Hepatitis B virus (HBV) dsRNA agent compositions and methods of use thereof

Abstract

The present specification relates to double-stranded RNA agents that target the hepatitis B virus (HBV) genome, and methods of using such agents to inhibit one or more HBV genes and to treat subjects with HBV infection or HBV-related disorders, such as chronic hepatitis B.

Description

[Technical field]

[0001] Description of sequence listing The sequence listing associated with this application is provided in text form in lieu of a paper copy and is incorporated herein by reference. The text file containing the sequence listing is named 930385_410WO_SEQUENCE_LISTING.txt. The text file is 83.1KB, was created on August 4, 2019, and has been submitted electronically via EFS-Web. [Background technology]

[0002] Over 400 million people worldwide are chronically infected with HBV and are therefore at high risk of developing serious liver diseases, including chronic hepatitis, cirrhosis, liver failure and hepatocellular carcinoma (HCC), which result in an estimated 600,000 deaths each year.

[0003] The natural evolution of chronic HBV infection involves four successive stages: (1) an initial "immune tolerance" stage, with high levels of viral replication and minimal hepatitis; (2) an immune response stage, with significant hepatitis and elevated serum aminotransferases; some patients progress to (3); (3) a "non-replicating" stage, with seroconversion to anti-HBe, undetectable or low levels of viremia (<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 the production of HBeAg 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 both HBeAg-positive and 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 and ranges from 2 to 5% in patients with established cirrhosis.

[0004] The primary goal of HBV treatment is to permanently inhibit HBV replication and ameliorate liver disease. Clinically important short-term goals are to achieve HBeAg-seroconversion, normalization of serum ALT and AST, resolution of hepatic inflammation, and prevent hepatic decompensation. The ultimate goal of treatment is to achieve a sustained response that prevents the development of cirrhosis and hepatocarcinoma and prolongs survival. HBV infection cannot be completely eradicated because a specific form of viral covalently closed circular DNA (cccHBV DNA) continues to persist in the nuclei of infected hepatocytes. However, treatment-induced serum HBsAg clearance is a marker for the termination of chronic HBV infection and is associated with the best long-term outcome.

[0005] The current standard methods of HBV treatment include immunotherapy based on interferon or thymosin alpha 1, and inhibition of viral production by inhibition of HBV polymerase. HBV polymerase inhibitors are effective in reducing viral production, but have little or no effect in rapidly reducing HBsAg, or can only slowly reduce HBsAg with long-term treatment in a limited number of patients (as with tenofovir disoproxil fumarate). Interferon-based immunotherapy can achieve both reduction of viral production and early removal of HBsAg from the blood, but only in a small proportion of treated subjects. The generally accepted role of HBsAg in the blood is to sequester anti-HBsAg antibodies and allow infectious viral particles to evade immune detection, which is presumed to be one of the reasons why HBV infection remains chronic. In addition, HBsAg, HBeAg and HBcAg all have immunoinhibitory properties and the persistence of these viral proteins in a patient's blood following administration of any of the currently available HBV treatments is likely to have a significant impact in preventing a patient from immunologically controlling their HBV infection.

[0006] All three major HBV proteins (HBsAg, HBeAg and HBcAg) have immune-inhibitory properties, but HBsAg accounts for the vast majority of HBV proteins in the circulation of HBV-infected subjects. In addition, while removal of HBeAg (through seroconversion) or reduction of serum viremia does not correlate with the occurrence of sustained HBV infection control upon treatment cessation, removal of serum HBsAg from the blood (and seroconversion) in HBV infection is a well-recognized prognostic indicator of antiviral response during treatment that may lead to HBV infection control upon treatment cessation (although this occurs in only a small proportion of patients undergoing immunotherapy). Thus, although reduction of all three major HBV proteins (HBsAg, HBeAg and HBcAg) may result in optimal removal of inhibitory effects, removal of HBsAg alone appears to be sufficient in itself to remove the majority of the virus-induced immune function inhibition in HBV-infected subjects.

[0007] Thus, while there is currently no treatment regimen that can restore immunological control of HBV in most patients, there is a need for an effective treatment for HBV infection that can inhibit viral replication and restore immunological control in the majority of patients.Therefore, there is a need in the art for alternative and combination therapies for subjects infected with HBV or with HBV-related diseases. Summary of the Invention

[0008] In some embodiments, the present disclosure provides double-stranded ribonucleic acid (dsRNA) agent compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of a Hepatitis B virus (HBV) gene. The HBV gene may be in a cell, for example, in a cell within a subject, such as a human.

[0009] Also provided herein are methods and therapies for treating subjects with HBV infection or a disorder, such as an HBV-related disease, e.g., chronic hepatitis B infection (CHB), that would benefit from inhibiting expression of the HBV gene, with dsRNA agent compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the HBV gene to inhibit expression of the HBV gene.

[0010] In one embodiment, the present disclosure provides a dsRNA agent for inhibiting the expression of HBV.For example, the present disclosure provides a dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand is 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16), 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18), 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20), 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23), 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24), 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25), or 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO:28): [wherein a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; dA, dC, dG and dT are 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxythymidine-3'-phosphate, respectively; (Agn) is adenosine glycol nucleic acid (GNA); and s is a phosphorothioate linkage. The present invention provides a dsRNA agent comprising a modified nucleotide sequence represented by:

[0011] In some embodiments, the sense strand has the modified nucleotide sequence: 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO:29). [wherein a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; and s is a phosphorothioate linkage. Includes.

[0012] In some embodiments, the antisense strand and the sense strand are (a) 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (b) 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (c) 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (d) 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (e) 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (f) 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); or (g) 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29): [wherein a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; dA, dC, dG and dT are 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxythymidine-3'-phosphate, respectively; (Agn) is adenosine glycol nucleic acid (GNA); and s is a phosphorothioate linkage. The modified nucleotide sequence is represented by:

[0013] In some embodiments, at least one strand of the dsRNA agent comprises a 3' overhang of at least one nucleotide. In some embodiments, at least one strand of the dsRNA agent comprises a 3' overhang of two nucleotides.

[0014] In some embodiments, the double-stranded region of the dsRNA agent is 19-21 nucleotide pairs in length.

[0015] In some embodiments, each strand of the dsRNA agent independently has between 19 and 23 nucleotides.In some embodiments, each strand of the dsRNA agent independently has between 19 and 21 nucleotides.

[0016] In some embodiments, the dsRNA agent further comprises a ligand. In some embodiments, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent. In some embodiments, the ligand is an N-acetylgalactosamine (GalNAc) derivative. In certain embodiments, the ligand is represented by the formula: [ka] As shown in the figure.

[0017] In some embodiments, the dsRNA agent has the formula: [ka] wherein X is O or S. In some embodiments, X is O.

[0018] In some embodiments, the antisense strand is 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16), 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18), 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20), 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23), 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24), 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25), or 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO:28): [wherein a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; dA, dC, dG and dT are 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxythymidine-3'-phosphate, respectively; (Agn) is adenosine glycol nucleic acid (GNA); and s is a phosphorothioate linkage. It consists of a modified nucleotide sequence represented by:

[0019] In some embodiments, the present disclosure provides sense and antisense dsRNA agents consisting of the following modified nucleotide sequences: (a) 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (b) 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (c) 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (d) 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (e) 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (f) 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); or (g) 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29): In the sequence, a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; dA, dC, dG and dT are 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxythymidine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and The 3' end of the sense strand is conjugated to an N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol (L96) ligand.

[0020] In other aspects, the present specification provides a cell containing a dsRNA agent disclosed herein.

[0021] The present specification also provides a pharmaceutical composition comprising a dsRNA agent described herein and a pharmaceutical excipient.

[0022] The present specification also provides a method for inhibiting Hepatitis B virus (HBV) gene expression in a cell, by contacting the cell with the dsRNA agent or pharmaceutical composition disclosed in the present invention, inhibiting HBV gene expression in the cell. In some embodiments, the cell is in a subject. In some embodiments, the subject is a human. In some embodiments, the subject suffers from HBV-related disease. In some embodiments, the cell is in vitro. In some embodiments, HBV gene expression is inhibited by at least 80%, 90%, 95% or 98%, or below the detection level of the assay.

[0023] The present disclosure provides a method for inhibiting the replication of Hepatitis B virus (HBV) in cells, comprising contacting the cells with the dsRNA agent or pharmaceutical composition disclosed herein to inhibit the replication of HBV in cells. In some embodiments, the cells are in a subject. In some embodiments, the subject is a human. In certain embodiments, the subject suffers from HBV-related disease. In some embodiments, the cells are in vitro. In certain embodiments, the replication of HBV in cells is inhibited by at least 80%, 90%, 95% or 98%, or below the detection level of assay.

[0024] Also provided herein is a method for reducing Hepatitis B virus (HBV) antigen level in a subject infected with HBV, comprising administering a therapeutically effective amount of a dsRNA agent or pharmaceutical composition disclosed herein to the subject, thereby reducing the HBV antigen level of the subject.In some embodiments, the HBV antigen is HBsAg.In some embodiments, the HBV antigen is HBeAg.In some embodiments, the HBV antigen is measured in the serum from the subject.In some embodiments, the subject is HBeAg positive.In some embodiments, the subject is HBeAg negative.In some embodiments, the HBV antigen level is reduced in serum by at least 1 log10, at least 2 log10, at least 3 log10 or at least 4 log10, or below the detection level of the assay.

[0025] The present specification also provides a method for reducing the viral load of Hepatitis B virus (HBV) in a subject infected with HBV, comprising administering a therapeutically effective amount of the dsRNA agent or pharmaceutical composition disclosed herein to the subject, thereby reducing the viral load of the subject's HBV.In some embodiments, the viral load of HBV is measured in the serum from the subject.In some embodiments, the subject is HBeAg positive.In some embodiments, the subject is HBeAg negative.In some embodiments, the viral load of HBV is reduced in serum by at least 1 log10, at least 2 log10, at least 3 log10 or at least 4 log10, or below the detection level of assay.

[0026] Also provided herein is a method for treating a subject with Hepatitis B virus (HBV) infection or HBV-related disorder, comprising administering a therapeutically effective amount of the dsRNA agent or pharmaceutical composition disclosed herein to the subject, thereby treating the subject.In some embodiments, the subject is HBeAg positive.In some embodiments, the subject is HBeAg negative.In some embodiments, the HBV-related disorder is chronic hepatitis, and the subject is HBeAg positive.In some embodiments, the HBV-related disorder is chronic hepatitis, and the subject is HBeAg negative.

[0027] In some embodiments of the above method, the dsRNA agent is administered to the subject at a dose of 0.01 mg / kg to 10 mg / kg, 0.5 mg / kg to 50 mg / kg, or 3 mg / kg to 10 mg / kg. In some embodiments of the method, the dsRNA agent is administered to the subject at a dose of 3 mg / kg to 10 mg / kg. In some embodiments of the method, the dsRNA agent is administered to the subject at a fixed dose of 50 mg to 200 mg.

[0028] In some embodiments of the above methods, the dsRNA agent is administered subcutaneously to the subject.

[0029] In some embodiments of the above methods, the dsRNA agent is administered to the subject in two or more doses.

[0030] In some embodiments of the above method, the dsRNA agent is administered to the subject once a month, once every two months, or once every three months. In some embodiments of the method, the dsRNA agent is administered to the subject more than once a month.

[0031] In some embodiments of the above method, the method further comprises administering to the subject additional therapeutic agent, for example, one or more additional therapeutic agents.Additional therapeutic agents include, but are not limited to, for example, antiviral agents, reverse transcriptase inhibitors, immunostimulants, therapeutic vaccines, virus 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 above.In some embodiments, the additional therapeutic agent is a reverse transcriptase inhibitor.

[0032] In some embodiments, more than one additional therapeutic agent is administered, and the additional therapeutic agent is a reverse transcriptase inhibitor and an immunostimulator.Reverse transcriptase inhibitors include, but are not limited to, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine and AGX-1009.Immunostimulators include, but are not limited to, pegylated interferon alpha 2a (PEG-IFN-alpha 2a), interferon alpha-2b, recombinant human interleukin-7, and toll-like receptor 7 (TLR7) agonist.

[0033] Also provided herein is a composition for carrying out any of the methods disclosed herein. In some embodiments, the present specification provides a dsRNA agent or pharmaceutical composition disclosed herein for use in treating Hepatitis B Virus (HBV) infection in a subject. In some embodiments, the present specification provides a dsRNA agent or pharmaceutical composition disclosed herein for treating Hepatitis B Virus (HBV)-related disorder in a subject. In some embodiments, the present specification provides a dsRNA agent or pharmaceutical composition disclosed herein for treating Hepatitis B Virus (HBV)-related disorder in a subject. In some embodiments, the HBV-related disorder is chronic hepatitis, and the subject is HBeAg positive. In some embodiments, the HBV-related disorder is chronic hepatitis, and the subject is HBeAg negative. In some embodiments, the subject is administered or has been administered an additional therapeutic agent, such as an antiviral agent, a reverse transcriptase inhibitor, an immunostimulant, a therapeutic vaccine, a viral entry inhibitor, an oligonucleotide that inhibits secretion or release of HbsAg, a capsid inhibitor, a covalently closed circular (ccc) HBV DNA inhibitor, or any combination of the above. In some embodiments, the additional therapeutic agent is a reverse transcriptase inhibitor, such as tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, or AGX-1009. In some embodiments, the additional therapeutic agent administered is a reverse transcriptase inhibitor (e.g., tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, or AGX-1009) and an immunostimulant (e.g., pegylated interferon alpha 2a (PEG-IFN-α2a), interferon alpha-2b, recombinant human interleukin-7, or a Toll-like receptor 7 (TLR7) agonist).

[0034] The present specification provides the use of a dsRNA agent or pharmaceutical composition disclosed herein to carry out any of the above methods.

[0035] The present specification also provides the use of a dsRNA agent disclosed herein for the preparation or manufacture of a therapeutic medicament for carrying out any of the above methods.

[0036] The present specification provides kits that contain the dsRNA agents or pharmaceutical compositions disclosed herein and, optionally, contain instructions for practicing the methods described herein. [Brief description of the drawings]

[0037] [Figure 1] Figure 1 shows the schematic structure of the approximately 3.2 kb double-stranded HBV genome. Replication of the HBV genome occurs via an RNA intermediate, producing four overlapping viral transcripts (an approximately 3.5 kb transcript, an approximately 2.4 kb transcript, an approximately 2.1 kb transcript and an approximately 0.7 kb transcript) that encode seven viral proteins (pre-S1, pre-S2, S, P, X, pre-C and C) that are translated across three reading frames.

[0038] [Diagram 2] Figures 2A-2B show (A) serum HBsAg concentrations (ng / mL) and (B) serum HBsAg levels compared to pre-treatment in an AAV-HBV mouse model. Figure 2A shows serum levels of HBsAg before (days -24, -2, 0) or after a single dose of AD-81890 at 0.3 mg / kg, 1 mg / kg, or 3 mg / kg (days 14, 21, 33, 47, 59, 74) in HBV-AAV mice. Each point represents the mean of n=6-9 animals, and bars indicate SD. Figure 2B shows serum levels of HBsAg in HBV-AAV mice compared to before AD-81890 administration (days -24, -2, 0) or after a single dose at 0.3 mg / kg, 1 mg / kg, or 3 mg / kg (days 14, 21, 33, 47, 59, 74). Each point represents the mean of n = 6–9 animals, and bars indicate SD.

[0039] [Diagram 3]Figure 3 shows serum HBsAg levels compared to pre-dosing on days -55, -27, -13, 0, 14, 28, 42, 56, 84, 112, and 140 in the AAV-HBV mouse model. Dosing regimens included control (PBS) Q2W x 6; AD-66810 (1 mg / kg; Q2W x 6); AD-81890 (1 mg / kg; Q2W x 6); AD-81890 (1 mg / kg; QM x 3); AD-81890 (3 mg / kg; Q2W x 6); AD-81890 (3 mg / kg; QM x 3); and AD-81890 (9 mg / kg; QM x 1). The first dose was administered on day 0. Each point represents the mean of n=4–6 animals, and bars indicate SD.

[0040] [Figure 4] Figure 4 shows box plots showing the log2 fold change (treated / control) of all genes significantly downregulated (adjusted p-value <0.05, log2 fold change <0) in AD-66810 or AD-81890. The thick horizontal line indicates the median log2 fold change, the vertical range of each box indicates the interquartile range (IQR), and the whiskers extend to + / -1.58 IQR / sqrt(N), where N indicates the number of genes in each group. Statistical significance was assessed using Welch's two-tailed two-sample t-test.

[0041] [Diagram 5] Figures 5A-5B show ALT levels over time in PXB mice following administration of AD-66810 (Figure 5A) or AD-81890 (Figure 5B) compared to mice receiving PBS. Mice were administered 12 mg / kg, 36 mg / kg or 100 mg / kg AD-66810, AD-81890 or PBS (control) by subcutaneous injection on days 0, 21, 28, 35 and 42 (n=4 per group). Blood was collected by retro-orbital bleeding twice weekly and serum was prepared using routine methods.

[0042] Detailed Description The present disclosure provides dsRNA agent compositions that affect the RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of Hepatitis B virus (HBV) gene. The gene may be in a cell, for example, in a cell within a subject, such as a human. These dsRNA agents can be used to target and degrade the mRNA of the corresponding gene (HBV gene) in a mammal.

[0043] The dsRNA agents described herein are designed to target regions in the HBV genome that are conserved across at least eight known genotypes of HBV. Furthermore, the dsRNA agents described herein are designed to inhibit all stages of the HBV life cycle, such as replication, assembly, viral secretion, and subviral antigen secretion, by inhibiting the expression of more than one HBV gene. In particular, since the HBV genome is transcribed into polycistronic overlapping RNAs, in some embodiments, a dsRNA agent targeting a single HBV gene will significantly inhibit the expression of most or all HBV transcripts. For example, since the HBV genome is transcribed into a single mRNA, a dsRNA agent described herein that targets the S gene will inhibit not only S gene expression, but also its "downstream" polymerase gene expression. Furthermore, the dsRNA agents described herein are designed to inhibit HBV viral replication by targeting HBV structural genes and HBV X genes, thereby allowing the subject's immune system to detect and respond to the presence of HBsAg and produce anti-HBV antibodies to clear HBV infection. Without intending to be limited to any particular theory, it is believed that the combination or subcombination of the above properties with specific target sites or specific modifications of the dsRNA agents herein confers improvements in efficacy, stability, safety, potency, and durability to the dsRNA agents herein.

[0044] The present inventors have demonstrated that dsRNA agents targeting HBV genes can strongly mediate RNAi and significantly inhibit the expression of more than one HBV gene using in vitro and in vivo assays.Therefore, the methods and compositions comprising the dsRNA agents herein are useful for treating subjects with HBV infection or HBV-related disease, such as chronic hepatitis B (CHB).

[0045] Thus, the present specification also provides methods of treating subjects having a disorder that may benefit from inhibiting or reducing expression of an HBV gene, e.g., an HBV-related disease, such as chronic hepatitis B virus infection (CHB), with a dsRNA agent composition that affects RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the HBV gene.

