disrupting the cytoskeleton. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0393] In certain embodiments, the ligands attached to the iRNAs described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in the backbone, are also suitable for use as ligands (e.g., PK-modulating ligands) in the present invention. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0394] Ligand-conjugated oligonucleotides of the invention can be synthesized by using oligonucleotides bearing reactive pendant functional groups, such as those derived from the attachment of a binding molecule to an oligonucleotide (described below). The reactive oligonucleotides can be reacted directly with commercially available ligands, synthesized ligands bearing any of a variety of protecting groups, or ligands having a binding moiety attached.

[0395] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely made by the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may be used in addition or instead. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0396] In the ligand-conjugated oligonucleotides and ligand molecules having sequence-specifically bound nucleosides of the present invention, the oligonucleotides and oligonucleosides can be assembled in a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already have a linking moiety, ligand-nucleotide or nucleoside conjugate precursors that already have a ligand molecule, or non-nucleoside ligand-containing building blocks.

[0397] When using a nucleotide conjugate precursor that already has a linking moiety, synthesis of the sequence-specific linked nucleoside is typically completed before the ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In certain embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizer using phosphoramidites derived from the ligand-nucleoside conjugates in addition to commercially available standard and non-standard phosphoramidites commonly used in oligonucleotide synthesis.

[0398] A. Lipid Conjugates In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecule preferably binds to serum protein, for example, human serum albumin (HSA). HSA-binding ligand allows the conjugate to be distributed to target tissues in the body, for example, non-renal target tissues. For example, the target tissue can be the liver, including liver parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligand can be used to (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) adjust the binding to serum protein, for example, HSA.

[0399] Lipid-based ligands can be used to inhibit, for example, 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 be targeted to the kidney and therefore less likely to be removed from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target conjugates to the kidney.

[0400] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, the lipid-based ligand binds to HSA with sufficient affinity such that the conjugate preferably distributes to non-renal tissues. However, the affinity is preferably not so strong that the HSA-ligand binding cannot be reversed.

[0401] In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate preferably distributes to the kidney. Other moieties that target kidney cells can also be used in place of or in addition to the lipid-based ligand.

[0402] In another embodiment, the ligand is a moiety, e.g., a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful, for example, for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by target cells, such as hepatocytes. Also included are HAS and low-density lipoprotein (LDL).

[0403] B. Cell-penetrating agents In another embodiment, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. Exemplary agents are peptides such as tat or antennopedia. If the agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudo-peptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent, which preferably has a lipophilic and lipophobic phase.

[0404] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, for example, by enhancing cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5-50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0405] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 43). RFGF analogs containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 44)) can also be targeting moieties. The peptide moiety can be a "delivery" peptide capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 45)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 46)) have been shown to be capable of functioning as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., 2004). al., Nature, 354:82-84, 1991). An example of a peptide or peptidomimetic attached to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is a peptide such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as to enhance stability or direct conformational properties. Any of the structural modifications described below can be used.

[0406] The RGD peptide moiety for use in the compositions and methods of the present invention can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissues. RGD-containing peptides and peptidomimetics can use D-amino acids, as well as synthetic RGD mimics. In addition to RGD, other moieties that target integrin ligands can be used. Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0407] A "cell-penetrating peptide" is capable of penetrating cells, e.g., microbial cells such as bacterial or fungal cells, or mammalian cells such as human cells. Peptides that penetrate microbial cells can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bisected amphipathic peptide such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0408] C. Carbohydrate conjugates In certain embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo nucleic acid delivery, as described herein, and the compositions are suitable for in vivo therapeutic uses. As used herein, "carbohydrate" refers to either a compound that is a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), each with an oxygen, nitrogen, or sulfur atom attached to it; or a compound that has as part thereof a carbohydrate moiety composed of one or more monosaccharide units, each of which has at least six carbon atoms (which may be linear, branched, or cyclic), each with an oxygen, nitrogen, or sulfur atom attached to it. Exemplary carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars of HBV or greater (e.g., HBV, C6, C7, or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., HBV, C6, C7, or C8).

[0409] In one embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is a monosaccharide. [ka] and other N-acetylgalactosamines.

[0410] In another embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention comprises: [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0411] Other exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to: [ka] and when one of X or Y is an oligonucleotide, the other is hydrogen.

[0412] In certain embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, including but not limited to, a PK modulator and / or a cell penetrating peptide.

[0413] Additional carbohydrate conjugates (and linkers) suitable for use in the present invention include those described in PCT Publication Nos. WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0414] D. Linker In certain embodiments, the conjugates or ligands described herein can be attached to the iRNA oligonucleotide using a variety of linkers, which can be cleavable or non-cleavable.

[0415] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, for example, covalently bonds the two parts of a compound. Linkers are typically a direct bond or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a group including, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkynyl, alkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclyl and alkylaryl, alkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl (wherein one or more methylenes can be interrupted or terminated by O, S, S(O), SO, N(R), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic); where R is hydrogen, acyl, aliphatic, or substituted aliphatic.In one embodiment, the linker is about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

[0416] A cleavable linking group is one that is sufficiently stable outside a cell, but is cleaved after entering a target cell to release the two moieties held together by the linker. In preferred embodiments, the cleavable linking group is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or more, or at least about 100-fold faster inside the target cell or under first reference conditions (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under second reference conditions (which may, for example, be selected to mimic or represent conditions found in blood or serum).

[0417] Cleavable linking groups are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are more prevalent or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include oxidizing or reducing enzymes present in cells or reducing agents such as mercaptans that can degrade redox-cleavable linking groups by reduction, which are selective for specific substrates or have no substrate specificity; esterases; agents that can form endosomes or acidic environments, such as agents that cause a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0418] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH, approximately 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell or into a desired compartment of the cell.

[0419] The linker may contain a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the target cell. For example, a liver-targeting ligand may be linked to a cationic lipid via a linker containing an ester group. Because hepatocytes are rich in esterases, this linker will be cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types that are rich in esterases include lung, renal cortex, and testicular cells.

[0420] Linkers containing peptide bonds can be used to target peptidase-rich cell types such as hepatocytes and synoviocytes.

[0421] In general, the suitability of a candidate cleavable binding group can be evaluated by testing the ability of a degradative agent (or degradative condition) to cleave the candidate binding group. It may also be desirable to test the ability of the candidate cleavable binding group to resist cleavage in blood or upon contact with other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between first and second conditions, the first selected to be indicative of cleavage within target cells, and the second selected to be indicative of cleavage in other tissues or biological fluids, such as blood or serum. This evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in a whole animal. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm with further evaluations in a whole animal. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0422] i. Redox-cleavable linking group In one embodiment, the cleavable linking group is a redox-cleavable linking group that is cleaved after reduction or oxidation. An example of a reductively cleavable linking group is a disulfide bond (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group," or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one may turn to the methods described herein. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art, which mimic the cleavage rate that can be observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In one embodiment, the candidate compound is cleaved at about 10% or less in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster inside cells (or under in vitro conditions selected to mimic intracellular conditions) compared to in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0423] ii. Phosphate-based cleavable linking groups In another embodiment, the cleavable linker comprises a phosphate-based cleavable linking group that is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves phosphate groups within a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups are -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-, -OP(S)(Rk)-S-. Preferred embodiments are -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-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0424] iii. Acid-cleavable linking groups In another embodiment, the cleavable linker comprises an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or less) or by an agent, such as an enzyme, that can act as a general acid. Within a cell, certain low-pH organelles, such as endosomes and lysosomes, may provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can be represented by the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0425] iv. Ester-based linking groups In another embodiment, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved by enzymes such as intracellular esterases and amylases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups are represented by the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0426] v. Peptide-Based Cleavage Groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. Peptide-based cleavable linking groups are cleaved by enzymes, such as intracellular peptidases and proteases. Peptide-based cleavable groups are peptide bonds formed between amino acids to give oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to give peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give peptides and proteins, but do not include all amide functional groups. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0427] In one embodiment, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the present invention include, but are not limited to: [ka] [ka] and when one of X or Y is an oligonucleotide, the other is hydrogen.

