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

Abstract

The present disclosure relates to double-stranded RNA agents that target the Hepatitis B virus (HBV) genome, and methods of using the agents to inhibit expression of one or more HBV genes and methods of treating subjects having an HBV infection or an HBV-related disorder, such as chronic hepatitis B virus infection.

Description

[0001] This application is a divisional application of invention patent application No. 201980053789.8, whose application date is August 12, 2019 and whose invention name is "Hepatitis B virus (HBV) dsRNA agent composition and its use method".

[0002] Statement regarding sequence listing

[0003] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is 930385_410WO_SEQUENCE_LISTING.txt. The text file is 83.1KB, created on August 4, 2019, and submitted electronically via EFS-Web. Background Art

[0004] Globally, more than 400 million people are chronically infected with HBV and are therefore at increased risk of developing severe liver disease such as chronic hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma (HCC), resulting in an estimated 600,000 deaths each year.

[0005] The natural evolution of chronic HBV infection consists of four sequential phases: (1) an early “immune tolerance” phase with high levels of viral replication and minimal liver inflammation; (2) an immune response phase with marked liver inflammation and elevated serum aminotransferases; some patients progress to (3) a “non-replicating” phase with seroconversion to anti-HBe, undetectable or low-level viremia (less than 2000 IU / ml by PCR-based assays), and resolution of liver inflammation; and (4) HBeAg-negative chronic hepatitis B due to the development of specific viral mutations that prevent HBeAg production but do not impede viral replication. This form of chronic hepatitis B (CHB) is characterized by fluctuating serum HBV DNA and serum aminotransferase (ALT and AST) levels and progressive liver disease. It is important to note that CHB may present as either HBeAg-positive or HBeAg-negative CHB. Longitudinal studies of patients with CHB have shown a 5-year cumulative incidence of cirrhosis of 8%-20%. The 5-year cumulative incidence of hepatic decompensation is approximately 20%. The incidence of HCC has increased worldwide and currently constitutes the fifth most common cancer. The annual incidence of HBV-related HCC is high, ranging from 2-5% at the time of cirrhosis.

[0006] The main goals of HBV treatment are to permanently suppress HBV replication and improve liver disease. Clinically important short-term goals are to achieve HBeAg-seroconversion, normalization of serum ALT and AST, resolution of liver inflammation, and prevention of liver decompensation. The ultimate goal of treatment is to obtain a durable response to prevent the development of cirrhosis and liver cancer and thus prolong survival. HBV infection cannot be completely eliminated due to the persistence of a specific form of viral covalently closed circular DNA (cccHBV DNA) in the nucleus of infected hepatocytes. However, clearance of serum HBsAg caused by treatment is a hallmark of termination of chronic HBV infection and is associated with the best long-term outcomes.

[0007] Current standard approaches to treating HBV include immunotherapy based on interferon or thymosin α1, as well as suppression of viral production by inhibiting HBV polymerase. HBV polymerase inhibitors are effective in reducing viral production, but have little effect in rapidly reducing HBsAg, or can slowly reduce HBsAg in a limited number of patients when treated for a long time (such as in the case of tenofovir disoproxil fumarate). Interferon-based immunotherapy can achieve a reduction in viral production and early removal of HBsAg from the blood, but only in a small proportion of treated subjects. The generally recognized role of HBsAg in the blood is to sequester anti-HBsAg antibodies and allow infectious viral particles to escape immune detection, which is likely one of the reasons why HBV infection remains a chronic condition. In addition, HBsAg, HBeAg, and HBcAg all have immunosuppressive properties, and the persistent presence of these viral proteins in the patient's blood after administration of any currently available HBV treatment may have an important impact on preventing patients from gaining immune control of their HBV infection.

[0008] Although all three major HBV proteins (HBsAg, HBeAg, and HBcAg) have immunosuppressive properties, HBsAg constitutes the vast majority of HBV proteins circulating in the blood of HBV-infected subjects. In addition, although clearance of HBeAg (by seroconversion) or reduction in serum viremia is not associated with the development of sustained control of HBV infection off treatment, clearance of serum HBsAg from the blood (and seroconversion) in HBV infection is a recognized prognostic indicator of antiviral response to treatment (which leads to control of HBV infection off treatment) (although this occurs in only a small proportion of patients receiving immunotherapy). Therefore, although reduction of all three major HBV proteins (HBsAg, HBeAg, and HBcAg) can result in optimal removal of suppressive effects, HBsAg removal alone may also be sufficient by itself to eliminate most viral suppression of immune function in HBV-infected subjects.

[0009] Therefore, in the absence of any current treatment regimen that can restore immune control of HBV in a large proportion of patients, there is a need for an effective treatment for HBV infection that can suppress viral replication and restore immune control in a large proportion of patients. Therefore, there is a need in the art for alternative and combination therapies for subjects infected with HBV or suffering from HBV-related diseases. Summary of the invention

[0010] In some embodiments, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) agent composition that affects RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of hepatitis B virus (HBV) genes. The HBV gene can be within a cell, such as within a cell in a subject (such as a human).

[0011] The present disclosure also provides methods and therapies for treating a subject with a disease that benefits from inhibition of HBV gene expression, such as HBV infection or HBV-related diseases, e.g., chronic hepatitis B infection (CHB), using a dsRNA agent composition that affects RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of HBV genes, thereby inhibiting expression of the HBV genes.

[0012] In one aspect, the present disclosure provides a dsRNA agent for inhibiting HBV expression. 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 comprises a modified nucleotide sequence as shown below:

[0013] 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16),

[0014] 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3'(SEQ ID NO:18),

[0015] 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20),

[0016] 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23),

[0017] 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO:24),

[0018] 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO:25), or

[0019] 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28),

[0020] where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively;

[0021] Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively;

[0022] dA, dC, dG, and dT are 2′-deoxyadenosine-3′-phosphate, 2′-deoxycytidine-3′-phosphate, 2′-deoxyguanosine-3′-phosphate, and 2′-deoxythymidine-3′-phosphate, respectively;

[0023] (Agn) is adenosine diol nucleic acid (GNA); and

[0024] s is a phosphorothioate linkage.

[0025] In some embodiments, the sense strand comprises a modified nucleotide sequence 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29),

[0026] where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively;

[0027] 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

[0028] s is a phosphorothioate linkage.

[0029] In some embodiments, the antisense strand and the sense strand comprise modified nucleotide sequences as shown below:

[0030] (a) 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29);

[0031] (b) 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29);

[0032] (c) 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29);

[0033] (d) 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29);

[0034] (e) 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29);

[0035] (f) 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO:25) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO:29); or

[0036] (g) 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29);

[0037] where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively;

[0038] Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively;

[0039] dA, dC, dG, and dT are 2′-deoxyadenosine-3′-phosphate, 2′-deoxycytidine-3′-phosphate, 2′-deoxyguanosine-3′-phosphate, and 2′-deoxythymidine-3′-phosphate, respectively;

[0040] (Agn) is adenosine diol nucleic acid (GNA); and

[0041] s is a phosphorothioate linkage.

[0042] In some embodiments, at least one strand of the dsRNA agent comprises a 3' overhang of at least 1 nucleotide. In some embodiments, at least one strand of the dsRNA agent comprises a 3' overhang of 2 nucleotides.

[0043] In some embodiments, the double-stranded region of the dsRNA agent is 19-21 nucleotide pairs in length.

[0044] In some embodiments, each strand of the dsRNA agent independently has 19-23 nucleotides. In some embodiments, each strand of the dsRNA agent independently has 19-21 nucleotides.

[0045] 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

[0046]

[0047] In some embodiments, as shown schematically below, the dsRNA agent is conjugated to a ligand.

[0048]

[0049] wherein X is O or S. In some embodiments, X is O.

[0050] In some embodiments, the antisense strand consists of a modified nucleotide sequence as shown below:

[0051] 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16),

[0052] 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3'(SEQ ID NO:18),

[0053] 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20),

[0054] 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23),

[0055] 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO:24),

[0056] 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO:25), or

[0057] 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28),

[0058] where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively;

[0059] Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively;

[0060] dA, dC, dG, and dT are 2′-deoxyadenosine-3′-phosphate, 2′-deoxycytidine-3′-phosphate, 2′-deoxyguanosine-3′-phosphate, and 2′-deoxythymidine-3′-phosphate, respectively;

[0061] (Agn) is adenosine diol nucleic acid (GNA); and

[0062] s is a phosphorothioate linkage.

[0063] In some embodiments, the present disclosure provides dsRNA agents, wherein the sense strand and the antisense strand consist of modified nucleotide sequences as shown below:

[0064] (a) 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29);

[0065] (b) 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29);

[0066] (c) 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29);

[0067] (d) 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29);

[0068] (e) 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29);

[0069] (f) 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO:25) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO:29); or

[0070] (g) 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29);

[0071] where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively;

[0072] Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively;

[0073] dA, dC, dG, and dT are 2′-deoxyadenosine-3′-phosphate, 2′-deoxycytidine-3′-phosphate, 2′-deoxyguanosine-3′-phosphate, and 2′-deoxythymidine-3′-phosphate, respectively;

[0074] (Agn) is adenosine diol nucleic acid (GNA);

[0075] s is a phosphorothioate linkage; and

[0076] The 3' end of the sense strand was conjugated to N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol (L96) ligand.

[0077] In another aspect, the present disclosure also provides cells comprising a dsRNA agent as disclosed herein.

[0078] The present disclosure also provides a pharmaceutical composition comprising the dsRNA agent described herein and a pharmaceutically acceptable excipient.

[0079] The present disclosure also provides a method for inhibiting hepatitis B virus (HBV) gene expression in a cell, the method comprising contacting the cell with a dsRNA agent or a pharmaceutical composition disclosed herein, thereby inhibiting the expression of HBV genes 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 an 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 is inhibited to a level below the detection level of the assay method.

[0080] The present disclosure also provides a method for inhibiting the replication of hepatitis B virus (HBV) in a cell, the method comprising contacting the cell with a dsRNA agent or a pharmaceutical composition disclosed herein, thereby inhibiting the replication of HBV in the cell. In some embodiments, the cell is in a subject. In some embodiments, the subject is a human. In certain embodiments, the subject suffers from an HBV-related disease. In some embodiments, the cell is in vitro. In certain embodiments, the replication of HBV in the cell is inhibited by at least 80%, 90%, 95% or 98%, or is inhibited to a detection level below the assay method.

[0081] Also provided herein is a method for reducing the level of hepatitis B virus (HBV) antigen in a subject infected with HBV, the method comprising administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition disclosed herein, thereby reducing the level of HBV antigen in 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 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 in serum is reduced by at least 1log10, at least 2log10, at least 3log10, or at least 4log10, or is reduced to a detection level below the assay method.

[0082] The present disclosure also provides a method for reducing the viral load of hepatitis B virus (HBV) in a subject infected with HBV, the method comprising administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition as disclosed herein, thereby reducing the HBV viral load of the subject. In some embodiments, the HBV viral load is measured in 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 viral load in serum is reduced by at least 1log10, at least 2log10, at least 3log10, or at least 4log10, or is reduced to a detection level below the assay method.

[0083] Also provided herein is a method for treating a subject suffering from hepatitis B virus (HBV) infection or HBV-related disorders, the method comprising administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition as disclosed herein, thereby treating the subject. In some embodiments, the subject is HBeAg positive. In some embodiments, the subject is HBeAg negative. In some embodiments, HBV-related disorders are chronic hepatitis, and the subject is HBeAg positive. In some embodiments, HBV-related disorders are chronic hepatitis, and the subject is HBeAg negative.

[0084] In some embodiments of the aforementioned methods, 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 methods, the dsRNA agent is administered to the subject at a dose of 3 mg / kg to 10 mg / kg. In some embodiments of the methods, the dsRNA agent is administered to the subject at a fixed dose of 50 mg to 200 mg.

[0085] In some embodiments of the foregoing methods, the dsRNA agent is administered subcutaneously to the subject.

[0086] In some embodiments of the foregoing methods, the dsRNA agent is administered to the subject in two or more doses.

[0087] In some embodiments of the aforementioned methods, 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 methods, the dsRNA agent is administered to the subject no more than once a month.

[0088] In some embodiments of the aforementioned methods, the method further comprises administering to the subject an additional therapeutic agent, such as one or more additional therapeutic agents. Additional therapeutic agents may include, but are not limited to, antiviral agents, reverse transcriptase inhibitors, immunostimulants, therapeutic vaccines, viral entry inhibitors, oligonucleotides that inhibit HBsAg secretion or release, capsid inhibitors, and covalently closed circular (ccc) HBV DNA inhibitors, and any combination thereof. In some embodiments, the additional therapeutic agent is a reverse transcriptase inhibitor.

[0089] In some embodiments, more than one additional therapeutic agent is administered, and the additional therapeutic agent is a reverse transcriptase inhibitor and an immunostimulant. Reverse transcriptase inhibitors may include but are not limited to tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, and AGX-1009. Immunostimulants may include but are not limited to pegylated interferon α2a (PEG-IFN-α2a), interferon α-2b, recombinant human interleukin 7, and Toll-like receptor 7 (TLR7) agonists.

[0090] Also provided herein is a composition for implementing any method disclosed herein. In some embodiments, the disclosure provides a dsRNA agent or pharmaceutical composition as disclosed herein, for treating a subject's hepatitis B virus (HBV) infection. In some embodiments, the disclosure provides a dsRNA agent or pharmaceutical composition as disclosed herein, for treating a subject's hepatitis B virus (HBV) related disorder. In some embodiments, HBV related disorders are chronic hepatitis, and the subject is HBeAg positive. In some embodiments, HBV related disorders are chronic hepatitis, and the subject is HBeAg negative. In some embodiments, the subject is or has been administered another therapeutic agent, such as an antiviral agent, a reverse transcriptase inhibitor, an immunostimulant, a therapeutic vaccine, a virus entry inhibitor, an oligonucleotide, a capsid inhibitor or a covalently closed circular (ccc) HBV DNA inhibitor that inhibits the secretion or release of HBsAg, or any combination thereof. 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 (tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, or AGX-1009) and an immunostimulant (e.g., pegylated interferon α2a (PEG-IFN-α2a), interferon α-2b, recombinant human interleukin 7, or a Toll-like receptor 7 (TLR7) agonist).

[0091] The present disclosure provides the use of a dsRNA agent or pharmaceutical composition as disclosed herein for carrying out any of the aforementioned methods.

[0092] The present disclosure also provides the use of a dsRNA agent as disclosed herein in the preparation or manufacture of a medicament for carrying out any of the aforementioned methods.

[0093] The present disclosure also provides kits comprising a dsRNA agent or pharmaceutical composition as disclosed herein, optionally with instructions for practicing the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 The structure of the approximately 3.2 kb double-stranded HBV genome is schematically depicted. The replication of the HBV genome occurs through an RNA intermediate and produces four overlapping viral transcripts (approximately 3.5 kb transcript, approximately 2.4 kb transcript, approximately 2.1 kb transcript, and approximately 0.7 kb transcript), encoding seven viral proteins (pre-S1, pre-S2, S, P, X, pre-C, and C) translated in three reading frames.

[0095] Figure 2A-2B Shown are (A) serum HBsAg concentration (ng / mL) and (B) serum HBsAg levels relative to pre-dose in the AAV-HBV mouse model. Figure 2A HBsAg serum levels in HBV-AAV mice before administration (day -24, day -2, day 0) or after a single dose of AD-81890 at 0.3, 1 or 3 mg / kg (day 14, day 21, day 33, day 47, day 59, day 74) are shown. Each point represents the mean of n = 6-9 animals, and the bars represent SD. Figure 2B HBsAg serum levels in HBV-AAV mice are shown relative to pre-dose (day -24, day -2, day 0) or after a single dose of AD-81890 at 0.3, 1 or 3 mg / kg (day 14, day 21, day 33, day 47, day 59, day 74). Each point represents the mean of n = 6-9 animals, and the bars represent SD.

[0096] Figure 3 Serum HBsAg levels relative to pre-dose in the AAV-HBV mouse model are shown at -55 days, -27 days, -13 days, 0 days, 14 days, 28 days, 42 days, 56 days, 84 days, 112 days, and 140 days. Dosing regimens included control (PBS) Q2W×6; AD-66810 (1 mg / kg; Q2W×6); AD-81890 (1 mg / kg; Q2W×6); AD-81890 (1 mg / kg; QM×3); AD-81890 (3 mg / kg; Q2W×6); AD-81890 (3 mg / kg; QM×3); and AD-81890 (9 mg / kg; QM×1). The first dose was administered on day 0. Each point represents the mean of n 4-6 animals, and the bars represent SD.

[0097] Figure 4 Box plots are shown, which are displayed in AD-66810 or AD-81890 significantly down-regulated (adjusted p value < 0.05, log2 fold change < 0) of all genes log2 fold change (treatment / control). Horizontal thick lines represent median log2 fold change, and the vertical range of each box represents the interquartile range (IQR), and must extend to + / -1.58IQR / sqrt (N), where N is the number of genes in each group. Statistical significance is assessed using Welch's two-sided two-sample t test.

[0098] Figure 5A-5B Compared with mice administered PBS, mice administered AD-66810 ( Figure 5A ) or AD-81890( Figure 5B ) after ALT levels in PXB mice over time. Mice were administered 12, 36 or 100 mg / kg of 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 a week, and serum was prepared using conventional methods. DETAILED DESCRIPTION

[0099] The present disclosure provides dsRNA agent compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of hepatitis B virus (HBV) genes. The gene can be in a cell, such as in a cell in a subject (such as a human). The use of these dsRNA agents enables targeted degradation of the mRNA of the corresponding gene (HBV gene) in mammals.

[0100] The dsRNA agents described herein have been designed to target regions in the HBV genome that are conserved in at least eight known HBV genotypes. In addition, the dsRNA agents disclosed herein have been designed to inhibit all steps of the HBV life cycle, such as replication, assembly, viral secretion, and secretion of subviral antigens, by inhibiting the expression of more than one HBV gene. In particular, since the transcription of the HBV genome produces polycistronic, overlapping RNAs, in some embodiments, the dsRNA agents targeting a single HBV gene significantly inhibit the expression of most or all HBV transcripts. For example, because the HBV genome is transcribed into a single mRNA, the dsRNA agents disclosed herein that target the S gene will result in not only inhibiting the expression of the S gene, but also inhibiting the expression of the "downstream" polymerase gene. In addition, the dsRNA agents disclosed herein have been designed to inhibit HBV viral replication by targeting HBV structural genes, and inhibiting HBV X genes, thereby allowing the subject's immune system to detect and respond to the presence of HBsAg to produce anti-HBV antibodies to clear HBV infection. Without intending to be limited by theory, it is believed that the combination or sub-combination of the aforementioned properties with specific target sites or specific modifications in these dsRNA agents imparts improved efficacy, stability, safety, effectiveness, and persistence to the dsRNA agents of the present disclosure.