[0046] The dsRNA agents herein comprise an RNA strand (antisense strand) having a complementary region of about 9-21 nucleotides in length, e.g., about 19 nucleotides in length, that is substantially complementary to at least a portion of an mRNA transcript of the HBV gene of at least one HBV genotype. It is understood that multiple HBV genotypes exist such that the dsRNA agents herein may have differing degrees of complementarity to different HBV genotypes.

[0047] In some embodiments, the sense strand and the antisense strand form a duplex of 19 to 21 consecutive nucleotides.

[0048] The following detailed description discloses how to make and use compositions containing dsRNA agents to inhibit expression of HBV genes, as well as compositions, uses, and methods for treating subjects with diseases and disorders that may benefit from inhibition or reduction of expression of HBV genes.

[0049] I. definition In order that this specification may be more readily understood, certain terms are first defined. Furthermore, it should be noted that whenever a value or range of values ​​for a parameter is described, values ​​and ranges intermediate to the described values ​​are also intended to be part of the present features.

[0050] Unless the context requires otherwise, throughout the specification and claims, the term "comprise" and its variations, such as "comprises" and "comprising" are to be construed in an open and inclusive sense, i.e., "including but not limited to." "Consisting of" shall mean excluding more than trace elements of other components or substantial method steps disclosed herein. For example, a polynucleotide consists of a sequence of nucleotides without any additional nucleotides, but does not exclude the incorporation of ligands, e.g., targeting ligands, or modifications. The term "consisting essentially of" limits the scope of the claim to specific materials or steps, or those that do not substantially affect the fundamental control of the claimed invention. For example, a pharmaceutical composition consisting essentially of the components defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharma- ceutical acceptable carriers, such as phosphate buffered saline, preservatives, etc. Similarly, a polynucleotide consists essentially of a nucleotide sequence if it contains additional nucleotides that contribute to at least 20% of the length of the polynucleotide and do not substantially affect the activity of the polynucleotide (e.g., alter the activity of the polynucleotide by less than 50%).Embodiments defined by each transitional term are within the scope of the invention.

[0051] 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 component, e.g., a plurality of components.

[0052] The term "including" is used herein to mean, and is used synonymously with, the phrase "including but not limited to."

[0053] The term "or" is used herein to mean and is used synonymously with the term "and / or," unless the context clearly indicates otherwise. For example, "sense strand or antisense strand" is understood as "sense strand or antisense strand, or sense strand and antisense strand."

[0054] The term "about" is used herein to mean within the typical range of acceptable range in the art.For example, "about" can be understood to be within 2 standard deviations from the mean value.When "about" is present before a series of numbers or ranges, it is understood that each number in the series of numbers or ranges can be modified.

[0055] The term "at least" before a number or a series of numbers is understood as each number in the series of numbers and all subsequent numbers or integers that can be logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule should be an integer. For example, "at least 18 nucleotides of 21 nucleic acid molecules" means that 18, 19, 20 or 21 nucleotides have the indicated property. When "at least" is present before a series of numbers or a range, it is understood that each number in the series or range can be modified.

[0056] As used herein, "not more than" or "less than" is understood as logical from the context to mean the number from the value adjacent to the phrase and logical lower value or integer to zero. For example, a duplex having an overhang of "not more than 2 nucleotides" has an overhang of 2, 1 or 0 nucleotides. When "not more than" occurs before a series of numbers or ranges, it is understood to modify each number in the series or range.

[0057] As used herein, ranges include both upper and lower limits.

[0058] In the event of a discrepancy between a sequence and its indicated site on the transcript or other sequence, the nucleotide sequence set forth herein shall prevail.

[0059] The various embodiments herein may be combined as appropriately determined by one of ordinary skill in the art.

[0060] As used herein, "Hepatitis B virus" is used interchangeably with the term "HBV" and refers to a well-known non-cytopathic hepatotropic DNA virus belonging to the family Hepadnaviridae.

[0061] The HBV genome is a partially double-stranded circular DNA with overlapping reading frames (see, eg, FIG. 1).

[0062] There are four size-based transcripts (sometimes referred to herein as "genes" or "open reading frames") encoded by the HBV genome. These contain open reading frames designated C, X, P, and S. The core protein is encoded by gene C (HBcAg). The Hepatitis B e antigen (HBeAg) is produced by proteolytic processing of the pre-core (pre-C) protein. The DNA polymerase is encoded by gene P. Gene S is the gene that codes for the surface antigen (HBsAg). The HBsAg gene is one long open reading frame that contains three in-frame "start" (ATG) codons that give rise to three different sized polypeptides (pre-S1+pre-S2+S, pre-S2+S, or S), designated large, middle, and small S antigens. In addition to decorating the HBV envelope, surface antigens are also part of subviral particles that are produced in excess compared to virion particles and play a role in immune tolerance and sequestering anti-HBsAg antibodies, thereby allowing infectious particles to escape immune detection. The function of the nonstructural protein encoded by gene X is not fully understood, but it may play a role in transcriptional transactivation and replication and may be related to the development of hepatocarcinoma.

[0063] 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 generation of an RNA intermediate, which is then reverse transcribed to produce the DNA viral genome.

[0064] When cells are infected with HBV, the relaxed circular DNA (rcDNA) of the viral genome 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 into 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 responsible for the establishment of infection and viral persistence.

[0065] HBV infection produces two distinct particles: 1) the infectious HBV virus itself (or Dane particle), which contains a viral capsid built from HBcAg and is covered by an envelope made of lipid membrane with HBV surface antigen, and 2) the subviral particle (or SVP), which contains the non-infectious small and intermediate forms of the Hepatitis B surface antigen HBsAg. For each viral particle produced, more than 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 pre-core protein called HBVe antigen (HBeAg).

[0066] Eight genotypes of HBV, designated A-H, have been sequenced, and two additional genotypes, I and J, have been proposed, each with a distinct geographic distribution. The virus is noncytopathic, and virus-specific cell-mediated immunity is the primary factor determining the outcome of exposure to HBV, whether it is acute infection with resolution of liver disease in 6 months, or chronic HBV infection with frequent progressive liver damage.

[0067] The term "HBV" includes any genotype (A-J) of HBV. The 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). The antisense sequences are shown in SEQ ID NO:2 and SEQ ID NO:4, respectively. The amino acid sequences of the C, X, P and S proteins can be found, for example, in NCBI accession numbers YP_009173857.1 (C protein) (SEQ ID NO: 37); YP_009173867.1 and BAA32912.1 (X protein) (SEQ ID NOs: 36 and 40); YP_009173866.1 and BAA32913.1 (P protein) (SEQ ID NOs: 32 and 38); and YP_009173869.1, YP_009173870.1, YP_009173871.1, and BAA32914.1 (S protein) (SEQ ID NOs: 33, 34, 35, 39). Protein and DNA sequences from HBV genotype D, strain ayw are shown in SEQ ID NOs: 36-37. Protein and DNA sequences from the HBV gene are shown in SEQ ID NOs: 38-39. Further examples of HBV protein and DNA sequences, or their reverse complements, are shown in SEQ ID NOs: 41-49.

[0068] Further examples of HBV mRNA sequences are readily available using publicly available databases such as GenBank, UniProt and OMIM. The International Repository of Hepatitis B Virus Strain Data can be accessed at http: / / www.hpa- bioinformatics.org.uk / HepSEQ / main.php.

[0069] As used herein, the term "HBV" also refers to naturally occurring DNA sequence variants of the HBV genome, such as genotypes A through J and variants thereof.

[0070] As used herein, "hepatitis D virus" is used synonymously with the term "HDV" and 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 herein by reference.Unless otherwise stated, HDV refers to all clades and variants of HDV.

[0071] HDV produces one protein, HDAg, which exists in two forms: the 27 kDa large HDAg (also called lHD, L-HDAg, and large HDV antigen), and the 24 kDa small HDAg (also called sHD, S-HDAg, and small HDV antigen). The N-terminus of these two forms is the same, and they differ by 19 amino acids at the C-terminus of the large HDAg. Both isoforms are produced from the same reading frame, which contains a UAG stop codon at codon 196 and normally produces only the small HDAg. However, when the stop codon is changed to UCG by editing with the cellular enzyme adenosine deaminase-1, the large HDAg is produced. Despite the fact that these two proteins have 90% identical sequences, they play different roles during the infection process. HDAg-S is produced early in infection and enters the nucleus to assist in viral replication. In contrast, HDAg-L is produced late during infection, acts as an inhibitor of viral replication, and is required for viral particle assembly.

[0072] Further examples of HDV mRNA sequences are readily available using publicly available databases, such as GenBank, uNIpROT, and OMIM.

[0073] As used herein, the term "HDV" also refers to naturally occurring DNA sequence variants of the HDV genome.

[0074] As used herein, the term "nucleotide analog" or "reverse transcriptase inhibitor" refers to an inhibitor of DNA replication that is structurally similar to a nucleotide or nucleoside and that specifically inhibits the replication of HBV cccDNA and does not significantly inhibit the replication of host (e.g., human) DNA. Such inhibitors include tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine, clevudine, ritonavir, dipivoxil, lobucavir, famvir, FTC, N-acetyl-cysteine ​​(NAC), PC1323, theradigm-HBV, thymosin-alpha, ganciclovir, besifovir (ANA-380 / LB-80380), and tenofvir-exaliade (TLX / CMX157). In certain embodiments, the nucleotide analog is entecavir (ETV). Nucleotide analogs are commercially available from a number of sources and may be used in the methods provided herein according to their labeling (e.g., typically administered orally at specific doses) or as determined by a skilled practitioner in the treatment of HBV.

[0075] "Target sequence" as used herein refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of an HBV gene, including the mRNA that is the product of RNA processing of a primary transcript.In some embodiments, the target portion of the sequence will be at least long enough to serve as a substrate for the targeted cleavage of a dsRNA agent at or near said target portion in the nucleotide sequence of an mRNA molecule formed during transcription of an HBV gene.

[0076] The target sequence can be about 19-21 nucleotides in length, for example, 19, 20 or 21 nucleotides in length.

[0077] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides represented by a sequence referenced using standard nucleotide nomenclature.

[0078] "G", "C", "A", "T" and "U" generally refer to nucleotides containing guanine, cytosine, adenine, thymidine and uracil as bases, respectively. However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, or surrogate replacement moieties (see, for example, Table 1), as described in more detail below. Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced by other moieties without substantially changing the base pairing 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 can be replaced by, for example, a nucleotide containing inosine in the nucleotide sequence of dsRNA characterized herein. In another example, adenine and cytosine anywhere in the oligonucleotide may be replaced by guanine and uracil, respectively, to form a GU wobble base pair with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured herein.

[0079] The terms "dsRNA agent", "RNAi agent", "iRNA agent", "iRNA" and "RNA interference agent" are used interchangeably herein and refer to an agent that contains RNA as defined herein and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. The dsRNA agent directs the sequence-specific degradation of mRNA by a process known as RNA interference (RNAi). The dsRNA agent regulates, e.g., inhibits, the expression of HBV genes (e.g., one or more HBV genes) in a cell, e.g., a cell in a subject, e.g., a mammalian subject.

[0080] The "dsRNA agent" for use in the compositions, uses and methods disclosed herein is double-stranded RNA, and is referred to herein as "dsRNA agent", "double-stranded RNA agent", "double-stranded RNA (dsRNA) molecule", "dsRNA", "iRNA", "iRNA agent", "dsRNAi agent", "RNAi agent" or "siRNA". The term "dsRNA" refers to a complex of ribonucleic acid molecules, which is a complex having a duplex structure comprising two antiparallel and substantially complementary strands of nucleic acid, meaning that they have a "sense" and "antisense" orientation with respect to a target RNA, i.e., the HBV gene. In some embodiments herein, the dsRNA induces the degradation of a target RNA, e.g., mRNA, through a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0081] Generally, each strand of a dsRNA molecule can contain ribonucleotides, but as described in detail herein, each strand or both strands can also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Furthermore, as used herein, a "dsRNA agent" can contain ribonucleotides with chemical modifications; a dsRNA agent can contain substantial modifications in multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide that has, independently, a modified sugar moiety, a modified internucleotide linkage, 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 linkage, sugar moiety, or nucleobase. Modifications suitable for use in the agents herein include all types of modifications disclosed herein or known in the art. Any such modifications used in dsRNA agent type molecules are encompassed by "dsRNA agent" for the purposes of this specification and claims.

[0082] As used herein, the term "inhibit" is used interchangeably with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes any level of inhibition. Preferably, "inhibit" includes statistically significant inhibition or clinically significant inhibition.

[0083] As used herein, the phrase "inhibiting expression of HBV" or "inhibiting expression of an HBV gene" includes inhibition of expression of an HBV gene (e.g., an HBV gene expressed from HBV in an HBV viral infection, an HBV gene expressed from an expression construct in a cell), and variants or mutants of the HBV gene that encode HBV proteins. This term includes knockdown of any of the HBV transcripts (e.g., the 3.5 kb, 2.4 kb, 2.1 kb or 0.7 kb transcripts) that encode one or more HBV viral proteins (e.g., pre-S1 / 2-S, pre-S, S, P, X, pre-C, and C, etc.), and variants or mutants of the HBV gene.

[0084] "Inhibiting the expression of HBV genes" includes inhibition of any level of HBV genes or transcripts, for example, partial inhibition of the expression of at least HBV genes, for example, HBV genes S, P, X or C, or any combination thereof, for example, S, P and C. The expression of HBV genes can be evaluated based on the level or variation of the level that can vary in relation to HBV genes, for example, HBV mRNA level or HBV protein level, or HBV cccDNA level. The level can be evaluated in an individual cell or cell group, including, for example, a sample from a subject, for example, the level can be monitored in serum. The inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level utilized in the art, for example, a baseline level before administration, or a level determined from a similar subject or population average from a suitable control subject, cell or sample that is untreated or treated with a control (for example, a buffer-only control or an inactive agent control).

[0085] In some embodiments of the methods herein, expression of HBV genes is inhibited by at least 80%, 85%, 90%, 95%, e.g., at least 1 log10, 2 log10, 3 log10, 4 log10, or to below the detection level of the assay in the subject. In preferred embodiments, inhibition of expression of HBV genes in a subject results in a clinically relevant level of inhibition of gene expression, e.g., when administered alone or in combination with other agents to promote or enhance the immune response, is sufficiently inhibited to allow an effective immune response against HBV proteins.

[0086] In in vitro cell-based assays or in vivo models of heterologous gene expression, such as the mouse AAV-hHBV model provided herein, an inhibition of at least 90% of total HBV expression is preferred, for example at least 1 log10, 2 log10 or 3 log10. In treating HBV-infected subjects, a reduction of at least 90% of HBV gene or protein levels, i.e., the difference between HBV gene or protein levels before and after treatment, is preferred. More than one administration may be required to achieve the desired level of inhibition.

[0087] Inhibition of HBV gene expression may be manifested by a reduction in the number of RNAs expressed by a first cell or group of cells (such cells may, for example, be present in a sample from a subject), where the HBV gene in the first cell or group of cells is transcribed, and the first cell or group of cells is treated (for example, by contacting the cells with a dsRNA agent herein, or by administering a dsRNA agent herein to a subject in which the cells are or were present) to inhibit expression of the HBV gene, compared to a second cell or group of cells (control cells) that is substantially the same as the first cell or group of cells, but is not so treated. In a preferred embodiment, inhibition is assessed by the rtPCR method provided in Example 2 of WO2016 / 077321 (incorporated herein by reference), using an in vitro assay performed in an appropriately adapted cell line with a 10 nM concentration of the duplex, and expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells, using the following formula:

number

[0088] Alternatively, inhibition of HBV gene expression can be assessed in terms of a reduction in a parameter functionally related to HBV gene expression. HBV gene silencing can be determined in any cell expressing the HBV gene, constitutively or by genome engineering, and by any assay known in the art.

[0089] Inhibition of the expression of HBV protein can be manifested by a reduction in the level of HBV protein expressed by a cell or cell group (e.g., the level of protein expressed in a sample derived from a subject).As explained above, for the assessment of mRNA inhibition, the inhibition of protein expression level in treated cell or cell group can also be expressed as a percentage of the protein level in control cell or cell group, or as a percentage of the protein level in serum.

[0090] The control cell or cell group that can be used to evaluate the inhibition of the expression of HBV gene includes a cell or cell group that is not contacted with the dsRNA material herein.For example, the control cell or cell group can be derived from an individual subject (e.g., a human or animal subject) before the subject is treated with the dsRNA material.In an alternative embodiment, the level can be compared with a suitable control sample, for example, a known population control sample.

[0091] The level of HBV RNA expressed by a cell or group of cells, or the level of HBV RNA in blood, can be determined using any method known in the art for evaluating mRNA expression, preferably using the rtPCR method provided in Example 2 of WO2016 / 077321. In some embodiments, the expression level of HBV genes (e.g., total HBV RNA, HBV transcripts, e.g., HBV 3.5 kb transcripts) in a sample is determined by detecting a transcribed polynucleotide or a portion thereof, e.g., the RNA of an HBV gene. RNA can be extracted from cells using RNA extraction techniques, including, for example, using acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kit (Qiagen®) or PAX gene (Preanalytix, Switzerland). Typical assay formats that utilize ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis. HBV mRNA in blood can be detected using the methods described in WO2012 / 177906, which is incorporated herein by reference.

[0092] In some embodiments, the expression level of HBV genes is measured using nucleic acid probes.The term "probe" as used herein means a molecule that can selectively bind to a specific HBV gene.Probes can be synthesized by those skilled in the art or derived from suitable biological preparations.Probes can also be specially designed to be labeled.Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, protein, antibody and organic molecules.

[0093] The isolated RNA can be used in hybridization or amplification assays, including but not limited to Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method of measuring mRNA levels involves contacting the isolated RNA with a nucleic acid molecule (probe) that can hybridize to HBV mRNA. In some embodiments, the mRNA is immobilized on a solid surface and contacted with a probe, for example, by running the isolated RNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose. In some other embodiments, the probe is immobilized on a solid surface and the mRNA is contacted with the probe, for example, in an Affymetrix® gene chip array. Those skilled in the art can easily adapt known mRNA detection methods for use in measuring HBV mRNA levels.

[0094] Alternative methods for measuring the expression level of HBV genes in a sample include, for example, RT-PCR (experimental embodiment described in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-propagating sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-β replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033), or other nucleic acid amplification methods, for example, includes a step of nucleic acid amplification of mRNA in a sample or reverse transcriptase (to prepare cDNA), followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for detection of nucleic acid molecules when such molecules are present in very low numbers. In certain embodiments herein, the level of expression of HBV genes is measured by quantitative fluorescent RT-PCR (i.e., TaqManTM system), for example, using the methods provided herein.

[0095] The expression level of HBV RNA can be monitored using membrane blots (such as those used in hybridization analysis such as Northern, dot, etc.), or microwells, sample tubes, gels, beads, fibers (or any solid support containing bound nucleic acid). For teachings related to such methods, see U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Measuring HBV expression levels can also include using nucleic acid probes in solution.