[0428] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0429] In one embodiment, the dsRNA of the present invention has the structure represented by formula (XXXII) to (XXXV): [ka] and conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of the following: During the ceremony: each occurrence of q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently represents 0 to 20, and the repeating units may be the same or different; P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4B, T4A, T5B, T5C are each, independently at each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CHO; Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C, each occurrence independently, is absent, alkylene, or substituted alkylene, wherein one or more methylenes are optionally interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R')=C(R"), C≡C, or C(O); R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, and R5C are each independently absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), -C(O)-CH(Ra)-NH-, CO, CH=NO, [ka] or heterocyclyl; L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B, and L5C represent ligands; i.e., each, independently at each occurrence, is a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and Ra is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives can be used with RNAi agents to form ligands of formula (XXXVI): [ka] are particularly useful for inhibiting the expression of target genes such as those of In the formula, L5A, L5B, and L5C represent monosaccharides such as GalNAc derivatives.

[0430] Examples of suitable divalent and trivalent branched linking groups for conjugation to GalNAc derivatives include, but are not limited to, the structures listed above as Formulas II, VII, XI, X, and XIII.

[0431] Representative patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 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 No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779 4,789,737; 4,824,941; 4,835,263; 4,876,335; Specification No. 4,904,582; Specification No. 4,958,013; Specification No. 5,082,830; Specification No. 5,112,963; Specification 5 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,27 Specification No. 2,250; Specification No. 5,292,873; Specification No. 5,317,098; Specification No. 5,371,241, Specification No. 5,391, Specification No. 723; Specification No. 5,416,203, Specification No. 5,451,463; Specification No. 5,510,475; Specification No. 5,512,66 No. 7; No. 5,514,785; No. 5,565,552; No. 5,567,810; No. 5,574,142; No. 5,585,481; No. 5,587,371; No. 5,595,726; No. 5,597,696; No. 5,599,923; No. 5,599,928 and No. 5,688,941; No. 6,294,664; No. 6,320,017; No. 6,576,752; No. 6,783,931; No. 6,900,297;Nos. 7,037,646 and 8,106,022, the entire contents of each of which are incorporated herein by reference.

[0432] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the above modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0433] In the context of the present invention, a "chimeric" iRNA compound or "chimera" refers to an iRNA compound, preferably a dsRNA, that contains two or more chemically distinct regions, each composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to the iRNA for the target nucleic acid. Additional regions of the iRNA may serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly increasing the efficiency of iRNA inhibition of gene expression. Consequently, when chimeric dsRNAs are used, comparable results can often be obtained with shorter iRNAs compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can typically be detected by gel electrophoresis and, if desired, by associated nucleic acid hybridization techniques known in the art.

[0434] In some cases, the RNA of an iRNA can be modified with a non-ligand group. Several non-ligand molecules have been conjugated to iRNAs to improve their activity, cellular distribution, or cellular uptake, and procedures for such conjugation are available in the scientific literature.Such non-ligand moieties include 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), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 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-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-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. al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic 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 US patents that teach the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA with an amino linker at one or more positions in the sequence. The amino group is then reacted with the conjugated molecule using an appropriate coupling or activating agent. The conjugation reaction can be carried out with the RNA still attached to a solid support or after cleavage of the RNA in solution phase. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0435] V. Delivery of iRNA of the Invention Delivery of an iRNA of the invention to a cell, e.g., a cell in a subject, such as a human subject (e.g., a subject in need of an iRNA agent, such as a subject with a disease, disorder, or condition associated with HBV infection), can be achieved in several different ways. For example, delivery can be achieved by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery can also be achieved directly by administering a composition containing an iRNA, e.g., a dsRNA, to the subject. Alternatively, in vivo delivery can be achieved indirectly by administering one or more vectors that encode and direct the expression of the iRNA. Examples of these alternatives are described further below.

[0436] Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the present invention (see, e.g., Akhtar S. and Julian R.L., (1992) Trends Cell. Biol. 2(5):139-144 and WO 94 / 02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider when delivering iRNA molecules include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, e.g., by direct injection or implantation into the tissue, or by administering the formulation locally. Local administration at the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may be adversely affected by or degrade the agent, and can reduce the total dose of the iRNA molecule administered. Several studies have demonstrated successful knockdown of gene products when iRNAs are administered locally. For example, intraocular delivery of VEGF dsRNA via intravitreal injection in cynomolgus monkeys (Tolentino, MJ. et al., (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ. et al., (2003) Mol. Vis. 9:210-216) have both been shown to prevent neovascularization in experimental models of age-related macular degeneration. Furthermore, direct intratumoral administration of dsRNA in mice can reduce tumor volume (Pille, J. et al., (2005) Mol. Ther. 11:267-274) and prolong the survival of tumor-bearing mice (Kim, WJ. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523).RNA interference can be delivered locally to the central nervous system by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P.H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, G.T. et al. (2004) Neuroscience 129:521-528; Thakker, E.R. et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y. et al. (2005) J. Neurophysiol. 93:594-602) and locally to the lung by intranasal administration (Howard, K.A. et al. (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55) have also demonstrated success. To administer iRNA systemically for disease treatment, the RNA can be modified or delivered using a drug delivery system; both methods serve to prevent rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of the RNA or pharmaceutical carrier can also enable targeting of iRNA compositions to target tissues and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups, such as cholesterol, to improve cellular uptake and prevent degradation. For example, iRNAs against ApoB conjugated to lipophilic cholesterol moieties were administered systemically to mice, resulting in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of iRNAs to aptamers has been shown to inhibit tumor growth and mediate tumor regression in mouse models of prostate cancer (McNamara, J. et al., (2006) Nat. Biotechnol. 24:1005-1015).In alternative embodiments, iRNAs can be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate binding of iRNA molecules (which are negatively charged) and also improve interaction with the negatively charged cell membrane, allowing for efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can be bound to iRNAs or induced to form vesicles or micelles that encapsulate iRNAs (see, e.g., Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents degradation of iRNAs when administered systemically. Methods for making and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, e.g., Sorensen, D.R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U.N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A.S. et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entireties).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, D.R., et al. (2003), supra; Verma, U.N. et al. (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T.S. et al. (2006) Nature 441:111-114), cardiolipin (Chien, P.Y. et al. (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al. (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M.E. et al. (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D. A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, iRNAs are complexed with cyclodextrins for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is incorporated herein by reference in its entirety.

[0437] A. Vector-encoded iRNA of the invention iRNAs targeting HBV genes can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., 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 (from a few hours to a few weeks) or sustained (for weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrating or non-integrating vectors. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0438] The individual strands of the iRNA can be transcribed from a promoter in an expression vector. Two separate expression vectors can be co-introduced into a target cell (e.g., by transfection or infection), where two separate strands are expressed to produce, for example, dsRNA. Alternatively, each individual strand of the dsRNA can be transcribed by a promoter located on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide joined by a linker polynucleotide sequence to form a stem-loop structure.

[0439] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for expressing the iRNAs described herein can be produced using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic expression vectors are well known in the art and are available from numerous commercial sources. Such vectors are typically provided containing convenient restriction sites for inserting the desired nucleic acid segment. Delivery of the iRNA expression vector can be systemic, for example, by intravenous or intramuscular administration, by administration to target cells transplanted from the patient and then reintroduced into the patient, or by any other means that allows for introduction into the desired target cells.