[0101] Using in vitro and in vivo assays, the inventors have demonstrated that dsRNA agents targeting HBV genes can potently mediate RNAi, thereby significantly inhibiting the expression of more than one HBV gene. Therefore, the methods and compositions comprising these dsRNA agents can be used to treat subjects suffering from HBV infection or HBV-related diseases, such as chronic hepatitis B (CHB).

[0102] Therefore, the present disclosure also provides a method for treating a subject suffering from a disorder that benefits from inhibiting or reducing the expression of HBV genes, such as HBV-related diseases, such as chronic hepatitis B virus infection (CHB), using a dsRNA agent composition that affects RNA-induced silencing complex (RISC)-mediated cleavage of HBV gene RNA transcripts.

[0103] The dsRNA agent of the present disclosure includes an RNA strand (antisense strand) having a complementary region, the complementary region length being about 19-21 nucleotides, for example, about 19 nucleotides in length, which is substantially complementary to at least a portion of the mRNA transcript of the HBV gene of at least one HBV genotype. It should be understood that there are a variety of HBV genotypes, so that the degree of complementarity of the dsRNA agent of the present disclosure to different HBV genotypes may vary.

[0104] In some embodiments, the sense strand and the antisense strand form a duplex of 19-21 consecutive nucleotides.

[0105] The following detailed description discloses how to prepare and use compositions containing dsRNA agents to inhibit the expression of HBV genes, as well as compositions, uses and methods for treating subjects suffering from diseases and disorders that benefit from inhibiting or reducing the expression of HBV genes.

[0106] I. definition

[0107] In order that the present invention may be more readily understood, certain terms are first defined.In addition, it should be noted that whenever a value or range of values ​​for a parameter is recited, values ​​and ranges intermediate to the recited values ​​are also intended to be part of the feature.

[0108] Unless the context requires otherwise, in this specification and claims, the word "comprising" and its variants (such as "including" and "containing") should be interpreted in an open, inclusive sense, i.e., "including, but not limited to". "Consisting of" means excluding other components more than trace elements or substantial method steps disclosed herein. For example, a polynucleotide consists of a nucleotide sequence when it does not include any other nucleotides but does not exclude the incorporation of a ligand (such as a targeting ligand) or a modification. The term "consisting essentially of" limits the scope of the claim to the specified materials or steps, or to those materials or steps that do not substantially affect the basic characteristics of the claimed invention. For example, a pharmaceutical composition consisting essentially of the elements defined herein will not exclude trace contaminants from separation and purification methods and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, etc. Similarly, a polynucleotide consists essentially of a nucleotide sequence when it includes additional nucleotides that constitute up to 20% of the length of the polynucleotide and does not substantially affect the activity of the polynucleotide (e.g., does not alter the activity of the polynucleotide by more than 50%). Embodiments defined by each of the transition terms are within the scope of this invention.

[0109] The articles "a" and "an" herein refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" refers to one element or more than one element, such as a plurality of elements.

[0110] The term "including" is used herein to refer to, and is used interchangeably with, the phrase "including but not limited to".

[0111] Unless the context clearly indicates otherwise, the term "or" is used herein to refer to the term "and / or" and is used interchangeably with the term. For example, "sense strand or antisense strand" is understood to be "sense strand or antisense strand or sense strand and antisense strand".

[0112] The term "about" as used herein refers to within the typical tolerance range in the art. For example, "about" can be understood to be within 2 standard deviations of the mean. When "about" is present before a series of numbers or ranges, it should be understood that "about" can modify each number in the series or range.

[0113] The term "at least" before a number or a series of numbers should be understood to mean each number in the series and all subsequent numbers or integers that can be logically included as clearly seen from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a nucleic acid molecule of 21 nucleotides" means that 18, 19, 20 or 21 nucleotides have the indicated properties. When at least appears before a series of numbers or ranges, it should be understood that "at least" can modify each number in the series or range.

[0114] As used herein, "not more than" or "less than" is understood to mean the value adjacent to the phrase and any logically lower value or integer that is logical from the context, 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" appears before a series of numbers or a range, it should be understood that "not more than" can modify each number in the series or range.

[0115] As used herein, ranges include upper and lower limits.

[0116] If there is a conflict between a sequence and its indicated site on a transcript or other sequence, the nucleotide sequence described in this specification shall prevail.

[0117] The various embodiments of the present disclosure may be combined as appropriately determined by one skilled in the art.

[0118] As used herein, "hepatitis B virus," which is used interchangeably with the term "HBV," refers to a well-known non-cytopathic, hepatotropic DNA virus belonging to the family Hepadnaviridae.

[0119] The HBV genome is a partially double-stranded, circular DNA with overlapping reading frames (see e.g. Figure 1 ).

[0120] There are four transcripts (which may be referred to herein as "genes" or "open reading frames") encoded by the HBV genome, depending on size. These transcripts contain open reading frames called C, X, P, and S. The core protein is encoded by gene C (HBcAg). Hepatitis B e antigen (HBeAg) is produced by proteolytic processing of pre-core (pre-C) protein. DNA polymerase is encoded by gene P. Gene S is the gene encoding the surface antigen (HBsAg). The HBsAg gene is a long open reading frame that contains three in-frame "start" (ATG) codons, thus producing three different sizes of polypeptides, respectively referred to as large, medium and small S antigens, pre-S1+pre-S2+S, pre-S2+S or S. In addition to decorating the envelope of HBV, the surface antigen is also part of the subviral particle, which is produced in great excess compared to the viral particle, and plays 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 plays a role in transcriptional transactivation and replication and has been implicated in the development of liver cancer.

[0121] HBV is one of the few DNA viruses that utilizes reverse transcriptase during replication, which involves multiple stages including entry, uncoating, and transfer of the viral genome to the cell nucleus. Initially, replication of the HBV genome involves the production of an RNA intermediate that is subsequently reverse transcribed to produce a DNA viral genome.

[0122] After HBV infects cells, the relaxed circular DNA (rcDNA) of the viral genome is transported into the nucleus and converted into free 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) is assembled with HBV polymerase and capsid proteins to form a nucleocapsid, where polymerase-catalyzed reverse transcription produces negative-strand DNA, which is then replicated into positive-strand DNA to form the progeny rcDNA genome. The mature nucleocapsid is then packaged with viral envelope proteins to appear as a virion, or shuttled into the nucleus to amplify the cccDNA pool through the intracellular cccDNA amplification pathway. cccDNA is an important component of the HBV replication cycle and is responsible for the establishment of infection and viral persistence.

[0123] HBV infection results in the production of two different particles: 1) the infectious HBV virus itself (or Dane particle), which includes a viral capsid assembled by HBcAg and is covered by an envelope consisting of a lipid membrane carrying HBV surface antigens, and 2) non-infectious subviral particles (or SVPs), which contain small or medium forms of the hepatitis B surface antigen HBsAg. For each viral particle produced, more than 10,000 SVPs are released into the blood. Therefore, SVPs (and the HBsAg proteins they carry) represent the vast majority of viral proteins in the blood. HBV-infected cells also secrete a soluble protein hydrolyzate of the pre-core protein, called HBV e-antigen (HBeAg).

[0124] Eight HBV genotypes have been identified, designated A to H, and two additional genotypes, I and J, have been proposed, each with a distinct geographic distribution. The virus is non-cytopathic, with virus-specific cellular immunity being the major determinant of the outcome of exposure to either acute HBV infection (6-month resolution of liver disease) or chronic HBV infection, which is often associated with progressive liver damage.

[0125] The term "HBV" includes any HBV genotype (A to J). The complete coding sequence of the HBV genome reference sequence 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 provided in SEQ ID NO:2 and SEQ ID NO:4, respectively. The amino acid sequences of C, X, P and S proteins can be found, for example, in NCBI Accession Nos. 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). SEQ ID NOs: 36-37 provide protein and DNA sequences from HBV genotype D, strain ayw. SEQ ID NOs: 38-39 provide protein and DNA sequences from HBV genotype C. SEQ ID NOs: 41-49 provide other examples of HBV protein and DNA sequences or their reverse complements.

[0126] Other examples of HBV mRNA sequences are readily available using public databases such as GenBank, UniProt and OMIM. The International Repository for Hepatitis B Virus Strain Data can be accessed at http: / / www.hpa-bioinformatics.org.uk / HepSEQ / main.php.

[0127] As used herein, the term "HBV" also refers to naturally occurring DNA sequence variations of the HBV genome, such as genotypes AJ and variants thereof.

[0128] As used herein, "hepatitis D virus", which is used interchangeably with the term "HDV", refers to a well-known non-cytopathic, hepatotropic DNA virus belonging to the Hepadnaviridae family. See, e.g., 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 indicated, HDV refers to all clades and variants of HDV.

[0129] HDV produces a protein, HDAg. It comes in two forms: the 27kDa large HDAg (also known as lHD, L-HDAg, and large HDV antigen) and the 24kDa small HDAg (also known as sHD, S-HDAg, and small HDV antigen). The N-termini of these two forms are identical; they differ in the 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 only produces small HDAg. However, editing by the cellular enzyme adenosine deaminase-1 changes this stop codon to UCG, allowing the production of large HDAg. Despite having 90% identical sequences, these two proteins play different roles during infection. HDAg-S is produced in the early stages of infection and enters the nucleus and supports viral replication. In contrast, HDAg-L is produced in the later stages of infection, acts as an inhibitor of viral replication, and is required for viral particle assembly.

[0130] Other examples of HDV mRNA sequences are readily available using public databases such as GenBank, UniProt, and OMIM.

[0131] As used herein, the term "HDV" also refers to naturally occurring DNA sequence variations of the HDV genome.

[0132] As used herein, the term "nucleotide (nucleoside) analog" or "reverse transcriptase inhibitor" is an inhibitor of DNA replication that is structurally similar to nucleotides or nucleosides and specifically inhibits the replication of HBV cccDNA without significantly inhibiting the replication of host (eg, human) DNA. Such inhibitors include tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine, clavudine, ritonavir, dipivoxil, lobucavir, famciclovir, FTC, N-acetyl-cysteine ​​(NAC), PC1323, hepatitis B therapeutic vaccine (theradigm-HBV), thymosin-α, ganciclovir, besifovir (ANA-380 / LB-80380) and tenofvir-exaliades (TLX / CMX157). In certain embodiments, the nucleotide (nucleoside) analog is entecavir (ETV). Nucleotide (nucleoside) analogs are commercially available from a number of sources and are used in the methods provided herein according to their label instructions (eg, usually administered orally at a specific dosage) or as determined by a practitioner skilled in the treatment of HBV.

[0133] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of an HBV gene, including mRNA that is a product of RNA processing of a primary transcription product. In some embodiments, the target portion of the sequence is at least long enough to serve as a substrate for dsRNA agent-directed cleavage of the nucleotide sequence of an mRNA molecule formed during transcription of an HBV gene at or near the portion.

[0134] The target sequence can be about 19 to 21 nucleotides in length, for example, 19, 20 or 21 nucleotides in length.

[0135] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides described by a sequence referred to using standard nucleotide nomenclature.

[0136] Typically, "G", "C", "A", "T" and "U" each represent a nucleotide containing guanine, cytosine, adenine, thymine and uracil as bases. However, it should be understood that the term "ribonucleotide" or "nucleotide" may also refer to a modified nucleotide, or a surrogate replacement moiety (see, e.g., Table 1), as further described below. It is well known to the skilled artisan that guanine, cytosine, adenine and uracil may be replaced by other moieties without substantially changing the base pairing properties of an oligonucleotide containing a nucleotide with such a replacement moiety. For example, but not limited to, a nucleotide containing inosine as its base may be base paired with a nucleotide containing adenine, cytosine or uracil. Thus, a nucleotide containing uracil, guanine or adenine may be replaced by a nucleotide containing, for example, inosine in the nucleotide sequence of a dsRNA characterized in the present disclosure. In another example, adenine and cytosine anywhere in an oligonucleotide may be replaced with guanine and uracil, respectively, to form a GU wobble base pair with a target mRNA. Sequences comprising such replacement moieties are suitable for use in the compositions and methods featured in this disclosure.

[0137] The terms "dsRNA agent", "RNAi agent", "iRNA agent", "iRNA" and "RNA interfering agent" used interchangeably herein refer to agents that contain RNA as defined herein and mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. dsRNA agents direct sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). dsRNA agents regulate (e.g., inhibit) expression of HBV genes (e.g., one or more HBV genes) in cells (e.g., cells in a subject (e.g., a mammalian subject)).

[0138] The "dsRNA agent" used in the compositions, uses and methods disclosed herein is a double-stranded RNA, and is referred to herein as a "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 having a duplex structure comprising two antiparallel and substantially complementary nucleic acid chains, which are referred to as having "sense" and "antisense" orientations relative to the target RNA (i.e., HBV gene). In some embodiments of the present disclosure, dsRNA triggers the degradation of a target RNA (e.g., mRNA) by a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).

[0139] Typically, each strand of a dsRNA molecule may contain ribonucleotides, but as described in detail herein, each strand or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. In addition, as used in this specification, "dsRNA agents" may include ribonucleotides with chemical modifications; dsRNA agents may include substantial modifications on multiple nucleotides. As used herein, the term "modified nucleotides" refers to nucleotides independently having modified sugar moieties, modified internucleotide bonds, or modified nucleobases. Therefore, the term modified nucleotides encompasses substitutions, additions, or removals of, for example, functional groups or atoms of internucleoside linkages, sugar moieties, or nucleobases. Modifications suitable for use in the present disclosure of agents include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modifications used in dsRNA dosage form molecules are covered by "dsRNA agents".

[0140] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any level of inhibition. Preferably, inhibition includes statistically significant inhibition or clinically significant inhibition.

[0141] As used herein, the phrase "inhibiting the expression of HBV" or "inhibiting the expression of HBV genes" includes inhibiting the expression of any HBV gene (e.g., HBV gene expressed by HBV in HBV viral infection, HBV gene expressed by expression construct in cells) and variants or mutants of HBV genes encoding HBV proteins. The term includes knockdown of any HBV transcript (e.g., 3.5kb, 2.4kb, 2.1kb or 0.7kb transcript) encoding one or more HBV viral proteins (e.g., preS1 / 2-S, preS, S, P, X, preC and C) and variants or mutants of HBV genes.

[0142] "Inhibiting the expression of HBV genes" includes inhibition of any level of HBV genes or transcripts, for example, at least partial inhibition of the expression of HBV genes (e.g., HBV genes S, P, X or C or any combination thereof, such as S, P and C). The expression of HBV genes can be assessed according to the level or level change of any variable related to HBV gene expression (e.g., HBV mRNA level or HBV protein level, or HBV cccDNA level). The level can be assessed in a single cell or a group of cells, including, for example, a sample derived from a subject, for example, the level in serum can be monitored. Inhibition can be assessed by a reduction 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 used in the art, such as a baseline level before administration, or a level determined from a similar subject or a population average of an appropriate control subject, cell or sample from an untreated or control (buffer-only control or inactive agent control) treated control.

[0143] In some embodiments of the disclosed methods, the expression of HBV genes is inhibited by at least 80%, 85%, 90%, 95%, for example, in a subject, by at least 1 log10, 2 log10, 3 log10 or 4 log10, or is inhibited to a level below the detection level of the assay method. In a preferred embodiment, the inhibition of HBV gene expression in a subject results in a clinically relevant inhibition of gene expression levels, for example, sufficient inhibition to allow an effective immune response to HBV proteins when administered alone or in combination with other agents that promote or enhance an immune response.

[0144] In an in vitro cell-based assay or in an in vivo model (e.g., a mouse AAV-hHBV model provided herein) in which a heterologous gene is expressed, preferably total HBV expression is inhibited by at least 90%, for example, by at least 1 log10, 2 log10, or 3 log10. When treating a subject with HBV infection, it is preferred to reduce the level of HBV gene or protein by at least 90%, that is, the difference between the level of HBV gene or protein before and after treatment. More than one dose may be required to achieve the desired level of inhibition.

[0145] The inhibition of HBV gene expression can be reflected by a reduction in the amount of RNA expressed by a first cell or cell group (e.g., these cells may be present in a sample derived from a subject), wherein the HBV gene is transcribed in the first cell or cell group and the first cell or cell group is treated (e.g., by contacting one or more cells with a dsRNA agent of the present invention, or by administering a dsRNA agent of the present invention to a subject in which the cells are present or have been present) so that the expression of HBV is inhibited compared to a second cell or cell group (control cells) that is substantially the same as the first cell or cell group but has not been treated in this way. In a preferred embodiment, inhibition is assessed by the rtPCR method provided in Example 2 of WO2016 / 077321 (the method is incorporated herein by reference) and in vitro assays performed with a 10 nM concentration of duplexes in appropriately matched cell lines. The mRNA level in the treated cells is expressed as a percentage of the mRNA level in the control cells using the following formula:

[0146]

[0147] Alternatively, inhibition of HBV gene expression may be assessed based on a reduction in a parameter functionally associated with HBV gene expression.HBV gene silencing may be determined in any cell expressing HBV genes constitutively or by genome engineering and by any assay known in the art.

[0148] The inhibition of HBV protein expression can be reflected by a decrease 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 described above, in order to assess mRNA inhibition, the inhibition of protein expression levels in treated cells or cell groups can be similarly expressed as a percentage of protein levels in control cells or cell groups or in serum.

[0149] Control cells or cell groups that can be used to evaluate the inhibition of HBV gene expression include cells or cell groups that have not yet been contacted with dsRNA agents of the present disclosure. For example, control cells or cell groups can be derived from a single subject (e.g., human or animal subject) before treating a subject with a dsRNA agent. In alternative embodiments, the level can be compared with an appropriate control sample, such as a known population control sample.

[0150] Any method known in the art for evaluating mRNA expression can be used, preferably using the rtPCR method provided in Example 2 of WO2016 / 077321, to determine the level of HBV RNA expressed by a cell or cell group, or the level of circulating HBV RNA. In some embodiments, the expression level of an HBV gene (e.g., total HBV RNA, HBV transcript, such as HBV 3.5kb transcript) in a sample is determined by detecting a transcribed polynucleotide or a portion thereof (e.g., RNA of an HBV gene). RNA can be extracted from cells using RNA extraction techniques, including, for example, extraction using acid phenol / guanidine isothiocyanate (RNAzol B; Biogenesis), RNeasy RNA preparation kit Or PAXgene (PreAnalytix, Switzerland). Typical assay formats using RNA 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. Circulating HBV mRNA can be detected using the method described in WO 2012 / 177906, which is incorporated herein by reference.