[0096] In a preferred embodiment, the level of RNA expression is assessed using real-time PCR (qPCR). The use of these methods is described and exemplified in Example 2 of WO2016 / 077321.

[0097] The level of HBV protein expression can be measured using any method known in the art for measuring protein levels, such as, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitation reactions, absorption spectroscopy, colorimetry, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, and the like.

[0098] dsRNA material The double-stranded region may be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and may range from about 19-21 base pairs in length, e.g., about 19, 20 or 21 base pairs in length. Exemplary dsRNA agents provided herein include duplexes that are 19-21 base pairs in length.

[0099] When the two substantially complementary strands of dsRNA are contained in separate RNA molecules, the molecules need not be, but may be, covalently linked. 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 forming a double-stranded structure, the linking structure is called a "linker". The two RNA strands can have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus the overhangs present in the double strand. In addition to the double-stranded structure, the dsRNA material can include one or more nucleotide overhangs.

[0100] The term "nucleotide overhang" as used herein refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of a double-stranded dsRNA material. 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 include an overhang of at least one nucleotide, or alternatively, the overhang can include two nucleotides. A nucleotide overhang can include or consist of a nucleotide / nucleoside analog, including a deoxynucleotide / nucleoside. An overhang can be present on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide of the overhang can be present on the 5' end, 3' end, or both ends of either the antisense strand or the sense strand of a dsRNA. In a preferred embodiment, a nucleotide overhang is present on the 3' end of the antisense strand.

[0101] " Blunt" or "blunt end" means that there is no unpaired nucleotide at the end of double-stranded dsRNA material, i.e., there is no nucleotide overhang. " Blunt end " dsRNA material is double-stranded throughout its entire length, i.e., there is no nucleotide overhang at either end of the molecule. In some embodiments, the dsRNA material herein comprises the dsRNA material with nucleotide overhang at one end (i.e., the dsRNA material with one overhang and one blunt end) or the dsRNA material with nucleotide overhang at both ends.

[0102] The term "antisense strand" or "guide strand" refers to the strand of a dsRNA agent that includes a region that is substantially complementary to a target sequence, e.g., HBV mRNA. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., an HBV nucleotide sequence, as defined herein. If the complementary region is not completely complementary to the target sequence, mismatches may exist in the internal or terminal regions of the molecule. Generally, most tolerated mismatches are within the terminal regions, e.g., 5, 4, 3, 2, or 1 nucleotide at the 5' and / or 3' ends of the dsRNA agent. In some embodiments, the dsRNA agent herein includes a nucleotide mismatch in the antisense strand. In some embodiments, the dsRNA agent herein includes a nucleotide mismatch in the sense strand. In some embodiments, the nucleotide mismatch is, e.g., within 5, 4, 3, 2, or 1 nucleotide from the 3' end of the dsRNA agent. In some embodiments, the nucleotide mismatch is, e.g., at the 3' terminal nucleotide of the dsRNA agent.

[0103] As used herein, the term "sense strand" or "passenger strand" refers to the strand of a dsRNA agent which includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0104] As used herein, the term "complementary" when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, as understood by those skilled in the art, unless otherwise specified, 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 certain conditions. Such conditions can be, for example, stringent conditions, where stringent conditions can 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, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions can be applied, such as physiologically relevant conditions that may occur inside an organism. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate application of the hybridized nucleotides.

[0105] A complementary sequence in a dsRNA material, for example, in a dsRNA described herein, includes base pairs over 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 are either fully complementary or, when hybridized to a duplex of up to 21 base pairs, while retaining the ability to hybridize under optimal conditions for their ultimate application, such as the inhibition of gene expression via the RISC pathway, they may form one or more, but generally no more than four, three or two mismatched base pairs. However, when two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs shall not be considered as mismatches for purposes of determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length can be referred to as "fully complementary" for purposes described herein if the longer oligonucleotide comprises a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide.

[0106] As used herein, "complementary" sequences may further include, or may be formed entirely of, non-Watson-Crick base pairs or base pairs formed from non-natural 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 Hoogstein base pairs.

[0107] The terms "complementary", "fully complementary" and "substantially complementary" herein can be used in reference to matching bases between the sense strand and antisense strand of dsRNA or between the antisense strand and target sequence of dsRNA material, as understood from the context of their use. It is understood that multiple HBV genotypes are known. Thus, a dsRNA material designed to be fully complementary to one HBV genotype may not be fully complementary to all HBV genotypes. A dsRNA material targeted to a specific site is effective in target knockdown across multiple genotypes, even if it is not fully complementary across all genotypes.

[0108] As used herein, a polynucleotide that is "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). For example, a polynucleotide is complementary to at least a portion of an HBV mRNA if the sequence of the polynucleotide is substantially complementary to a contiguous portion of an mRNA encoding an HBV gene.

[0109] Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target HBV sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target HBV sequence, and include a contiguous nucleotide sequence that is at least 80% complementary over the entire length of the nucleotide sequence of SEQ ID NO:1, or an equivalent region of a fragment of SEQ ID NO:1, e.g., at least 80%, 85%, 90% or 95% complementary.

[0110] In some embodiments, the dsRNA agents herein comprise a sense strand that is 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 over its entire length, e.g., at least 85%, 90% or 95% complementary to the equivalent region of the nucleotide sequence of SEQ ID NO:2.

[0111] In some embodiments, the dsRNA agent comprises an antisense strand that is substantially complementary to a target HBV sequence and comprises a contiguous nucleotide sequence that is at least 80% complementary over its entire length to the nucleotide sequence of any one of the sense nucleotide sequences in Table 2, or an equivalent region of a fragment of any one of the sense nucleotide sequences in Table 2, e.g., at least 85%, 90% or 95% complementary thereto.

[0112] As described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides.Furthermore, dsRNA material may contain ribonucleotides with chemical modifications.Such modifications may include all types of modifications disclosed herein or known in the art.Any of such modifications used in dsRNA material may be encompassed by "dsRNA material" for the purpose of this specification and claims.

[0113] In some embodiments, the majority of the nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides.In addition, dsRNA material can contain ribonucleotides with chemical modifications.Such modifications can include all types of modifications disclosed herein or known in the art.Any of such modifications used in dsRNA material are encompassed by "dsRNA material" for the purpose of this specification and claims.

[0114] The term "lower" in the context of the level of HBV gene expression or HBV protein production, or disease marker or symptom in a subject, refers to a statistically significant reduction in such level. Reduction can be, for example, at least 80%, at least 85%, at least 90%, at least 95%, or below the level of detection of the detection method, or toward or toward normal level (which may or may not be zero). In monitoring HBV infection, the log10 scale is generally used to describe the level of antigenemia (e.g., serum HBsAg level) or the level of viremia (serum HBV DNA level). It is understood that a reduction of 1 log10 is a 90% reduction (10% remaining), a reduction of 2 log10 is a 99% reduction (1% remaining), and so on. In certain embodiments, disease markers are reduced to below the level of detection. In certain embodiments, the method includes a clinically relevant inhibition of the expression of HBV, for example, as evidenced by a clinically relevant outcome after treating a control with a substance that reduces the expression of HBV. In some embodiments, at least partial inhibition of expression of an HBV gene is assessed by a reduction in the number of HBV mRNAs that can be isolated from a first cell or population of cells in which the HBV gene is transcribed or the cells are treated to inhibit expression of the HBV gene compared to a second cell or population of cells that is substantially the same as the first cell or population of cells but has not been so treated (control cells).

[0115] In certain embodiments, "lower" or "reduced" is understood to lower or reduce the level toward or to a normal level, i.e., normalize the level. In certain embodiments, the expression of the target is normalized to a level recognized as being within the normal range of an individual without such disorder, e.g., the level of a disease marker such as ALT or AST is reduced to a level recognized as being within the normal range of an individual without such disorder. If the disease-associated level is elevated from the normal level, the change is calculated from the upper limit of normal (ULN). If the disease-associated level is reduced from the normal level, the change is calculated from the lower limit of normal (LLN). The reduction refers to the percent difference in the change between the subject value and the normal value. For example, the normal level of AST may be reported as 10 to 40 units per liter. A subject with a pre-treatment AST level of 200 units per liter, or 5 times the ULN and 160 units per liter above the upper limit of normal, and a post-treatment AST level of 120 units per liter, or 3 times the ULN and 80 units per liter above the upper limit of normal, would have a 50% (80 / 160) reduction in elevated AST toward normal.

[0116] As another example, normal levels of ALT are generally considered to be between 7 and 55 units per liter (U / L), with 55 U / L being the upper limit of normal. A subject with a pre-treatment ALT level of 100 U / L (45 U / L above the upper limit of normal) and a post-treatment ALT level of 75 U / L (a 25 U / L reduction) will have a 55% (25 / 45 x 100%) reduction in ALT toward normal. As used herein, when a disease is associated with an elevated symptom value, "normal" is considered to be the upper limit of normal. When a disease is associated with a reduced symptom value, "normal" is considered to be the lower limit of normal.

[0117] The phrase "contacting a cell with a dsRNA material", for example, contacting with dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with a dsRNA material includes contacting a cell with a dsRNA material in vitro or contacting a cell with a dsRNA material in vivo. Contacting can be performed directly or indirectly. Thus, for example, the dsRNA material can be physically contacted with the cell by the person performing the method, or the dsRNA material can be placed in a situation that allows or subsequently causes it to contact with the cell.

[0118] The contacting of cells in vitro can be carried out, for example, by incubating the cells with dsRNA material.The contacting of cells in vivo can be carried out, for example, by injecting the dsRNA material into or near the tissue where the cells are located, or by injecting the dsRNA material into another area, for example, the bloodstream or subcutaneous space, so that the material then reaches the tissue where the cells to be contacted are located.For example, the dsRNA material can contain or be coupled to a ligand, for example, GalNAc3, which directs the dsRNA material to the site of interest, for example, the liver.A combination of in vitro and in vivo contact methods is also possible.For example, the cells can be contacted with the dsRNA material in vitro and then transplanted into a subject.

[0119] In some embodiments, contacting a cell with a dsRNA agent includes "introducing a dsRNA agent into a cell" or "delivering a dsRNA agent into a cell" by promoting or causing uptake or absorption into the cell. The uptake or uptake of the dsRNA agent can be performed via spontaneous diffusion or activation cellular steps, or by auxiliary agents or devices. The introduction of the dsRNA agent into a cell can be performed in vitro or in vivo. For example, for in vivo introduction, the dsRNA agent can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or known in the art.

[0120] As used herein, a "subject" is any mammal that can be infected with HBV, such as a mammal, including a primate (e.g., a human, a non-human primate, e.g., a monkey or a chimpanzee), or an animal that is considered an acceptable clinical model for HBV infection, such as the HBV-AAV mouse model (see, e.g., Yang et al. (2014) Cell and Mol Immunol 11:71) or the HBV 1.3xfs transgenic mouse model (Guidotti et al. (1995) J. Virol. 69:6158). In some embodiments, the subject has a hepatitis B virus (HBV) infection. In some embodiments, the subject has both a hepatitis B virus (HBV) infection and a hepatitis D virus (HDV) infection. In some embodiments, the subject is a human, such as a human with an HBV infection, particularly a chronic hepatitis B virus (HBV) infection.

[0121] As used herein, the term "treat" or "treatment" refers to, but is not limited to, one or more signs or symptoms associated with unwanted HBV gene expression or HBV replication, such as, but not limited to, the presence of serum or liver HBV ccc DNA; the presence of HBV DNA in serum; the presence of serum or liver HBV antigens, such as HBsAg or HBeAg; elevated ALT; elevated AST (normal range is generally considered to be about 10-34 U / L); 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; eating disorders; nausea; vomiting, low-grade fever; muscle aches; fatigue. ;taste and smell disorders (food and tobacco aversion);right upper quadrant and epigastric pain (intermittent, mild to moderate);hepatic encephalopathy;somnolence;disturbed sleep patterns;mental confusion;coma;ascites;gastrointestinal bleeding;coagulation disorders;jaundice;hepatomegaly (mildly enlarged, soft liver);splenomegaly;palmar erythema;spider nevi;muscle wasting;spider angiomas;vasculitis;variceal hemorrhage;peripheral edema;gynecomastia;testicular atrophy;abdominal collateral veins (caput medusa) medusa); ALT levels higher than AST levels; elevated gamma-glutamyl transpeptidase (GGT) (normal range is generally considered to be about 8-65 U / L) and alkaline phosphatase (ALP) levels (normal range is generally considered to be about 44-147 IU / L (international units per liter), not to exceed three 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 life span; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); presence of serum 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) or HBV presence of DNA; increased bilirubin levels; hyperglobulinemia; presence of tissue non-specific antibodies such as anti-smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA) (10-20%); presence of tissue specific antibodies such as antibodies against the thyroid gland (10-20%); elevated rheumatoid factor (RF) levels; reduced platelet and white blood cell counts; concomitant lobular inflammation with degenerative and reparative hepatocyte changes;and reduction or amelioration of predominantly centrilobular necrosis, whether detectable or undetectable. The likelihood of developing liver fibrosis is reduced, for example, when an individual with one or more risk factors for liver fibrosis, such as chronic hepatitis B infection, does not develop liver fibrosis or develops liver fibrosis with a lower severity compared to a population with the same risk factors and not treated as described herein. Non-development of a disease, disorder or condition, or reduction (e.g., by a clinically relevant amount) in the onset of signs or symptoms associated with such disease, disorder or condition, or delay (e.g., by days, weeks, months, or years) of signs or symptoms, is considered effective prevention. "Treatment" can also mean extending survival compared to expected survival in the absence of treatment. Prevention may require more than one administration.;

[0122] In a preferred embodiment, treatment of HBV infection results in a "functional cure" of hepatitis B. As used herein, functional cure is understood as the clearance of blood HBsAg, preferably accompanied by conversion to a state where HBsAg antibodies are detectable using a clinically relevant assay. For example, detectable antibodies may include a signal higher than 10 mIU / ml as measured by chemiluminescent microparticle immunoassay (CMIA) or any other immunoassay, referred to as anti-HBs seroconversion. Functional cure does not require clearance of all replicative forms of HBV (e.g., cccDNA from the liver). Anti-HBs seroconversion occurs naturally in approximately 0.2-1% of chronically infected individuals per year. However, even after anti-HBs seroconversion, persistence of low levels of HBV is observed for decades, indicating that functional cure occurs rather than complete cure. Without being bound by mechanism, it has been proposed that the immune system is capable of inhibiting HBV. Functional cure allows treatment of HBV infection to be discontinued. However, it is understood that a "functional cure" for HBV infection may not be sufficient to prevent or treat diseases or conditions resulting from HBV infection, such as liver fibrosis, HCC, cirrhosis, and the like.

[0123] 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 or replication. The term "HBV-related disease" includes diseases, disorders or conditions that benefit from reducing HBV gene expression 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.

[0124] In some embodiments, the HBV-related disease is hepatitis D virus infection. Hepatitis D virus or delta virus (HDV) is a human pathogen. However, this virus is defective and depends on the essential helper function provided by hepatitis B virus (HBV) to spread; in fact, HDV requires related or existing HBV infection, specifically the viral envelope containing the surface antigen of hepatitis B, to become infectious and grow. HDV can lead to severe acute and chronic liver disease associated with HBV. Hepatitis D infection or delta hepatitis is highly endemic in some African countries, the Amazon region, and the Middle East, while its prevalence is low in industrialized countries, except in the Mediterranean region.

[0125] HDV transmission can occur through coinfection with HBV (coinfection) or superimposed on chronic hepatitis B or hepatitis B colonization (superinfection). Both superinfection and coinfection with HDV lead to more severe complications compared to infection with HBV alone. These complications include liver failure in acute infections, and rapid progression to cirrhosis in chronic infections, as well as a higher likelihood of experiencing the development of liver cancer. In combination with hepatitis B virus, hepatitis D has the highest case fatality rate of 20% of all hepatitis infections.

[0126] In some embodiments, the HBV-related disease is acute hepatitis B. Acute hepatitis B includes inflammation of the liver that lasts less than 6 months. Typical symptoms of acute hepatitis B are fatigue, anorexia, nausea and vomiting. Extremely high aminotransferase levels (>1000U / L) and hyperbilirubinemia are often observed. Severe cases of acute hepatitis B can rapidly progress to acute liver failure, characterized by insufficient liver synthetic function. 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 develop into chronic hepatitis B.

[0127] In some embodiments, the HBV-related disease is chronic hepatitis. Chronic hepatitis B (CHB) includes inflammation of the liver that lasts for more than 6 months. Subjects with CHB are HBsAg positive and have either high viremia (≧104 HBV-DNA copies / ml of blood) or low viremia (<103 HBV-DNA copies / ml of blood). In some embodiments, the subject has been infected with HBV for at least 5 years. In some embodiments, the subject has been infected with HBV for at least 10 years. In some embodiments, the subject was infected with HBV at birth. Subjects with chronic hepatitis B disease may have immune tolerance or have inactive chronic infection with no evidence of active disease, and they may also be asymptomatic. Patients with chronic active hepatitis may have symptoms similar to those of acute hepatitis, especially during the replicative state. Subjects with chronic hepatitis B disease may have active chronic infection with necroinflammatory liver disease, or may have increased hepatocyte turnover in the absence of detectable necroinflammation, or may have inactive chronic infection without any evidence of active disease, and may also be asymptomatic. The persistence of HBV infection in CHB subjects is due to ccc HBV DNA. In some embodiments, the subject with CHB is HBeAg positive. In another embodiment, the subject with CHB is HBeAg negative. The subject with CHB has a serum HBV DNA level of less than 105, and persistent elevation of transaminases, such as ALT, AST and γ-glutamyltransferase. The subject with CHB may have a liver biopsy score (e.g., necroinflammatory score) of less than 4.

[0128] In some embodiments, the HBV-related disease is acute fulminant hepatitis B. Subjects with acute fulminant hepatitis B have the symptoms of acute hepatitis, as well as the additional symptoms of confusion or coma (due to the liver being unable to detoxify chemicals) and purpura or bleeding (due to the lack of blood clotting factors).

[0129] The subject with HBV infection, such as CHB, may develop liver fibrosis.Therefore, in some embodiments, the HBV-related disease is liver fibrosis.Liver fibrosis or cirrhosis is histologically defined as diffuse liver lesions characterized by fibrosis (excessive fibrous connective tissue) and structural changes from normal liver structure to abnormal nodules.

[0130] The subject with HBV infection, such as CHB, can develop end-stage liver disease.Therefore, in some embodiments, the HBV-related disease is end-stage liver disease.For example, liver fibrosis can progress to the point that the body can no longer compensate for, for example, the reduction in liver function, as a result of liver fibrosis (i.e., decompensated liver), causing, for example, mental and neurological symptoms and liver failure.

[0131] Subjects with HBV infection, such as CHB, can develop hepatocellular carcinoma (HCC), also called malignant liver cancer.Therefore, in some embodiments, the HBV-related disease is HCC.HCC often develops in subjects with CHB, and can be fibrolamellar, pseudoglandular (adenoid), pleomorphic (giant cell type) or clear cell.

[0132] "HDV-associated disorder" or "hepatitis D virus-associated disorder" is a disease or disorder associated with the expression 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.