[0440] iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdown targeting different regions of the target RNA over a period of one week or more are also contemplated by the present invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be indicated using a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable transfection of cells ex vivo can be ensured using a marker that confers resistance to certain environmental factors (e.g., antibiotics and drugs) on the transfected cells, such as hygromycin B resistance.

[0441] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) orthopox, e.g., vaccinia virus vectors, or avian pox, e.g., canarypox or fowlpox, poxvirus vectors; and (j) helper-dependent or attenuated adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors may or may not integrate into the cellular genome. The constructs may optionally include viral sequences for transfection. Alternatively, the constructs may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are discussed further below.

[0442] Vectors useful for delivery of iRNA will contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide for either constitutive or regulatable / inducible expression.

[0443] Expression of iRNA can be precisely regulated, for example, by using inducible regulatory sequences that are sensitive to specific physiological regulators, such as blood glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Suitable inducible expression systems for controlling dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in the art will be able to select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene.

[0444] Viral vectors containing nucleic acid sequences encoding iRNAs can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for proper packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding the iRNAs are cloned into one or more vectors, which facilitate delivery of the nucleic acid to a patient. Further details about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references demonstrating the use of retroviral vectors in gene therapy are Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV-based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference.

[0445] Adenoviruses are also contemplated for use in delivering iRNAs of the present invention. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, respiratory epithelia. Adenoviruses naturally infect respiratory epithelia, causing a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), provide a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994), demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other examples of the use of adenovirus in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155; Mastrangeli et al. (1992), J. Clin. Invest. 91:225-234 (1993); PCT Publication WO 94 / 12649; and Wang et al., Gene Therapy 2:775-783 (1995). AV vectors suitable for expressing iRNAs featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0446] Adeno-associated virus (AAV) vectors can also be used to deliver the iRNAs of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNAs can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNA featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J.Virol. 61:3096-3101; Fisher KJ et al. (1996), J.Virol, 70:520-532; Samulski R et al. (1989), J.Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Patent Application No. WO 94 / 13788; and International Patent Application No. WO 93 / 24641, the entire disclosures of which are incorporated herein by reference.

[0447] Another viral vector suitable for delivery of the iRNA of the invention is a poxvirus, such as a vaccinia virus, e.g., an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, or an avian pox, such as fowlpox or canarypox.

[0448] The tropism of viral vectors can be modified by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins as needed. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors can be engineered to target different cells by engineering the vector to express different capsid protein serotypes. See, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0449] The pharmaceutical preparation of the vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0450] VI. Pharmaceutical Compositions of the Present Invention The present invention also includes pharmaceutical compositions and formulations comprising the iRNA of the present invention. In one embodiment, a pharmaceutical composition containing the iRNA described herein and a pharmaceutically acceptable carrier is also provided herein. Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with HBV gene expression or activity. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral administration, e.g., subcutaneous (SC) or intravenous (IV) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma, e.g., by injection into the brain, such as by continuous pump infusion. The pharmaceutical compositions of the present invention can be administered at a dosage sufficient to inhibit HBV gene expression.

[0451] In one embodiment, an iRNA agent of the invention is administered to a subject as a weight-based dose. A "weight-based dose" (e.g., a dose in mg / kg) is a dose of an iRNA agent that can vary depending on the subject's weight. In another embodiment, an iRNA agent is administered to a subject as a flat dose. A "flat dose" (e.g., a dose in mg) means that one dose of an iRNA agent is used for all subjects, regardless of any specific subject-related factors, such as weight. In a particular embodiment, the flat dose of an iRNA agent of the invention is based on a predetermined weight or age.

[0452] In general, suitable doses of iRNAs of the present invention can range from about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, generally from about 1 to 50 mg per kilogram of body weight per day. For example, dsRNAs can be administered at about 0.01 mg / kg, about 0.05 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg per single dose.

[0453] For example, the dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0454] In another embodiment, the dsRNA is about 0.1 to about 50 mg / kg, about 0.25 to about 50 mg / kg, about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / kg, about 1.5 to about 50 mg / kg, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, or about 20 to about 50 mg / kg , about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.1 to about 45 mg / kg, about 0.25 to about 45 mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / kg, about 1.5 to about 45 mg / kg, about 2 to about 45 mg / kg, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about About 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.1 to about 40 mg / kg, about 0.25 to about 40 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / kg, about 1.5 to about 40 mg / kg, about 2 to about 40 mg / kg kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / kg, about 1.5 to about 30 mg / kg, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30mg / kg, about 5 to about 30mg / kg, about 7.5 to about 30mg / kg, about 10 to about 30mg / kg, about 15 to about 30mg / kg, about 20 to about 30mg / kg, about 20 to about 30mg / kg, about 25 to about 30mg / kg, about 0.1 to about 20m g / kg, about 0.25 to about 20 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / kg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the present invention.

[0455] For example, the dsRNA may have a concentration of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5 , 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0456] In another embodiment, the dsRNA is about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / kg, about 1.5 to about 50 mg / kg, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, or about 25 to about 50 mg / kg. 0mg / kg, about 30 to about 50mg / kg, about 35 to about 50mg / kg, about 40 to about 50mg / kg, about 45 to about 50mg / kg, about 0.5 to about 45mg / kg, about 0.75 to about 45mg / kg, about 1 to about 45mg / kg, about 1.5 to about 45mg / kg, about 2 to about 45mg / k g, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / kg kg, about 1.5 to about 40 mg / kg, about 2 to about 40 mg / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 ~40mg / kg, 15~40mg / kg, 20~40mg / kg, 20~40mg / kg, 25~40mg / kg, 25~40mg / kg, 30~40mg / kg, 35~40mg / kg, 0.5~30mg / kg, 0.75~30 mg / kg, about 1 to about 30 mg / kg, about 1.5 to about 30 mg / kg, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.It is administered at a dose of 5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / kg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. In one embodiment, the dsRNA is administered at a dose of about 10 mg / kg to about 30 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0457] For example, targets may include approximately 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.7...

Claims

1. A double-stranded RNAi agent for inhibiting expression of hepatitis B virus (HBV) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and the antisense strand comprising at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides; substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and A double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

2. 2. The double-stranded RNAi agent of claim 1, wherein one or more of the three nucleotide differences in the nucleotide sequence of the antisense strand are nucleotide mismatches in the antisense strand.

3. 2. The double-stranded RNAi agent of claim 1, wherein one or more of the three nucleotide differences in the nucleotide sequence of the antisense strand are nucleotide mismatches in the sense strand.

4. 2. The double-stranded RNAi agent of claim 1, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

5. 2. The double-stranded RNAi agent of Claim 1, wherein the sense strand and the antisense strand comprise a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the sequences listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26.

6. 6. The double-stranded RNAi agent of any one of claims 1-5, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxynucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxyly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising an unnatural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, and a nucleotide comprising a 5'-phosphate mimic.

7. 10. The double-stranded RNAi agent of claim 1, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

8. 10. The double-stranded RNAi agent of claim 1, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

9. 2. The double-stranded RNAi agent of claim 1, wherein the double-stranded region is 15 to 30 nucleotide pairs in length.

10. 2. The double-stranded RNAi agent of claim 1, wherein the double-stranded region is 17 to 23 nucleotide pairs in length.

11. 2. The double-stranded RNAi agent of claim 1, wherein the double-stranded region is 17 to 25 nucleotide pairs in length.

12. 2. The double-stranded RNAi agent of claim 1, wherein the double-stranded region is 23 to 27 nucleotide pairs in length.