[0151] In some embodiments, the expression level of HBV gene is determined using a nucleic acid probe. As used herein, the term "probe" refers to any molecule that can selectively bind to a specific HBV gene. The probe can be synthesized by a person skilled in the art, or derived from an appropriate biological product. The probe can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0152] The isolated RNA can be used in hybridization or amplification assays, including but not limited to Northern blot analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize with HBV mRNA. In some embodiments, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and 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, on a In gene chip arrays. Technicians can easily adopt known mRNA detection methods to measure the level of HBV mRNA.

[0153] Alternative methods for determining the level of HBV gene expression in a sample involve amplification of nucleic acids such as mRNA in the sample or reverse transcriptase (to prepare cDNA) by, for example, RT-PCR (protocols are shown in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustaining sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification system (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. (1991) Proc. Natl. Acad. Sci. USA 88:189-193), and reverse transcriptase (Richardson et al. (1992) Proc. Natl. Acad. Sci. USA 89:1174-1178). al., U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, and then detecting the amplified molecules using techniques well known to those skilled in the art. If the nucleic acid molecules are present in very low quantities, these detection schemes are particularly useful for the detection of such molecules. In certain aspects of the present disclosure, for example, by using the methods provided herein, by quantitative fluorescent RT-PCR (i.e., TaqMan TM System) to measure the expression level of HBV genes.

[0154] The expression level of HBV RNA can be monitored using membrane blotting (e.g., for hybridization analysis, such as Northern blotting, dot blotting, etc.) or microwells, sample tubes, gels, beads or fibers (or any solid support containing bound nucleic acids). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference to provide teachings related to such methods. The determination of HBV expression levels may also include the use of nucleic acid probes in solution.

[0155] 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.

[0156] HBV protein expression level can be measured using any method known in the art for measuring protein level. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitation reaction, absorption spectroscopy, colorimetry, spectrophotometry, flow cytometry, immunodiffusion (single or double direction), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc.

[0157] dsRNA Agents

[0158] The duplex region can have any length that allows specific degradation of the desired target RNA by the RISC pathway, and the length can be in the range of about 19-21 base pairs, for example, about 19, 20 or 21 base pairs in length. Exemplary dsRNA agents provided herein include duplex lengths of 19-21 base pairs.

[0159] If the two basic complementary chains of dsRNA are composed of separate RNA molecules, these molecules need not but can be covalently linked. If the two chains are covalently linked in a manner other than an uninterrupted nucleotide chain between the 3' end of one chain and the 5' end of the other chain to form a double-stranded structure, the connection structure is called a "joint". RNA chains can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest chain of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the dsRNA agent may include one or more nucleotide overhangs.

[0160] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a double-stranded dsRNA agent. For example, a nucleotide overhang is present when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand or vice versa. The dsRNA may comprise an overhang having at least one nucleotide; alternatively, the overhang may comprise two nucleotides. The nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang may be present at either the 5'-end, the 3'-end, or both ends of the antisense strand or the sense strand of the dsRNA. In a preferred embodiment, the nucleotide overhang is at the 3' end of the antisense strand.

[0161] "Blunt" or "flat end" refers to the absence of unpaired nucleotides at that end of a double-stranded dsRNA agent, i.e., no nucleotide overhangs. A "flat-ended" dsRNA agent is a double-stranded dsRNA over its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. In some embodiments, the dsRNA agents of the present disclosure include dsRNA agents having nucleotide overhangs at one end (i.e., dsRNA agents having one overhang and one flat end) or dsRNA agents having nucleotide overhangs at both ends.

[0162] The term "antisense strand" or "guide strand" refers to a strand in a dsRNA agent that includes a region that is substantially complementary to a target sequence (e.g., HBV mRNA). As used herein, the term "complementarity region" refers to a region on the antisense strand that is substantially complementary to a sequence defined herein, such as a target sequence (e.g., HBV nucleotide sequence). In the case where the complementary region is not completely complementary to the target sequence, the mismatch may be in the interior or terminal region of the molecule. Typically, the most tolerated mismatch is in the terminal region, such as within 5, 4, 3, 2, or 1 nucleotides at the 5'- or 3'-end of the dsRNA agent. In some embodiments, the dsRNA agent of the present disclosure includes a nucleotide mismatch in the antisense strand. In some embodiments, the dsRNA agent of the present disclosure includes a nucleotide mismatch in the sense strand. In some embodiments, the nucleotide mismatch is within 5, 4, 3, 2, or 1 nucleotides at the 3' end of the dsRNA agent, for example. In some embodiments, the nucleotide mismatch is, for example, in the 3'-terminal nucleotides of the dsRNA agent.

[0163] As used herein, the term "sense strand" or "passenger strand" refers to the strand of a dsRNA agent that comprises a region that is substantially complementary to a region of the antisense strand as defined herein.

[0164] As used herein, and unless otherwise indicated, as understood by those skilled in the art, when describing a first nucleotide sequence relative to a second nucleotide sequence, the term "complementary" refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising a second nucleotide sequence under certain conditions. Such conditions can be, for example, stringent conditions, wherein stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours, followed by washing (e.g., see "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 be encountered in an organism. The skilled person will be able to determine the set of conditions that are most suitable for testing the complementarity of two sequences based on the ultimate application of the hybridizing nucleotides.

[0165] The complementary sequence in a dsRNA agent (e.g., dsRNA agent as described herein) includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence on the total length of one or two nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. However, when the first sequence is referred to herein as "substantially complementary" relative to the second sequence, the two sequences may be fully complementary, or for a duplex of up to 21 base pairs, one or more may be formed during their hybridization, but generally no more than 4, 3 or 2 mismatched base pairs, while retaining the ability to hybridize under conditions most relevant to its final application (e.g., inhibiting gene expression by RISC pathways). However, in the case where two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs should not be considered as mismatches in terms of determining complementarity. For example, a dsRNA agent comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can still be referred to as "fully complementary" for the purposes described herein.

[0166] As used herein, "complementary" sequences may also include or be formed entirely of non-Watson-Crick base pairs or base pairs formed by non-natural and modified nucleotides, as long as the above requirements for their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogstein base pairing.

[0167] It can be understood from the context of its use that the terms "complementary", "completely complementary" and "substantially complementary" herein can be used for base matching between the sense strand and the antisense strand of a dsRNA or between the antisense strand of a dsRNA agent and a target sequence. It is understood that a variety of HBV genotypes are known. Therefore, a dsRNA agent designed to be completely complementary to one HBV genotype may not be completely complementary to all HBV genotypes. dsRNA agents targeting specific sites are effective in knocking down targets on multiple genotypes without being completely complementary on all genotypes.

[0168] 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 continuous portion of a target mRNA (e.g., an mRNA encoding an HBV gene). For example, if its sequence is substantially complementary to a non-interrupted portion of an mRNA encoding an HBV gene, then the polynucleotide is complementary to at least a portion of an HBV mRNA.

[0169] Therefore, in some embodiments, the antisense strand polynucleotide disclosed herein is completely complementary to the target HBV sequence. In some other embodiments, the antisense strand polynucleotide disclosed herein is substantially complementary to the target HBV sequence and comprises a continuous nucleotide sequence that is at least 80% complementary to the equivalent region of the nucleotide sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 over its entire length, for example, at least 80%, 85%, 90% or 95% complementary.

[0170] In some embodiments, the dsRNA agents of the present disclosure include a sense strand that is substantially complementary to an antisense polynucleotide, which in turn is complementary to a target HBV sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least 80% complementary over its entire length to an equivalent region of the nucleotide sequence of SEQ ID NO: 2, e.g., at least 85%, 90%, or 95% complementary.

[0171] In some embodiments, the dsRNA agent includes an antisense strand that is substantially complementary to the target HBV sequence and comprises a contiguous nucleotide sequence that is at least 80% complementary over its entire length to an equivalent region of a nucleotide sequence of any sense nucleotide sequence in Table 2 or a fragment of any sense nucleotide sequence in Table 2, e.g., at least 85%, 90% or 95% complementary.

[0172] As described in detail herein, each chain or two chains also include one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. In addition, dsRNA agents may include ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modification as used in dsRNA agents may be covered by "dsRNA agents".

[0173] In some embodiments, the majority of nucleotides in each chain are ribonucleotides, but as described in detail herein, each or both chains may also include one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. In addition, dsRNA agents may include ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modifications as used in dsRNA agents may be covered by "dsRNA agents" and "dsRNA reagents".

[0174] In the case of HBV gene expression or HBV protein production level or disease marker or symptom in a subject, the term "lower" refers to a statistically significant reduction in such level. The reduction can be, for example, at least 80%, 85%, 90% or 95%, or below the detection level of the detection method, or more toward or reach a normal level (which may be zero or non-zero). When monitoring HBV infection, the log10 scale is generally used to describe the antigenemia level (e.g., HBsAg level in serum) or the viremia level (HBV DNA level in serum). It is understood that a reduction of 1log10 is a 90% reduction (10% remaining), a reduction of 2log10 is a 99% reduction (1% remaining), and the like. In certain embodiments, disease markers are reduced to below the detection level. In certain embodiments, the method includes the inhibition of clinically relevant HBV expression, for example, as demonstrated by clinically relevant results after treating a subject with an agent that reduces HBV expression. In some embodiments, at least partial inhibition of HBV gene expression is assessed by a reduction in the amount of HBV mRNA, which can be isolated from or detected in a first cell or group of cells in which the HBV gene is transcribed or which has been treated such that expression of the HBV gene is inhibited compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but has not been treated (control cells).

[0175] In certain embodiments, "reduction" or "reduction" is understood to reduce or decrease the level toward the normal level or reduce or decrease the level to the normal level, that is, normalize the level. In certain embodiments, the expression of the target is normalized to a level within the normal range of individuals who are generally recognized as not having the disorder, for example, the level of disease markers such as ALT or AST. When the disease-related level increases from the normal level, the change is calculated from the upper limit of normal (ULN). When the disease-related level decreases from the normal level, the change is calculated from the lower limit of normal (LLN). Lowering is the percentage difference between the change in the object value and the normal value. For example, normal AST levels can be reported as 10-40 units / liter. If the subject with an AST level of 200 units / liter (i.e., 5 times of ULN, higher than the upper limit of normal 160 units / liter) before treatment has an AST level of 120 units / liter (i.e., 3 times of ULN, 80 units / liter higher than the upper limit of normal) after treatment, the elevated AST is reduced by 50% (80 / 160) toward normal.

[0176] As another example, a normal ALT level is generally considered to be 7 to 55 units / liter (U / L), resulting in an upper normal limit level of 55 U / L. A subject's ALT level was 100 U / L (45 U / L above the upper normal limit) before treatment and 75 U / L (25 U / L lower) after treatment, and the subject's ALT was reduced by 55% (25 / 45×100%) toward normal levels. As used herein, if a disease is associated with an elevated value for symptoms, "normal" is considered to be the upper normal limit level. If a disease is associated with a reduced value for symptoms, "normal" is considered to be the lower normal limit level.

[0177] As used herein, the phrase "contacting a cell with a dsRNA agent," such as a dsRNA, includes contacting the cell by any possible means. Contacting the cell with a dsRNA agent includes contacting the cell with the dsRNA agent in vitro or contacting the cell with the dsRNA agent in vivo. The contact may be made directly or indirectly. Thus, for example, the dsRNA agent may be brought into physical contact with the cell by the individual performing the method, or the dsRNA agent may be placed in a condition that permits or causes its subsequent contact with the cell.

[0178] In vitro contact with cells can be carried out, for example, by incubating the cells with dsRNA agents. In vivo contact with cells can be carried out, for example, by injecting dsRNA agents into the tissue where the cells are located or in the vicinity thereof, or by injecting dsRNA agents into another region (e.g., bloodstream or subcutaneous space) so that the agent subsequently reaches the tissue where the cells to be contacted are located. For example, dsRNA agents can include or be coupled to a ligand, such as GalNAc3, which guides the dsRNA agent to a target site, such as the liver. A combination of in vitro and in vivo contact methods is also possible. For example, cells can also be contacted with dsRNA agents in vitro, and then transplanted into a subject.

[0179] In some embodiments, contacting a cell with a dsRNA agent includes "introducing" or "delivering a dsRNA agent into a cell" by promoting or enabling uptake or absorption into the cell. The absorption or uptake of the dsRNA agent can occur by unassisted diffusive or active cellular processes, or by an adjuvant or device. The introduction of the dsRNA agent into the cell can be 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 cells includes methods known in the art, such as electroporation and lipofection. Further methods are described below or are known in the art.

[0180] As used herein, "subject" is an animal, such as a mammal, including any mammal that can be infected by HBV, such as a primate (e.g., a human, a non-human primate, such as a monkey or a chimpanzee), or an animal that is considered to be an acceptable clinical model of HBV infection, a HBV-AAV mouse model (see, e.g., Yang et al. (2014) Cell and Mol Immunol 11: 71) or a HBV 1.3xfs transgenic mouse model (Guidotti et al. (1995) J. Virol. 69: 6158). In some embodiments, the subject suffers from hepatitis B virus (HBV) infection. In some embodiments, the subject suffers from hepatitis B virus (HBV) infection and hepatitis D virus (HDV) infection. In some embodiments, the subject is a human, such as a human suffering from HBV infection, particularly a chronic hepatitis B virus (HBV) infection.

[0181] As used herein, the terms "treating" or "treatment" refer to beneficial or desired results, including but not limited to alleviating or ameliorating one or more signs or symptoms associated with undesirable HBV gene expression or HBV replication, such as the presence of serum or liver HBV cccDNA; the presence of serum HBV DNA; the presence of serum or liver HBV antigens (e.g., HBsAg or HBeAg); elevated ALT; elevated AST (generally considered to be in the normal range of about 10-34 U / L); lack of or low levels of anti-HBV antibodies, liver damage; cirrhosis; hepatitis D, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma; serum sickness syndrome (serum sickness–like syndrome); anorexia; nausea; vomiting, low-grade fever; myalgia; easy fatigability; dysregulated taste and smell sensitivity (aversion to food and cigarettes); right upper quadrant and epigastric pain (intermittent, mild to moderate); hepatic encephalopathy; somnolence; disturbed sleep pattern; confusion; coma; ascites; gastrointestinal bleeding; coagulopathy; jaundice; hepatomegaly (mild enlargement, soft liver); splenomegaly; palmar erythema; spider angiomas; muscular atrophy; spider angiomas; vasculitis; venous hemorrhage; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (caput medusa); ALT level higher than AST level; 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 more than 3 times the ULN); slightly low albumin level; elevated serum iron level; leukopenia (i.e., granulocytopenia); lymphocytosis; elevated erythrocyte sedimentation rate (ESR); shortened red blood cell survival; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); presence of serum or liver HBsAg, HBeAg, or hepatitis B core antibody (anti-HBc) immunoglobulin M (IgM); hepatitis B surface antibody (anti-HBs), hepatitis B e antibody (anti-HBe), or HBV The presence of DNA; elevated bilirubin levels; hyperglobulinemia; the presence of tissue nonspecific antibodies, such as anti-smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA) (10-20%); the presence of tissue-specific antibodies, such as antibodies to the thyroid gland (10-20%); elevated rheumatoid factor (RF) levels; low platelet and white blood cell counts; lobular, degenerative and regenerative hepatocellular changes, with associated inflammation; and predominantly centrilobular necrosis, either detectable or undetectable.For example, when individuals with one or more risk factors for liver fibrosis (e.g., chronic hepatitis B infection) are unable to develop liver fibrosis or develop liver fibrosis with a lower severity relative to a group with the same risk factors but not receiving treatment as described herein, the likelihood of developing liver fibrosis is reduced. No disease, disorder, or condition occurs, or the occurrence of signs or symptoms associated with such disease, disorder, or condition is reduced (e.g., clinically relevant amounts); or delayed signs or symptoms that show delay (e.g., delay for a few days, weeks, months, or years) are considered effective prevention. "Treatment" can also mean an extended survival period compared to the expected survival in the absence of treatment. Prevention may require the administration of more than one dose.

[0182] In a preferred embodiment, the treatment of HBV infection results in a "functional cure" of hepatitis B. As used herein, functional cure is understood to be the removal of circulating HBsAg, and is preferably accompanied by a transition to a state in which HBsAg antibodies become undetectable using a clinically relevant assay. For example, detectable antibodies may include a signal greater than 10mIU / ml, as measured by chemiluminescent microparticle immunoassay (CMIA) or any other immunoassay known as anti-HB seroconversion. Functional cure does not require the removal of all replicated forms of HBV (e.g., cccDNA from the liver). Anti-HB seroconversion occurs spontaneously in approximately 0.2-1% of chronically infected patients each year. However, even after anti-HB seroconversion, low levels of HBV have been observed to persist for decades, indicating that functional cure rather than complete cure has occurred. Without being limited to the mechanism, it is proposed that the immune system is able to keep HBV under control. Functional cure allows termination of any treatment for HBV infection. However, it should be understood that a "functional cure" for HBV infection may not be sufficient to prevent or treat diseases or conditions caused by HBV infection, such as liver fibrosis, HCC, cirrhosis.

[0183] As used herein, the term "hepatitis B virus-related disease" or "HBV-related disease" is 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 reduced HBV gene expression or replication. Non-limiting examples of HBV-related diseases include, for example, hepatitis D virus infection, hepatitis D, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0184] In some embodiments, the HBV-related disease is hepatitis D virus infection. Hepatitis D virus or hepatitis D virus (HDV) is a human pathogen. However, the virus is defective and relies on the specialized helper functions provided by hepatitis B virus (HBV) for transmission; in fact, HDV requires an associated or pre-existing HBV infection to become infectious and active, in particular a viral envelope containing hepatitis B surface antigen. HDV can cause severe acute and chronic liver disease forms associated with HBV. Hepatitis D infection or hepatitis D is very prevalent in several African countries, the Amazon region and the Middle East, while the prevalence in industrialized countries (except the Mediterranean) is low.

[0185] Transmission of HDV can occur either through simultaneous HBV infection (coinfection) or superimposed on chronic hepatitis B or hepatitis B carrier status (superinfection). Superinfection and coinfection with HDV both lead to more severe complications compared to HBV infection alone. These complications include a greater likelihood of developing liver failure and rapid progression to cirrhosis in acute infection, and an increased chance of developing liver cancer in chronic infection. In combination with hepatitis B virus, hepatitis D has the highest mortality rate of all hepatitis infections at 20%.