[0133] As used herein, a "therapeutically effective amount" is intended to include an amount of dsRNA material sufficient to cause disease treatment (e.g., by attenuating or maintaining an existing disease or one or more symptoms of the disease) when administered to a patient for treating a subject with HBV infection or HBV-related disease. A "therapeutically effective amount" may vary depending on the dsRNA material, the method of administration of the material, the disease and its severity, medical history, age, weight, family history, genetic makeup, the stage of the pathological process mediated by HBV gene expression, the type of previous or concomitant treatment, if any, and other individual characteristics of the patient to be treated. A therapeutically effective amount may require more than one administration.

[0134] "Therapeutically effective amount" also includes the amount of dsRNA agent that produces some desired effect at a reasonable benefit / risk ratio applicable to any treatment. The dsRNA agent used in the methods herein can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0135] As used herein, the term "sample" refers to a sample of a similar fluid, cell, or tissue isolated from a subject, as well as a sample of a fluid, cell, or tissue present within a subject. Collection Examples of biological fluids include blood, serum, and serous fluid, plasma, lymph, urine, saliva, etc. 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 a specific part of the liver, or a specific type of cell in the liver, such as hepatocytes). In a preferred embodiment, "sample from subject" refers to plasma or serum obtained from blood taken from a subject. In a further embodiment, "sample from subject" refers to liver tissue (or a subcomponent thereof) or blood tissue (or a subcomponent thereof, such as serum) from a subject.

[0136] II. dsRNA material Provided herein are dsRNA agents that inhibit the expression of one or more HBV genes. In some embodiments, the dsRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of HBV genes in a cell, such as a cell in a subject, such as a mammal, such as a human, having an HBV-related disease, such as chronic hepatitis B. The dsRNA agent comprises an antisense strand having a complementary region that is complementary to at least a portion of an mRNA formed during expression of the HBV gene. The complementary region is about 19-21 nucleotides in length. Upon contact with a cell expressing an HBV gene, the dsRNA agent inhibits the expression of the HBV gene by at least 80% 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. In a preferred embodiment, the percentage of inhibition is measured using the real-time PCR method provided in Example 2, using the cell lines provided therein, with the dsRNA agent used in transfection at a concentration of 10 nM.

[0137] dsRNA comprises two RNA strands, which are complementary and hybridize to form a double-stranded structure under the conditions in which dsRNA is used. One strand of dsRNA (antisense strand) comprises a complementary region that is substantially complementary to the target sequence, and generally completely complementary. However, due to sequence differences between HBV genotypes, dsRNA may be completely complementary to some, but not all, HBV genotypes. The target sequence may be derived from the sequence of the mRNA formed during the expression of HBV genes. The other strand (sense strand) comprises a region that is complementary to the antisense strand, such that the two strands hybridize to form a double-stranded structure when combined under appropriate conditions. As described elsewhere herein and known in the art, the complementary sequence of dsRNA may also be contained as a self-complementary region of one nucleic acid molecule, as opposed to being on a separate oligonucleotide.

[0138] Generally, the double-stranded structure is 19-21 base pairs in length, for example 19, 20 or 21 base pairs in length.

[0139] Similarly, the complementary region of the target sequence is 19-23 nucleotides in length, for example, 19-23, 19-22, 19-21, 19-20, 20-23, 20-22, 20-21, 21-22, 19, 20, 21, 22 or 23 nucleotides in length. In some embodiments, the complementary region is 21 nucleotides in length. As those skilled in the art will also recognize, the region of RNA targeted for cleavage is most often a part of a larger RNA molecule, often an mRNA molecule. Where relevant, a "part" 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).

[0140] One of skill in the art will also recognize that a double-stranded region is a primary functional portion of a dsRNA, e.g., a double-stranded region of about 19-21 base pairs, e.g., 19-21, 19-20, 20-21, 19, 20, or 21 base pairs. In some embodiments, the double-stranded region is 19 base pairs.

[0141] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, for example, 1 or 2 nucleotides.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 nucleotides of the overhangs may be present on the 5'-end, 3'-end, or both ends of one or both of the antisense strand or the sense strand of the dsRNA.In some embodiments, the dsRNA comprises a 2-nucleotide overhang on the 3'-end of the antisense strand.

[0142] dsRNA can be prepared, for example, using an automated DNA synthesizer (e.g., Biosearch, Applied Biosystems TM dsRNA can be synthesized by standard methods known in the art, as described further below, by use of dsRNAs commercially available from Sigma-Aldrich, Inc. Methods for synthesizing dsRNA for use in pharmaceutical compositions are also known in the art.

[0143] The dsRNA substance compound 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 dsRNA substance compound can be prepared using solution phase or solid phase organic synthesis or both. Organic synthesis offers the advantage that oligonucleotide strands containing non-natural or modified nucleotides can be easily prepared. The single-stranded oligonucleotide of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0144] In some embodiments, the dsRNA agent herein comprises at least two nucleotide sequences, sense strand sequence and antisense strand sequence.The sense strand can comprise a nucleotide sequence selected from the group consisting of any one of the sense strand nucleotide sequences of the duplex in any one of Table 2.The antisense strand can comprise a nucleotide sequence selected from the group consisting of any one of the antisense strand nucleotide sequences of the duplex in any one of Table 2.In some embodiments, the dsRNA agent is not AD-66810.

[0145] In some embodiments, the dsRNA agents herein comprise, consist essentially of, or consist of the sense and antisense strands shown in Table 2.

[0146] III. modified dsRNA material In certain embodiments, the RNA of the dsRNA material herein is unmodified and does not include, for example, chemical modifications or conjugations known in the art and described herein.In another embodiment, the RNA of the dsRNA material herein is chemically modified to improve stability or other beneficial properties.In some embodiments, the nucleotide of the dsRNA material herein is substantially all modified.In some embodiments herein, all of the nucleotides of the dsRNA material or substantially all of the nucleotides of the dsRNA material are modified, i.e., there are no more than 5, 4, 3, 2 or 1 unmodified nucleotides in the strand of the dsRNA material.

[0147] The nucleic acids patented in the present invention can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, end modifications, such as 5' end modifications (phosphorylation, conjugation, reverse linkage) or 3' end modifications (conjugation, DNA nucleotides, reverse linkage, etc.); base modifications, such as replacement with stable bases, unstable bases, or bases that base pair with an expanding repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or replacement of sugars; or backbone modifications, including modification or replacement of phosphodiester linkages. Specific examples of dsRNA agent compounds useful in the embodiments described herein include, but are not limited to, RNAs that contain modified backbones or that do not contain natural internucleoside linkages. The RNA with modified backbone includes in particular those that do not have phosphorus atom in backbone.For the purpose of this specification, as sometimes referred to in the art, the modified RNA that does not have phosphorus atom in internucleoside backbone can also be considered as oligonucleoside.In some embodiments, the modified dsRNA material has phosphorus atom in its internucleoside backbone.

[0148] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates, including 3'-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linkage analogs, and those with reverse polarity, where 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.

[0149] Representative United States patents which teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. 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; 5,321,131; 5,321,132; 5,321,133; 5,321,134; 5,321,135; 5,321,136; 5,321,137; 5,321,138; 5,321,139; 5,421,243; 5,517,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,321,132; 5,421,243; 5,517,195 ... ,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; Same No. 5,536,821; Same No. 5,541,316; Same No. 5,550,111; Same No. 5,563,253; Same No. 5,571,799; Same No. 5,587,361; Same No. 5,625,050 No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6,169,170; No. 6,172,209; No. 6,239,265; No. 6,277, No. 603; No. 6,326,199; No. 6,346,614; No. 6,444,423; No. 6,531,590; No. 6,534,639; No. 6,608,035; No. 6,68 Nos. 3,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent No. RE39464, each of which is incorporated herein by reference for its teachings related to such preparation methods.

[0150] Modified RNA backbones that do not contain internal phosphorus atoms have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain 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 component moieties.

[0151] Representative United States patents which 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; 5,489,677; 5,541, Nos. 5,677,437 and 5,677,439, each of which is incorporated herein by reference for its teachings related to such methods.

[0152] Suitable RNA mimics are contemplated for use in the dsRNA material provided herein, in which both the sugar and the internucleoside linkage, i.e., the backbone of the nucleotide unit, are replaced by novel groups. The base unit is maintained for hybridization with a suitable nucleic acid target compound. One such 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 by an amide-containing backbone, in particular an aminoethylglycine backbone. Nucleic acid bases are retained and are directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference for teachings related to such methods. Further suitable PNA compounds for use in the dsRNA agents herein are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0153] Some embodiments of the present invention include RNA with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as methylene (methylimino) or MMI backbones], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- [wherein the natural phosphodiester backbone is represented as --O--P--O--CH2--] of the above-mentioned US Patent No. 5,489,677, and the amide backbones of the above-mentioned US Patent No. 5,602,240. In some embodiments, the RNA patented herein has the morpholino backbone structure of the above-mentioned US Patent No. 5,034,506.

[0154] Modified RNAs may also contain one or more substituted sugar moieties. The dsRNA agents patented herein may comprise 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 alkyl, alkenyl and alkynyl are substituted or unsubstituted C1-C6 10 Alkyl or C2-C 10 An exemplary suitable modification is O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON [(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA comprises at the 2' position, 10The modification includes one of the following: 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 cleavage group, reporter group, intercalator, group that improves the pharmacokinetic properties of dsRNA agent, or group that improves the pharmacodynamic properties of dsRNA agent, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504), i.e., alkoxy-alkoxy 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 herein below in the Examples. Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides, (both R and S isomers of these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0155] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of a dsRNA agent, particularly at the 3' position of the sugar on the 3' terminal nucleotide or in a 2'-5' linked dsRNA and at the 5' position of the 5' terminal nucleotide. A dsRNA agent can also have a sugar mimic, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative United States patents which 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; 5,567,811; 5,5 Nos. 76,427; 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 commonly owned with the present application. The contents of each of the above patent publications are incorporated herein by reference for their teachings related to such methods.

[0156] dsRNA materials may 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 other synthetic and natural nucleobases, such as deoxy-thymine (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. These include uracil, 5-uracil (pseudouracil), 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 adenine, 8-azaguanine and adenine, 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, pages 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, pages 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 patented in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. The 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and is an exemplary base substitution, especially when combined with a 2'-O-methoxyethyl sugar modification.

[0157] Representative United States patents which teach the preparation of some of the above and other modified nucleobases include, but are not limited to, U.S. Pat. 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; 5,594,121; 5,596,09 Nos. 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, each of which is incorporated herein by reference for its teachings related to such methods.

[0158] In some embodiments, the RNA of the dsRNA agent may also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a bridge of two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a bridge linking two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge links the 4'-carbon and the 2'-carbon of the sugar ring. Thus, in some embodiments, the agent herein may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, where the ribose moiety includes an additional bridge linking the 2' and 4' carbons. In other words, an LNA is a nucleotide that includes a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in a 3'-endo structural configuration. Adding a fixed nucleic acid to a dsRNA agent has been shown to increase the stability of the dsRNA agent 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 herein include, but are not limited to, nucleosides that contain a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents herein include one or more bicyclic nucleosides that contain a 4'-2' bridge.Examples of such 4'-2' bridged bicyclic nucleosides include 4'-(CH2)--O-2' (LNA); 4'-(CH2)--S-2'; 4'-(CH2)2--O-2' (ENA); 4'-CH(CH3)--O-2' (also referred to as "constrained ethyl" or "cEt") and 4'-CH(CH2OCH3)--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. 8,278,283); 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 Publication No. 2004 / 0171570); 4'-CH2--N(R)-O-2' (where R is H, C1-C. 12 alkyl), or protecting groups (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). Each of the above related to modified nucleic acids is incorporated herein by reference.

[0159] Additional representative U.S. patents and U.S. patent publications which teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Pat. 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; and 7,427,672. Nos. 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; U.S. Patent Publication No. 2008 / 0039618; and U.S. Patent Publication No. 2009 / 0012281, which are hereby incorporated by reference for their teachings related to such methods.

[0160] For example, any of the bicyclic nucleosides described above can be prepared having one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0161] The RNA of the dsRNA agent can also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid that includes a bicyclic sugar moiety that includes a 4'-CH(CH3)-0-2' bridge. In some embodiments, the constrained ethyl nucleotide is in the S configuration, referred to herein as "S-cEt."

[0162] The dsRNA agents herein may also include 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. CRNs lock the ribose ring into a stable conformation and increase hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity, resulting in less puckering of the ribose ring.

[0163] Representative publications that teach the preparation of some of the above CRNs include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383; and PCT Publication No. WO2013 / 036868, which are incorporated by reference herein for their teachings related to such methods.

[0164] In some embodiments, the dsRNA agent herein comprises one or more monomers that are UNA (non-locked nucleic acid) nucleotides. UNA is a non-locked acyclic nucleic acid in which any bond of the sugar is removed to form a non-locked "sugar" residue. In one example, UNA also encompasses monomers in which the bond between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) is removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) is removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference for teachings related to non-locked nucleic acid nucleotides).

[0165] Representative U.S. patent publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, which are incorporated by reference herein for their teachings related to such methods.

[0166] Potentially stable modifications to the ends 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'-O-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 is found in PCT Publication No. WO2011 / 005861.

[0167] In certain embodiments, the dsRNA agent is modified to include one or more adenosine-glycol nucleic acids ("GNAs"). The term "GNAs" refers to glycol nucleic acids, which are polymers similar to DNA or RNA, but differ in composition in their "backbone," which consists of repeating glycerol units linked by phosphodiester bonds: [ka] A description of adenosine-GNAs can be found, for example, in Zhang, et al. (JACS 127(12):4174-75 (2005).

[0168] Other modifications of the nucleotide of the dsRNA material disclosed herein include 5' phosphate or 5' phosphate mimic, for example, 5' terminal phosphate or phosphate mimic for the antisense strand of the dsRNA material.Suitable phosphate mimics are disclosed in US Patent Publication No. 2012 / 0157511, which is incorporated herein by reference for the teachings related to such modifications.

[0169] Further modified dsRNA agents targeting HBV are provided, for example, in WO2016 / 077321, which is incorporated herein by reference for its teachings related to such modifications.

[0170] In some embodiments, the dsRNA agent comprises, consists essentially of, or consists of the modified sense and antisense strands shown in Table 2.

[0171] IV. Ligand-conjugated dsRNA agents The dsRNA material herein may be conjugated with a ligand.Ligand can change the distribution, targeting or life span of the dsRNA material in which it is incorporated.In a preferred embodiment, the ligand improves the affinity to a selected target, for example, a molecule, a cell or cell type, a compartment, for example, a cell or organ compartment, a tissue, an organ or an area of ​​the body, for example, compared to the material species in which such ligand is not present.Preferred ligands do not interfere with the pairing of the double strands in double-stranded nucleic acid.

[0172] The ligand-conjugated oligonucleotides herein can be synthesized by using an oligonucleotide having a pendant reactive functional group that is derivatized with a linking molecule (described below). The reactive oligonucleotide can be reacted directly with a commercially available ligand, a synthetic ligand having any of a variety of protecting groups, or a ligand having a linking moiety attached thereto.

[0173] In the ligand-conjugated oligonucleotides and ligand molecules having sequence-specific linked nucleosides herein, the oligonucleotides and oligonucleosides can be assembled by 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.

[0174] When using a nucleotide-conjugate precursor that already has a linking moiety, typically, a sequence-specific linked nucleoside is synthesized, and then a ligand molecule is reacted with the linking moiety to form a ligand-conjugated oligonucleotide.In some embodiments, the oligonucleotide or linked nucleoside of the present invention is synthesized by an automatic synthesizer using the phosphoramidite derived from the ligand-nucleoside conjugate, together with the commercially available standard phosphoramidite and non-standard phosphoramidite that are commonly used in oligonucleotide synthesis.

[0175] Representative United States patents which 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; 5,486,603; 5,51 No. 2,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735 ; Same No. 4,667,025; Same No. 4,762,779; Same No. 4,789,737; Same No. 4,824,941; Same No. 4,83 No. 5,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 5,082,830 ; Same No. 5,112,963; Same No. 5,214,136; Same No. 5,082,830; Same No. 5,112,963; Same No. 5,21 No. 4,136; No. 5,245,022; No. 5,254,469; No. 5,258,506; No. 5,262,536 ; Same No. 5,272,250; Same No. 5,292,873; Same No. 5,317,098; Same No. 5,371,241, Same No. 5,3 No. 91,723; No. 5,416,203, No. 5,451,463; No. 5,510,475; No. 5,512,667 No. 5,514,785; No. 5,565,552; No. 5,567,810; No. 5,574,142; No. 5,5 Nos. 85,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, which are incorporated by reference herein for their teachings related to such methods.

[0176] A. Carbohydrate Conjugates In a preferred embodiment of the compositions and methods herein, the dsRNA agent oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated dsRNA agents are advantageous for compositions suitable for in vivo nucleic acid delivery and in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to either a compound that is a carbohydrate itself, which has at least six carbon atoms (which may be linear, branched or cyclic), and is composed of one or more monosaccharide units, each of which has an oxygen, nitrogen or sulfur atom attached to it, or a compound that has as a part a carbohydrate moiety, which has at least six carbon atoms (which may be linear, branched or cyclic), and is composed of one or more monosaccharide units, each of which has an oxygen, nitrogen or sulfur atom attached to it. Representative 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. Particular monosaccharides include sugars of C5 and above (e.g., C5, C6, C7 or C8), and disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7 or C8).

[0177] In some embodiments, the carbohydrate conjugate for use in the compositions and methods herein is a monosaccharide. In some embodiments, the carbohydrate conjugate for use in the compositions and methods herein is a monosaccharide. [ka] [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0178] In some embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). In some embodiments, the carbohydrate comprises multiple N-acetylgalactosamine units, e.g. [ka] Includes.

[0179] Another exemplary carbohydrate conjugate that may be used in the embodiments described herein is [ka] where when one X or Y is an oligonucleotide, the other is hydrogen.

[0180] In some embodiments herein, the dsRNA material herein is linked with GalNAc or GalNAc derivative via a monovalent linker.In some embodiments herein, the dsRNA material herein is linked with GalNAc or GalNAc derivative via a bivalent linker.In some embodiments herein, the dsRNA material herein is linked with GalNAc or GalNAc derivative via a trivalent linker.In some embodiments herein, the carbohydrate ligand comprises three N-acetylgalactosamine units ("GalNAc3") linked with a trivalent linker.

[0181] In some embodiments, the double-stranded dsRNA material comprises one GalNAc or GalNAc derivative attached to the dsRNA material.In some embodiments, the double-stranded dsRNA material comprises multiple (e.g., 2, 3, 4, 5 or 6) GalNAc or GalNAc derivatives, each of which is independently attached to multiple nucleotides of the double-stranded dsRNA material via multiple monovalent linkers.

[0182] Further carbohydrate conjugates suitable for use herein include those described in PCT Publications WO2014 / 179620 and WO2014 / 179627, which are incorporated by reference herein for their teachings related to such conjugates.