13. 2. The double-stranded RNAi agent of claim 1, wherein the double-stranded region is 19 to 21 nucleotide pairs in length.

14. 2. The double-stranded RNAi agent of claim 1, wherein the double-stranded region is 21 to 23 nucleotide pairs in length.

15. The double-stranded RNAi agent of claim 1, wherein each strand has 15 to 30 nucleotides.

16. The double-stranded RNAi agent of claim 1, wherein each strand has 19 to 30 nucleotides.

17. The ligand is 【Chemistry 1】 The double-stranded RNAi agent of claim 1, wherein

18. 10. The RNAi agent according to claim 1, wherein the RNAi agent is 【Chemistry 2】 wherein X is O or S.

19. 2. The double-stranded RNAi agent of claim 1, wherein the RNAi agent is selected from the group of RNAi agents listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26.

20. A double-stranded RNAi agent for inhibiting expression of hepatitis B virus (HBV) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and the antisense strand comprises 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and A double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

21. A double-stranded RNAi agent for inhibiting expression of hepatitis B virus (HBV) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and the antisense strand comprises 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and A double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

22. A double-stranded RNAi agent for inhibiting expression of hepatitis B virus (HBV) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-UCGUGGUGGACUUCUCUCUCA-3' (SEQ ID NO: 5), and the antisense strand comprises 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and A double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

23. A double-stranded RNAi agent for inhibiting expression of hepatitis B virus (HBV) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), and the antisense strand comprises 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and A double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

24. A double-stranded RNAi agent for inhibiting expression of hepatitis B virus (HBV) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-CGUGGUGGACUUCUCUCUCAAUU-3' (SEQ ID NO: 9), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and A double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

25. A double-stranded RNAi agent for inhibiting expression of hepatitis B virus (HBV) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-CGUGGUGGUCUUCUCUCUAAAUU-3' (SEQ ID NO: 37), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and A double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

26. The double-stranded RNAi agent of any one of claims 20 to 25, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

27. 25. The double-stranded RNAi agent of any one of claims 20-24, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxynucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxyly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, and a nucleotide comprising a 5'-phosphate mimic.

28. 28. The double-stranded RNAi agent of claim 27, wherein the 5'-phosphate mimic is 5'-vinyl phosphate (5'-VP).

29. 23. The double-stranded RNAi agent of claim 22, wherein the sense strand comprises 5'-uscsguGfgUfGfGffacuucucucuca-3' (SEQ ID NO: 13) and the antisense strand comprises 5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

30. 23. The double-stranded RNAi agent of claim 22, wherein the sense strand comprises 5'-uscsguGfgUfGfGffacuucucucuca-3' (SEQ ID NO: 15) and the antisense strand comprises 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

31. 24. The double-stranded RNAi agent of claim 23, wherein the sense strand comprises 5'-gsusgcacUfuCfGfCfuucacccucua-3' (SEQ ID NO: 17) and the antisense strand comprises 5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

32. 24. The double-stranded RNAi agent of claim 23, wherein the sense strand comprises 5'-gsusgcacUfuCfGfCfuucacccucua-3' (SEQ ID NO: 19) and the antisense strand comprises 5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

33. 25. The double-stranded RNAi agent of claim 24, wherein the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) and the antisense strand comprises 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

34. 25. The double-stranded RNAi agent of claim 24, wherein the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO:23) and the antisense strand comprises 5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO:24), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

35. 26. The double-stranded RNAi agent of claim 25, wherein the sense strand comprises 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) and the antisense strand comprises 5'-asdAsuugagagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; dA, dC, dG, and dT are deoxyribose A, C, G, and T; and s is a phosphorothioate linkage.

36. 22. The double-stranded RNAi agent of claim 21, wherein the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) and the antisense strand comprises 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

37. 22. The double-stranded RNAi agent of claim 21, wherein the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO:27) and the antisense strand comprises 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO:28), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

38. 21. The double-stranded RNAi agent of claim 20, wherein the sense strand comprises 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) and the antisense strand comprises 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

39. The ligand is 【Transformation 3】 The double-stranded RNAi agent according to any one of claims 20 to 38, wherein

40. 10. The RNAi agent according to claim 1, wherein the RNAi agent is 【Chemistry 4】 wherein X is O or S.

41. 38. The double-stranded RNAi agent of any one of claims 30, 32, 34, and 37, wherein P is a 5'-phosphate mimetic.

42. 38. The double-stranded RNAi agent of any one of claims 30, 32, 34, and 37, wherein the 5'-phosphate mimic is 5'-vinyl phosphate (5'-VP).

43. 1. A composition for inhibiting expression of Hepatitis B virus (HBV) in a cell, comprising two or more double-stranded RNAi agents, each double-stranded RNAi agent independently comprising a sense strand and an antisense strand forming a double-stranded region, wherein each of the sense strands independently comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, and each of the antisense strands independently comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides; substantially all of each nucleotide of the sense strand and substantially all of each nucleotide of the antisense strand are, independently, modified nucleotides; each of the sense strands is independently conjugated to a ligand attached at its 3' end; and The composition, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

44. 44. The composition of claim 43, wherein one or more of the three nucleotide differences in the nucleotide sequence of the antisense strand is a nucleotide mismatch in the antisense strand.

45. 44. The composition of claim 43, wherein one or more of the three nucleotide differences in the nucleotide sequence of the antisense strand is a nucleotide mismatch in the sense strand.

46. 44. The composition of claim 43, wherein all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

47. 44. The composition of claim 43, wherein the sense strand and the antisense strand comprise a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the sequences listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26.

48. 48. The composition of any one of claims 43 to 47, wherein at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyly modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

49. 1. A composition for inhibiting expression of Hepatitis B virus (HBV) in a cell, comprising: (a) a first double-stranded RNAi agent comprising a first sense strand and a first antisense strand forming a double-stranded region; substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides; the first sense strand is conjugated to a ligand attached at its 3' end; and a first double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; and (b) a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a double-stranded region, substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides; the second sense strand is conjugated to a ligand attached at its 3' end; and a second double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; wherein the first and second sense strands each independently comprise: 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5), 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), 5'-CGUGGUGGACUUCUCUCUCAAUU-3' (SEQ ID NO: 9), 5'-CGUGGUGGUCUUCUCUCUAAAUU-3' (SEQ ID NO: 37), 5'-GGUGGACUUCUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and The first and second antisense strands each independently comprise: 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6), 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8), 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10), 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38), 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12) and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40).

50. 50. The composition of claim 49, wherein all of the nucleotides of the first and second sense strands and / or all of the nucleotides of the first and second antisense strands comprise a modification.

51. 50. The composition of claim 49, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxynucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxyly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, and a nucleotide comprising a 5'-phosphate mimic.

52. the first and second RNAi agents are 5'-uscsguGfgUfGfGfGfacuucucuca-3' (SEQ ID NO: 13) 5′-usGfsagaGfaAfGfuccaCfcAfcgasusu-3′ (SEQ ID NO: 14); 5'-uscsguGfgUfGfGfGfacuucucuca-3' (SEQ ID NO: 15) 5′-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3′ (SEQ ID NO: 16); 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17) 5′-usAfsgagGfugaagcgAfaGfugcacsusu-3′ (SEQ ID NO: 18); 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19) 5′-PusAfsgagGfugaagcgAfaGfugcacsusu-3′ (SEQ ID NO: 20); 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) 5′-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3′ (SEQ ID NO: 22); 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23) 5′-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3′ (SEQ ID NO: 24); 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) 5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36); 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) 5′-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3′ (SEQ ID NO: 26); 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27) 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) 50. The composition of claim 49, wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf are 2'-fluoro A, G, C, or U; dA, dC, dG, and dT are deoxyribose A, C, G, and T; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

53. the first and second RNAi agents are 5'-uscsguGfgUfGfGfGfacuucucuca-3' (SEQ ID NO: 15) 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16); and 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22) 50. The composition of claim 49, wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

54. the first and second RNAi agents are 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) 50. The composition of claim 49, wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

55. A double-stranded RNAi agent comprising an RNAi agent listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26.

56. 26. A vector comprising the double-stranded RNAi agent of any one of claims 1 and 20 to 25.