[0186] In some embodiments, HBV related diseases are acute hepatitis B. Acute hepatitis B includes liver inflammation that lasts less than six months. Typical symptoms of acute hepatitis B are fatigue, anorexia, nausea and vomiting. Very high transaminase values ​​(>1000U / L) and hyperbilirubinemia are often observed. Severe cases of acute hepatitis B may rapidly develop into acute liver failure, which is marked by poor liver synthetic function. In the absence of previous liver disease, this is usually defined as a prothrombin time (PT) of 16 seconds or an international normalized ratio (INR) of 1.5. Acute hepatitis B can evolve into chronic hepatitis B.

[0187] In some embodiments, the HBV-related disease is chronic hepatitis. Chronic hepatitis B (CHB) includes inflammation of the liver that persists for more than six months. Subjects with CHB are HBsAg positive and have high viremia (≥10 4 HBV-DNA copies / ml blood) or low viremia (<10 3HBV-DNA copies / ml blood). In some embodiments, the subject has been infected with HBV for at least five years. In some embodiments, the subject has been infected with HBV for at least ten years. In some embodiments, the subject was infected with HBV at birth. Subjects with chronic hepatitis B disease may be immune tolerant or have inactive chronic infection without any evidence of active disease, and they are also asymptomatic. Patients with chronic active hepatitis, especially during the replicative state, may have symptoms similar to acute hepatitis. Subjects with chronic hepatitis B disease may have active chronic infection with necroinflammatory liver disease, increased liver cell turnover in the absence of detectable necroinflammation, or have inactive chronic infection without any evidence of active disease, and they are also asymptomatic. The persistence of HBV infection in CHB subjects is the result of cccHBVDNA. In some embodiments, subjects with CHB are HBeAg positive. In another embodiment, subjects with CHB are HBeAg negative. The serum HBV DNA level of subjects with CHB is less than 10 5 , and transaminases (eg, ALT, AST, and γ-glutamyl transferase) remain elevated. Subjects with CHB may have a liver biopsy score (eg, necroinflammatory score) of less than 4.

[0188] In some embodiments, the HBV-related disease is acute fulminant hepatitis B. Subjects with acute fulminant hepatitis B have symptoms of acute hepatitis and additional symptoms of unconsciousness or coma (due to the liver's inability to detoxify chemicals) and bruising or bleeding (due to lack of clotting factors).

[0189] Subjects with HBV infection (e.g., CHB) may develop liver fibrosis. Therefore, in some embodiments, the HBV-related disease is liver fibrosis. Liver fibrosis or cirrhosis is histologically defined as a diffuse liver process characterized by fibrosis (excessive fibrous connective tissue) and the transformation of normal liver structure into structurally abnormal nodules.

[0190] Subjects with HBV infection (e.g., CHB) may develop end-stage liver disease. Thus, in some embodiments, the HBV-related disease is end-stage liver disease. For example, liver fibrosis may progress to a point where the body may no longer be able to compensate for, for example, a decrease in liver function (i.e., liver decompensation) due to liver fibrosis, and lead to, for example, psychiatric and neurological symptoms and liver failure.

[0191] Subjects with HBV infection (e.g., CHB) may develop hepatocellular carcinoma (HCC), also known as malignant liver tumors. Therefore, in some embodiments, the HBV-related disease is HCC. HCC usually occurs in subjects with CHB and may be fibrolamellar, pseudoglandular (adenoid), pleomorphic (giant cell), or clear cell.

[0192] An "HDV-associated disorder" or "hepatitis D virus-associated disorder" is a disease or disorder associated with HDV expression. 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.

[0193] As used herein, "therapeutically effective amount" is intended to include the amount of dsRNA agents sufficient to effectively treat the disease (e.g., by reducing or maintaining one or more symptoms of an existing disease or disease) when administered to a patient for the treatment of a patient with HBV infection or HBV-related disease. "Therapeutically effective amount" may vary according to the dsRNA agent, how the agent is administered, the disease and its severity, and medical history, age, weight, family history, genetic makeup, the stage of the pathological process mediated by HBV gene expression, the type of previous or concomitant therapy (if any), and other individual characteristics of the patient to be treated. A therapeutically effective amount may require administration of more than one dose.

[0194] "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 disclosed method can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0195] As used herein, the term "sample" includes similar fluids, cells or tissues separated from a subject, and the collection of fluids, cells or tissues present in a subject. The example of a biological fluid includes blood, serum and serosal fluid, plasma, lymph, urine, saliva, etc. Tissue samples can include samples from tissues, organs or local areas. For example, a sample can be derived from a specific organ, a part of an organ or the fluid or cells in those organs. In certain embodiments, a sample can be derived from liver (cells of certain types in certain sections of the whole liver or liver or liver, such as hepatocytes). In a preferred embodiment, "sample derived from a subject" refers to the blood extracted from a subject, or plasma or serum obtained from blood. In a further embodiment, "sample derived from a subject" refers to liver tissue (or its subcomponents) or blood tissue (or its subcomponents, such as serum) derived from a subject.

[0196] II. dsRNA Agents

[0197] The present disclosure provides dsRNA agents that inhibit the expression of one or more HBV genes. In some embodiments, the dsRNA agent includes a double-stranded ribonucleic acid (dsRNA) molecule, which is used to inhibit the expression of HBV genes in cells (e.g., cells in subjects (e.g., mammals, such as people with HBV-related diseases, such as chronic hepatitis B)). The dsRNA agent includes an antisense strand with a complementary region, which is complementary to at least a portion of the mRNA formed in the expression of the HBV gene. The length of the complementary region is about 19-21 nucleotides. When contacted with cells expressing HBV genes, the dsRNA agent inhibits the expression of the HBV gene by at least 80%, such as by, for example, PCR or a branched DNA (bDNA)-based method or by a protein-based method (e.g., by immunofluorescence analysis), using, for example, Western blotting or flow cytometry techniques. In a preferred embodiment, the real-time PCR method provided in Example 2 is used to determine the percentage of inhibition using a cell line provided therein with a dsRNA agent (used at a concentration of 10 nM in transfection).

[0198] DsRNA comprises two complementary and hybridized RNA chains to form a duplex structure under the conditions of dsRNA use. One chain (antisense strand) of dsRNA comprises a complementary region, which is substantially complementary to the target sequence and is usually completely complementary. However, due to the sequence variation between the HBV genotypes, dsRNA may be completely complementary to some but not all HBV genotypes. The target sequence can be derived from the sequence of the mRNA formed during HBV gene expression. Another chain (sense strand) includes a region complementary to the antisense strand so that the two chains hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and known in the art, in contrast to on a single oligonucleotide, the complementary sequence of dsRNA can also be included as the self-complementary region of a single nucleic acid molecule.

[0199] Typically, the duplex structure is 19-21 base pairs in length, such as 19, 20 or 21 base pairs in length.

[0200] Similarly, the length of the complementary region to the target sequence is 19-23 nucleotides, such as 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 length of the complementary region is 21 nucleotides. It will also be appreciated by those of ordinary skill that the RNA region targeted for cleavage is most often a portion of a larger RNA molecule (usually an mRNA molecule). In relevant cases, a "portion" of an mRNA target is a continuous sequence of an mRNA target of sufficient length so that it can serve as a substrate for RNAi-guided cleavage (i.e., cleavage by a RISC approach).

[0201] Those skilled in the art will also recognize that the duplex region is the main functional portion of the dsRNA, such as a duplex region of about 19-21 base pairs, such as 19-21, 19-20, 20-21, 19, 20 or 21 base pairs. In some embodiments, the duplex region is 19 base pairs.

[0202] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, such as 1 or 2 nucleotides. The nucleotide overhangs may comprise or be composed of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhangs may be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhangs may be present at the 5' end, 3' end, or both ends of the antisense strand or the sense strand of the dsRNA. In some embodiments, the dsRNA comprises an overhang of 2-nucleotides on the 3' end of the antisense strand.

[0203] dsRNA can be synthesized by standard methods known in the art as discussed further below, for example, by using an automated DNA synthesizer such as those available from, for example, Biosearch, Applied Biosystems, TM , Inc. Methods for synthesizing dsRNA for use in pharmaceutical compositions are also known in the art.

[0204] The dsRNA agent compound of the present disclosure can be prepared using a two-step process. First, prepare the single strands of the double-stranded RNA molecule respectively. Then, anneal the component strands. The single strands of the dsRNA agent compound can be prepared using solution phase or solid phase organic synthesis or both. The advantage of organic synthesis is that oligonucleotide chains containing non-natural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the present disclosure can be prepared using solution phase or solid phase organic synthesis or both.

[0205] In some embodiments, the dsRNA agent of the present disclosure includes at least two nucleotide sequences, a sense strand sequence and an antisense strand sequence. The sense strand may comprise a nucleotide sequence selected from any sense strand nucleotide sequence of any duplex in Table 2. The antisense strand may comprise a nucleotide sequence selected from any antisense strand nucleotide sequence of any duplex in Table 2. In some embodiments, the dsRNA agent is not AD-66810.

[0206] In some embodiments, the dsRNA agents of the present disclosure comprise, consist essentially of, or consist of a sense strand and an antisense strand as shown in Table 2.

[0207] III. Modified dsRNA Agents

[0208] In certain embodiments, the RNA of the dsRNA agent of the present disclosure is unmodified and does not include chemical modifications or conjugations such as those known in the art and described herein. In other embodiments, the RNA of the dsRNA agent of the present disclosure is chemically modified to enhance stability or other beneficial properties. In some embodiments of the present disclosure, substantially all nucleotides of the dsRNA agent of the present disclosure are modified. In some embodiments of the present disclosure, all nucleotides of the dsRNA agent or substantially all nucleotides of the dsRNA agent are modified, that is, no more than 5, 4, 3, 2 or 1 unmodified nucleotides are present in the chain of the dsRNA agent.

[0209] The nucleic acids featured in the present disclosure can be synthesized or modified by well-established methods in the art, for example, those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse connection) or 3'-terminal modifications (conjugation, DNA nucleotides, reverse connection, etc.); base modifications, such as replacement with stabilized bases, destabilized bases, or bases that pair with an extended partner library, removal of bases (abasic nucleotides) or conjugated bases; sugar modifications (e.g., at the 2'-position or 4'-position) or replacement of sugars; or backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of dsRNA agent compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or not containing natural internucleoside linkages. The RNA with modified main chain especially includes those RNA without phosphorus atom in the main chain.For the purpose of this specification, and as sometimes mentioned in this area, the modified RNA without phosphorus atom in the internucleoside main chain can also be considered as oligonucleoside.In some embodiments, the dsRNA agent of modification has phosphorus atom in its internucleoside main chain.

[0210] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates include 3'-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates include 3'-phosphoramidates and aminoalkylphosphonates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and boranophosphates, which have normal 3'-5' bonds, 2'-5'-connected analogs of these esters and those with reversed polarity, wherein adjacent nucleoside unit pairs are 3'-5' to 5'-3' connected or 2'-5' to 5'-2' connected. Various salts, mixed salts and free acid forms are also included.

[0211] Representative U.S. patents that teach the preparation of the above-described phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,58 7,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,6 39; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. No. RE39464, each of which is hereby incorporated by reference herein for teachings relating to such methods of preparation.

[0212] The modified RNA backbones that do not contain phosphorus atoms have backbones formed by short-chain alkyl or cycloalkyl nucleoside interlinkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside interlinkages, or one or more short-chain heteroatoms or heterocyclic nucleoside interlinkages. These include backbones with morpholine linkages (partially formed by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; acetyl and thioacetyl backbones; methyleneacetyl and thioacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and those with other backbones of mixed N, O, S and CH2 components.

[0213] Representative U.S. patents that teach the preparation of the above oligonucleotides 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; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is hereby incorporated by reference for teachings relating to such methods.

[0214] It is envisioned that suitable RNA mimics will be used in the dsRNA agents provided herein, wherein the sugar and internucleoside connections of the nucleotide units, i.e., the main chain, are replaced by new groups. The base units are maintained to hybridize with appropriate nucleic acid target compounds. One such oligomeric compound is referred to as peptide nucleic acid (PNA), in which RNA mimics have been shown to have good hybridization properties. In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing main chain, particularly an aminoethylglycine main chain. The nucleobase is retained and directly or indirectly bound to the aza nitrogen atom of the amide portion of the main chain. Representative U.S. 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 to provide teachings related to such methods. Other PNA compounds suitable for dsRNA agents of the present disclosure are described in, for example, Nielsen et al., Science, 1991, 254, 1497-1500.

[0215] Some embodiments characterized in the present disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [referred to 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--] in the above-mentioned U.S. Patent No. 5,489,677, the amide backbone in the above-mentioned U.S. Patent No. 5,602,240. In some embodiments, the RNA characterized herein has the morpholinyl backbone structure of the above-mentioned U.S. Patent No. 5,034,506.

[0216] The modified RNA may also contain one or more substituted sugar moieties. The dsRNA agents characterized herein may include one of the following at the 2'-position: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, wherein n and m are 1 to about 10. In other embodiments, the dsRNA includes one of the following at the 2' position: C 1- C 10Lower 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 for improving the pharmacokinetic properties of dsRNA agent or group for improving 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, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, as described in the Examples below. Further exemplary modifications include: 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers in these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0217] 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 of the RNA of the dsRNA agent, particularly at the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and at the 5' position of the 5' terminal nucleotide. The dsRNA agent can also have a sugar mimetic (e.g., a cyclobutyl moiety) instead of the furanosyl sugar. Representative U.S. patents that teach 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; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which have common owners with the present application. The contents of each of the above patent disclosures are hereby incorporated by reference into this document to provide teachings related to such methods.

[0218] dsRNA agents can also include modifications or replacements of nucleobases (often referred to in the art as "bases"). 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 deoxythymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propyne uracil and cytosine, 6-azouracil, Cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Other nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present disclosure. These nucleobases include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are an exemplary base substitution, even more so when combined with 2'-O-methoxyethyl sugar modifications.

[0219] Representative U.S. patents that teach the preparation of some of the above-mentioned modified nucleobases as well as other modified nucleobases include, but are not limited to, the above-mentioned U.S. Patent Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594, 7,427,672; and 7,495,088, each of which is hereby incorporated by reference herein for teachings relating to such methods.

[0220] In some embodiments, the RNA of dsRNA agent can also be modified to include one or more bicyclic sugar moieties." Bicyclic sugar " is a furanosyl ring modified by the bridging of two atoms. " Bicyclic nucleoside " (" BNA ") is a nucleoside with a bridge comprising two carbon atoms connecting a sugar ring to form a sugar moiety of a bicyclic system. In certain embodiments, the bridge connects 4'-carbon and 2'-carbon of a sugar ring. Therefore, in some embodiments, the agent of the present disclosure may include one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with modified ribose moieties, wherein the ribose moiety comprises an extra bridge connecting 2' and 4' carbon. In other words, LNA is a nucleotide comprising a bicyclic sugar moiety, and the bicyclic sugar moiety comprises a 4'-CH2-O-2' bridge. This structure effectively "locks" ribose into a 3'-endo structural conformation. It has been demonstrated that adding locked nucleic acid to dsRNA agents improves the stability of dsRNA agents in serum and reduces off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33 (1): 439-447; Mook, OR. et al., (2007) Mol Canc Ther 6 (3): 833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31 (12): 3185-3193). Examples of bicyclic nucleosides for use in polynucleotides of the present disclosure include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the present disclosure include one or more bicyclic nucleosides comprising a 4'-2' bridge.Examples of such 4'-2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also known as "constrained ethyl" nucleosides). ethyl)" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and the like; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and the like; see, e.g., U.S. Pat. No. 8,278,283); 4'-CH2-N(OCH3)-2' (and the like; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH2-O-N(CH3)-2' (see, e.g., U.S. Pat. Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2', wherein R is H, C1-C12 alkyl or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al. al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2—C(═CH2)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The contents of the above-mentioned documents related to modified nucleic acids are each incorporated herein by reference.

[0221] Other representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US2008 / 0039618; and US2009 / 0012281, which are hereby incorporated by reference into this document for teachings related to such methods.

[0222] Any of the foregoing bicyclic nucleosides may be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0223] The RNA of dsRNA agent can also be modified to include one or more constrained ethyl nucleotides. As used herein, "constrained ethyl nucleotides" or "cEt" are locked nucleic acids comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-O-2' bridge. In some embodiments, the constrained ethyl nucleotide is an S configuration, referred to herein as "S-cEt".

[0224] The dsRNA agents of the present disclosure may also comprise one or more "conformationally restricted nucleotides" ("CRNs"). CRNs are nucleotide analogs having 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 length of the linker is sufficient to place the oxygen in a position that is optimal for stability and affinity, resulting in less puckering of the ribose ring.

[0225] Representative publications that teach the preparation of certain of the above-described CRNs include, but are not limited to, US Patent Publication No. 2013 / 0190383; and PCT Publication WO 2013 / 036868, which are hereby incorporated by reference herein for teachings related to such methods.

[0226] In some embodiments, the dsRNA agent of the present invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which any bond of a sugar is removed to form an unlocked "sugar" residue. In one example, UNA also includes monomers in which the bond between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between C1' and C4' carbon) is removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between C2' and C3' carbon) 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 to provide teachings related to unlocked nucleic acid nucleotides).

[0227] Representative U.S. publications that teach UNA preparation include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, which are hereby incorporated by reference herein for teachings related to such methods.

[0228] Potential stabilizing modifications to the ends of RNA molecules may include N-(acetylaminohexanoyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyprolinol) (Hyp-C6), N-(acetyl-4-hydroxyprolinol) (Hyp-NHAc), thymidine 2'-O-deoxythymidine (ether), N-(aminohexanoyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosahexaenoyl-uridine-3'-phosphate, inverted base dT (idT) and others. The disclosure of this modification can be found in PCT Publication No. WO2011 / 005861.

[0229] In certain embodiments, the dsRNA agent is modified to include one or more adenosine-diol nucleic acids ("GNA"). The term "GNA" refers to diol nucleic acid, which is a polymer similar to DNA or RNA, but the composition of its "backbone" differs from that of DNA or RNA in that it is composed of repeating glycerol units linked by phosphodiester bonds:

[0230] A description of adenosine-GNA can be found, for example, in Zhang, et al. (JACS 127(12):4174-75 (2005)).

[0231] Other modifications of the nucleotides of the dsRNA agents disclosed herein include 5' phosphates or 5' phosphate mimetics, such as 5'-terminal phosphates or phosphate mimetics on the antisense strand of the dsRNA agent. Suitable phosphate mimetics are disclosed in, for example, U.S. Patent Publication No. 2012 / 0157511, which is hereby incorporated herein by its teachings related to such modifications.

[0232] Additional modified dsRNA agents targeting HBV are provided, for example, in WO2016 / 077321, which is incorporated herein by reference for teachings related to modifications.