[0183] Non-limiting examples of dsRNA agent carbohydrate conjugates with linkers that can be used in the compositions and methods disclosed herein include: [ka] [ka] [ka] where when one X or Y is an oligonucleotide, the other is hydrogen.

[0184] In some embodiments of the compositions and methods disclosed herein, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0185] In some embodiments, the dsRNA agent disclosed herein has the formula (XXXI)-(XXXIV): [ka] [ka] [In the formula, q 2A , q 2B , q 3A , q 3B , q 4A , q 4B , q 5A , q 5B and q 5C represents independently at each occurrence 0 to 20, and the repeat units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C each occurrence is independently absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently at each occurrence absent, alkylene, substituted alkylene, where one or more methylenes are optionally interrupted or terminated by one or more O, S, S(O), SO2, N(RN), C(R')=C(R''), C≡C, or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R5C Each occurrence is independently absent or NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; i.e., each, independently at each occurrence, is a monosaccharide (e.g., GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; and R a is H or an amino acid side chain] The conjugated bivalent or trivalent branched linker is selected from the group of structures shown in any of the following: The trivalent conjugated GalNAc derivative is used with a dsRNA agent to have the formula (XXXIV): [ka] [In the formula, L 5A , L 5B and L 5C represents a monosaccharide such as a GalNAc derivative. The present invention is particularly useful for inhibiting the expression of target genes such as those

[0186] Examples of GalNAc derivatives conjugated with suitable divalent and trivalent branched linker groups include, but are not limited to, the structures listed above as Formulas I, VI, X, IX and XII.

[0187] B. Linker In some embodiments, the dsRNA agent oligonucleotide can be attached to a conjugate or ligand as described herein by a variety of linkers, which can be cleavable or non-cleavable.

[0188] The term "linker" or "linking group" means an organic moiety that links two parts of a compound, e.g., covalently bonds two parts of a compound.A linker typically comprises a direct bond, or may be an atom, such as oxygen or sulfur, a unit, such as NR, C(O), C(O)NH, SO, SO, SONH, or a chain of atoms, 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 ... Arylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl , alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, including those in which one or more methylenes may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic (wherein R8 is hydrogen, acyl, aliphatic, or substituted aliphatic).In some embodiments, the linker is about 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

[0189] The cleavable linking group is sufficiently stable outside a cell, but once inside a target cell, it is cleaved to release the two moieties that the linker holds together. In some embodiments, the cleavable linking group is cleaved at least about 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster, inside the target cell or under a first reference condition (e.g., which can be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (e.g., which can be selected to mimic or represent conditions found in blood or serum).

[0190] Cleavable linking groups are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are more widely present or found at higher levels or activity inside cells compared to serum or blood. Examples of such degrading agents include, for example, oxidizing or reducing enzymes present in cells, or redox agents that are selected for specific substrates or have no substrate specificity, including reducing agents such as mercaptans that can degrade redox-cleavable linking groups by reduction; esterases; endosomes, or substances that can form an acidic environment, such as substances that produce a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which can be substrate specific) and phosphatases.

[0191] 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, around 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.

[0192] 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 cell to be targeted. For example, a liver targeting ligand may be linked to a cationic lipid via a linker that contains an ester group. Since liver cells are rich in esterase, this linker will be cleaved more efficiently in liver cells compared to cell types that are not rich in esterase. Other cell types that are rich in esterase include lung, renal cortex and testis cells.

[0193] Linkers containing peptide bonds can be used in targeting cell types rich in peptidases, such as hepatocytes and synovial cells.

[0194] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or degrading condition) to cleave the candidate linking group. It may also be desirable to test the ability of the candidate cleavable linking group to resist cleavage in blood or upon contact with other non-target tissues. Thus, the relative susceptibility to cleavage between a first condition and a second condition can be determined, where the first condition is selected to indicate cleavage in target cells, and the second condition is selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. The 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 evaluation in a cell-free or culture condition and confirm with further evaluation in a whole animal. In preferred embodiments, useful candidate compounds are cleaved at least about 2-fold, 4-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or about 100-fold faster within 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).

[0195] i. Redox-cleavable linking groups In some embodiments, 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 linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a specific dsRNA material portion and a specific targeting agent, the methods described herein can be looked at. For example, the candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agent using a reagent known in the art, which mimics the cleavage rate that can be observed in cells, for example, target cells. The candidate can also be evaluated under conditions selected to mimic blood or serum conditions. In some embodiments, the candidate compound is cleaved in blood at a maximum of about 10%. In some embodiments, useful candidate compounds are degraded at least about 2-fold, 4-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than 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 intracellular media and compared to conditions selected to mimic extracellular media.

[0196] ii. Phosphate-Based Cleavable Linkers In some embodiments, the cleavable linker comprises a phosphate-based cleavable linker. The phosphate-based cleavable linker is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves the phosphate group within a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups include: -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. In some embodiments, the phosphate-based linking group is -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-, or -OP(S)(H)-S-. In some embodiments, the phosphate-based linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0197] iii. Acid-Cleavable Linking Groups In some embodiments, the cleavable linker is an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In some embodiments, the acid-cleavable linking group is cleaved in an acidic environment with 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. Intracellularly, certain low pH organelles such as endosomes and lysosomes can 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 have the general formula -C=NN-, C(O)O, or -OC(O). In some embodiments, the carbon attached 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.

[0198] iv. Ester-Based Linking Groups In some embodiments, the cleavable linker comprises an ester-based cleavable linking group. The ester-based cleavable linking group is cleaved by enzymes such as esterase and amylase in cells. 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.

[0199] v. Peptide-Based Cleavage Groups In some embodiments, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. The peptide-based cleavable linking group is a peptide bond formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not include an amide group (-C(O)NH-). An amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to produce peptides and proteins. The peptide-based cleaving group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to produce peptides and proteins, and does not include all amide functional groups. The peptide-based cleavable linking group is represented by the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0200] V. Delivery of dsRNA material Delivery of the dsRNA material of the present invention to cells, for example, cells in a subject, such as a human subject, can be achieved in several different ways.For example, delivery can be performed by contacting cells with the dsRNA material herein, either in vitro or in vivo.In vivo delivery can also be performed directly by administering a composition comprising the dsRNA material to a subject.Alternatively, in vivo delivery can be performed indirectly by administering one or more vectors that code for and induce the expression of the dsRNA material.These alternatives are discussed further below.

[0201] Generally, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the dsRNA agents of the present invention (see, for example, Akhtar S. and Julian RL., (1992) Trends Cell.Biol.2(5):139-144 and WO94 / 02595, which are incorporated herein by reference for teachings related to such delivery methods).For in vivo delivery, factors to be considered for delivering dsRNA agent molecules include, for example, the biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue.

[0202] In order to administer dsRNA agent systemically for the treatment of disease, RNA can be modified or can be delivered using drug delivery system instead; both methods act to prevent dsRNA from being rapidly degraded by endonuclease and exonuclease in vivo.Modification of RNA, pharmaceutical carrier or pharmaceutical excipient can also allow dsRNA agent composition to be targeted to target tissue and avoid undesirable off-target effects.dsRNA agent molecule can be modified by chemical conjugation, for example, carbohydrate conjugate as described above.

[0203] VI. Pharmaceutical Compositions The present specification also includes pharmaceutical compositions and formulations that include the dsRNA agent of the present specification. In some embodiments, the present specification provides pharmaceutical compositions that contain the dsRNA agent described herein, pharma- ceutically acceptable carriers. The pharmaceutical compositions that contain the dsRNA agent are useful for treating diseases or disorders related to the expression or activity of HBV genes. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition that is formulated for systemic administration via parenteral delivery, for example, subcutaneous (SC), intramuscular (IM) or intravenous (IV) delivery. In certain embodiments, the present specification provides compositions that are formulated for organ-specific (e.g., hepatic) intra-arterial, intratumoral, intradermal, intravitreal injection, ocular topical, ocular (eye drops), spray, ocular topical or other topical route, suppository, or oral administration. In a preferred embodiment, the composition is administered subcutaneously.

[0204] The pharmaceutical composition of the present specification can be administered at a dose sufficient to inhibit the expression of HBV genes. In some embodiments, the dosage is 0.5 mg / kg to 50 mg / kg, 0.3 mg / kg to 20 mg / kg, or 3 mg / kg to 10 mg / kg per administration, or preferably 3 mg / kg to 10 mg / kg per administration. For example, the dsRNA is administered at about 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg per single administration. In some embodiments, the dsRNA agent is administered at a dose of 50 mg to 900 mg.

[0205] The composition can also be prepared and packaged into a fixed dose that is independent of body weight for the subject.Exemplary dosage levels can be calculated by multiplying the body weight per kilogram by the body weight of an average subject.For example, an average adult is usually considered to be about 70 kg.

[0206] Repeated administration regimen can include administering therapeutic amount of dsRNA substance periodically, such as once a month, once every two months, or once every three months.In a preferred embodiment, dsRNA substance is administered no more than once a month.After the first treatment regimen, treatment can be administered less frequently.

[0207] The pharmaceutical composition can be administered for an indefinite period of time, for example, to a subject having one or more signs or symptoms of HBV infection, e.g., detectable HBV antigen or HBV DNA, including HBV cccDNA. In some embodiments, treatment with the dsRNA agent is discrete or over a period of time and provides a functional cure.

[0208] Those skilled in the art will recognize that certain factors, including but not limited to disease or disease severity, previous treatment, overall health or age of subject, and other existing diseases, can affect the dosage and time required to effectively treat subject.Furthermore, the treatment of subject with therapeutically effective amount of composition can include a single treatment or a series of treatments.Effective dosage and in vivo half-life for each dsRNA agent encompassed by this specification can be estimated by using conventional methodology or based on the in vivo test using suitable animal model as described elsewhere herein.

[0209] A. Excipients A "pharmaceutical carrier" or "pharmaceutical excipient" is a pharma- ceutical acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Such agents are well known in the art.

[0210] B. Other components The compositions herein may also contain other auxiliary components that are conventionally found in pharmaceutical compositions, at their use levels established in the art.Thus, for example, the compositions may contain additional compatible pharma- ceutically active materials, such as antipruritic agents, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials that are useful for physically formulating the various dosage forms of the compositions herein, such as preservatives, antioxidants and stabilizers.However, such materials must not excessively interfere with the biological activity of the components of the compositions herein when added.The formulation may be sterilized, and if necessary, may be mixed with auxiliary agents that do not adversely interact with the nucleic acid of the formulation, such as preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, or buffers.

[0211] In some embodiments, the pharmaceutical compositions patented in the present invention comprise (a) one or more dsRNA agent compounds and (b) one or more agents that function by a non-iRNA mechanism and are useful for treating HBV-related disorders. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-adipose agents, anti-viral agents, and anti-fibrotic agents.

[0212] In addition, other substances that are generally used for liver protection, such as silymarin, can also be used in combination with the dsRNA substances described herein.Other drugs that are useful for treating liver disease include telbivudine, entecavir, and protease inhibitors, such as telaprevir, and others, such as those disclosed in US2005 / 0148548, US2004 / 0167116, US2003 / 0144217 and US2004 / 0127488.

[0213] The toxicity and therapeutic effect of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective in 50% of the population).The dose ratio between toxicity and therapeutic effect is therapeutic index, and can be expressed as LD50 / ED50 ratio.In some embodiments, compounds that show high therapeutic index are preferred.

[0214] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of the compositions patented herein in this disclosure is generally within a range of circulating concentrations that includes the ED50 with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the methods patented herein, a therapeutically effective dose can be initially estimated from cell culture assays. A dose can be formulated to achieve in an animal model a circulating plasma concentration range of the compound, or, where appropriate, the polypeptide product of the target sequence (e.g., achieve a reduction in the concentration of the polypeptide), that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as measured in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0215] In addition to the above-mentioned administration, the dsRNA agent described herein can be administered in combination with other known drugs that are effective in treating HBV infection.In any case, the administering physician can adjust the dosage and administration time of the dsRNA agent based on observed results, using standard effectiveness measures known in the art or described herein.

[0216] VII. method The present specification also provides a method for inhibiting the expression of HBV in a cell, comprising contacting the cell with an amount of a dsRNA agent, e.g., a double-stranded dsRNA agent, effective to inhibit the expression of HBV in the cell, thereby inhibiting the expression of HBV in the cell.

[0217] The contact of cells with dsRNA material, for example, double-stranded dsRNA material, can be performed in vitro or in vivo. Contacting cells with dsRNA material in vivo includes contacting a cell or a group of cells in a subject, for example, a human subject, with dsRNA material. A combination of in vitro and in vivo methods of contacting cells is also possible. As discussed above, contacting cells can be direct or indirect. Furthermore, contacting cells can be performed via a targeting ligand, including any of the ligands described herein or known in the art. In a preferred embodiment, the targeting ligand is a carbohydrate moiety, for example, a GalNAc3 ligand, or any other ligand that directs dsRNA material to a significant site.

[0218] In some embodiments of the method herein, dsRNA material is administered to subject and delivered to specific site in subject.The inhibition of HBV gene expression is evaluated by measuring the level or variation of HBV mRNA or HBV protein in the sample obtained from body fluid or tissue from specific site in subject.In a preferred embodiment, the site is selected from liver and blood.The site can also be a subsection or subgroup of cells or body fluids prepared from any one of the above sites.

[0219] In some embodiments, the methods disclosed herein are useful for treating subjects with HBV infection, such as subjects who benefit from reducing HBV gene expression or HBV replication. In one embodiment, the present specification provides a method for reducing the level of hepatitis B virus ccc DNA in a subject infected with HBV. In another embodiment, the present specification provides a method for reducing the level of HBV antigen, such as HBsAg or HBeAg, in a subject infected with HBV. In another embodiment, the present specification provides a method for reducing the HBV viral load in a subject infected with HBV. The present specification also provides a method for reducing the level of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in a subject infected with HBV (although a transient increase in ALT or AST may be associated with viral clearance). In one embodiment, the present specification provides a method for increasing the level of anti-HBV antibodies in a subject infected with HBV. In another embodiment, the present specification provides a method for treating a subject with HBV infection. In one aspect, the present disclosure provides a method for treating subjects with HBV-related diseases, such as hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; or hepatocellular carcinoma.In addition, because HDV infection depends on the essential helper function provided by HBV for transmission, and subjects with HBV infection can also have HDV infection, in some embodiments, the treatment method described herein is also useful for treating subjects with HDV infection or HDV-related disorders, such as hepatitis B virus infection, chronic hepatitis B infection (CHB), chronic hepatitis B infection (CHB), liver cirrhosis, liver failure and hepatocellular carcinoma (HCC).In some embodiments, the treatment method (and use) described herein comprises administering to a subject, such as a human, a pharmaceutical composition comprising a therapeutically effective amount of the dsRNA agent described herein that targets HBV gene or a dsRNA agent described herein that targets HBV gene.

[0220] In one aspect, the present disclosure provides a method for detecting at least one symptom in a subject having HBV infection, such as, for example, the presence of serum or liver HBV ccc DNA; the presence of serum HBV DNA; the presence of serum or liver HBV antigen, such as HBsAg or HBeAg; elevated ALT; elevated AST; absence 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; eating disorders; nausea; vomiting, low-grade fever; muscle pain; fatigue; impaired taste and smell (eating disorders). and tobacco aversion); right upper quadrant 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 hemorrhage; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (caput medusa); ALT levels higher than AST levels; elevation of gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) levels not more than 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 life span; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); presence of serum 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) or HBV The present invention provides a method for preventing the following: DNA; increased bilirubin levels; hyperglobulinemia; the presence of tissue non-specific antibodies (10-20%), such as anti-smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA); the presence of tissue specific antibodies (10-20%), such as antibodies against the thyroid gland; elevated levels of rheumatoid factor (RF); reduced platelet and white blood cell counts; inflammation accompanied by and accompanied by lobular, degenerative and reparative hepatocyte changes; or primarily centrilobular necrosis, whether detectable or undetectable.The method includes administering to a subject a dsRNA agent of the invention, e.g., a dsRNA, or a pharmaceutical composition comprising a therapeutically effective amount of the dsRNA agent, thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced HBV gene expression, such as a subject having HBV infection or a subject having both HBV and HDV infection.

[0221] In another aspect, the present specification provides a use of a therapeutically effective amount of a dsRNA agent of the present specification to treat a subject who may benefit from reducing or inhibiting HBV gene expression, such as a subject having an HBV infection or a subject having both HBV and HDV infection.

[0222] In a further aspect, the specification provides the use of a dsRNA agent, e.g., a dsRNA, or a pharmaceutical composition comprising a dsRNA agent, targeting an HBV gene, herein, in the manufacture of a therapeutic medicament for the treatment of a subject, e.g., a subject having an HBV infection or a subject having both HBV and HDV infection, who would benefit from reduced HBV gene expression or HBV replication, and a subject having a disorder that would benefit from reduced HBV gene expression, e.g., an HBV-related disease.

[0223] In another aspect, the present specification provides a use of a dsRNA agent described herein for preventing at least one symptom in a subject suffering from a disorder that would benefit from reducing or inhibiting HBV gene expression or HBV replication.

[0224] In a further aspect, the present specification provides the use of a dsRNA agent described herein in the manufacture of a therapeutic medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from reducing or inhibiting HBV gene expression or HBV replication, such as an HBV-related disease.

[0225] In some embodiments, when a dsRNA agent is administered to a subject, the dsRNA agent targeting HBV is administered to a subject having an HBV infection, or having both HBV and HDV infections, or having an HBV-related disease, such that, for example, expression of one or more HBV genes, HBV ccc DNA levels, HBV antigen levels, HBV viral load levels, ALT or AST in the subject's cells, tissues, blood, other tissues or bodily fluids is reduced or normalized by at least 80%, 85%, 90%, 95%, 98% or toward standard.

[0226] In some embodiments, the methods and uses herein include administering a composition as described herein to reduce target HBV gene expression, for example, for one month. In some embodiments, the expression of the target HBV gene is reduced for an extended period of time, for example, at least two months, three months or more. In some embodiments, the methods and uses herein include administering a composition as described herein to provide a functional cure.

[0227] The administration of dsRNA by the methods and uses described herein results in the reduction of the severity, signs, symptoms or markers of HBV infection or both HBV and HDV infection, or HBV-related disease in patients with such disease or disorder.In this context, "reduction" refers to the clinically significant reduction of such level.Reduction can be, for example, at least 80%, 85%, 90%, 95% or 98%, or below detection level.

[0228] In some embodiments, the effectiveness of the methods herein can be monitored by detecting or monitoring a reduction in symptoms of HBV-related disease, which can be assessed in vitro or in vivo using any method known in the art.