57. 26. A cell comprising the double-stranded RNAi agent of any one of claims 1 and 20-25.

58. A pharmaceutical composition comprising the double-stranded RNAi agent of any one of claims 1 and 20 to 25, or the composition of claim 43 or 49, or the vector of claim 56.

59. 59. The pharmaceutical composition of claim 58, wherein the double-stranded RNAi agent is administered in a non-buffered solution.

60. 60. The pharmaceutical composition of claim 59, wherein the non-buffered solution is saline or water.

61. 59. The pharmaceutical composition of claim 58, wherein the double-stranded RNAi agent is administered with a buffer.

62. 62. The pharmaceutical composition of claim 61, wherein the buffer comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

63. 63. The pharmaceutical composition of claim 62, wherein the buffer is phosphate buffered saline (PBS).

64. 1. A method for inhibiting Hepatitis B virus (HBV) gene expression in a cell, comprising: (a) contacting the cell with the double-stranded RNAi agent of any one of claims 1 and 20 to 25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58 to 63; (b) maintaining the cells produced in step (a) for a time sufficient to achieve degradation of mRNA transcripts of the HBV gene, thereby inhibiting expression of the HBV gene in the cells.

65. 65. The method of claim 64, wherein the HBV gene is selected from the group consisting of C, X, P, S, and combinations thereof.

66. 1. A method for inhibiting replication of hepatitis B virus (HBV) in a cell, comprising: (a) contacting the cell with the double-stranded RNAi agent of any one of claims 1 and 20 to 25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58 to 63; (b) maintaining the cells produced in step (a) for a time sufficient to achieve degradation of mRNA transcripts of HBV genes, thereby inhibiting replication of the HBV in the cells.

67. 67. The method of claim 64 or 66, wherein the cell is in a subject.

68. 68. The method of claim 67, wherein the subject is a human.

69. 69. The method of claim 68, wherein the subject is suffering from a disease associated with HBV.

70. 65. The method of claim 64, wherein the HBV gene expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

71. 67. The method of claim 66, wherein replication of HBV in the cells is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

72. 64. A method for reducing hepatitis B virus (HBV) covalently closed circular (ccc) DNA levels in a subject infected with HBV, comprising the step of administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of any one of claims 1 and 20 to 25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58 to 63, thereby reducing the level of covalently closed circular HBV DNA in the subject.

73. 64. A method for reducing hepatitis B virus (HBV) antigen levels in a subject infected with HBV, comprising the step of administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of any one of claims 1 and 20 to 25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58 to 63, thereby reducing the HBV antigen levels in the subject.

74. 74. The method of claim 73, wherein the HBV antigen is HBsAg.

75. 74. The method of claim 73, wherein the HBV antigen is HBeAg.

76. 64. A method for reducing hepatitis B virus (HBV) viral load in a subject infected with HBV, comprising the step of administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of any one of claims 1 and 20 to 25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58 to 63, thereby reducing the HBV viral load in the subject.

77. 64. A method for reducing alanine aminotransferase (ALT) levels in a subject infected with HBV, comprising the step of administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of any one of claims 1 and 20 to 25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58 to 63, thereby reducing the ALT level in the subject.

78. 64. A method for reducing aspartate aminotransferase (AST) levels in a subject infected with HBV, comprising the step of administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of any one of claims 1 and 20 to 25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58 to 63, thereby reducing the AST levels in the subject.

79. 64. A method for increasing an anti-hepatitis B virus (HBV) antibody level in a subject infected with HBV, comprising the step of administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of any one of claims 1 and 20 to 25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58 to 63, thereby increasing the anti-HBV antibody level in the subject.

80. 64. A method of treating a subject having a hepatitis B virus (HBV) infection, comprising the step of administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of any one of claims 1 and 20-25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58-63, thereby treating the subject.

81. 64. A method of treating a subject having a hepatitis B virus (HBV)-associated disorder, comprising the step of administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of any one of claims 1 and 20-25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58-63, thereby treating the subject.

82. 72. The method of claim 71, wherein the HBV-related disorder is selected from the group consisting of hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma.

83. 72. The method of claim 71, wherein the HBV-associated disorder is chronic hepatitis and the subject is HBeAg positive.

84. 72. The method of claim 71, wherein the HBV-associated disorder is chronic hepatitis and the subject is HBeAg negative.

85. A method of treating a subject having a hepatitis B virus (HBV) infection, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-UCGUGGUGGACUUCUCUCUCA-3' (SEQ ID NO: 5), and the antisense strand comprises 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

86. A method of treating a subject having a hepatitis B virus (HBV)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-UCGUGGUGGACUUCUCUCUCA-3' (SEQ ID NO: 5), and the antisense strand comprises 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

87. A method of treating a subject having a hepatitis B virus (HBV) infection, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), and the antisense strand comprises 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

88. A method of treating a subject having a hepatitis B virus (HBV)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), and the antisense strand comprises 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

89. 1. A method of treating a subject having a hepatitis B virus (HBV) infection, comprising administering to a subject a therapeutically effective amount of a double-stranded RNAi agent; wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-CGUGGUGGACUUCUCUCUCAAUU-3' (SEQ ID NO: 9), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

90. A method of treating a subject having a hepatitis B virus (HBV)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-CGUGGUGGACUUCUCUCUCAAUU-3' (SEQ ID NO: 9), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

91. A method of treating a subject having a hepatitis B virus (HBV) infection, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-CGUGGUGGUCUUCUCUCUAAAUU-3' (SEQ ID NO: 37), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

92. A method of treating a subject having a hepatitis B virus (HBV)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-CGUGGUGGUCUUCUCUCUAAAUU-3' (SEQ ID NO: 37), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

93. A method of treating a subject having a hepatitis B virus (HBV) infection, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and the antisense strand comprises 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

94. A method of treating a subject having a hepatitis B virus (HBV)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and the antisense strand comprises 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

95. A method of treating a subject having a hepatitis B virus (HBV) infection, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and the antisense strand comprises 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

96. A method of treating a subject having a hepatitis B virus (HBV)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and the antisense strand comprises 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

97. 97. The method of any one of claims 85 to 96, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

98. 97. The method of any one of claims 85 to 96, wherein at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 3' terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyly modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, and nucleotides comprising 5'-phosphate mimetics.

99. 99. The method of claim 98, wherein the 5'-phosphate mimic is 5'-vinyl phosphate (5'-VP).

100. 87. The method of claim 85 or 86, wherein the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13) and the antisense strand comprises 5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

101. 87. The method of claim 85 or 86, wherein the sense strand comprises 5'-uscsguGfgUfGfGffacuucucucuca-3' (SEQ ID NO: 15) and the antisense strand comprises 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

102. 89. The method of claim 87 or 88, wherein the sense strand comprises 5'-gsusgcacUfuCfGfCfuucacccucua-3' (SEQ ID NO: 17) and the antisense strand comprises 5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

103. 89. The method of claim 87 or 88, wherein the sense strand comprises 5'-gsusgcacUfuCfGfCfuucacccucua-3' (SEQ ID NO: 19) and the antisense strand comprises 5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

104. 91. The method of claim 89 or 90, wherein the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) and the antisense strand comprises 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

105. 91. The method of claim 89 or 90, wherein the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23) and the antisense strand comprises 5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

106. 93. The method of claim 91 or 92, wherein the sense strand comprises 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) and the antisense strand comprises 5'-asdAsuugagagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; dA, dC, dG, and dT are deoxyribose A, C, G, and T; and s is a phosphorothioate linkage.