[0233] In some embodiments, the dsRNA agent comprises, consists essentially of, or consists of a modified sense strand and a modified antisense strand as shown in Table 2.

[0234] IV. dsRNA agent conjugated to a ligand

[0235] The dsRNA agents disclosed herein can be conjugated with ligands. The ligands can change the distribution, targeting or life span of the dsRNA agents into which they are incorporated. In preferred embodiments, for example, such ligands provide enhanced affinity to selected targets (e.g., molecules, cells or cell types, compartments such as cells or organ compartments, tissues, organs or regions of the body) compared to species lacking ligands. Preferred ligands will not participate in duplex pairing in double-stranded nucleic acids.

[0236] The ligand-conjugated oligonucleotides of the present disclosure can be synthesized by using oligonucleotides with pendant reactive functional groups, such as those derived from linker molecules attached to the oligonucleotides (described below). The reactive oligonucleotides can react directly with commercially available ligands, synthetic ligands with any of a variety of protecting groups, or ligands with linker moieties attached thereto.

[0237] In the ligand-conjugated oligonucleotides and ligand-carrying sequence-specific linked nucleosides molecules of the present disclosure, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already carry a linking portion, ligand-nucleotide or nucleoside-conjugate precursors that already carry a ligand molecule, or building blocks with non-nucleoside ligands.

[0238] When using nucleotide-conjugate precursors that already carry a linking moiety, the synthesis of sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form a ligand-conjugated oligonucleotide. In some embodiments, in addition to standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis, phosphoramidites derived from ligand-nucleoside conjugates are also used to synthesize the oligonucleotides or linked nucleosides disclosed herein by an automated synthesizer.

[0239] Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,48 6,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5 ,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241,5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,81 0; 5,574,142; 5,585,481; 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; 8,106,022, which are hereby incorporated herein by their teachings relating to such methods of preparation.

[0240] A. Carbohydrate conjugates

[0241] In the preferred embodiment of the compositions and methods disclosed herein, the dsRNA agent oligonucleotide further comprises carbohydrates. As described herein, the dsRNA agent conjugated with carbohydrates is conducive to the in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a carbohydrate composed of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) and oxygen, nitrogen or sulfur atoms bonded to each carbon atom, or a compound having a carbohydrate moiety composed of one or more monosaccharide units as a part thereof, each monosaccharide unit having at least 6 carbon atoms (which can be linear, branched or cyclic) and oxygen, nitrogen or sulfur atoms bonded to each carbon atom. 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 gum. Specific monosaccharides include sugars of C5 and above (eg, C5, C6, C7, or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (eg, C5, C6, C7, or C8).

[0242] In some embodiments, the carbohydrate conjugate used in the compositions and methods of the present disclosure is a monosaccharide. In some embodiments, the carbohydrate conjugate used in the compositions and methods of the present disclosure is selected from:

[0243]

[0244]

[0245]

[0246]

[0247]

[0248] In some embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). In some embodiments, the carbohydrate comprises multiple N-acetylgalactosamine units, such as

[0249]

[0250] Another representative carbohydrate conjugate useful in the embodiments described herein includes, but is not limited to,

[0251]

[0252]

[0253] Wherein when one of X or Y is an oligonucleotide, the other is hydrogen.

[0254] In some embodiments of the present disclosure, GalNAc or a GalNAc derivative is connected to a dsRNA agent of the present disclosure via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is connected to a dsRNA agent of the present disclosure via a divalent linker. In some embodiments of the present disclosure, GalNAc or a GalNAc derivative is connected to a dsRNA agent of the present disclosure via a trivalent linker. In some embodiments, the carbohydrate ligand comprises three N-acetylgalactosamine units ("GalNAc3") connected via a trivalent linker.

[0255] In some embodiments, the double-stranded dsRNA agent comprises a GalNAc or GalNAc derivative connected to the dsRNA agent. In some embodiments, the double-stranded dsRNA agent comprises a plurality of (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each of which is independently connected to a plurality of nucleotides of the double-stranded dsRNA agent via a plurality of monovalent linkers.

[0256] Additional carbohydrate conjugates suitable for use in the present disclosure include those described in PCT Publication Nos. WO2014 / 179620 and WO2014 / 179627, which are incorporated herein by reference for their teachings related to such conjugates.

[0257] Non-limiting examples of dsRNA agent carbohydrate conjugates with linkers that can be used in the compositions and methods disclosed herein include, but are not limited to,

[0258]

[0259]

[0260]

[0261] Wherein when one of X or Y is an oligonucleotide, the other is hydrogen.

[0262] 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.

[0263] In some embodiments, the dsRNA agents disclosed herein are conjugated to a divalent or trivalent branched linker selected from the structures shown in any one of Formulas (XXXI)-(XXXIV):

[0264]

[0265] in:

[0266] q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C are independently 0-20 at each occurrence, wherein the repeating units may be the same or different;

[0267] 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 CHO;

[0268] Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C Each occurrence is independently absent, alkylene, substituted alkylene, wherein one or more methylene groups may be replaced by O, S, S(O), SO2, N(R N ), C(R')=C(R"), C≡C or C(O);

[0269] R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C Each occurrence is independently absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, or a heterocyclic group;

[0271] L2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; that is, each occurrence is independently a monosaccharide (eg, GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; and R a Is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are particularly useful when used with dsRNA agents for inhibiting the expression of target genes, such as those of formula (XXXIV):

[0272]

[0273] Where L 5A , L 5B and L 5C represents a monosaccharide, such as a GalNAc derivative.

[0274] Examples of suitable divalent and trivalent branched linker group conjugated GalNAc derivatives include, but are not limited to, the structures described above as Formulas I, VI, X, IX and XII.

[0275] B. Connectors

[0276] In some embodiments, the conjugates or ligands described herein can be linked to the dsRNA agent oligonucleotide through various linkers, cleavable or non-cleavable.

[0277] The term "linker" or "linking group" refers to an organic moiety that connects two parts of a compound, such as covalently attaching two parts of a compound. The linker typically comprises a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, 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, Alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, whose one or more methylene groups are interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle; 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.

[0278] A cleavable linking group is a linking group that is sufficiently stable outside the cell, but is cleaved after entering the target cell to release the two parts held together by the linker. In some embodiments, the cleavable linking group is cleaved in the target cell or under a first reference condition than in the blood of the subject or under a second reference condition (which can, for example, be selected to simulate or represent the conditions present in blood or serum) by 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.

[0279] Cleavable linking groups are susceptible to the influence of cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Typically, cleavage agents are more prevalent or present at higher levels or activities in cells than in serum or blood. Examples of such degrading agents include: redox agents that are selected for specific substrates or have no substrate specificity, including, for example, oxidative or reductases or reductants present in cells, such as thiols, which can degrade redox cleavable linking groups by reduction; esterases; endosomes or agents that can produce an acidic environment, such as those that result in a pH of 5 or less; enzymes, peptidases (which can be substrate-specific), and phosphatases that can hydrolyze or degrade acid-cleavable linking groups by acting as generalized acids.

[0280] Cleavable linking groups, such as disulfide bonds, can be sensitive to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, about 7.1-7.3. Endosomes are more acidic, with a pH range of 5.5-6.0, while lysosomes have an even more acidic pH of about 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand or into the desired cellular compartment within the cell.

[0281] The linker can include a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker can depend on the cell to be targeted. For example, a liver-targeted part can be connected to a cationic lipid by a linker that includes an ester group. Hepatocytes are rich in esterases, so compared to cell types that are not rich in esterases, the linker is more efficiently cut in hepatocytes. Other cell types that are rich in esterases include cells of the lung, kidney cortex, and testis.

[0282] When targeting cell types rich in peptidases (eg, hepatocytes and synoviocytes), linkers containing peptide bonds can be used.

[0283] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradation agent (or condition) to cut a candidate linking group. It is also desirable to test the ability of a candidate cleavable linking group to resist cutting in blood or when contacting with other non-target tissues. Therefore, the relative cutting susceptibility between the first condition and the second condition can be determined, wherein the first condition is selected as the cutting in the target cell indicated, and the second condition is selected as the cutting in other tissues or biological fluids (e.g., blood or serum). It can be evaluated in a cell-free system, in a cell, in a cell culture, in an organ or tissue culture, or in a whole animal. It may be useful to conduct a preliminary assessment under cell-free or culture conditions and to confirm by further assessment in a whole animal. In a preferred embodiment, the available candidate compound has a cutting speed block of at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times in a cell (or under in vitro conditions selected as simulated intracellular conditions) and serum or blood (or under in vitro conditions selected as simulated extracellular conditions).

[0284] i. Redox cleavable linker

[0285] In some embodiments, the cleavable linking group is a redox cleavable linking group that is cleaved when reduced or oxidized. The example of a reduction cleavable linking group is a disulfide bond linking group (-SS-). In order to determine whether a candidate cleavable linking group is a suitable "reduction cleavable linking group", or whether it is suitable for use with a specific dsRNA agent part and a specific targeting agent, reference can be made to the methods described herein. For example, a candidate can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art, which simulate the cleavage rate observed in cells (such as target cells). Candidates can also be evaluated under conditions selected to simulate blood or serum conditions. In some embodiments, candidate compounds are cut up to about 10% in blood. In some embodiments, available candidate compounds are at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90 or about 100 times faster than blood (or under in vitro conditions selected to simulate extracellular conditions) in cells (or under in vitro conditions selected to simulate intracellular conditions). The rate of cleavage of a candidate compound can be determined using standard enzyme kinetic assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

[0286] ii. Phosphate-based cleavable linkers

[0287] In some embodiments, the cleavable linker comprises a cleavable linking group based on phosphate. The cleavable linking group based on phosphate is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents that cleave phosphate groups in cells are enzymes, such as phosphatases in cells. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. In some embodiments, examples of phosphate-based linking groups are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, 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.

[0288] iii. Acid cleavable linking group

[0289] In some embodiments, the cleavable linking group comprises 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 of a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or less), or is cleaved by a reagent such as an enzyme that can act as a general acid. In cells, specific low pH organelles (e.g., endosomes and lysosomes) can provide a cleavage environment for acid cleavable linking groups. Examples of acid cleavable linking groups include, but are not limited to, esters of hydrazones, esters, and amino acids. An acid cleavable group may have the general formula -C=NN-, C(O)O, or -OC(O). In some embodiments, the carbon of the oxygen (alkoxy) connected to the ester is an aryl, substituted alkyl, or tertiary alkyl, such as a dimethylpentyl or tert-butyl group. These candidates can be evaluated using methods similar to those described above.

[0290] iv. Ester-based linking groups

[0291] 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 esterases and amidases in the cell. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene. The ester-cleavable linking group has the general formula -C (O) O- or -OC (O) -. These candidates can be evaluated using methods similar to those described above.

[0292] v. Peptide-based cleavage groups

[0293] 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-). The amide group can be formed between any alkylene, alkenylene or alkynylene. The peptide bond is a specific type of amide bond formed between amino acids to produce peptides and proteins. The peptide-based cleavage group is generally limited to the peptide bond (i.e., amide bond) formed between the amino acids producing peptides and proteins, and does not include the entire amide functional group. The general formula of the peptide-based cleavable linking group is-NHCHRAC(O)NHCHRBC(O)-, wherein RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using a method similar to that described above.

[0294] V. Delivery of dsRNA agents

[0295] The dsRNA agents of the present invention can be delivered to cells, such as cells in a subject such as a human subject, in a variety of different ways. For example, delivery can be carried out by contacting the cells with the dsRNA agents of the present invention in vitro or in vivo. In vivo delivery can also be carried out directly by applying a composition comprising the dsRNA agent to the subject. Alternatively, in vivo delivery can be carried out indirectly by administering one or more vectors that encode and direct the expression of the dsRNA agent. These alternatives will be discussed further below.

[0296] 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 disclosure (see, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5): 139-144 and WO94 / 02595, which are incorporated herein by reference to provide teachings related to such delivery methods). For in vivo delivery, factors to be considered for the delivery of dsRNA agent molecules include, for example, biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue.

[0297] In order to administer dsRNA agents systemically to treat diseases, RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent endonucleases and exonucleases from rapidly degrading dsRNA in vivo. Modification of RNA or drug carriers or pharmaceutical excipients can also allow dsRNA agent compositions to be targeted to target tissues and avoid adverse off-target effects. dsRNA agent molecules can be modified by chemical conjugation, such as carbohydrate conjugates as described above.

[0298] VI. Pharmaceutical composition

[0299] The present invention also includes pharmaceutical compositions and preparations comprising dsRNA agents disclosed herein. In some embodiments, pharmaceutical compositions comprising dsRNA agents as described herein and pharmaceutically acceptable carriers are provided herein. Pharmaceutical compositions comprising dsRNA agents can be used to treat diseases or conditions related to the expression or activity of HBV genes. Such pharmaceutical compositions are formulated based on delivery methods. An example is a composition formulated for systemic administration by parenteral delivery (e.g., by subcutaneous (SC), intramuscular (IM) or intravenous (IV) delivery). In certain embodiments, the present disclosure provides compositions formulated for organ-specific (e.g., liver) intraarterial, intratumoral, intradermal, intravitreal injection, ocular topical, ophthalmic (eye drops), atomization, ocular topical or other local routes, suppositories or oral administration. In a preferred embodiment, the composition is administered subcutaneously.

[0300] The pharmaceutical composition of the present disclosure can be administered with a dosage sufficient to inhibit HBV gene expression. In some embodiments, the dsRNA agent is administered with the following dosage: about 0.5mg / kg to 50mg / kg per dose, or 0.3mg / kg to 20mg / kg, or 3mg / kg to 10mg / kg, or preferably 3mg / kg to 10mg / kg per dose. For example, dsRNA can be administered with a dosage of about 0.5mg / kg, 1mg / kg, 1.5mg / kg, 2mg / kg, 3mg / kg, 10mg / kg, 20mg / kg, 30mg / kg, 40mg / kg or 50mg / kg per single dose. In some embodiments, the dsRNA agent is administered with a dosage of 50mg to 900mg.

[0301] Compositions can also be prepared and packaged in fixed doses for subjects that are independent of body weight. Exemplary dosage levels can be calculated by multiplying per kilogram of body weight by the average subject's body weight. For example, the average body weight of a typical adult is considered to be about 70 kilograms.

[0302] Repeated dosage regimens can include regularly administering a therapeutic amount of dsRNA agent, such as once a month, once every other month, or once every three months. In a preferred embodiment, the dsRNA agent is administered at a frequency no more than once a month. After the initial treatment regimen, treatment can be administered at a lower frequency.

[0303] The pharmaceutical composition can be administered indefinitely, for example, in subjects with one or more signs or symptoms of HBV infection, such as detectable HBV antigen or HBV DNA (including HBV cccDNA). In some embodiments, treatment with a dsRNA agent is performed for a discontinuous or limited period of time and provides a functional cure.

[0304] The skilled person will appreciate that certain factors may affect the dosage and timing required for effective treatment of a subject, including but not limited to the severity of the disease or condition, previous treatment, the subject's overall health or age, and other diseases present. In addition, treatment of a subject with a therapeutically effective amount of a composition may include a single treatment or a series of treatments. As described elsewhere herein, conventional methods or in vivo tests based on the use of appropriate animal models may be used to estimate the effective dosage and in vivo half-life of a single dsRNA agent encompassed by the present invention.

[0305] A. excipient

[0306] A "pharmaceutical carrier" or "pharmaceutical excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmaceutically inert vehicle for delivering one or more nucleic acids to an animal. Such agents are well known in the art.

[0307] B. Other components

[0308] Compositions disclosed herein can additionally include other auxiliary components conventionally present in pharmaceutical compositions with the use level established in this area.Therefore, for example, said composition can include other, compatible pharmaceutically active substances, such as antipruritic, astringent, local anesthetic or anti-inflammatory, or can include other materials of various dosage forms that can be used for physical preparation of compositions disclosed herein, such as preservative, antioxidant and stabilizer.However, when adding, such materials should not inappropriately interfere with the biological activity of the components of compositions disclosed herein.Preparation can be sterilized, and if necessary, can be mixed with auxiliary agents that can not be harmfully interacted with the nucleic acid of preparation, such as preservative, stabilizer, wetting agent, emulsifier, salt or buffer agent that affect osmotic pressure etc.

[0309] In some embodiments, the pharmaceutical compositions characterized in the present disclosure include (a) one or more dsRNA agent compounds and (b) one or more agents that function through non-RNAi mechanisms and can be used to treat HBV-related disorders. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-fat degeneration agents, anti-viral agents, and anti-fibrotic agents.

[0310] In addition, other substances commonly used to protect the liver, such as silymarin, can also be used in combination with the dsRNA agents described herein. Other agents that can be used to treat liver diseases include telbivudine, entecavir and protease inhibitors, such as telaprevir and other drugs disclosed in, for example, US2005 / 0148548, US2004 / 0167116, US2003 / 0144217 and US2004 / 0127488.

[0311] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., for determining LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population)). The dose ratio between toxicity and therapeutic efficacy is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. In some embodiments, compounds that exhibit high therapeutic indices are preferred.

[0312] The data obtained from cell culture assays and animal studies can be used to formulate dosage ranges for humans. The dosage of the compositions characterized herein is generally within the range of circulating concentrations including the ED50 with little or no toxicity. The dosage can vary within this range according to the dosage form used and the route of administration used. For any compound used in the method characterized in the present disclosure, the therapeutically effective dose can be initially estimated from the cell culture assay. The dosage can be formulated in an animal model to achieve a circulating plasma concentration range (e.g., to achieve a reduced polypeptide concentration) of the polypeptide product of the compound or (when appropriate) target sequence, the circulating plasma concentration range includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine the available dosage for humans. The level in plasma can be measured, for example, by high performance liquid chromatography.

[0313] As described above, in addition to their administration, the dsRNA agents characterized herein can be administered in combination with other known agents effective in treating HBV infection. In any case, the administering physician can adjust the amount and timing of dsRNA agent administration based on the results observed using standard efficacy measurements known in the art or described herein.

[0314] VII. method

[0315] The present disclosure also provides a method for inhibiting HBV expression in a cell. The method comprises contacting the cell with a dsRNA agent (eg, a double-stranded dsRNA agent) in an amount effective to inhibit HBV expression in the cell, thereby inhibiting HBV expression in the cell.

[0316] The contact of cells with dsRNA agents (e.g., double-stranded dsRNA agents) can be carried out in vitro or in vivo. Contacting cells with dsRNA agents in vivo includes contacting cells or cell groups in subjects such as human subjects with dsRNA agents. Combinations of in vitro and in vivo methods of cell contact are also possible. As described above, contacting cells can be direct or indirect. In addition, cell contact can be achieved by targeting ligands, including any ligands described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, such as a GalNAc3 ligand, or any other ligand that directs the dsRNA agent to the target site.