[0229] The effectiveness of disease treatment may be assessed, for example, by measuring disease progression, disease remission, symptom severity, pain reduction, quality of life, the dose of drug required to sustain the treatment effect, the level of disease markers, or other measurable parameters appropriate for the given disease being treated. It is well within the ability of one of ordinary skill in the art to monitor the effectiveness of treatment by measuring any one of such parameters, or any combination of parameters. For example, the effectiveness of treatment of CHB may be assessed, for example, by periodic monitoring of viral load and transaminase levels. Comparison of early and later readings provides the physician with an indication of whether the treatment is effective. It is well within the ability of one of ordinary skill in the art to monitor the effectiveness of treatment by measuring any one of such parameters, or any combination of parameters. In connection with administration of a dsRNA agent or pharmaceutical composition thereof targeting HBV, "effective against" an HBV-related disease indicates that administration in a clinically relevant manner will result in a beneficial effect in at least a statistically significant percentage of patients, such as amelioration of symptoms, cure, reduction of disease, extension of life span, improvement in quality of life, or other effect generally recognized as favorable by physicians knowledgeable in the treatment of HBV infection or HBV-related disease and related causes.

[0230] Treatment effect is evident when there is a clinically significant improvement in one or more parameters of disease state, or when there is no worsening or otherwise expected symptoms.As an example, at least 50%, preferably at least 70% or more favorable changes in measurable parameters of disease may indicate effective treatment.The efficacy of a given dsRNA substance drug or its formulation can also be determined using experimental animal models for a given disease, as known in the art.When using experimental animal models, the efficacy of treatment is demonstrated when a statistically significant reduction in signs or symptoms is observed.

[0231] Administration of a dsRNA agent can, for example, reduce the presence of serum or liver HBV cccDNA in the patient's cells, tissues, blood, urine or other compartments, the presence of serum or liver HBV antigens, such as HBsAg or HBeAg; or normalized ALT or AST levels, toward or to the upper limit of normal for clinical laboratory values, by at least 70%, 75%, 80%, 85%, 90%, 95%, or to below the level of detection of the assay.

[0232] Administration of a dsRNA agent can, for example, cause the presence of serum or liver anti-HBV antibodies, e.g., anti-HBsAg antibodies, in the patient's cells, tissues, blood or other compartments to become detectable or increase the presence by at least 80%, 85%, 90%, 95% or more, or cause the antibodies to become detectable where none were detected prior to treatment.

[0233] Due to the inhibitory effect of HBV expression, in some embodiments, the compositions herein or pharmaceutical compositions prepared therefrom can improve quality of life.

[0234] Subjects who would benefit from reducing or inhibiting HBV gene expression are those with HBV infection or an HBV-related disease or disorder described herein.

[0235] Treatment of subjects who would benefit from reducing or inhibiting HBV gene expression includes therapeutic and prophylactic treatments.

[0236] The present specification further provides methods and uses of the dsRNA agents or pharmaceutical compositions thereof for treating subjects who would benefit from reducing or inhibiting HBV gene expression, e.g., subjects with HBV-related diseases, in combination with other pharmaceutical agents or other therapies, e.g., known pharmaceutical agents or known therapies, e.g., those currently used to treat these disorders.

[0237] For example, in some embodiments, dsRNA agents targeting one or more HBV genes are administered in combination with agents useful for treating HBV-related diseases, for example, as described herein.For example, suitable additional therapeutic agents and therapies for treating subjects who may benefit from a reduction in HBV expression, such as subjects with HBV-related diseases, include dsRNA agents targeting different parts of the HBV genome, antiviral agents, a nucleotide analogs, nucleoside analogs, reverse transcriptase inhibitors (e.g., tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine, clevudine, ritonavir, dipivoxil, lobucavir, famvir, FTC, N-acetyl-cysteine ​​(NAC), PC1323, theradigm-HBV, thymosin-alpha, and ganciclovir), immunostimulatory agents, and the like. These include substances (e.g., pegylated interferon alpha 2a (PEG-IFN-α2a), interferon alpha-2b, recombinant human interleukin-7, and toll-like receptor 7 (TLR7) agonists), therapeutic vaccines (e.g., GS-4774, DV-601, and TG1050), viral entry inhibitors (e.g., Myrcludex), oligonucleotides that inhibit the secretion or release of HbsAg (e.g., REP9AC), capsid inhibitors (e.g., Bay41-4109 and NVR-1221), ccc DNA inhibitors (e.g., IHVR-25), or other therapeutic substances or manipulations to treat HBV-related disease, such as liver transplantation or chemotherapy, or a combination of any of the above.

[0238] The subject who receives the dsRNA agent of the present invention is also administered one or more other therapeutic agents that function by non-RNAi mechanism and are useful for treating HBV infection.The exemplary therapeutic agents that can be used in combination with the therapy of the present invention include, for example, immune modulators that stimulate immune system by enhancing T cell helper activity, maturing B lymphocytes, inhibiting T cell suppressor, and HLA type I expression.Suitable immune modulators include interferons that have various properties, including antiviral effect, immunomodulatory effect, and antiproliferative effect.

[0239] For example, the current treatment for chronic hepatitis B is interferon therapy, which is administered to subjects with confirmed HBV infection for at least six months, elevated liver enzymes (AST and ALT), and actively dividing virus in their blood (HBeAg or HBV DNA positive test). Interferon-α therapy produces long-term, sustained remission of the disease in approximately 35% of chronic hepatitis B individuals, with normalization of liver enzymes and disappearance of the three markers of active infection (HBeAg, HBV DNA, and HBsAg). Subjects with acute HBV infection, end-stage cirrhosis, or other significant medical problems are generally not treated with interferon.

[0240] Furthermore, interferon therapy for patients with HBV-related cirrhosis significantly reduces the rate of hepatocellular carcinoma (HCC), especially in patients with high amounts of serum HBV DNA. In patients with HBeAg-positive compensated cirrhosis, virologic and biochemical remission after interferon therapy is associated with improved survival. In patients with chronic HBV infection, clearance of HBeAg after treatment with interferon-α is associated with improved clinical outcomes. The standard treatment duration is considered to be 16 weeks. Patients who exhibit low levels of viral replication at the end of the standard regimen benefit most from long-term treatment.

[0241] In some embodiments, the methods herein include administering a reverse transcriptase inhibitor to a subject with HBV infection or an HBV-related disease. In some embodiments, the methods herein include administering a reverse transcriptase inhibitor and an immunostimulatory agent to a subject with HBV infection or an HBV-related disease.

[0242] The dsRNA agent and the additional therapeutic agent or treatment may be administered at the same time or in the same combination, e.g., parenterally, or the additional therapeutic agent may be administered as part of a separate composition, may be administered at a separate time, or may be administered by other methods known in the art or described herein.

[0243] The present specification also provides a method for reducing or inhibiting HBV expression in a cell using a dsRNA agent described herein or a composition containing the dsRNA agent described herein. In yet another embodiment, the present specification provides a use of a dsRNA agent described herein or a composition containing the dsRNA agent described herein for the manufacture of a therapeutic medicament for reducing or inhibiting HBV gene expression in a cell. In yet another embodiment, the present specification provides a use of a dsRNA agent described herein or a composition containing the dsRNA agent described herein for the manufacture of a medicament for reducing or inhibiting HBV replication in a cell. In yet another embodiment, the present specification provides a use of a dsRNA agent described herein or a composition containing the dsRNA agent described herein for the manufacture of a medicament for reducing or inhibiting HBV replication in a cell. The method and use include contacting a cell with a dsRNA agent as disclosed herein and maintaining the cell for a sufficient time to obtain degradation of the mRNA transcript of the HBV gene, thereby inhibiting the expression of the HBV gene in the cell or inhibiting HBV replication.

[0244] In the above methods and uses, the cells may be contacted in vitro or in vivo, i.e. the cells may be within a subject.

[0245] The cell suitable for treatment with the method disclosed herein can be any cell expressing HBV gene, for example, a cell infected with HBV, a cell containing an expression vector containing a part of HBV genome or HBV gene, or a transgenic mouse expressing HBV gene.The cell suitable for use in the method and use disclosed herein can be a mammalian cell, for example, a primate cell (human cell or non-human primate cell, such as monkey cell or chimpanzee cell) or a non-primate cell (mouse cell, rat cell, or other mammalian cell).In certain embodiments, the cell is a cell that can be infected with HBV.In certain embodiments, the cell is a human cell, for example, a human hepatocyte.

[0246] HBV gene expression can be inhibited in cells by at least 80%, 85%, 90%, 95% or more, eg, to below the detection level of the assay.

[0247] HBV replication may be inhibited in the cells by at least 80%, 85%, 90%, 95% or more, eg, to below the detection level of the assay.

[0248] The in vivo method and use disclosed herein may include administering a composition containing a dsRNA agent comprising a nucleotide sequence that is complementary to at least a portion of the RNA transcript of the HBV gene of the mammal to be treated. When the organism to be treated is a human, the composition may be administered by any means known in the art, including but not limited to subcutaneous, intravenous or intramuscular administration. In some embodiments, the composition is administered by subcutaneous injection. In some embodiments, the dsRNA agent is formulated to administer the entire dose as a single injection. In some embodiments, the present disclosure provides compositions that are formulated for organ-specific (e.g., hepatic) intra-arterial, intratumoral, intradermal, intravitreal injection, ocular topical, ocular (eye drops), spray, ocular topical or other topical route, suppository, or oral administration.

[0249] In one embodiment, the present specification also provides a method for inhibiting the expression of HBV gene in mammals, such as humans.The present specification also provides a composition comprising the dsRNA material that targets the HBV gene in mammalian cells, for use in inhibiting the expression of HBV gene in mammals.In another embodiment, the present specification provides the use of the dsRNA material that targets the HBV gene in mammalian cells in the manufacture of therapeutic medicaments for inhibiting the expression of HBV gene in mammals.

[0250] The methods and uses include administering to a mammal, e.g., a human, a composition comprising a dsRNA agent that targets an HBV gene in cells in the mammal, and maintaining the mammal for a sufficient time to obtain degradation of mRNA transcripts of the HBV gene, thereby inhibiting expression of the HBV gene in the mammal.

[0251] In certain embodiments, gene expression reduction can be evaluated in the peripheral blood sample of the subject administered with dsRNA material by any method known in the art, for example, qRT-PCR as described herein.Protein production reduction can be evaluated by any method known in the art and by methods described herein, for example, ELISA or Western blotting.Clinically accepted methods for measuring gene and protein expression levels are appropriately used.In certain embodiments, liver puncture biopsy sample serves as tissue material for monitoring HBV gene or protein expression reduction.In some other embodiments, blood sample serves as tissue material for monitoring HBV gene or protein expression reduction.

[0252] In some embodiments, validation of RISC medicinal cleavage of the target in vivo following administration of the dsRNA agent is performed by 5'-RACE or modifications of protocols known in the art (Lasham A et al., (2010) Nucleic Acid Res., 38 (3) p-e19) (Zimmermann et al. (2006) Nature 441: 111-4).

[0253] This invention is further illustrated by the following examples which should not be construed as limiting. The present disclosure relates, for example, to the following: [Section 1] A double-stranded ribonucleic acid (dsRNA) material comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand being 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16), 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18), 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20), 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23), 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24), 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25), or 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO:28): [wherein a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; dA, dC, dG and dT are 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxythymidine-3'-phosphate, respectively; (Agn) is adenosine glycol nucleic acid (GNA); and s is a phosphorothioate linkage. A dsRNA agent comprising a modified nucleotide sequence as shown in [Section 2] The sense strand has the modified nucleotide sequence: 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO:29). [wherein a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; and s is a phosphorothioate linkage. 2. The dsRNA agent of claim 1, comprising: [Section 3] The antisense strand and the sense strand are (a) 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (b) 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (c) 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (d) 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (e) 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (f) 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); or (g) 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29): [wherein a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; dA, dC, dG and dT are 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxythymidine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); and s is a phosphorothioate linkage. 2. The dsRNA agent of claim 1, comprising a modified nucleotide sequence as shown in [Section 4] 4. The dsRNA agent of any one of paragraphs 1 to 3, wherein at least one strand comprises a 3' overhang of at least one nucleotide. [Section 5] 4. The dsRNA agent of any one of paragraphs 1 to 3, wherein at least one strand comprises a 3' overhang of two nucleotides. [Section 6] 6. The dsRNA agent of any one of claims 1 to 5, wherein the double-stranded region is 19 to 21 nucleotide pairs in length. [Section 7] 7. The dsRNA agent of any one of paragraphs 1 to 6, wherein each strand independently has from 19 to 23 nucleotides. [Section 8] The dsRNA agent of paragraph 7, wherein each strand independently has between 19 and 21 nucleotides. [Section 9] 9. The dsRNA agent of any one of paragraphs 1 to 8, further comprising a ligand. [Section 10] The dsRNA agent of paragraph 9, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent. [Section 11] 11. The dsRNA agent of paragraph 9 or 10, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative. [Section 12] The ligand has the formula: [C1] TIFF0007625512000021.tif64102 12. The dsRNA agent of claim 11, wherein the dsRNA agent is [Section 13] formula: [C2] TIFF0007625512000022.tif70117 "Wherein, X is O or S" 13. The dsRNA agent of claim 12, wherein the dsRNA agent is conjugated to a ligand, [Section 14] The dsRNA agent of paragraph 13, wherein X is O. [Section 15] The antisense strand is 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16), 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18), 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20), 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23), 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24), 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25), or 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO:28): "In the sequence, a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; dA, dC, dG and dT are 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxythymidine-3'-phosphate, respectively; (Agn) is adenosine glycol nucleic acid (GNA); and s is a phosphorothioate linkage." 2. The dsRNA agent of claim 1, comprising a modified nucleotide sequence represented by: [Section 16] The antisense strand and the sense strand are (a) 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); (b) 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); (c) 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); (d) 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); (e) 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); (f) 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); or (g) 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10): "In the sequence, a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; dA, dC, dG and dT are 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxythymidine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.” 2. The dsRNA agent of claim 1, comprising a modified nucleotide sequence represented by: [Section 17] The antisense and sense strands were 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); "In the sequence, a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; dA, dC, dG and dT are 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxythymidine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.” 2. The dsRNA agent of claim 1, comprising a modified nucleotide sequence represented by: [Section 18] 18. The dsRNA agent according to any one of items 1 to 17, wherein the dsRNA agent inhibits expression of Hepatitis B Virus (HBV) in a cell. [Section 19] 19. A cell comprising a dsRNA agent according to any one of items 1 to 18. [Section 20] 20. A pharmaceutical composition comprising a dsRNA agent according to any one of items 1 to 19 and a pharmaceutical excipient. [Section 21] A method for inhibiting Hepatitis B virus (HBV) gene expression in a cell, comprising contacting the cell with a dsRNA substance according to any one of items 1 to 18, or the pharmaceutical composition according to item 20, thereby inhibiting HBV gene expression in the cell. [Section 22] 22. The method of claim 21, wherein HBV gene expression is inhibited by at least 80%, 90%, 95% or 98%, or to below the detection level of the assay method. [Section 23] A method for inhibiting the replication of Hepatitis B virus (HBV) in a cell, comprising contacting the cell with a dsRNA agent according to any one of items 1 to 18, or with the pharmaceutical composition according to item 20, thereby inhibiting the replication of HBV in a cell. [Section 24] 24. The method of claim 23, wherein the replication of HBV in the cell is inhibited by at least 80%, 90%, 95% or 98%, or to below the detection level of the assay method. [Section 25] 25. The method according to any one of items 21 to 24, wherein the cell is within a subject. [Section 26] 26. The method of claim 25, wherein the subject is a human. [Section 27] The method of claim 26, wherein the subject is suffering from an HBV-related disease. [Section 28] 25. The method according to any one of items 21 to 24, wherein the cell is in vitro. [Section 29] A method for reducing the level of Hepatitis B virus (HBV) antigen in a subject infected with HBV, comprising administering to said subject a therapeutically effective amount of a dsRNA agent according to any one of items 1 to 18, or a pharmaceutical composition according to item 20, thereby reducing the level of HBV antigen in the subject. [Section 30] 30. The method of claim 29, wherein the HBV antigen is HBsAg. [Section 31] 30. The method of claim 29, wherein the HBV antigen is HBeAg. [Section 32] The method of claim 29, wherein the antigen is measured in serum from the subject. [Section 33] 30. The method of claim 29, wherein the subject is HBeAg positive. [Section 34] The method of claim 29, wherein the subject is HBeAg negative. [Section 35] 35. The method of any one of clauses 29 to 34, wherein HBV antigen levels are reduced in serum by at least 1 log10, at least 2 log10, at least 3 log10 or at least 4 log10, or below the detection level of the assay. [Section 36] A method for reducing the viral load of Hepatitis B virus (HBV) in a subject infected with HBV, comprising administering to said subject a therapeutically effective amount of a dsRNA agent according to any one of items 1 to 18, or the pharmaceutical composition according to item 20, thereby reducing the viral load of HBV in the subject. [Section 37] The method of claim 36, wherein the HBV viral load is measured in serum from the subject. [Section 38] 38. The method of claim 36 or 37, wherein the subject is HBeAg positive. [Section 39] 38. The method of claim 36 or 37, wherein the subject is HBeAg negative. [Section 40] 40. The method of any one of clauses 36 to 39, wherein the HBV viral load is reduced in serum by at least 1 log10, at least 2 log10, at least 3 log10 or at least 4 log10, or to below the detection level of the assay. [Section 41] A method of treating a subject having a Hepatitis B virus (HBV) infection, comprising administering to the subject a therapeutically effective amount of a dsRNA agent according to any one of items 1 to 18, or a pharmaceutical composition according to item 20, thereby treating the subject. [Section 42] A method of treating a subject having a Hepatitis B virus (HBV)-related disorder, comprising administering to the subject a therapeutically effective amount of a dsRNA agent according to any one of items 1 to 18, or a pharmaceutical composition according to item 20, thereby treating the subject. [Section 43] 43. The method of claim 42, wherein the HBV-related disorder is chronic hepatitis and the subject is HBeAg positive. [Section 44] 43. The method of claim 42, wherein the HBV-related disorder is chronic hepatitis and the subject is HBeAg negative. [Section 45] 45. The method of any one of paragraphs 21 to 44, wherein the dsRNA agent is administered to the subject at a dose of 0.01 mg / kg to 10 mg / kg, 0.5 mg / kg to 50 mg / kg, or 3 mg / kg to 10 mg / kg. [Section 46] 46. ​​The method of claim 45, wherein the dsRNA agent is administered to the subject at a dose of 3 mg / kg to 10 mg / kg. [Section 47] 45. The method of any one of paragraphs 21 to 44, wherein the dsRNA agent is administered to the subject at a fixed dose of 50 mg to 200 mg or 50 mg to 900 mg. [Section 48] 48. The method of any one of paragraphs 21 to 47, wherein the dsRNA agent is administered subcutaneously to the subject. [Section 49] 49. The method of any one of paragraphs 21 to 48, wherein the dsRNA agent is administered to the subject in two or more doses. [Section 50] 50. The method of any one of paragraphs 21 to 49, wherein the dsRNA agent is administered to the subject once a month, once every two months, or once every three months. [Section 51] 50. The method of any one of paragraphs 21 to 49, wherein the dsRNA agent is administered to the subject no more than once a month. [Section 52] 52. The method of any one of paragraphs 21 to 51, further comprising administering to the subject one or more additional therapeutic agents. [Section 53] 53. The method of claim 52, wherein the additional therapeutic agent is an antiviral agent, a reverse transcriptase inhibitor, an immunostimulant, a therapeutic vaccine, a viral entry inhibitor, an oligonucleotide that inhibits secretion or release of HBsAg, a capsid inhibitor or a covalently closed circular (ccc) HBV DNA inhibitor, or any combination thereof. [Section 54] 53. The method of claim 52, wherein the additional therapeutic agent is a reverse transcriptase inhibitor. [Section 55] 53. The method of claim 52, wherein the additional therapeutic agents are a reverse transcriptase inhibitor and an immunostimulant. [Section 56] 56. The method of claim 54 or 55, wherein the reverse transcriptase inhibitor is tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine or AGX-1009. [Section 57] 56. The method of claim 55, wherein the immunostimulant is pegylated interferon alpha 2a (PEG-IFN-α2a), interferon alpha-2b, recombinant human interleukin-7 or a Toll-like receptor 7 (TLR7) agonist. [Section 58] A pharmaceutical composition for carrying out any of the methods according to any of items 21 to 57, comprising a dsRNA agent according to any of items 1 to 18 and a pharmaceutical excipient. [Section 59] 21. The dsRNA agent of any one of clauses 1 to 18, or the pharmaceutical composition of clause 20, for use in treating Hepatitis B Virus (HBV) infection in a subject. [Section 60] 21. The dsRNA agent of any one of clauses 1 to 18, or the pharmaceutical composition of clause 20, for use in treating a Hepatitis B Virus (HBV)-related disorder in a subject. [Section 61] 61. The dsRNA agent or pharmaceutical composition for use according to clause 60, wherein the HBV-related disorder is chronic hepatitis and the subject is HBeAg positive. [Section 62] 61. The dsRNA agent or pharmaceutical composition for use according to clause 60, wherein the HBV-related disorder is chronic hepatitis and the subject is HBeAg negative. [Section 63] 63. A dsRNA agent or pharmaceutical composition for use according to any one of clauses 59 to 62, wherein one or more additional therapeutic agents are being or have been administered to the subject. [Section 64] 64. The dsRNA agent or pharmaceutical composition for use according to claim 63, wherein the additional therapeutic agent is 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 or a covalently closed circular (ccc) HBV DNA inhibitor, or any combination thereof. [Section 65] 64. The dsRNA agent or pharmaceutical composition for use according to paragraph 63, wherein the additional therapeutic agent is a reverse transcriptase inhibitor. [Section 66] 64. The dsRNA agent or pharmaceutical composition for use according to paragraph 63, wherein the additional therapeutic agent is a reverse transcriptase inhibitor and an immunostimulatory agent. [Section 67] 67. The dsRNA agent or pharmaceutical composition for use according to clause 65 or 66, wherein the reverse transcriptase inhibitor is tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine or AGX-1009. [Section 68] 67. The dsRNA agent or pharmaceutical composition for use according to clause 66, wherein the immunostimulatory agent is pegylated interferon alpha 2a (PEG-IFN-α2a), interferon alpha-2b, recombinant human interleukin-7 or a Toll-like receptor 7 (TLR7) agonist. [Section 69] 58. Use of a dsRNA agent according to any one of clauses 1 to 18 or a pharmaceutical composition according to clause 20 for carrying out any method according to any one of clauses 21 to 57. [Section 70] 58. Use of a dsRNA agent according to any one of clauses 1 to 18 or a pharmaceutical composition according to clause 20 for the preparation of a medicament for carrying out any method according to any one of clauses 21 to 57. EXAMPLES