107. 95. The method of claim 93 or 94, wherein the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) and the antisense strand comprises 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

108. 95. The method of claim 93 or 94, wherein the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27) and the antisense strand comprises 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

109. 97. The method of claim 95 or 96, wherein the sense strand comprises 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) and the antisense strand comprises 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

110. The ligand is 【Transformation 5】 The method according to any one of claims 85 to 96, wherein

111. 10. The RNAi agent according to claim 1, wherein the RNAi agent is 【Transformation 6】 wherein X is O or S.

112. 97. The method of any one of claims 86, 88, 90, 92, 94, and 96, wherein the HBV-related disorder is selected from the group consisting of hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma.

113. 97. The method of any one of claims 86, 88, 90, 92, 94, and 96, wherein the HBV-related disorder is chronic hepatitis and the subject is HBeAg positive.

114. 97. The method of any one of claims 86, 88, 90, 92, 94, and 96, wherein the HBV-related disorder is chronic hepatitis and the subject is HBeAg negative.

115. 1. A method of treating a subject having a hepatitis B virus (HBV) infection, comprising: (a) a first double-stranded RNAi agent comprising a first sense strand and a first antisense strand forming a double-stranded region; substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides; a first double-stranded RNAi agent, wherein the first sense strand is conjugated to a ligand attached at its 3′ end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; and (b) a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a double-stranded region, substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides; the second sense strand is conjugated to a ligand attached at its 3' end; and a second double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; wherein the first and second sense strands each independently comprise: 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5), 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), 5'-CGUGGUGGACUUCUCUCUCAAUU-3' (SEQ ID NO: 9), 5'-CGUGGUGGUCUUCUCUCUAAAUU-3' (SEQ ID NO: 37), 5'-GGUGGACUUCUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and the first and second antisense strands each independently comprise: 5′-UGAGAGAAGUCCACCACGAUU-3′ (SEQ ID NO: 6); 5′-UAGAGGUGAAGCGAAGUGCACUU-3′ (SEQ ID NO: 8); 5′-AAUUGAGAGAAGUCCACCAGCAG-3′ (SEQ ID NO: 10); 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38), 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40) to the subject, thereby treating the subject.

116. 1. A method of treating a subject having a hepatitis B virus (HBV)-associated disorder, comprising: a composition for inhibiting expression of hepatitis B virus (HBV) in a cell, the composition comprising: (a) a first double-stranded RNAi agent comprising a first sense strand and a first antisense strand forming a double-stranded region; substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides; a first double-stranded RNAi agent, wherein the first sense strand is conjugated to a ligand attached at its 3′ end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; and (b) a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a double-stranded region, substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides; the second sense strand is conjugated to a ligand attached at its 3' end; and a second double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; wherein the first and second sense strands each independently comprise: 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5), 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), 5'-CGUGGUGGACUUCUCUCUCAAUU-3' (SEQ ID NO: 9), 5'-CGUGGUGGUCUUCUCUCUAAAUU-3' (SEQ ID NO: 37), 5'-GGUGGACUUCUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and The first and second antisense strands each independently comprise: 5′-UGAGAGAAGUCCACCACGAUU-3′ (SEQ ID NO: 6); 5′-UAGAGGUGAAGCGAAGUGCACUU-3′ (SEQ ID NO: 8); 5′-AAUUGAGAGAAGUCCACCAGCAG-3′ (SEQ ID NO: 10); 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38), 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40) to the subject, thereby treating the subject.

117. 117. The method of claim 115 or 116, wherein all of the nucleotides of the first and second sense strands and all of the nucleotides of the first and second antisense strands comprise a modification.

118. 117. The method of claim 115 or 116, wherein at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 3' terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyly modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, and nucleotides comprising 5'-phosphate mimetics.

119. the first and second RNAi agents are 5'-uscsguGfgUfGfGfGfacuucucuca-3' (SEQ ID NO: 13) 5′-usGfsagaGfaAfGfuccaCfcAfcgasusu-3′ (SEQ ID NO: 14); 5'-uscsguGfgUfGfGfGfacuucucuca-3' (SEQ ID NO: 15) 5′-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3′ (SEQ ID NO: 16); 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17) 5′-usAfsgagGfugaagcgAfaGfugcacsusu-3′ (SEQ ID NO: 18); 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19) 5′-PusAfsgagGfugaagcgAfaGfugcacsusu-3′ (SEQ ID NO: 20); 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) 5′-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3′ (SEQ ID NO: 22); 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23) 5′-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3′ (SEQ ID NO: 24); 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) 5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36); 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) 5′-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3′ (SEQ ID NO: 26); 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27) 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf are 2'-fluoro A, G, C, or U; dA, dC, dG, and dT are deoxyribose A, C, G, and T; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

120. the first and second RNAi agents are 5'-uscsguGfgUfGfGfGfacuucucuca-3' (SEQ ID NO: 15) 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16); and 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22) wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

121. the first and second RNAi agents are 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

122. The ligand is 【Transformation 7】 117. The method of claim 115 or 116, wherein:

123. 10. The RNAi agent according to claim 1, wherein the RNAi agent is 【Transformation 8】 wherein X is O or S.

124. 117. The method of claim 115 or 116, wherein the subject is a human.

125. The method of claim 116, wherein the HBV-related disorder is selected from the group consisting of hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

126. 117. The method of claim 116, wherein the HBV-associated disorder is chronic hepatitis and the subject is HBeAg positive.

127. 117. The method of claim 116, wherein the HBV-associated disorder is chronic hepatitis and the subject is HBeAg negative.

128. 128. The method of any one of claims 85-127, wherein the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg.

129. 129. The method of Claim 128, wherein said double-stranded RNAi agent is administered at a dose of about 10 mg / kg to about 30 mg / kg.

130. 129. The method of claim 128, wherein the double-stranded RNAi agent is administered at a dose of about 3 mg / kg.

131. 129. The method of claim 128, wherein the double-stranded RNAi agent is administered at a dose of about 10 mg / kg.

132. 129. The method of Claim 128, wherein said double-stranded RNAi agent is administered at a dose of about 0.5 mg / kg twice a week.

133. 121. The method of any one of claims 82-120, wherein the double-stranded RNAi agent is administered at a fixed dose of about 50 mg to 200 mg.

134. 128. The method of any one of claims 85-127, wherein the double-stranded RNAi agent is administered subcutaneously.

135. 128. The method of any one of claims 85-127, wherein the double-stranded RNAi agent is administered intravenously.

136. 128. The method of any one of claims 85-127, wherein the RNAi agent is administered in two or more doses.

137. 128. The method of any one of claims 85-127, wherein the RNAi agent is administered at an interval selected from the group consisting of about once every 12 hours, about once every 24 hours, about once every 48 hours, about once every 72 hours, and about once every 96 hours.

138. 128. The method of any one of claims 85-127, wherein the RNAi agent is administered twice weekly.

139. 128. The method of any one of claims 85-127, wherein the RNAi agent is administered every two weeks.

140. 128. The method of any one of claims 85 to 127, further comprising administering to the subject a further therapeutic agent.

141. 141. The method of claim 140, wherein the additional therapeutic agent is selected from the group consisting of antiviral agents, reverse transcriptase inhibitors, immunostimulants, therapeutic vaccines, viral entry inhibitors, oligonucleotides that inhibit the secretion or release of HBsAg, capsid inhibitors, covalently closed circular (ccc) HBV DNA inhibitors, and any combination of the foregoing.