[0317] In some embodiments of the method of the present disclosure, the dsRNA agent is administered to the subject so that the dsRNA agent is delivered to a specific site in the subject. The inhibition of HBV gene expression can be evaluated by measuring the level or level of HBV mRNA or HBV protein in a sample of a fluid or tissue derived from a specific site in the subject. In a preferred embodiment, the site is selected from liver and blood. The site can also be a segmentation or subgroup of cells or fluids prepared from any of the above-mentioned sites.

[0318] In some embodiments, the methods disclosed herein can be used to treat subjects with HBV infection, such as subjects who benefit from reduced HBV gene expression or HBV replication. In one aspect, the present disclosure provides a method for reducing the level of hepatitis B virus cccDNA in subjects infected with HBV. On the other hand, the present disclosure provides a method for reducing the level of HBV antigens (such as HBsAg or HBeAg) in subjects infected with HBV. On the other hand, the present disclosure provides a method for reducing the HBV viral load in subjects infected with HBV. The present disclosure also provides a method for reducing the level of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in subjects infected with HBV (although a transient increase in ALT or AST may be associated with viral clearance). In one aspect, the present disclosure provides a method for increasing the level of anti-HBV antibodies in subjects infected with HBV. On the other hand, the present disclosure provides a method for treating a subject infected with HBV. In one aspect, the present disclosure provides a method for treating a subject with an HBV-related disease, such as hepatitis D virus infection, hepatitis D, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; or hepatocellular carcinoma. In addition, since HDV infection relies on the specialized auxiliary function provided by HBV for transmission, and subjects with HBV infection may also suffer from HDV infection, in some embodiments, the treatment methods described herein can also be used to treat subjects with HDV infection or HDV-related disorders, such as hepatitis B virus infection, chronic hepatitis B infection (CHB), chronic hepatitis B infection (CHB), cirrhosis, liver failure and hepatocellular carcinoma (HCC). In some embodiments, the treatment methods (and uses) of the present disclosure include administering to a subject, such as a human, a therapeutically effective amount of a dsRNA agent targeting an HBV gene of the present disclosure or a pharmaceutical composition comprising a dsRNA agent targeting an HBV gene of the present disclosure.

[0319] In one aspect, the present disclosure provides a method for preventing at least one symptom in a subject with HBV infection, e.g., the presence of serum or liver HBV cccDNA; the presence of serum HBV DNA; the presence of serum or liver HBV antigens (such as HBsAg or HBeAg); elevated ALT; elevated AST; lack of or low levels of anti-HBV antibodies; liver damage; cirrhosis; hepatitis D, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma; serum sickness-like syndrome; anorexia; nausea; vomiting, low-grade fever; myalgia; fatigue; taste sensitivity and olfactory disturbances (aversion to food and cigarettes); right upper quadrant and epigastric pain (intermittent, mild to moderate); hepatic encephalopathy; lethargy; disturbed sleep pattern; confusion; coma; ascites; gastrointestinal bleeding; coagulopathy; jaundice; hepatomegaly (mild enlargement, soft liver); splenomegaly; palmar erythema; spider nevi; muscle atrophy; spider angiomas; Vasculitis; ruptured bleeding from veins; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (sea snakehead); ALT levels higher than AST levels; elevated gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) levels (no more than 3 times the ULN); slightly low albumin levels; elevated serum iron levels; leukopenia (i.e., granulocytopenia); lymphocytosis; elevated erythrocyte sedimentation rate (ESR); shortened red blood cell survival; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); presence of serum or liver HBsAg, HBeAg, or hepatitis B core antibody (anti-HBc) immunoglobulin M (IgM); hepatitis B surface antibody (anti-HB), hepatitis B e antibody (anti-HBe), or HBV The presence of DNA; elevated bilirubin levels; hyperglobulinemia; the presence of tissue nonspecific antibodies, such as anti-smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA) (10-20%); the presence of tissue specific antibodies, such as antibodies to the thyroid gland (10-20%); elevated rheumatoid factor (RF) levels; low platelet and leukocyte counts; lobular, degenerative and regenerative hepatocyte changes, accompanied by inflammation; and major central lobular necrosis, whether detectable or undetectable. The method comprises administering to the subject a therapeutically effective amount of a dsRNA agent, such as a dsRNA, or a pharmaceutical composition comprising a dsRNA agent, thereby preventing at least one symptom in a subject having a disease that benefits from reduced HBV gene expression, such as a subject having HBV infection or a subject having both HBV and HDV infection.

[0320] In another aspect, the disclosure provides for use of a therapeutically effective amount of a dsRNA agent of the disclosure for treating a subject, such as a subject that would benefit from reduction or inhibition of HBV gene expression, such as a subject with HBV infection or a subject with both HBV and HDV infection.

[0321] In yet another aspect, the present invention provides use of a dsRNA agent of the present disclosure, such as a dsRNA, targeting an HBV gene, or a pharmaceutical composition comprising a dsRNA agent targeting an HBV gene in the preparation of a medicament for treating a subject, for example, a subject who benefits from a reduction in HBV gene expression or HBV replication, such as a subject with HBV infection or a subject with both HBV and HDV infection, and a subject with a disorder that benefits from a reduction in HBV gene expression (e.g., an HBV-related disease).

[0322] In another aspect, the present disclosure provides for use of a dsRNA agent described herein for preventing at least one symptom in a subject having a disorder that would benefit from reduction or inhibition of HBV gene expression or HBV replication.

[0323] In yet another aspect, the present disclosure provides use of a dsRNA agent as described herein in the preparation of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from reduction or inhibition of HBV gene expression or HBV replication, such as an HBV-related disease.

[0324] In some embodiments, a dsRNA agent targeting HBV is administered to a subject having HBV infection or having both HBV infection and HDV infection, or having an HBV-related disease, such that when the dsRNA agent is administered to the subject, for example, the 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 or other tissues or fluids is reduced by at least 80%, 85%, 90%, 95%, 98% or more to tend toward normal or be normalized.

[0325] In some embodiments, the methods and uses of the present disclosure include administering a composition as described herein, such that the expression of the target HBV gene is reduced, for example, for about 1 month. In some embodiments, the target HBV gene expression is reduced for an extended duration, for example, at least two months, three months or longer. In some embodiments, the methods and uses of the present disclosure include administering a composition as described herein to cause functional cure.

[0326] Administration of dsRNA according to the methods and uses described herein may result in a reduction in the severity, signs, symptoms or markers of such diseases or disorders in patients with HBV infection or concurrent HBV and HDV infection or HBV-related diseases. In this case, "reduction" refers to a clinically significant reduction in such levels. The reduction may be, for example, at least 80%, 85%, 90%, 95% or 98%, or to below the level of detection.

[0327] In some embodiments, the efficacy of the disclosed methods can be monitored by detecting or monitoring a reduction in symptoms of HBV-related disease. These symptoms can be assessed in vitro or in vivo using any method known in the art.

[0328] The therapeutic efficacy of the disease can be assessed by, for example, measuring disease progression, disease regression, symptom severity, pain relief, quality of life, drug dosage required to maintain the therapeutic effect, disease marker levels, or any other measurable parameters suitable for a given disease being treated. It is entirely within the capabilities of those skilled in the art to monitor the therapeutic efficacy by measuring any one of these parameters or any combination of parameters. For example, the therapeutic efficacy of CHB can be assessed by regularly monitoring viral load and transaminase levels. The comparison of subsequent readings with initial readings provides the physician with an indication of whether the treatment is effective. It is entirely within the capabilities of those skilled in the art to monitor the therapeutic efficacy by measuring any one of these parameters or any combination of parameters. In combination with the administration of a dsRNA agent targeting HBV or its pharmaceutical composition, "effectively counteracting" HBV-related diseases indicates that the administration in a clinically appropriate manner produces a beneficial effect on at least a statistically significant portion of patients, such as improvement of symptoms, cure, alleviation of the disease, extension of life span, improvement of quality of life, or other effects considered to be positive by doctors familiar with HBV infection or HBV-related diseases and related etiologies.

[0329] When one or more parameters of the disease state are clinically significantly improved, or by failure or aggravation or occurrence of symptoms as originally expected, the therapeutic effect is evident. For example, a favorable change of at least 50%, preferably at least 70% or more, in a measurable disease parameter can indicate an effective treatment. The efficacy of a given dsRNA agent drug or a preparation of the drug can also be judged using an experimental animal model of the given disease known in the art. When using an experimental animal model, if a statistically significant reduction of signs or symptoms is observed, then the therapeutic efficacy is demonstrated.

[0330] Administration of the dsRNA agent can reduce the presence of serum or liver HBV cccDNA, the presence of serum or liver HBV antigens (e.g., HBsAg or HBeAg), in, for example, cells, tissues, blood, urine, or other compartments of the patient; or normalize ALT levels or AST levels by at least 70%, 75%, 80%, 85%, 90% or 95% to below the detection level of the assay method, thereby approaching or reaching the upper limit of normal for the experimental value.

[0331] Administration of the dsRNA agent can render the presence of serum or liver anti-HBV antibodies (e.g., anti-HBsAg antibodies) detectable or increase it by at least 80%, 85%, 90%, 95% or more, e.g., in patient cells, tissues, blood, urine or other compartments; or render the antibodies detectable when they were not detected prior to treatment.

[0332] Due to the inhibitory effect on HBV expression, in some embodiments, the composition according to the present disclosure or the pharmaceutical composition prepared therefrom can improve the quality of life.

[0333] A subject that would benefit from reduction or inhibition of HBV gene expression is a subject suffering from HBV infection or an HBV-related disease or disorder as described herein.

[0334] Treatment of subjects who would benefit from reduction or inhibition of HBV gene expression includes both therapeutic and prophylactic treatments.

[0335] The present disclosure further provides methods and uses of dsRNA agents or pharmaceutical compositions thereof in combination with other drugs or other treatment methods for treating subjects who benefit from reduction or inhibition of HBV gene expression (e.g., subjects suffering from HBV-related diseases), such as in combination with known drugs or known treatment methods, such as those currently used to treat these disorders.

[0336] For example, in some embodiments, a dsRNA agent targeting one or more HBV genes is co-administered with, for example, an agent useful for treating an HBV-related disease as described herein. For example, additional therapeutic agents and treatments suitable for treating subjects who benefit from reduced HBV expression (e.g., subjects with HBV-related diseases) include dsRNA agents targeting different parts of the HBV genome, antiviral agents, nucleotide analogs, nucleoside analogs, reverse transcriptase inhibitors (e.g., tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine, clavudine, ritonavir, dipivoxil, lobcavir, famciclovir, FT C, N-acetyl-cysteine ​​(NAC), PC1323, HBV therapeutic vaccine (radigm-HBV), thymosin alpha and ganciclovir), immunostimulants (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., REP 9AC), capsid inhibitors (e.g., Bay41-4109 and NVR-1221), cccDNA inhibitors (e.g., IHVR-25), or other therapeutic agents or procedures for treating HBV-related diseases, such as liver transplantation or chemotherapy, or a combination of any of the above.

[0337] The subject of the dsRNA agent of the present disclosure can further be administered one or more other therapeutic agents that work by non-RNAi mechanisms and can be used to treat HBV infection. Exemplary therapeutic agents that can be used for the combination therapy of the present disclosure include immunomodulators, which stimulate the immune system by, for example, enhancing helper T cell activity, maturation of B lymphocytes, inhibiting T cell inhibitors, and enhancing HLA type I expression. Suitable immunomodulators include interferons, which have multiple properties including antiviral, immunomodulatory and antiproliferative effects.

[0338] For example, the current treatment for chronic hepatitis B is interferon therapy, which is administered to subjects who have demonstrated at least six months of HBV infection, elevated liver enzymes (AST and ALT), and active viral division in the blood (positive test for HBeAg or HBV DNA). Interferon-α therapy can produce long-term, sustained disease remission in about 35% of chronic hepatitis B patients, normalizing liver enzymes and losing three markers of active infection (HBeAg, HBV DNA, and HBsAg). Subjects with acute HBV infection, end-stage cirrhosis, or other major medical problems are generally not treated with interferon.

[0339] In addition, interferon treatment of patients with HBV-related cirrhosis significantly reduces the rate of hepatocellular carcinoma (HCC), especially in patients with high serum HBV DNA amounts. In patients with HBeAg-positive compensated cirrhosis, virologic and biochemical remission after interferon treatment is associated with improved survival. In patients with chronic HBV infection, clearance of HBeAg after interferon-α treatment is associated with improved clinical outcomes. The standard duration of treatment is 16 weeks. Patients who demonstrate low levels of viral replication at the end of the standard treatment regimen benefit most from extended treatment.

[0340] In some embodiments, the methods of the present disclosure include administering a reverse transcriptase inhibitor to a subject suffering from HBV infection or HBV-related disease. In some embodiments, the methods of the present disclosure include administering a reverse transcriptase inhibitor and an immunostimulant to a subject suffering from HBV infection or HBV-related disease.

[0341] The dsRNA agent and the additional therapeutic agent or treatment can be administered simultaneously or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at a separate time or by another method known in the art or described herein.

[0342] The present disclosure also provides a method for reducing or inhibiting HBV expression in a cell using a dsRNA agent as described herein or a composition comprising a dsRNA agent. In other aspects, a dsRNA agent as described herein or a composition comprising a dsRNA agent is provided for preparing a medicine for reducing or inhibiting HBV gene expression in a cell. In other aspects, the present disclosure provides a dsRNA agent disclosed herein or a composition comprising a dsRNA agent for reducing or inhibiting HBV replication in a cell. In other aspects, a dsRNA agent disclosed herein or a composition comprising a dsRNA agent is provided for preparing a medicine 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 a cell sufficient to degrade the mRNA transcript of the HBV gene for a time, thereby inhibiting the expression of the HBV gene in the cell or inhibiting HBV replication.

[0343] In the above methods and uses, the cells may be contacted in vitro or in vivo, ie, the cells may be within the body of a subject.

[0344] Cells suitable for processing using the methods disclosed herein can be any cells expressing HBV genes, such as cells infected with HBV, cells containing expression vectors containing parts of HBV genomes or HBV genes, or transgenic mice expressing HBV genes. Cells suitable for use in methods and uses disclosed herein can be mammalian cells, such as primate cells (such as human cells or non-human primate cells, such as monkey cells or chimpanzee cells) or non-primate cells (such as mouse cells, rat cells or other mammalian cells). In certain embodiments, cells are cells that can be infected by HBV. In certain embodiments, cells are human cells, such as human hepatocytes.

[0345] HBV gene expression may be inhibited in cells by at least 80%, 85%, 90% or 95% or more, for example, to a level below detection by an assay.

[0346] HBV replication can be inhibited in cells by at least 80%, 85%, 90% or 95% or more, for example, to a level below detection by an assay.

[0347] In vivo methods and uses disclosed herein may include administering to a subject a composition comprising a dsRNA agent, wherein the dsRNA agent comprises a nucleotide sequence 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 in a single injection. In some embodiments, the disclosure provides compositions formulated for organ-specific (e.g., liver) intraarterial, intratumoral, intradermal, intravitreal injection, ocular topical, intraocular (eye drops), atomization, ocular topical or other local routes, suppositories or oral administration.

[0348] In one aspect, the present disclosure also provides a method for inhibiting HBV gene expression in mammals, such as humans. The present disclosure also provides a composition comprising a dsRNA agent targeting an HBV gene in a mammalian cell, which is used to inhibit the expression of the HBV gene in a mammal. On the other hand, the present disclosure provides the use of a dsRNA agent targeting an HBV gene in a mammalian cell in the preparation of a drug for inhibiting HBV gene expression in a mammal.

[0349] The method and use include administering to a mammal, such as a human, a composition comprising a dsRNA agent targeting an HBV gene in cells of the mammal, and maintaining the mammal for a time sufficient to degrade mRNA transcripts of the HBV gene, thereby inhibiting the expression of the HBV gene in the mammal.

[0350] In certain embodiments, the reduction of gene expression in the peripheral blood sample of the subject of dsRNA agent administration can be assessed by any method known in the art, such as qRT-PCR as described herein. The reduction of protein production can be assessed by any method known in the art and the method described herein (such as ELISA or Western blot). Clinically acceptable methods for determining gene and protein expression levels can be used as appropriate. In certain embodiments, puncture liver biopsy samples are used as tissue materials for monitoring HBV gene or protein expression reduction. In some other embodiments, blood samples are used as tissue materials for monitoring HBV gene or protein expression reduction.

[0351] In some embodiments, verification of RISC-mediated cleavage of the target in vivo following administration of the dsRNA agent is accomplished by performing 5'-RACE or refined 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).

[0352] The present invention is further illustrated by the following examples, which should not be construed as limiting.

[0353] Example

[0354] Example 1

[0355] dsRNA synthesis

[0356] Reagent Source

[0357] In the absence of specific sources of reagents herein, these reagents can be obtained from any molecular biology reagent supplier at quality / purity standards applicable to molecular biology.

[0358] dsRNA agent design

[0359] As described in WO / 2016 / 077321, the choice of dsRNA design targeting HBV was driven by two main factors: a) efficacy, and b) the desire to use an agent with a near-perfect match and greater than 90% local coverage of a large number of public HBV sequences of all known genotypes (A to H). The coordinates for RNA agent selection were determined relative to the NCBI HBV reference genome sequence NC_003977.1 (GenBank Accession No. GI: ​​21326584 (SEQ ID NO: 1)). The first group of RNA agents was designed, synthesized and screened in vitro, which contained structure-activity modifications (including various 2'-O-methyl and 2'-fluoro substitution patterns) centered on two adjacent regions in the HBV genome encoding surface antigen (HbSAg) and HBV polymerase. A second group of RNA agents was also designed, synthesized and screened in vitro, which targeted other regions in the HBV genome, particularly positions 1581-1599 of SEQ ID NO: 1, encoding HbSAg, polymerase and X gene regions. The selected sequences were further chemically modified and tested. These duplex designs are provided in WO2016 / 077321 (the entire contents of which are incorporated herein by reference); a detailed list of unmodified HBV sense and antisense strand nucleotide sequences is provided in Tables 3, 6, 12, 22 and 25 of WO2016 / 077321, and a detailed list of modified HBV sense and antisense strand nucleotide sequences is provided in Tables 4, 7, 13, 23 and 26 of WO2016 / 077321. The results of screening assays performed with these agents are also provided.

[0360] These studies identified a duplex, AD-66810, having an antisense strand with a modified nucleotide sequence of 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 13) and a sense strand with a modified nucleotide sequence of 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: NO: 29), wherein a, c, g and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate and 2'-O-methyluridine-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 the N-acetylgalactosamine moiety N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol (also referred to as (Hyp-(GalNAc-alkyl)3) or referred to herein as L96) is covalently attached to the 3' end of the sense strand.