[0254] Example 1 Synthesis of DSRNA material Reagent Suppliers If the source of a reagent is not specifically indicated herein, such reagents may be obtained from any molecular biology reagent supplier of quality / purity standards for molecular biology applications.

[0255] Design of dsRNA agents As described in WO / 2016 / 077321, the selection of dsRNA designs targeting HBV was based on two fundamental factors: a) the potency of the material to match almost perfectly and cover more than 90% of the fragments of the numerous published HBV sequences of all known genotypes (A-H), and b) the desire to use it. The coordinates for selecting the RNA material were determined against the NCBI HBV reference genome sequence NC_003977.1 (GenBank accession number GI:21326584 (SEQ ID NO:1)). A first set of RNA materials containing structure-activity modifications, including various 2'-O-methyl and 2'-fluoro substitution patterns, centered on two adjacent regions of the HBV genome encoding the surface antigen (HbSAg) and the HBV polymerase, were designed, synthesized, and screened in vitro. A second set of agents was also designed, synthesized and screened in vitro targeting additional regions in the HBV genome, specifically positions 1581-1599 of SEQ ID NO: 1, the region coding for the HbSAg, polymerase and X genes. Selected sequences were further chemically modified and tested. These duplex designs are described in WO2016 / 077321, the entire contents of which are incorporated herein by reference, with detailed listings of unmodified HBV sense and antisense strand nucleotide sequences in Tables 3, 6, 12, 22 and 25 of WO2016 / 077321 and detailed listings of modified HBV sense and antisense strand nucleotide sequences in Tables 4, 7, 13, 23 and 26 of WO2016 / 077321. Results from screening assays performed with these agents are also described therein.

[0256] These studies demonstrated that the duplex AD-66810 has an antisense strand with the modified nucleotide sequence: 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 13) and a sense strand with the modified nucleotide sequence: 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29) [wherein a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively; and s is a phosphorothioate linkage; and wherein an N-acetylgalactosamine moiety, N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol ((also known as Hyp-(GalNAc-alkyl)3) or referred to herein as L96), is covalently attached to the 3'-end of the sense strand.

[0257] Further studies described herein have identified potent and specific dsRNA agent molecules based on the previously identified sequence AD-66810, which targets the X transcript of human Hepatitis B virus (HBV; U95551). To this end, a series of chemically modified dsRNA agents were designed, synthesized, and tested for activity in vitro using Dual-Luc reporter-based assays and HepG2.2.15 cell lines. All compounds were conjugated to a ternary N-acetylgalactosamine (GalNac) ligand (L96) covalently attached to the 3' end of their sense strand.

[0258] Synthesis of dsRNA material HBV sense and antisense strand sequences were synthesized on a 1 μmol scale on a Mermade 192 synthesizer (BioAutomation) using solid support-mediated phosphoramidite chemistry. The solid supports were controlled pore glass (500A) or universal supports (AM biochemical) loaded with custom GalNAc ligands. Ancillary synthesis reagents, 2'-F and 2'-O-methyl RNA, and deoxyphosphoramidites were obtained from Thermo-FisherTM (Milwaukee, WI) and Hongene (China). 2'F2'-O-methyl, GNA (glycol nucleic acid), 5'-phosphate and abasic modifications were introduced using the corresponding phosphoramidites. Synthesis of 3' GalNAc-conjugated single strands was performed on GalNAc-modified CPG supports. Custom CPG universal solid supports were used for the synthesis of antisense single strands. Coupling times for all phosphoramidites (100 mM in acetonitrile) were 5 min, and 5-ethylthio-1H-tetrazole (ETT) was used as the activator (0.6 M in acetonitrile). Phosphorothioate linkages were generated using a 50 mM solution of 3-((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Wilmington, MA, USA) in anhydrous acetonitrile / pyridine (1:1 v / v). Oxidation times were 3 min. All sequences were synthesized with final removal of the DMT group ("DMT off").

[0259] After completion of solid-phase synthesis, oligoribonucleotides were cleaved from the solid support and deprotected with 200 μL methylamine water-soluble reagent in a sealed 96-deep-well plate for 20 min at 60 °C. At the end of the cleavage and deprotection steps, the synthesis plate was brought to room temperature and precipitated by adding 1 mL of acetontile:ethanol mixture (9:1). The plate was cooled at -80 °C for 2 h and the supernatant was carefully decanted using a multichannel pipette. The oligonucleotide pellet was resuspended in 20 mM NaOAc buffer and desalted using a 5 mL HiTrapTM size exclusion column (GE HealthcareTM) on an AKTA purification system equipped with an A905 autosampler and a Frac 950 fraction collector. The desalted samples were collected in a 96-well plate. Samples from each sequence were analyzed by LC-MS to confirm identity and quantified by UV (260 nm) and IEX chromatography to measure the purity of a select set of samples.

[0260] Annealing of single strands was performed on a Tecan liquid handling robot. Equimolar mixtures of sense and antisense single strands were combined and annealed in a 96-well plate. After combining the complementary single strands, the 96-well plate was tightly sealed and heated in an oven at 100°C for 10 minutes and allowed to slowly come to room temperature over a period of 2-3 hours. The concentration of each duplex was normalized to 1 μM in 1XPBS.

[0261] Abbreviations for the modified nucleotide monomers disclosed herein are provided in Table 1. Table 2 shows the AD-66810 and HBV dsRNA agents synthesized using the methods described above.

[0262] Table 1. Nucleotide monomer abbreviations used in modified nucleic acid sequence representations Unless otherwise specified, these monomers, when present in an oligonucleotide, will be understood to be linked to each other by 5'-3'-phosphodiester bonds. [Table 1-1] [Table 1-2]

[0263] [Table 2]

[0264] Example 2 In vitro screening of DSRNA substance duplexes Dual-Glo® Luciferase Assay: Cos7 cells (ATCC®, Manassas, VA) were grown to near confluence in DMEM (ATCC) supplemented with 10% FBS at 37°C in a 5% CO2 atmosphere and then detached from the plates by trypsinization. Five μl of RNA duplex and 5 μl of psiCHECK2-HBV (or psiCHECK2-HBV genotypes A, C, E, or F) plasmid per well were added to 5 μl Opti-MEM® plus 0.1 μl Lipofectime. TM RNAiMax (Invitrogen TM Cos7 cells were transfected with the double-stranded material and psiCHECK2-HBV (an expression vector containing a nucleotide sequence encoding a portion of the matching wild-type HBV sequence; SEQ ID NO:5), or with the double-stranded material and psiCHECK2 vector expressing the nucleotide sequence of HBV genotypes A, C, E, or F (SEQ ID NO:6, 7, 8, or 9, respectively) by adding 100 μl of ...

[0265] 28 hours after transfection of dsRNA material and psiCHECK2 plasmid, Firefly (transfection control) and Rinella (fused to HBV target sequence) luciferase were measured. First, the medium was removed from the cells. Then, Firefly luciferase activity was measured by adding 20 μl of Dual-Glo® Luciferase Reagent equal to the medium volume to each well and mixed. The mixture was incubated at room temperature for 30 minutes, and then luminescence (500 nm) was measured on a Spectramax® (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 20 μl of room temperature Dual-Glo® Stop&Glo® Reagent to each well, and the plate was incubated for 10-15 minutes, after which luminescence was measured again to determine the Renilla luciferase signal. Dual-Glo® Stop&Glo® Reagent stops the firefly luciferase signal and maintains the luminescence of the Renilla luciferase reaction. dsRNA agent activity was determined by normalizing the Renilla (HBV) signal to the Firefly (control) signal within each well. The magnitude of dsRNA agent activity was then assessed relative to cells transfected with the same vector but not treated with dsRNA agent or treated with a non-targeting dsRNA agent. All transfections were performed with n=2 or more.

[0266] The results of these assays, using the agents listed in Table 2, are provided in Table 3.

[0267] HepG2.2.15 and PLC in vitro screening: HepG2.2.15 and PLC (human hepatoma cells; ATCC® CRL-8024) cells were grown to near confluence at 37° C. in a 5% CO2 atmosphere in DMEM or EMEM medium (ATCC), respectively, supplemented with 10% FBS (ATCC), and then detached from the plate by trypsinization. 5 μl of dsRNA material duplex was added per well to a 96-well plate, 14.8 μl Opti-MEM® plus 0.2 μl Lipofectamine per well. TM RNAiMax (Invitrogen TM Reverse transfection was performed by adding 100 μl of 100% PBS (Cat # 13778-150, Carlsbad CA) and incubating for 15 min at room temperature. Then, 80 μl of complete growth medium without antibiotics containing 2x104 HepG2.2.15 or PLC cells was added. Cells were incubated for 24, 48 and 72 hours before RNA purification.

[0268] The results of these assays using the substances listed in Table 2 are provided in Table 4.

[0269] Total RNA isolation using MagMAX-96 Total RNA Isolation Kit (Applied Biosystem, Forer City CA, part #: AM1830): Cells were harvested and lysed in 140 μl of lysis / binding solution, then mixed for 1 min at 850 rpm using an Eppendorf TMTermomixer (mixing speed was the same throughout the process). 20 μl of magnetic beads and lysis / binding enhancer mix were added to the cell lysate and mixed for 5 min. The magnetic beads were captured using a magnetic stand, and the supernatant was removed without disturbing the beads. After removing the supernatant, the magnetic beads were washed using wash solution 1 (with isopropanol added) and mixed for 1 min. The beads were captured again and the supernatant was removed. Next, the beads were washed using 150 μl of wash solution 2 (with ethanol added), captured, and the supernatant was removed. Next, 50 μl of DNase mix (MagMax turbo DNase buffer and Turbo DNase) was added to the beads, and these were mixed for 10-15 min. After mixing, 100 μl of RNA rebinding solution was added and mixed for 3 min. The supernatant was removed, the magnetic beads were washed again with 150 μl of washing solution 2, mixed for 1 min, and the supernatant was completely removed. The magnetic beads were mixed and dried for 2 min, after which the RNA was eluted with 50 μl of water.

[0270] cDNA synthesis using the ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat #4368813): 2 μl 10X buffer, 0.8 μl 25X dNTPs, 2 μl random primers, 1 μl reverse transcriptase, 1 μl RNase inhibitor master mix, and 3.2 μl H2O per reaction were added to 10 μl total RNA. cDNA was generated using a Bio-Rad® C-1000 or S-1000 thermal cycler (Hercules, CA) with the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

[0271] Real-time PCR: 2 μl of cDNA was added to a master mix containing 0.5 μl of human GAPDH TaqMan probe (Applied Biosystems Cat # 4319413E), 1 μl of SORF2 specific TaqMan® probe and 5 μl of LightCycler480 probe master mix (Roche Cat # 04887301001) per well of a 384-well plate (Roche cat # 04887301001). Real-time PCR was performed on a LightCycler480 Real-time PCR System (Roche) using the DDCt(RQ) assay. To calculate relative fold changes, real-time data was analyzed using the DDCt method and normalized to assays performed on AD-1955-transfected or mock-transfected cells.

[0272] To calculate relative fold changes, real-time data was analyzed using the DDCt method and normalized to assays performed in cells transfected with a non-targeting control dsRNA agent.

[0273] Example 3 Transfection of GALNAC-conjugated HBV-targeting DSRNA agents in the DUAL-LUC system The silencing of HBV mRNA after transfection with each conjugated dsRNA agent is shown in Table 3. Each dsRNA agent was tested by transfection with 50nM, 10nM and 0.1nM dsRNA agent concentrations in Cos7 cells. Based on the screening results, 7 of 20 dsRNA agents showed the same efficacy as AD-66810 parent. [Table 3]

[0274] Example 4 Transfection of GALNAC-conjugated HBV-targeting DSRNA agents in HEPG2.2.15 cells Silencing of HBV mRNA (PORF1 and SORF2 mRNA determined using forward and reverse primers and TaqMan probes) in HepG2.2.15 cells after transfection with each conjugated dsRNA agent is shown in Table 4. Overall, more robust silencing of PORF1 and SORF2 viral transcripts was observed in HepG2.2.15 cells compared to the silencing observed in the Dual-Luc overexpression system, with the 10 dsRNA agents having comparable potency to the parent molecule AD-66810. Similar dsRNA agent activity was observed against PORF1 and SORF2 mRNA, which was expected since the target sites of the dsRNA agents are present in both transcripts. [Table 4]

[0275] Based on the above studies, the double-stranded AD-81890 was selected for further analysis.

[0276] Example 5 Pharmacological evaluation of AD-81890 following a single subcutaneous injection in an adeno-associated HBV mouse model The pharmacology of AD-81890 was assayed in an adeno-associated HBV (HBV-AAV) mouse model. AAV8-HBV (SignaGen Laboratories) was diluted in 1x PBS to a final concentration of 2x1012 GC / mL. Male C57BL / 6 mice aged 6-8 weeks were intravenously injected with 2x1011 GC / mouse in a fixed volume of 100 μL via the lateral tail vein.

[0277] AD-81890 was diluted in sterile 1× PBS and administered in a variable volume of 10 μl / g. Animals were administered a single SC dose of AD-81890 at 0.3 mg / kg, 1 mg / kg, or 3 mg / kg as described in Table 5, and bled via the retro-orbital sinus (ROS) on days −24, −2, 0, 14, 21, 33, 47, 59, and 74 post-dose. [Table 5]

[0278] After collection, blood was allowed to clot for 30 minutes and then spun in a microcentrifuge at 13,000 rpm for 10 minutes at 4° C. Serum was aspirated and stored at −20° C.

[0279] Hepatitis B virus surface antigen protein levels were assessed via ELISA (BioTang, Waltham, MA). Standard curves were generated using US Biologics (Memphis, TN) HBsAg protein. Serum samples were diluted 1:2000 or 1:500 in 1x PBS (Gibco, Gaithersburg, MD) and assessed using the ELISA protocol with minor modifications. Briefly, 50 μL / well of diluted serum or standard was loaded into the plate and incubated for 1 hour at 37°C. After this incubation, 50 μL / well of enzyme conjugate was added to each well and the plate was incubated for 30 minutes at 37°C. The plate was washed three times with 300 μL / well of 1x wash buffer, then blotted until all liquid was removed from the wells, 100 μL / well of substrate was added, and the plate was incubated for 30 minutes at 37°C. Finally, an additional 100 μL / well of stop solution was added and absorbance was measured at a wavelength of 450 nm. If the calculated HBsAg level was below the lower limit of quantification (LLOQ) of the assay, the value was recorded as the LLOQ (i.e., 313 ng / mL).

[0280] The mean ± SD serum HBsAg concentrations after a single subcutaneous (SC) dose of AD-81890 in HBV-AAV mice are shown in Figure 2A. The mean ± SD serum HBsAg levels relative to baseline in HBV-AAV mice are shown in Figure 2B. A single SC injection of AD-81890 at 0.3 mg / kg, 1 mg / kg, or 3 mg / kg potently and sustainably reduced serum HBsAg concentrations in HBV-AAV mice, with a maximal reduction of 92% observed at day 7 in the highest (3 mg / kg) AD-81890 dose group. The maximal reduction level was maintained until day 33 in the highest dose group, after which HBsAg concentrations began to return to baseline (Figure 2B). Intermediate reductions in serum HBsAg concentrations were observed in the 0.3 mg / kg and 1 mg / kg AD081890 dose groups, with a maximal reduction of 23% and 72%, respectively, at day 7. HBsAg levels in both the 0.3 mg / kg and 1 mg / kg AD-81890 treatment groups returned to baseline levels by the end of the study (day 74).

[0281] Example 6 Pharmacological evaluation of AD-81890 in an adeno-associated HBV mouse model after multiple subcutaneous injections The pharmacology of AD-81890 was assayed in an adeno-associated HBV (HBV-AAV) mouse model. AAV8-HBV (SignaGen Laboratories) was diluted in 1x PBS to a final concentration of 2x1012 GC / mL. Male C57BL / 6 mice aged 6-8 weeks were intravenously injected with 2x1011 GC / mouse in a fixed volume of 100 μL via the lateral tail vein.