142. 128. The method of any one of claims 85 to 127, further comprising administering to the subject a reverse transcriptase inhibitor.

143. 128. The method of any one of claims 85 to 127, further comprising administering to the subject a reverse transcriptase inhibitor and an immunostimulant.

144. 144. The method of claim 142 or 143, wherein the reverse transcriptase inhibitor is selected from the group consisting of tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, and AGX-1009.

145. 144. The method of claim 143, wherein the immunostimulant is selected from the group consisting of pegylated interferon alpha 2a (PEG-IFN-α2a), interferon alpha-2b, recombinant human interleukin-7, and a Toll-like receptor 7 (TLR7) agonist.

146. A method of treating a subject having a hepatitis B virus (HBV)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:29 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:30 by no more than 3 nucleotides; substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

147. 1. A method of treating a subject having a hepatitis B virus (HBV) infection, comprising: (a) a first double-stranded RNAi agent comprising a first strand and a first antisense strand forming a double-stranded region; substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides; a first double-stranded RNAi agent, wherein the first sense strand is conjugated to a ligand attached at its 3′ end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; and (b) a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a double-stranded region, substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides; the second sense strand is conjugated to a ligand attached at its 3' end; and a second double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; wherein the first sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and the first antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides; wherein the second sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:29, and the second antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:30, thereby treating the subject.

148. the first sense strand comprises: 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5), 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), 5'-CGUGGUGGACUUCUCUCUCAAUU-3' (SEQ ID NO: 9), 5'-CGUGGUGGUCUUCUCUCUAAAUU-3' (SEQ ID NO: 37), 5'-GGUGGACUUCUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and the second antisense strand comprises: 5′-UGAGAGAAGUCCACCACGAUU-3′ (SEQ ID NO: 6); 5′-UAGAGGUGAAGCGAAGUGCACUU-3′ (SEQ ID NO: 8); 5′-AAUUGAGAGAAGUCCACCAGCAG-3′ (SEQ ID NO: 10); 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38), 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 147. The method of claim 146, comprising a sequence selected from the group consisting of: 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40).

149. 149. The method of any one of claims 146 to 148, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

150. 150. The method of any one of claims 146 to 149, wherein at least one of the modified nucleotides is selected from the group consisting of dexoy-nucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyly modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, and nucleotides comprising 5'-phosphate mimetics.

151. The ligand is 【Chemistry 9】 The method of any one of claims 146 to 149, wherein

152. 10. The RNAi agent according to claim 1, wherein the RNAi agent is 【Chemistry 10】 wherein X is O or S.

153. 1. A method for inhibiting replication of hepatitis D virus (HDV) in a cell, comprising: (a) contacting the cell with the double-stranded RNAi agent of any one of claims 1 and 20 to 25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58 to 63; (b) maintaining the cells produced in step (a) for a time sufficient to achieve degradation of mRNA transcripts of HBV genes, thereby inhibiting replication of the HDV in the cells.

154. 154. The method of claim 153, wherein the cells are within the subject's body.

155. 155. The method of claim 154, wherein the subject is a human.

156. 64. A method for reducing hepatitis D virus (HDV) antigen levels in a subject infected with HDV, comprising the step of administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of any one of claims 1 and 20 to 25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58 to 63, thereby reducing the HDV antigen levels in the subject.

157. 157. The method of claim 156, wherein the HDV antigen is S-HDAg.

158. 157. The method of claim 156, wherein the HDV antigen is L-HDAg.

159. 64. A method for reducing hepatitis D virus (HDV) viral load in a subject infected with HBV, comprising the step of administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of any one of claims 1 and 20 to 25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58 to 63, thereby reducing the HDV viral load in the subject.

160. 64. A method of treating a subject having a hepatitis D virus (HDV) infection, comprising the step of administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of any one of claims 1 and 20-25, or the composition of claim 43 or 49, or the vector of claim 56, or the pharmaceutical composition of any one of claims 58-63, thereby treating the subject.

161. A method of treating a subject having a hepatitis D virus (HDV) infection, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-UCGUGGUGGACUUCUCUCUCA-3' (SEQ ID NO: 5), and the antisense strand comprises 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

162. A method of treating a subject having a hepatitis D virus (HDV)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-UCGUGGUGGACUUCUCUCUCA-3' (SEQ ID NO: 5), and the antisense strand comprises 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

163. A method of treating a subject having a hepatitis D virus (HDV) infection, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), and the antisense strand comprises 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

164. A method of treating a subject having a hepatitis D virus (HDV)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), and the antisense strand comprises 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

165. A method of treating a subject having a hepatitis D virus (HDV) infection, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-CGUGGUGGACUUCUCUCUCAAUU-3' (SEQ ID NO: 9), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

166. A method of treating a subject having a hepatitis D virus (HDV)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-CGUGGUGGACUUCUCUCUCAAUU-3' (SEQ ID NO: 9), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

167. A method of treating a subject having a hepatitis D virus (HDV) infection, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-CGUGGUGGUCUUCUCUCUAAAUU-3' (SEQ ID NO: 37), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

168. A method of treating a subject having a hepatitis D virus (HDV)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-CGUGGUGGUCUUCUCUCUAAAUU-3' (SEQ ID NO: 37), and the antisense strand comprises 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

169. A method of treating a subject having a hepatitis D virus (HDV) infection, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and the antisense strand comprises 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

170. A method of treating a subject having a hepatitis D virus (HDV)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and the antisense strand comprises 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

171. A method of treating a subject having a hepatitis D virus (HDV) infection, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and the antisense strand comprises 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

172. A method of treating a subject having a hepatitis D virus (HDV)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent. wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and the antisense strand comprises 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40); substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the sense strand is conjugated to a ligand attached at the 3' end; and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker to the subject, thereby treating the subject.

173. 173. The method of any one of claims 161 to 172, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

174. 173. The method of any one of claims 161 to 172, wherein at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 3' terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyly modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, and nucleotides comprising 5'-phosphate mimetics.

175. 175. The method of claim 174, wherein said 5'-phosphate mimic is 5'-vinyl phosphate (5'-VP).

176. The method of claim 161 or 162, wherein the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13) and the antisense strand comprises 5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14) [wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage].

177. 163. The method of claim 161 or 162, wherein the sense strand comprises 5'-uscsguGfgUfGfGffacuucucucuca-3' (SEQ ID NO: 15) and the antisense strand comprises 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

178. The method of claim 163 or 164, wherein the sense strand comprises 5'-gsusgcacUfuCfGfCfuucacccucua-3' (SEQ ID NO: 17) and the antisense strand comprises 5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18) [wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage].

179. 165. The method of claim 163 or 164, wherein the sense strand comprises 5'-gsusgcacUfuCfGfCfuucacccucua-3' (SEQ ID NO: 19) and the antisense strand comprises 5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

180. The method of claim 165 or 166, wherein the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) and the antisense strand comprises 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22) [wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage].

181. 167. The method of claim 165 or 166, wherein the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23) and the antisense strand comprises 5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

182. The method of claim 167 or 168, wherein the sense strand comprises 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) and the antisense strand comprises 5'-asdAsuugagagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; dA, dC, dG, and dT are deoxyribose A, C, G, and T; and s is a phosphorothioate linkage.

183. The method of claim 169 or 170, wherein the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) and the antisense strand comprises 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

184. 171. The method of claim 169 or 170, wherein the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27) and the antisense strand comprises 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

185. The method of claim 171 or 172, wherein the sense strand comprises 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) and the antisense strand comprises 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42), wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; and s is a phosphorothioate linkage.