[0361] Based on the previously identified sequence AD-66810 targeting the X transcript of human hepatitis B virus (HBV; U95551), further studies are provided herein to identify effective and specific dsRNA agent molecules. To achieve this goal, a series of chemically modified dsRNA agents were designed, synthesized, and tested for in vitro activity using a Dual-Luc reporter-based assay and HepG2.2.15 cell line. All compounds were conjugated to a triantennary N-acetylgalactosamine (GalNAc) ligand (L96) covalently attached to the 3' end of the sense strand.

[0362] dsRNA synthesis

[0363] HBV sense and antisense sequences were synthesized on a Mermade 192 synthesizer (BioAutomation) using solid support-mediated phosphoramidite chemistry at a 1 μmol scale. The solid support was either controlled pore glass (500A) loaded with custom GalNAc ligands or a universal solid support (AM biochemical). Ancillary synthesis reagents, 2′-F and 2′-O-methyl RNA, and deoxyphosphoramidites were obtained from Thermo-Fisher, respectively. TM (Milwaukee, WI) and Hongene (China). 2'F 2'-O-methyl, GNA (diol nucleic acid), 5'-phosphate and abasic modifications were introduced using the corresponding phosphoramidites. The synthesis of 3'GalNAc-conjugated single strands was performed on a GalNAc-modified CPG support. A custom CPG universal solid support was used to synthesize antisense single strands. 5-Ethylthio-1H-tetrazole (ETT) was used as an activator (0.6 M in acetonitrile) and the coupling time for all phosphoramidites (100 mM in acetonitrile) was 5 minutes. A 50 mM solution of 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, MA, USA)) in anhydrous acetonitrile / pyridine (1:1 v / v) was used to generate phosphorothioate linkages. The oxidation time was 3 minutes. All sequences were synthesized with the DMT group finally removed ("DMToff").

[0364] After solid phase synthesis is complete, the oligoribonucleotides are cleaved from the solid support and deprotected in a sealed 96-well deep well plate using 200 μL of aqueous methylamine reagent at 60°C for 20 minutes. At the end of the cleavage and deprotection steps, the synthesis plate is allowed to reach room temperature and precipitated by adding 1 mL of an acetonitrile: ethanol mixture (9:1). The plate is cooled at -80°C for 2 hours and the supernatant is carefully removed with the aid of a multichannel pipette. The oligonucleotide pellet is resuspended in 20 mM NaOAc buffer and precipitated using a 5 mL HiTrap HPLC system equipped with an A905 autosampler and a Frac 950 fraction collector. TM Size exclusion column (GE Healthcare TM ) desalted. Desalted samples were collected in 96-well plates. Samples from each sequence were analyzed by LC-MS to confirm identity, quantified by UV (260 nm), and the purity of a selected set of samples was determined by IEX chromatography.

[0365] Annealing of HBV single strands was performed on a Tecan liquid handling robot. An equimolar mixture of sense and antisense single strands was 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 min and allowed to slowly warm to room temperature over 2-3 h. The concentration of each duplex was normalized to 10 μM in 1× PBS.

[0366] Abbreviations for the modified nucleomonomers disclosed herein are shown in Table 1. Table 2 shows AD-66810 and HBV dsRNA agents synthesized using the above methods.

[0367] Table 1. Abbreviations of nucleotide monomers used in the representation of modified nucleic acid sequences .

[0368] It is understood that, unless otherwise specified, when present in an oligonucleotide, these monomers are linked to each other via 5'-3' phosphodiester bonds.

[0369]

[0370]

[0371] Table 2. Modified nucleotide sequences of GalNAc-conjugated HBV dsRNA agents

[0372]

[0373]

[0374]

[0375] Example 2

[0376] In vitro screening of dsRNA duplexes

[0377] Luciferase assay:

[0378] Cos7 cells ( Manassas, VA) were grown to near confluence at 37°C in a 5% CO2 atmosphere and then released from the plates by trypsinization. Cos7 cells were transfected with the duplex agent and psiCHECK2-HBV (an expression vector comprising a nucleotide sequence encoding a portion of the consensus wild-type HBV sequence, SEQ ID NO: 5), or the duplex agent and a psiCHECK2 vector expressing a nucleotide sequence of HBV genotype A, C, E, or F (SEQ ID NO: 6, 7, 8, or 9, respectively) by adding 5 μl of RNA duplex and 5 μl of psiCHECK2-HBV (or psiCHECK2-HBV genotype A, C, E, or F) plasmid per well and 5 μl of Add 0.1 μl Lipofectamine TM RNAiMax (Invitrogen TM , Carlsbad CA. cat# 13778-150) and then incubated at room temperature for 15 minutes. The mixture was then added to the wells, which were resuspended in 35 μl of fresh complete medium. The transfected cells were incubated at 37° C. in a 5% CO 2 atmosphere.

[0379] 48 hours after transfection of dsRNA agents and psiCHECK2 plasmid; Firefly (transfection control) and Rinella (fused to HBV target sequence) luciferase were measured. First, the culture medium was removed from the cells. Then 20 μl of culture medium volume was added to each well and the mixture was The mixture was incubated at room temperature for 30 minutes and then The Firefly luciferase signal was detected by measuring luminescence (500 nm) on a 4% paraformaldehyde (Molecular Devices) plate. Renilla luciferase activity was detected by adding 20 μl of room temperature Stop& Reagent and incubate the plate for 10-15 minutes before measuring luminescence again to determine the Renilla luciferase signal. Stop& Reagent quenches the Firefly luciferase signal and maintains the luminescence of the Renilla luciferase reaction. The activity of the dsRNA agent is determined by standardizing the Renilla (HBV) signal in each well to the Firefly (control) signal. Then, the size of the dsRNA agent activity is assessed relative to cells transfected with the same vector but not treated with dsRNA agents or treated with non-targeted dsRNA agents. All transfections were performed with n=2 or more.

[0380] Table 3 provides the results of these assays performed using the dsRNA agents listed in Table 2.

[0381] HepG2.2.15 and PLC in vitro screening:

[0382] HepG2.2.15 and PLC (human hepatoma cells; CRL-8024) cells were grown in DMEM or EMEM medium (ATCC) supplemented with 10% FBS at 37°C in a 5% CO2 atmosphere to near confluence and then released from the plate by trypsinization. Reverse transfection was performed as follows: 5 μl of dsRNA agent duplex was added to each well of a 96-well plate and 14.8 μl of Add 0.2 μl Lipofectamine TM RNAiMax (Invitrogen TM , Carlsbad CA. cat# 13778-150) and incubated at room temperature for 15 minutes. Eighty microliters of complete growth medium without antibiotics was then added, containing 2×10 4 HepG2.2.15 or PLC cells were incubated for 24, 48 and 72 hours before RNA purification.

[0383] Table 4 provides the results of these assays performed using the dsRNA agents listed in Table 2.

[0384] The total RNA was isolated using the MagMAX-96 Total RNA Isolation Kit (Applied Biosystem, ForerCity CA, Part #: Total RNA isolation:

[0385] Cells were harvested and lysed in 140 μl of lysis / binding solution and then centrifuged using an Eppendorf TMThermomixer was mixed at 850rpm for 1 minute (mixing speed was the same throughout the process). Twenty microliters of magnetic beads and lysis / binding enhancer (Lysis / Binding Enhancer) mixture were added to the cell lysate and mixed for 5 minutes. The magnetic beads were captured using a magnetic rack and the supernatant was removed without disturbing the beads. After removing the supernatant, the magnetic beads were washed with washing solution 1 (adding isopropanol) and mixed for 1 minute. The beads were captured again and the supernatant was removed. The beads were then washed with 150μl washing solution 2 (adding ethanol), captured and the supernatant was removed. Then 50μl DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was added to the magnetic beads and mixed for 10 to 15 minutes. After mixing, 100μl RNA rebinding solution was added and mixed for 3 minutes. The supernatant was removed and the magnetic beads were washed again with 150μl washing solution 2, and mixed for 1 minute and the supernatant was completely removed. The beads were mixed for 2 min to dry, and the RNA was then eluted with 50 μl of water.

[0386] The cDNA was reverse transcribed using the ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat# 4368813) for cDNA synthesis:

[0387] A master mix of 2 μl 10× buffer, 0.8 μl 25× dNTPs, 2 μl random primers, 1 μl reverse transcriptase, 1 μl RNase inhibitor and 3.2 μl H2O was added to 10 μl total RNA per reaction. cDNA was generated using a C-1000 or S-1000 thermal cycler (Hercules, CA) by the following steps: 25°C for 10 min, 37°C for 120 min, 85°C for 5 sec, and 4°C hold.

[0388] Real-time PCR:

[0389] 2 μl of cDNA was added to a 384-well plate (Roche cat#04887301001) containing 0.5 μl of human GAPDH TaqMan probe (Applied Biosystems Cat#4319413E), 1 μl of SORF2-specific Probe and 5 μ l Lightcycler 480 probe master mix (Roche Cat#04887301001) master mix. Use DDCt (RQ) determination method to carry out real-time PCR in LightCycler480 real-time PCR system (Roche). In order to calculate relative fold change, use DDCt method to analyze real-time data, and relative to the determination standardization of cells transfected with AD-1955 or simulation (mock) transfected cells.

[0390] To calculate relative fold changes, real-time data were analyzed using the DDCt method and normalized to assays performed in cells transfected with a non-targeting control dsRNA agent.

[0391] Example 3

[0392] Transfection of GalNAc-conjugated HBV-targeting dsRNA agents in the Dual-Luc system

[0393] The silencing of HBV mRNA after transfection with each conjugated dsRNA agent is shown in Table 3. Each dsRNA agent was tested by transfection at 50nM, 10nM and 0.1nM dsRNA agent concentrations in Cos7 cells. According to the screening results, 7 of the 20 dsRNA agents showed efficacy comparable to the AD-66810 parent.

[0394] Table 3. Conjugates transfected at 50 nM, 10 nM or 1 nM using the Dual-Luc assay Screening results of HBV dsRNA agents

[0395]

[0396]

[0397] Abbreviations: ID = identity; SD = standard deviation

[0398] a Fraction of relative HBV expression remaining 48 hours after transfection with the screening group's HBV dsRNA agent (relative to control transfected cells)

[0399] Example 4

[0400] Transfection of GalNAc-conjugated HBV-targeted dsRNA agents in HepG2.2.15 cells

[0401] Table 4 shows the silencing of HBV mRNA (PORF1 and SORF2 mRNA as determined using forward and reverse primers and TaqMan probes) in HepG2.2.15 cells after transfection with each conjugated dsRNA agent. Overall, stronger silencing of PORF1 and SORF2 viral transcripts was observed in HepG2.2.15 compared to silencing in the Dual-Luc overexpression system when 10 dsRNA agents with comparable efficacy to the parental molecule were used. Similar dsRNA agent activity was observed for PORF1 and SORF2 mRNA, which is expected because the dsRNA agent target site is present in both transcripts.

[0402] Table 4. Conjugated HBV transfected with HepG2.2.15 at 50 nM, 10 nM or 1 nM Screening results of dsRNA agents

[0403]

[0404] Abbreviation: SD = standard deviation

[0405] a Fraction of relative HBV expression remaining 24 hours after transfection of the screening set of HBV dsRNA agents (relative to control transfected cells). Based on the above studies, duplex AD-81890 was selected for further analysis.

[0406] Example 5

[0407] Evaluation of AD-81890 pharmacology in the gland-associated HBV mouse model following a single subcutaneous injection

[0408] The pharmacology of AD-81890 was determined in the adeno-associated HBV (HBV-AAV) mouse model. AAV8-HBV (SignaGen Laboratories) was diluted in 1× PBS to a final concentration of 2×10 12 Male C57BL / 6 mice aged 6-8 weeks were injected intravenously via the lateral tail vein with 2×10 11 GC / mouse.

[0409] AD-81890 was diluted with sterile 1× PBS and administered in a variable volume of 10 μl / g. Animals received a single SC dose of AD-81890 at 0.3, 1, or 3 mg / kg as described in Table 5, and blood was collected via the retro-orbital sinus on days -24, -2, 0, 14, 21, 33, 47, 59, and 74 post-dose.

[0410] Table 5. AD-81890 Single-Dose AAV-HBV Study Design

[0411]

[0412] Abbreviations: No. = number; PBS = phosphate buffered saline

[0413] a On day 47, n = 8 animals; on day 59, n = 6; on day 72, n = 5, animals were euthanized due to fighting injuries

[0414] After collection, blood was allowed to clot for 30 minutes and then centrifuged in a microcentrifuge at 13,000 rpm for 10 minutes at 4° C. Serum was aspirated and stored at -20° C.

[0415] Hepatitis B virus surface antigen protein levels were evaluated by ELISA (BioTang, Waltham, MA). A standard curve was generated using USBiologics (Memphis, TN) HBsAg protein. Serum samples were diluted 1:2000 or 1:500 in 1×PBS (Gibco, Gaithersburg, MD) and evaluated using a slightly modified ELISA protocol. In brief, 50 μL / well of diluted serum or standard was loaded into the plate and incubated at 37°C for 1 hour. After this incubation, 50 μL / well enzyme conjugate was added to each well, and the plate was incubated at 37°C for 30 minutes. The plate was washed 3 times with 300 μL / well of 1× wash buffer, then blotted until all liquid was removed from the wells, 100 μL / well of substrate was added, and the plate was incubated at 37°C for 30 minutes. Finally, 100 μL / well of stop solution was added, and the absorbance was measured at a wavelength of 450 nm. In any case where the calculated HBsAg level was below the lower limit of quantitation (LLOQ) of the assay, the value was recorded as the LLOQ (ie, 313 ng / mL).

[0416] Mean ± SD serum HBsAg concentrations in HBV-AAV mice after a single subcutaneous (SC) dose of AD-81890 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 0.3, 1, or 3 mg / kg of AD-81890 resulted in effective and sustained reductions in serum HBsAg concentrations in HBV-AAV mice, with a maximum reduction of 92% observed on day 7 in the highest (3 mg / kg) AD-81890 dose group. In the highest dose group, the highest level of reduction was maintained until day 33, after which HBsAg levels began to return to baseline ( Figure 2B ). In the 0.3mg / kg and 1mg / kg AD081890 dose groups, a moderate reduction in serum HBsAg concentration was observed, with the highest reductions of 23% and 72% on Day 7, respectively. At the completion of the study (Day 74), HBsAg levels in the 0.3mg / kg and 1mg / kg AD-81890 dose groups returned to baseline levels.

[0417] Example 6

[0418] Evaluation of AD-81890 pharmacology in the gland-associated HBV mouse model following multiple subcutaneous injections

[0419] The pharmacology of AD-81890 was determined in the adeno-associated HBV (HBV-AAV) mouse model. AAV8-HBV (SignaGen Laboratories) was diluted in 1× PBS to a final concentration of 2×10 12 Male C57BL / 6 mice aged 6-8 weeks were injected intravenously via the lateral tail vein with 2×10 11 GC / mouse.

[0420] AD-66810 and AD-81890 were diluted with sterile 1×DPBS and administered in a variable volume of 10 μl / g. Animals received AD-66810 given at 1 mg / kg, Q2Wx6, or received a single dose of AD-81890 at 9 mg / kg, or received multiple doses of AD-81890 at 1 or 3 mg / kg, Q2Wx6 or QMx3. Serum was collected from animals at multiple time points as described in Table 6. Blood was collected via retro-orbital sinus as described in Table 6.

[0421] Table 6. AD-81890 Multiple-Dose AAV-HBV Study Design

[0422]

[0423] 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 with standard deviations are shown in Table 7. Figure 3 shown.

[0424]

[0425] Dose-responsive serum HBsAg levels were observed in the AAV8-HBV mouse model after AD-81890 administration. A maximum HBsAg reduction of approximately 2.7 log10 was observed after a single 9 mg / kg dose or 3 mg / kg q2w x 6 of AD-81890. Animals receiving 3 mg / kg q2w x 6 had a sustained HBsAg reduction of greater than 2 log10 for a total of approximately 8 weeks.

[0426] These in vivo studies demonstrated that AD-81890 effectively reduced HBsAg in the serum of the HBV-AAV mouse model.

[0427] Example 7

[0428] Specific off-target analysis of AD-81890

[0429] A combination of in silico bioinformatics and in vitro methods were used to evaluate the potential off-target activities of the antisense strand of AD-81890.

[0430] Bioinformatics

[0431] A set of dsRNA agents targeting the "X" gene (GenBank nucleotide ID U95551; NCBI Gene ID: 7276; SEQ ID NO: 49) of hepatitis B virus (HBV) subtype ayw was designed using custom R and Python scripts. The circular U95551 HBV genome is 3182 bases in length, and positions 1376-1840 in the NCBI record encode the "X" gene CDS region. Details of the dsRNA agent design and screening methods are provided above and in WO2016 / 077321.

[0432] Transfection Screening of GalNAc-Conjugated dsRNA Agents for Off-Target Detection in HepG2.2.15 Cells

[0433] To measure off-target inhibition, responses of endogenously expressed transcripts were tested by qPCR in the hepatocyte cell line HepG2.2.15. In 96-well plates, cells (2 × 10 cells / well) were transfected with AD-81890 using Lipofectamine RNAiMax (ThermoFisher) at concentrations ranging from 50 nM to 5 fM. 4 After 24 hours, MagMAX TM -96 Total RNA Isolation Kit (ThermoFisher); and cDNA was generated using the ABI High Capacity cDNA Reverse Transfection Kit (ThermoFisher). Samples were assayed for HBV mRNA inhibition and potential off-target silencing. For quantification by qPCR, HBV expression was assessed using two different custom TaqMan assays (PORF-1 and SORF-2), which recognize different regions of the HBV viral transcript.

[0434] To assess off-target silencing, quantification was performed using TaqMan probes specific for each potential off-target (Table 8). qPCR was performed using a LightCycler 480 real-time PCR instrument (Roche).

[0435] Table 8. Potential off-target sequences of AD-81890

[0436]

[0437]

[0438] a Mismatch positions are defined in the 5'-3' direction relative to the antisense strand, so position 1 corresponds to the base complementary to the 5'-most nucleotide of the AD-81890 antisense strand.

[0439] bIf multiple RefSeq IDs are associated with the same target gene and off-target profile, both IDs are listed.

[0440] In order to determine the degree of inhibition of target (HBV) and potential off-target genes, relative RNA levels were determined by expressing relative to human GAPDH RNA from the same sample. The results were compared with the non-specific dsRNA control of transfection, and the error was expressed as standard deviation. The IC50 of AD-81890 for PORF1 transcript target was 0.803nM, and for SORF2 transcript target was 0.766nM. Even at the highest AD-81890 concentration, no significant target knockdown was observed for SIDT2 and ZBTB2 transcripts.