[0282] AD-66810 and AD-81890 were each diluted in sterile 1XDPBS and administered at a variable volume of 10ul / g. Animals were administered AD-66810 at 1mg / kg by specific Q2Wx6 or AD-81890 at a single dose of 9mg / kg or multiple doses of 1mg / kg or 3mg / kg by specific Q2Wx6 or QMx3. Serum was collected from animals at multiple time points as described in Table 6. Blood was collected from the retro-orbital sinus as described in Table 6. [Table 6]

[0283] Blood samples were processed and ELISA assays were performed as described in the previous examples. The results are shown in Table 7 below. The results, including standard deviations, are shown in Figure 3. [Table 7]

[0284] Dose-responsive serum HBsAg levels were observed in the AAV8-HBV mouse model following administration of AD-81890. A maximum HBsAg reduction of approximately 2.7 log10 was observed following a single dose of AD-81890 at 9 mg / kg or 3 mg / kg q2w x 6. Animals dosed at 3 mg / kg q2w x 6 maintained a >2 log10 HBsAg reduction for a total of approximately 8 weeks.

[0285] These in vivo studies demonstrated that AD-81890 was effective in reducing serum HBsAg in the HBV-AAV mouse model.

[0286] Example 7 Specific off-target analysis of AD-81890 A combination of in silico bioinformatics and in vitro methods were used to assess potential off-target activity of the antisense strand of AD-81890.

[0287] Bioinformatics Custom R and Python scripts were used to design a series of dsRNA agents targeting the "X" gene of Hepatitis B virus (HBV) subtype ayw (GenBank nucleotide ID U95551; NCBI gene ID: 7276; SEQ ID NO: 49). The circular U95551 HBV genome has a length of 3182 bases, and the CDS region of the "X" gene encodes at positions 1376-1840 in the NCBI record. Details of the design and screening methods for dsRNA agents are described above and in WO2016 / 077321.

[0288] Transfection screen of GalNAc-conjugated dsRNA agents for off-target detection in HepG2.2.15 cells To measure off-target inhibition, the response of endogenously expressed transcripts by qPCR was tested in the hepatocyte cell line HepG2.2.15. Cells were transfected in 96-well plates (2 × 104 cells per well) with AD-81890 at concentrations ranging from 50 nM to 5 fM using Lipofectamine RNAiMax (ThermoFisher). After 24 h, MagMAX TM RNA was extracted from cells using the -96 Total RNA Isolation Kit (ThermoFisher) and cDNA was synthesized using the ABI High Capacity cDNA Reverse Transfection Kit (ThermoFisher). Samples were assayed for inhibition of HBV mRNA and potential off-target silencing. For quantification by qPCR, HBV expression was assessed using PORF-1 and SORF-2, two different custom TaqMan assays that recognize different regions of the HBV viral transcript.

[0289] To assess off-target silencing, TaqMan probes specific for each potential off-target (Table 8) were used for quantification. qPCR was performed using a LightCycler 480 real-time PCR machine (Roche). [Table 8]

[0290] To determine the degree of on-target (HBV) and potential off-target gene inhibition, relative RNA levels were determined by normalization to human GAPDH RNA expression from the same samples. Results were compared to a control group of transfected nonspecific dsRNA material, and errors were expressed as standard deviations. The IC50 of AD-81890 was 0.803 nM for the PORF1 transcript target and 0.766 nM for the SORF2 transcript target. No significant target knockdown was observed for SIDT2 and ZBTB2 transcripts, even at the highest concentration of AD-81890.

[0291] The extent of off-target inhibition by AD-81890 was assessed in a dose-response screen of two potential off-targets from endogenously expressed transcripts in HepG2.2.15. AD-81890 did not inhibit expression of SIDT2 or ZBTB2 at any dose tested, but HBV inhibition was dose responsive. Fitting the dose-response data using a four-parameter fit model (XLfit) yielded IC50 values ​​of 803 pM and 766 pM for PORF-1 or SORF-2, respectively.

[0292] Example 8 In vitro analysis of AD-81890 specificity using RNA-SEQ in HEPG2.2.15 cells We used transcriptome-wide changes in expression levels using RNA-Seq to measure the impact of chemical modifications of dsRNA materials comparing AD-66810 and AD-81890 in the HBV-expressing HepG2.2.15 cell line. The AD-66810 and AD-81890 molecules have the same nucleotide sequence but differ by the substitution of a single glycol nucleic acid (GNA) at position 6 from the 5' end of the molecule in the antisense strand (see Table 2).

[0293] HepG2.2.15 cells, a HepG2-derived cell line stably transfected with whole genome HBV, were diluted in culture medium to a final concentration of 187,500 cells / mL and 80 μL was pipetted into a 96-well collagen-coated plate (BD Biocoat, Cat#356407) to give a final concentration of 15,000 cells / well.

[0294] dsRNA agent stocks were diluted in 1XDPBS to a concentration of 1,000nM or 100nM. RNAiMAX (ThermoFisher, Cat#13778150) was diluted in Opti-MEM (ThermoFisher Cat#31985062) to a concentration of 0.3μL RNAiMAX / 10μL Opti-MEM and incubated at room temperature for 5 minutes. After incubation, 10μL / well was added to each well of a 96-well collagen-coated plate (BD Biocoat, Cat#356407) along with 10μL / well of the appropriate dsRNA agent dilution, mixed gently, and incubated at room temperature for 20 minutes. 80μL of the prepared cell suspension was added to each well to give a final cell density of 15,000 cells / well and final dsRNA agent concentrations of 100nM and 10nM. The cells were incubated for 16-22 hours in a 37°C incubator containing 5% CO2. Cells were plated so that each experimental condition had 16 wells, and experiments were performed in duplicate.

[0295] RNA was isolated using ThermoFisher RNAqueous-96 total RNA isolation kit according to the protocol. Briefly, after 16–22 h, the supernatant was aspirated from each well and 100 μL / well of 1× DPBS was added to wash off the remaining medium, then aspirated. 200 μL lysis / binding solution was added to each well in columns 1 and 5 of each plate, pipetted up and down several times, and transferred to subsequent columns such that 4 wells / condition were pooled, ensuring sufficient RNA recovery. This resulted in four replicates per condition. 100 μL of 100% ethanol was added to each well of the culture plate containing the lysate, mixed several times, and transferred to the wells of the filter plate. Samples were washed with the provided wash solution, treated with DNase reagent, and eluted in 100 μL of nuclease-free water by a series of centrifugation steps (1,900 × g, 1 min). RNA concentration was measured with a NanoDrop 8000 spectrophotometer (ThermoFisher).

[0296] The RNA was further treated with TURBO DNase (Ambion). Each RNA sample (≦10 μg RNA / sample) was mixed with 2 μL DNase, 10 μL 10X buffer, and nuclease-free water to a total volume of 100 μL and incubated at 37° C. for 30 min. Following DNase treatment, the RNA was further treated with TURBO DNase (Ambion). Purification was performed using the RNeasy MinElute Cleanup Kit (Qiagen). RNA was eluted in 30 μL of nuclease-free water and RNA concentration was measured with a NanoDrop 8000 spectrophotometer. RNA was stored at -80°C. This RNA was subsequently used for cDNA library preparation using the TruSeq standard total RNA library preparation kit (Illumina) and sequenced on a NextSeq500 desktop sequencer (Illumina), all according to the manufacturer's instructions. Two experimental replicates were performed.

[0297] HepG2.2.15 cells were transfected in quadruplicate with 10 nM or 100 nM AD-66810 or AD-81890 and cultured for 24 h alongside untreated controls. RNA extracted with Purelink RNA kit (ThermoFisher) was used for cDNA library preparation using TruSeq standard total RNA library preparation kit (Illumina) with Ribo-Zero human / mouse / rat for rRNA removal and sequenced on a NextSeq500 desktop sequencer (Illumina), all according to the manufacturer's instructions. A total of 40 samples were pooled per NextSeq 500 / 550 high output v2 (75 cycles) flow cell (Illumina). Experimental repeats were performed twice.

[0298] Raw RNA-Seq read data were filtered with a minimum average quality score of 25 and a minimum residual length of 36 using fastq-mcf. The filtered read data were simultaneously aligned to the human (hg19 / GRCh37) and HBV (GenBank nucleotide ID U95551; NCBI gene ID: 7276) genomes using STAR (version 2.4.2a). Due to the circular structure of the HBV genome, 46 base pairs are repeated at the ends of the linearized version of the HBV sequence, allowing reads to map at the breakpoints. Specific aligned reads mapping to exons were counted by featureCounts (version 1.5.0). All samples had >5M mapped reads. Differential gene expression analysis was performed in R (version 3.4.1) using package DESeq2 (version 1.16.1). Multiple testing correction to obtain adjusted p-values ​​was performed by DESeq2 using the method of Benjamini & Hochberg, 1995.

[0299] MA plots were used to visualize both on-target HBV knockdown and off-target effects. Analysis of GNA chemistry to mitigate global off-target effects was restricted to downregulated genes (log2 fold change < 0). Upregulated genes (log2 fold change > 0) were considered secondary effects. Evaluation of off-target effects was restricted to the lowest (10 nM) dose since near-maximal HBV knockdown was achieved. To compare transcriptomic noise, significantly downregulated (adjusted p-value < 0.05) genes were identified in AD-66810 and / or AD-81890. The degree of downregulation was visualized by box plots of log2 fold change (Figure 4), with statistical differences between AD-66810 and AD-81890 assessed using Welch's two-tailed, two-sample t-test (Table 9). [Table 9]

[0300] A consistent reduction in off-target effects was observed with AD-81890 compared to AD-66810 in both experimental replicates (Figure 4, Table 9). At 10 nM, AD-81890 showed a 52% reduction (single replicate) or a 54% reduction (double replicate) in the mean log2 fold change of significantly downregulated genes compared to AD-66810 (Table 9). At 100 nM, AD-81890 showed a 71% reduction (single replicate) or a 43% reduction (double replicate) in the mean log2 fold change compared to AD-66810 (Table 9). In all cases, the observed reduction in log2 fold change was statistically significant. Thus, AD-81890 had a substantially lower level of transcriptomic noise.

[0301] Example 9 Evaluation of human-specific hepatotoxicity in PXB-mice PXB-mouse is a chimeric mouse (PhoenixBio) with a humanized liver that is highly repopulated with human hepatocytes. The mouse is a urokinase-type plasminogen activator (uPA) / severe combined immunodeficiency (SCID) mouse transplanted with human hepatocytes (humanized liver uPA / SCID mouse) (Mercer et al., Nat. Med. 7:927-933, 2001). The reported humanized liver uPA / SCID mouse has a replacement index (RI), which is the percentage of human hepatocytes in the liver, of more than 70%. The mouse can be used as a model for predicting human drug metabolism, pharmacokinetics, and hepatotoxicity (Naritomi et al., Drug Metab Pharmacokinet. 33:31-39, 2018).

[0302] A study was performed in PBX-mice to compare the hepatotoxicity of AD-66810 and AD-81890. Mice were administered 12 mg / kg, 36 mg / kg or 100 mg / kg of AD-66810, AD-81890 or PBS (control) by subcutaneous injection (n=4 per group) on days 0, 21, 28, 35 and 42. Clinical observations and body weights were performed twice weekly until day 49. Blood was collected twice weekly by retro-orbital bleeding and serum was prepared using routine methods. A terminal bleed was performed and animals were sacrificed by cardiac puncture and exsanguination. Necropsy was performed after exsanguination. Livers were harvested and weighed. Livers were isolated and stored in RNAlater solution (Ambion) before embedding in paraffin, storing in formalin and snap-frozen.

[0303] All animals maintained body weights greater than 80% of initial levels throughout the study period. Furthermore, the lowest arithmetic mean in the compound-treated groups was higher than in the control PBS-treated group.

[0304] Blood human serum albumin levels were monitored throughout the study, and all surviving animals maintained blood h-Alb concentrations above 7.0 mg / ml for the life of the study.

[0305] Liver enzymes were monitored throughout the course of the study. Specifically, ALT, AST, ALP, GGT, TBIL, and TG were monitored throughout the study. Enzyme levels measured on day 49 for mice administered AD-66810, AD-81890, or PBS are shown below in Table 10. Figures 5A and 5B show ALT levels over time following administration of AD-66810 (Figure 5A) or AD-81890 (Figure 5B) compared to administration of PBS. [Table 10]

[0306] In both test compound treatment groups, ALT, AST, ALP and GGT showed increases compared to the control group. Furthermore, dose-dependent changes in ALT, AST and GGT were demonstrated in the AD-66810 treatment group.

[0307] Necropsy revealed no test compound specific findings in any of the groups. There were no obvious changes in relative (liver / body weight) liver weights in compound-treated animals when compared to the control group.

[0308] equivalent weight Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein which are intended to be encompassed by the scope of the following claims.

[0309] While particular embodiments have been illustrated and described, it will be readily understood that the various implementations described above can be combined to provide further embodiments, and that various changes can be made without departing from the spirit and scope of the invention.

[0310] All figures, U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein or set forth in the Application Data Sheet, including U.S. Provisional Patent Application No. 62 / 718,314, filed August 13, 2018, are incorporated herein by reference in their entirety unless otherwise noted. Aspects of the embodiments can be modified where necessary to employ concepts from the various patents, applications, and publications to provide further embodiments.

[0311] These and other modifications can be made to the examples in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present specification.

Claims

1. A double-stranded ribonucleic acid (dsRNA) material comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO:29) and the antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO:16); In the sequence, a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; A ligand is conjugated to the 3' end of the sense strand, said ligand comprising: 【Chemistry 1】 and The dsRNA agent inhibits the expression of Hepatitis B virus (HBV) in cells.

2. 10. The dsRNA agent of claim 1, wherein at least one strand comprises a 3' overhang of at least one nucleotide or a 3' overhang of two nucleotides.

3. 3. The dsRNA agent of claim 1 or 2, wherein the double-stranded region is 19-21 nucleotides in length or each strand independently has 19-23 nucleotides.

4. A dsRNA agent having the formula: 【Chemistry 2】 "Wherein, X is O or S" 4. The dsRNA agent of any one of claims 1 to 3, conjugated to a ligand as shown in

5. 5. The dsRNA agent of claim 4, wherein X is O.

6. A double-stranded ribonucleic acid (dsRNA) material comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand consisting of 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10) and the antisense strand consisting of 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16); In the sequence, a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and The L96 is 【Chemistry 3】 That is, dsRNA material.

7. A cell containing a dsRNA agent according to any one of claims 1 to 6.

8. A pharmaceutical composition comprising a dsRNA agent according to any one of claims 1 to 6, and a pharmaceutical excipient.

9. 9. An in vitro method for inhibiting Hepatitis B Virus (HBV) gene expression or inhibiting Hepatitis B Virus (HBV) replication in a cell, by contacting said cell with a dsRNA agent according to any one of claims 1 to 6, or with a pharmaceutical composition according to claim 8.

10. 9. The dsRNA agent according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 8 for use in a method for reducing the level of Hepatitis B Virus (HBV) antigen or reducing the viral load of HBV in a subject infected with HBV.

11. 11. The dsRNA agent or pharmaceutical composition of claim 10, wherein the HBV antigen is HBsAg or HBeAg.

12. 12. The dsRNA agent or pharmaceutical composition of claim 10 or 11, wherein the subject is HBeAg positive.

13. 12. The dsRNA agent or pharmaceutical composition of claim 10 or 11, wherein the subject is HBeAg negative.

14. 9. The dsRNA agent of any one of claims 1 to 6 or the pharmaceutical composition of claim 8 for use in treating a subject with a Hepatitis B Virus (HBV) infection or a Hepatitis B Virus (HBV) related disease or disorder.

15. 15. The dsRNA agent or pharmaceutical composition of claim 14, wherein the HBV-related disorder is chronic hepatitis and the subject is HBeAg positive.

16. 15. The dsRNA agent or pharmaceutical composition of claim 14, wherein the HBV-related disorder is chronic hepatitis and the subject is HBeAg negative.

17. 17. The dsRNA agent or pharmaceutical composition of any one of claims 10 to 16, wherein the dsRNA agent is administered to the subject at a dose of 0.01 mg / kg to 10 mg / kg, 0.5 mg / kg to 50 mg / kg or 3 mg / kg to 10 mg / kg or at a fixed dose of 50 mg to 200 mg or 50 mg to 900 mg.

18. The dsRNA agent or pharmaceutical composition of any one of claims 10 to 17, wherein the dsRNA agent is administered subcutaneously to the subject.

19. The dsRNA agent or pharmaceutical composition of any one of claims 10 to 18, wherein the dsRNA agent is administered to a subject in two or more doses.

20. 20. The dsRNA agent or pharmaceutical composition of any one of claims 10-19, wherein the dsRNA agent is administered to a subject once a month, once every two months, or once every three months, or no more than once a month.

21. 21. The dsRNA agent or pharmaceutical composition of any one of claims 10 to 20, wherein the subject is administered or has been administered one or more additional therapeutic agents.

22. 22. The dsRNA agent or pharmaceutical composition of claim 21, wherein the additional therapeutic agent is an antiviral agent, a reverse transcriptase inhibitor, an immunostimulatory agent, a therapeutic vaccine, a viral entry inhibitor, an oligonucleotide that inhibits the secretion or release of HbsAg, a capsid inhibitor or a covalently closed circular (ccc) HBV DNA inhibitor, or any combination thereof.

23. 23. The dsRNA agent or pharmaceutical composition of claim 22, wherein the additional therapeutic agent is tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, pegylated interferon alpha 2a (PEG-IFN-α2a), interferon alpha-2b, recombinant human interleukin-7 or a toll-like receptor 7 (TLR7) agonist.

24. The dsRNA agent or pharmaceutical composition according to any one of claims 14 to 23, wherein the HBV-related disease is Hepatitis D Virus (HDV) infection.

25. 24. The dsRNA agent or pharmaceutical composition of any one of claims 14 to 23, wherein the HBV related disease is delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end stage liver disease; or hepatocellular carcinoma.

26. A double-stranded ribonucleic acid (dsRNA) material comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10) and the antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16); In the sequence, a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and The L96 is 【Chemistry 4】 and L96 is a compound of the formula: 【Chemistry 5】 "Wherein, X is O" and is conjugated to the 3' end of the sense strand as shown in The dsRNA agent inhibits the expression of Hepatitis B virus (HBV) in cells.

27. 27. The dsRNA agent of claim 26 for use in treating a subject having a Hepatitis B Virus (HBV) infection or a Hepatitis B Virus (HBV)-related disease or disorder.

28. 28. The dsRNA agent of claim 27, wherein the HBV-related disease is Hepatitis D Virus (HDV) infection.

29. Use of the dsRNA agent of any one of claims 1 to 6 or the pharmaceutical composition of claim 8 for the manufacture of a medicament for treating a subject with a Hepatitis B Virus (HBV) infection or a Hepatitis B Virus (HBV) related disease or disorder.

30. a) a dsRNA agent according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 8; and b) Instructions for use according to any one of claims 10 to 25. Kit including:

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