186. The ligand is 【Chemistry 11】 The method according to any one of claims 161 to 172, wherein

187. 10. The RNAi agent according to claim 1, wherein the RNAi agent is 【Chemistry 12】 wherein X is O or S.

188. 1. A method of treating a subject having a hepatitis D virus (HDV) infection, comprising: a composition for inhibiting expression of hepatitis B virus (HBV) in a cell, the composition comprising: (a) a first double-stranded RNAi agent comprising a first sense strand and a first antisense strand forming a double-stranded region; substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides; a first double-stranded RNAi agent, wherein the first sense strand is conjugated to a ligand attached at its 3′ end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; and (b) a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a double-stranded region, substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides; the second sense strand is conjugated to a ligand attached at its 3' end; and a second double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; wherein the first and second sense strands each independently comprise: 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5), 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), 5'-CGUGGUGGACUUCUCUCUCAAUU-3' (SEQ ID NO: 9), 5'-CGUGGUGGUCUUCUCUCUAAAUU-3' (SEQ ID NO: 37), 5'-GGUGGACUUCUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and the first and second antisense strands each independently comprise: 5′-UGAGAGAAGUCCACCACGAUU-3′ (SEQ ID NO: 6); 5′-UAGAGGUGAAGCGAAGUGCACUU-3′ (SEQ ID NO: 8); 5′-AAUUGAGAGAAGUCCACCAGCAG-3′ (SEQ ID NO: 10); 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38), 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40) to the subject, thereby treating the subject.

189. 189. The method of claim 188, wherein all of the nucleotides of the first and second sense strands and all of the nucleotides of the first and second antisense strands comprise a modification.

190. 189. The method of claim 188, wherein at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 3' terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyly modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, and nucleotides comprising 5'-phosphate mimetics.

191. the first and second RNAi agents are 5'-uscsguGfgUfGfGfGfacuucucuca-3' (SEQ ID NO: 13) 5′-usGfsagaGfaAfGfuccaCfcAfcgasusu-3′ (SEQ ID NO: 14); 5'-uscsguGfgUfGfGfGfacuucucuca-3' (SEQ ID NO: 15) 5′-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3′ (SEQ ID NO: 16); 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17) 5′-usAfsgagGfugaagcgAfaGfugcacsusu-3′ (SEQ ID NO: 18); 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19) 5′-PusAfsgagGfugaagcgAfaGfugcacsusu-3′ (SEQ ID NO: 20); 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) 5′-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3′ (SEQ ID NO: 22); 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23) 5′-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3′ (SEQ ID NO: 24); 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) 5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36); 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) 5′-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3′ (SEQ ID NO: 26); 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27) 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf are 2'-fluoro A, G, C, or U; dA, dC, dG, and dT are deoxyribose A, C, G, and T; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

192. the first and second RNAi agents are 5'-uscsguGfgUfGfGfGfacuucucuca-3' (SEQ ID NO: 15) 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16); and 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22) wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

193. the first and second RNAi agents are 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf, or Uf is 2'-fluoro A, G, C, or U; s is a phosphorothioate linkage; and P is a 5'-phosphate or a 5' phosphate mimic.

194. The ligand is 【Chemistry 13】 The method of claim 188, wherein

195. 10. The RNAi agent according to claim 1, wherein the RNAi agent is 【Chemistry 14】 wherein X is O or S.

196. 189. The method of claim 188, wherein the subject is a human.

197. 197. The method of any one of claims 161-196, wherein the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg.

198. 200. The method of claim 197, wherein said double-stranded RNAi agent is administered at a dose of about 10 mg / kg to about 30 mg / kg.

199. 198. The method of claim 197, wherein the double-stranded RNAi agent is administered at a dose of about 3 mg / kg.

200. 198. The method of claim 197, wherein the double-stranded RNAi agent is administered at a dose of about 10 mg / kg.

201. 200. The method of claim 199, wherein said double-stranded RNAi agent is administered at a dose of about 0.5 mg / kg twice a week.

202. 197. The method of any one of claims 161-196, wherein the double-stranded RNAi agent is administered in a fixed dose of about 50 mg to 200 mg.

203. 197. The method of any one of claims 161-196, wherein the double-stranded RNAi agent is administered subcutaneously.

204. 197. The method of any one of claims 161-196, wherein the double-stranded RNAi agent is administered intravenously.

205. 197. The method of any one of claims 161-196, wherein the RNAi agent is administered in two or more doses.

206. 200. The method of any one of claims 161-196, wherein the RNAi agent is administered at an interval selected from the group consisting of about once every 12 hours, about once every 24 hours, about once every 48 hours, about once every 72 hours, and about once every 96 hours.

207. 197. The method of any one of claims 161-196, wherein the RNAi agent is administered twice weekly.

208. 197. The method of any one of claims 161-196, wherein the RNAi agent is administered every two weeks.

209. 197. The method of any one of claims 161 to 196, further comprising administering to the subject a further therapeutic agent.

210. 210. The method of claim 209, wherein the additional therapeutic agent is selected from the group consisting of antiviral agents, reverse transcriptase inhibitors, immunostimulants, therapeutic vaccines, viral entry inhibitors, oligonucleotides that inhibit the secretion or release of HBsAg, capsid inhibitors, covalently closed circular (ccc) HBV DNA inhibitors, and any combination of the foregoing.

211. 197. The method of any one of claims 161 to 196, further comprising administering to the subject a reverse transcriptase inhibitor.

212. 197. The method of any one of claims 161 to 196, further comprising administering to the subject a reverse transcriptase inhibitor and an immunostimulant.

213. 213. The method of claim 211 or 212, wherein the reverse transcriptase inhibitor is selected from the group consisting of tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, and AGX-1009.

214. 214. The method of claim 213, wherein the immunostimulant is selected from the group consisting of pegylated interferon alpha 2a (PEG-IFN-α2a), interferon alpha-2b, recombinant human interleukin-7, and a Toll-like receptor 7 (TLR7) agonist.

215. 1. A method of treating a subject having a hepatitis D virus (HDV) infection, comprising: a composition for inhibiting expression of hepatitis D virus (HDV) in a cell, the composition comprising: (a) a first double-stranded RNAi agent comprising a first strand and a first antisense strand forming a double-stranded region; substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides; a first double-stranded RNAi agent, wherein the first sense strand is conjugated to a ligand attached at its 3′ end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; and (b) a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a double-stranded region, substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides; the second sense strand is conjugated to a ligand attached at its 3' end; and a second double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; wherein the first sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and the first antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides; wherein the second sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:29, and the second antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:30, thereby treating the subject.

216. the first sense strand comprises: 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5), 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), 5'-CGUGGUGGACUUCUCUCUCAAUU-3' (SEQ ID NO: 9), 5'-CGUGGUGGUCUUCUCUCUAAAUU-3' (SEQ ID NO: 37), 5'-GGUGGACUUCUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and the second antisense strand comprises: 5′-UGAGAGAAGUCCACCACGAUU-3′ (SEQ ID NO: 6); 5′-UAGAGGUGAAGCGAAGUGCACUU-3′ (SEQ ID NO: 8); 5′-AAUUGAGAGAAGUCCACCAGCAG-3′ (SEQ ID NO: 10); 5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38), 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 216. The method of claim 215, comprising a sequence selected from the group consisting of: 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40).

217. 217. The method of claim 215 or 216, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

218. 217. The method of claim 215 or 216, wherein at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyly modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, and nucleotides comprising 5'-phosphate mimetics.

219. The ligand is 【Chemistry 15】 The method of claim 215 or 216,

220. 10. The RNAi agent according to claim 1, wherein the RNAi agent is 【Chemistry 16】 wherein X is O or S.

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