[0441] The degree of off-target inhibition of AD-81890 for 2 potential off-targets of endogenously expressed transcripts in HepG2.2.15 was evaluated in a dose-response screen. AD-81890 did not inhibit the expression of SIDT2 or ZBTB2 at any dose tested, while HBV inhibition was dose-responsive. Fitting the dose-response data using a four-parameter fitting model (XLfit) yielded IC50s of 803pM and 766pM for PORF-1 or SORF-2, respectively.

[0442] Example 8

[0443] In vitro analysis of AD-81890 specificity in HepG2.2.15 cells using RNA-Seq

[0444] Using changes in transcriptome-wide expression levels and RNA-Seq, the effects of chemical changes in dsRNA agents were measured by comparing AD-66810 and AD-81890 in HepG2.2.15 cells expressing HBV. The AD-66810 and AD-81890 molecules have the same nucleotide sequence, but differ in the substitution of a single glycol nucleic acid (GNA) at position 6 from the 5' end of the antisense strand molecule (see Table 2).

[0445] HepG2.2.15 cells (HepG2-derived cell line stably transfected with full genome HBV) were diluted with culture medium to a final concentration of 187500 cells / mL, and 80 μL was pipetted into a 96-well collagen-coated plate (BD Biocoat, catalog number 356407) to give a final concentration of 15000 cells / well.

[0446] The dsRNA agent stock solution was diluted to the following concentrations in 1×DPBS: 1,000nM or 100nM. RNAiMAX (ThermoFisher, catalog number 13778150) was diluted with Opti-MEM (ThermoFisher, catalog number 31985062) at 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, catalog number 356407), as well as 10μL / well of the appropriate dsRNA agent dilution, gently mixed and incubated at room temperature for 20 minutes. 80μL of the prepared cell suspension was added to each well to a final cell density of 15,000 cells / well, and the final dsRNA agent concentration was 100nM and 10nM. The cells were incubated in a 37°C incubator containing 5% CO2 for 16-22 hours. Cells were seeded to give 16 wells per experimental condition and the experiments were performed in duplicate.

[0447] RNA was isolated using the ThermoFisher RNAqueous-96 Total RNA Isolation Kit according to the experimental protocol. Briefly, after 16-22 hours, the supernatant was aspirated from each well, 100 μL / well of 1× DPBS was added to rinse off the residual culture medium, and then aspirated. 200 μL of lysis / binding solution was added to each well of rows 1 and 5 of each plate, pipetted up and down several times, and transferred to subsequent rows to combine 4 wells / conditions to ensure 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. Through a series of centrifugation steps (1900×g, 1 min), the samples were washed with the provided wash solution, treated with DNase reagent, and eluted into 100 μL of nuclease-free water. RNA concentration was determined using a NanoDrop 8000 spectrophotometer (ThermoFisher).

[0448] RNA was further treated with TURBO DNase (Ambion). Each RNA sample (≤10 μg RNA / sample) was mixed with 2 μL DNase, 10 μL 10× buffer, and nuclease-free water to a total volume of 100 μL and then incubated at 37°C for 30 minutes. After DNase treatment, RNA was further purified using the RNeasy MinElute Cleanup kit (Qiagen) according to the experimental protocol. RNA was eluted in 30 μL nuclease-free water, and RNA concentration was determined using a NanoDrop 8000 spectrophotometer. RNA was stored at -80°C. Subsequently, the RNA was used for cDNA library preparation using the TruSeq Stranded Total RNA Library Preparation Kit (Illumina) and sequenced on a NextSeq500 desktop sequencer (Illumina), all according to the manufacturer's instructions. The experiment was repeated twice.

[0449] HepG2.2.15 cells were transfected with 10nM or 100nM AD-66810 or AD-81890 in quadruplicate and cultured for 24 hours with untreated controls. RNA extracted with Purelink RNA kit (ThermoFisher) was prepared using TruSeq Stranded Total RNA library preparation kit with Ribo-Zero Human / Mouse / Rat for rRNA depletion and Ribo-Zero Human / Mouse / Rat (Illumina) for rRNA depletion, all according to the manufacturer's instructions. Each NextSeq 500 / 550 High Output v2 (75 cycles) flow cell (Illumina) merged a total of 40 samples. Two experimental replicates were performed.

[0450] Raw RNA-Seq reads were filtered with a minimum average quality score of 25 and a minimum residual length of 36 using fastq-mcf. The filtered reads were aligned simultaneously to the human (hg19 / GRCh37) and HBV (GenBank nucleotide ID U95551; NCBI GeneID: 7276) genomes using STAR (version 2.4.2a). Due to the circular structure of the HBV genome, 46 base pairs were repeated at the ends of the linearized form of the HBV sequence to allow the reads to be mapped at the breakpoints. Unique aligned reads mapped 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 the software package DESeq2 (version 1.16.1). Multiple testing correction was performed by DESeq2 using the method of Benjamini & Hochberg, 1995 to obtain adjusted p-values.

[0451] MA plots were used to visualize on-target HBV knockdown and off-target effects. Analysis of GNA chemistry in terms of reducing overall off-target effects was limited to downregulated genes (log2 fold change <0). Upregulated genes (log2 fold change >0) were considered secondary effects. Assessment of off-target effects was limited to the lowest dose (10nM) as near-maximal HBV knockdown was obtained. To compare transcriptome noise, significantly downregulated genes were identified in AD-66810 and / or AD-81890 (adjusted p-value <0.05). The extent of downregulation was visualized by box plots of log2 fold change ( Figure 4 ), and the statistical difference between AD-66810 and AD-81890 was evaluated using Welch's two-sided two-sample t-test (Table 9).

[0452] Table 9. Statistical tests for significantly down-regulated genes

[0453]

[0454] exp: experimental replicates; Mean.AD-66810.IFC: mean log2 fold change of genes significantly down-regulated in AD-66810 and / or AD-81890; Mean.AD-81890.IFC: mean log2 fold change of genes significantly down-regulated in AD-81890 and / or AD-66810; t.statistic: t statistic of Welch's two-sided two-sample t-test; p.value: p-value of Welch's two-sided two-sample t-test; dof: degrees of freedom; N: number of genes significantly down-regulated (p<0.05) in AD-66810 and / or AD-81890

[0455] Consistent reduction in off-target effects was observed for AD-81890 compared to AD-66810 in both experimental replicates ( Figure 4 , Table 9). At 10nM, AD-81890 shows a 52% (repeat 1) or 54% (repeat 2) reduction (Table 9) of the average log2 fold change of significantly downregulated genes compared to AD-66810. At 100nM, AD-81890 shows a 71% (repeat 1) or 43% (repeat 2) reduction (Table 9) of the average log2 fold change compared to AD-66810. In all cases, the reduction of the observed log2 fold change is highly significant statistically. Therefore, the transcriptome noise level of AD-81890 is significantly lower.

[0456] Example 9

[0457] Evaluation of human-specific hepatotoxicity in PXB mice

[0458] The PXB mouse is a chimeric mouse with a humanized liver that is highly repopulated with human hepatocytes (PhoenixBio). 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 replacement index (RI) of the humanized liver uPA / SCID mouse (i.e., the percentage of human hepatocytes in the liver) exceeds 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).

[0459] Study was conducted in PBX mice to compare the hepatotoxicity of AD-66810 and AD-81890. Mice were administered 12, 36 or 100 mg / kg of AD-66810, AD-81890 or PBS (control) by subcutaneous injection on days 0, 21, 28, 35 and 42 (n=4 per group). General condition observations and body weight measurements were performed twice a week until the 49th day. Blood was collected by retroorbital bleeding twice a week, and serum was prepared using conventional methods. The last blood draw was performed, and the animals were killed by cardiac puncture and exsanguination. Autopsy was performed after exsanguination. The liver was harvested and weighed. The liver was divided for storage in RNAlater solution (Ambion), placed in formalin before paraffin embedding, and flash frozen.

[0460] The body weight of all animals remained above 80% of the initial level throughout the study period. In addition, the lowest arithmetic mean body weight was higher in the compound-treated groups than in the control PBS-treated group.

[0461] The level of human serum albumin in the blood was monitored throughout the study. The blood h-Alb concentration of all surviving animals remained above 7.0 mg / ml during the life of the study.

[0462] Liver enzymes were monitored throughout the study. In particular, ALT, AST, ALP, GGT, TBIL and TG were monitored throughout the study. Table 10 below shows the enzyme levels measured on day 49 in mice receiving AD-66810, AD-81890 or PBS. Figure 5A and 5B It was shown that administration of AD-66810 ( Figure 5A ) or AD-81890( Figure 5B ) relative to the ALT levels over time after PBS administration.

[0463] Table 10. Liver enzyme levels

[0464]

[0465] Compared with the control group, ALT, AST, ALP and GGT were increased in both test compound treated groups. In addition, dose-dependent changes were shown in ALT, AST and GGT in the AD-66810 treated group.

[0466] At necropsy, there were no test compound specific findings in either group. There were no significant changes in the relative (liver / body weight) liver weights of animals in the compound treated groups when compared to the control group.

[0467] Equivalent solution

[0468] 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. Such equivalents are intended to be encompassed by the scope of the appended claims.

[0469] While specific embodiments have been illustrated and described, it should be readily appreciated that the various embodiments described above may be combined to provide further embodiments and that various changes may be made without departing from the spirit and scope of the invention.

[0470] Unless otherwise indicated, 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 in this specification or listed in the Application Data Sheet are incorporated herein by reference in their entirety, including U.S. Provisional Patent Application No. 62 / 718,314 filed on August 13, 2018. Aspects of the embodiments can be modified, if necessary, to employ concepts of the various patents, applications, and publications to provide yet further embodiments.

[0471] These and other changes can be made to the embodiments in light of the above detailed description. Generally, in the following claims, the terms used should not be interpreted as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted to include all possible embodiments and the full range of equivalent methods claimed by these claims. Therefore, the claims are not limited by the present disclosure.

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a modified nucleotide sequence as shown below: 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), where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-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 diol nucleic acid (GNA); and s is a phosphorothioate linkage.

2. The dsRNA agent of claim 1, wherein the sense strand comprises a modified nucleotide sequence 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29), where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-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.

3. The dsRNA agent of claim 1, wherein the antisense strand and the sense strand comprise a modified nucleotide sequence as shown below: (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); where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-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 diol nucleic acid (GNA); and s is a phosphorothioate linkage.

4. The dsRNA agent of any one of claims 1-3, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.

5. The dsRNA agent of any one of claims 1-3, wherein at least one strand comprises a 3' overhang of 2 nucleotides.

6. The dsRNA agent of any one of claims 1-5, wherein the double-stranded region is 19-21 nucleotide pairs in length.

7. The dsRNA agent of any one of claims 1-6, wherein each strand independently has 19-23 nucleotides.

8. The dsRNA agent of claim 7, wherein each strand independently has 19-21 nucleotides.

9. The dsRNA agent of any one of claims 1-8, further comprising a ligand.

10. The dsRNA agent of claim 9, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

11. The dsRNA agent according to claim 9 or 10, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

12. The dsRNA agent of claim 11, wherein the ligand is 13. The dsRNA agent according to claim 12, wherein the dsRNA agent is conjugated to the ligand as shown schematically below: Wherein X is O or S.

14. The dsRNA agent of claim 13, wherein X is O.

15. The dsRNA agent of claim 1, wherein the antisense strand consists of a modified nucleotide sequence as shown below: 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), where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-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 diol nucleic acid (GNA); and s is a phosphorothioate linkage.

16. The dsRNA agent of claim 1, wherein the antisense strand and the sense strand consist of a modified nucleotide sequence as shown below: (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); where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-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 diol nucleic acid (GNA); s is a phosphorothioate linkage; and L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

17. The dsRNA agent of claim 1, wherein the antisense strand and the sense strand consist of a modified nucleotide sequence as shown below: 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-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 diol nucleic acid (GNA); s is a phosphorothioate linkage; and L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

18. The dsRNA agent of any one of claims 1-17, wherein the dsRNA agent inhibits the expression of hepatitis B virus (HBV) in a cell.

19. A cell comprising the dsRNA agent of any one of claims 1-18.

20. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1-19 and a pharmaceutically acceptable excipient.

21. A method for inhibiting the expression of a hepatitis B virus (HBV) gene in a cell, the method comprising contacting the cell with the dsRNA agent of any one of claims 1 to 18 or the pharmaceutical composition of claim 20, thereby inhibiting the expression of the HBV gene in the cell.

22. The method of claim 21, wherein the HBV gene expression is inhibited by at least 80%, 90%, 95% or 98%, or is inhibited to below the detection level of an assay.

23. A method for inhibiting the replication of hepatitis B virus (HBV) in a cell, the method comprising contacting the cell with the dsRNA agent of any one of claims 1-18 or the pharmaceutical composition of claim 20, thereby inhibiting the replication of the HBV in the cell.

24. The method of claim 23, wherein the replication of HBV in the cells is inhibited by at least 80%, 90%, 95% or 98%, or is inhibited to below the detection level of an assay.

25. The method of any one of claims 21-24, wherein the cell is in a subject.

26. The method of claim 25, wherein the subject is a human.

27. The method of claim 26, wherein the subject suffers from an HBV-related disease.

28. The method of any one of claims 21-24, wherein the cell is in vitro.

29. A method for reducing the level of HBV antigen in a subject infected with hepatitis B virus (HBV), the method comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1-18 or the pharmaceutical composition of claim 20, thereby reducing the level of the HBV antigen in the subject.

30. The method of claim 29, wherein the HBV antigen is HBsAg.

31. The method of claim 29, wherein the HBV antigen is HBeAg.

32. The method of claim 29, wherein the antigen is measured in serum from the subject.

33. The method of claim 29, wherein the subject is HBeAg positive.

34. The method of claim 29, wherein the subject is HBeAg negative.

35. The method of any one of claims 29-34, wherein the HBV antigen level in serum is reduced by at least 1 log10, at least 2 log10, at least 3 log10, or at least 4 log10, or is reduced to below the detection level of an assay method.

36. A method of reducing the viral load of hepatitis B virus (HBV) in a subject infected with HBV, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1 to 18 or the pharmaceutical composition of claim 20, thereby reducing the viral load of HBV in the subject.

37. The method of claim 36, wherein the HBV viral load is measured in serum from the subject.

38. The method of claim 36 or 37, wherein the subject is HBeAg positive.

39. The method of claim 36 or 37, wherein the subject is HBeAg negative.

40. The method of any one of claims 36 to 39, wherein the HBV viral load in serum is reduced by at least 1 log10, at least 2 log10, at least 3 log10, or at least 4 log10, or is reduced to below the detection level of an assay method.

41. A method of treating a subject suffering from hepatitis B virus (HBV) infection, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1-18 or the pharmaceutical composition of claim 20, thereby treating the subject.

42. A method of treating a subject suffering from a hepatitis B virus (HBV)-related disorder, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1-18 or the pharmaceutical composition of claim 20, thereby treating the subject.

43. The method of claim 42, wherein the HBV-related disorder is chronic hepatitis and the subject is HBeAg positive.

44. The method of claim 42, wherein the HBV-related disorder is chronic hepatitis and the subject is HBeAg negative.

45. The method of any one of claims 21-44, 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.

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.

47. The method of any one of claims 21-44, wherein the dsRNA agent is administered to the subject at a fixed dose of 50 mg to 200 mg or 50 to 900 mg.

48. The method of any one of claims 21-47, wherein the dsRNA agent is administered subcutaneously to the subject.

49. The method of any one of claims 21-48, wherein the dsRNA agent is administered to the subject in two or more doses.

50. The method of any one of claims 21-49, wherein the dsRNA agent is administered to the subject once a month, once every two months, or once every three months.

51. The method of any one of claims 21-49, wherein the dsRNA agent is administered to the subject no more than once a month.

52. The method of any one of claims 21-51, further comprising administering one or more additional therapeutic agents to the subject.

53. A method according to 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 the secretion or release of HBsAg, a capsid inhibitor or a covalently closed circular (ccc) HBV DNA inhibitor, or any combination of the above.

54. The method of claim 52, wherein the additional therapeutic agent is a reverse transcriptase inhibitor.

55. The method of claim 52, wherein the additional therapeutic agent is a reverse transcriptase inhibitor and an immunostimulant.

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.

57. The method of claim 55, wherein the immunostimulatory agent is pegylated interferon α2a (PEG-IFN-α2a), interferon α-2b, recombinant human interleukin 7, or a Toll-like receptor 7 (TLR7) agonist.

58. A pharmaceutical composition for practicing the method of any one of claims 21-57, comprising the dsRNA agent of any one of claims 1-18 and a pharmaceutically acceptable excipient.

59. The dsRNA agent of any one of claims 1-18 or the pharmaceutical composition of claim 20, for use in treating hepatitis B virus (HBV) infection in a subject.

60. The dsRNA agent of any one of claims 1-18 or the pharmaceutical composition of claim 20, for use in treating a hepatitis B virus (HBV)-related disorder in a subject.

61. The dsRNA agent or pharmaceutical composition for use according to claim 60, wherein the HBV-related disorder is chronic hepatitis and the subject is HBeAg positive.

62. The dsRNA agent or pharmaceutical composition for use according to claim 60, wherein the HBV-related disorder is chronic hepatitis and the subject is HBeAg negative.

63. The dsRNA agent or pharmaceutical composition for use according to any one of claims 59-62, wherein the subject is being or has been administered one or more additional therapeutic agents.

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 of the foregoing.

65. The dsRNA agent or pharmaceutical composition for use according to claim 63, wherein the additional therapeutic agent is a reverse transcriptase inhibitor.

66. The dsRNA agent or pharmaceutical composition for use according to claim 63, wherein the additional therapeutic agent is a reverse transcriptase inhibitor and an immunostimulant.

67. The dsRNA agent or pharmaceutical composition for use according to claim 65 or 66, wherein the reverse transcriptase inhibitor is tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine or AGX-1009.

68. The dsRNA agent or pharmaceutical composition for use according to claim 66, wherein the immunostimulatory agent is pegylated interferon α2a (PEG-IFN-α2a), interferon α-2b, recombinant human interleukin 7, or a Toll-like receptor 7 (TLR7) agonist.

69. Use of the dsRNA agent of any one of claims 1-18 or the pharmaceutical composition of claim 20 in carrying out the method of any one of claims 21-57.

70. Use of the dsRNA agent of any one of claims 1-18 or the pharmaceutical composition of claim 20 in the preparation of a medicament for practicing the method of any one of claims 21-57.

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