HEPATITIS B VIRUS (HBV) dsRNA AGENT COMPOSITIONS AND METHODS OF USE THEREOF

Abstract

To provide effective alternative therapies and combination therapies for subjects having an HBV-associated disease.SOLUTION: The present disclosure relates to double stranded RNA agents targeting the hepatitis B virus (HBV) genome, methods of using such agents to inhibit one or more HBV genes, and methods of treating subjects having an HBV infection or HBV-associated disorder, e.g., chronic hepatitis B.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Description of the Sequence Listing The sequence listing related to this application is provided in text format instead of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 930385_410WO_Sequence_LISTING.txt. The text file is 83.1 KB, was created on August 4, 2019, and was electronically submitted via EFS-Web.

Background Art

[0002] More than 400 million people worldwide are chronically infected with HBV, and thus are at high risk of developing severe liver diseases such as chronic hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma (HCC), with an estimated 600,000 deaths per year.

[0003] The natural course of chronic HBV infection includes four consecutive stages: (1) an initial "immune tolerance" stage, with high levels of viral replication and minimal hepatitis; (2) an immune response stage, with severe hepatitis and elevated serum aminotransferases; some patients progress to (3); (3) a "non-replicative" stage, with seroconversion to anti-HBe, undetectable or low-level viremia (less than 2000 IU / ml by PCR-based assay), and resolution of liver inflammation; and (4) HBeAg-negative chronic hepatitis B due to the emergence of specific viral mutations, which block the production of HBeAg but do not prevent viral replication. This form of chronic hepatitis B (CHB) is characterized by fluctuations in serum HBV DNA and serum aminotransferase (ALT and AST) levels, as well as progressive liver disease. It is important to note that CHB can exist as either HBeAg-positive CHB or HBeAg-negative CHB. Longitudinal studies of CHB patients have shown that the 5-year cumulative incidence of cirrhosis development ranges from 8 to 20%. The 5-year cumulative incidence of liver decompensation is approximately 20%. The worldwide incidence of HCC is increasing and is currently the fifth most common cancer. The annual incidence of HBV-related HCC is high and ranges from 2 to 5% in patients with established cirrhosis.

[0004] The main goal of HBV treatment is to permanently inhibit 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 achieve a sustained response that prevents the development of cirrhosis and hepatocellular carcinoma and prolongs survival. HBV infection cannot be completely eradicated because a specific form of viral covalently closed circular DNA (cccHBV DNA) persists in the nuclei of infected hepatocytes. However, serum HBsAg clearance induced by treatment is a marker of the end of chronic HBV infection and is associated with the best long-term outcomes.

[0005] Current standard HBV treatment methods include immunotherapy based on interferon or thymosin α1, and antiviral therapy that inhibits viral production by inhibiting HBV polymerase. HBV polymerase inhibitors are effective in reducing viral production but have little or no effect on rapidly reducing HBsAg, or (as in the case of tenofovir disoproxil fumarate) can only slowly reduce HBsAg in the long-term treatment of a limited number of patients. Interferon-based immunotherapy can achieve both reduction of viral production and initial removal of HBsAg from the blood, but this is only possible 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 avoid immune detection, which is hypothesized to be one of the reasons why HBV infection remains chronic. In addition, HBsAg, HBeAg, and HBcAg all have immunosuppressive properties, and the persistence of these viral proteins in the blood of patients after administration of any of the currently available HBV treatments is thought to have a significant impact on preventing immunological control of HBV infection in patients.

[0006] All three major HBV proteins (HBsAg, HBeAg, and HBcAg) have immunosuppressive properties, but HBsAg accounts for the overwhelming majority of HBV proteins in the circulation of HBV-infected subjects. In addition, while the removal of HBeAg (via seroconversion) or reduction of viremia does not correlate with the development of sustained HBV infection control upon treatment discontinuation, the removal (and seroconversion) of serum HBsAg from the blood in HBV infection is a well-recognized prognostic indicator of antiviral response during treatment that can lead to HBV infection control upon treatment discontinuation (although this occurs in only a small fraction of patients receiving immunotherapy). Thus, reduction of all three major HBV proteins (HBsAg, HBeAg, and HBcAg) can result in optimal removal of the inhibitory effect, but removal of HBsAg alone seems to be sufficient by itself to remove most of the viral immunosuppression of the immune function of HBV-infected subjects.

[0007] Thus, in the absence of a treatment regimen that can currently restore immunological control of HBV in most patients, there is a need for an effective treatment for HBV infection that can inhibit viral replication and restore immunological control in the majority of patients. Accordingly, there is a need in the art for alternative and combination therapies for subjects infected with HBV or having HBV-related diseases. SUMMARY OF THE INVENTION

[0008] In some embodiments, the present specification 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 may be intracellular, for example, within cells of a subject such as a human.

[0009] This specification also provides a method and therapy for treating a subject having a disorder such as an HBV infection or an HBV-related disease, e.g., chronic hepatitis B infection (CHB), which would benefit from inhibition of HBV gene expression, with a dsRNA substance composition that affects RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of an HBV gene to inhibit expression of the HBV gene.

[0010] In one aspect, this specification provides a dsRNA substance for inhibiting expression of HBV. For example, this specification provides a dsRNA substance comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand is 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16), 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18), 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20), 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23), 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24), 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25), or 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28): [In the sequence, 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-glycol nucleic acid (GNA); and s is a phosphorothioate linkage.] To provide a dsRNA substance comprising a modified nucleotide sequence represented by.

[0011] In some embodiments, the sense strand has the modified nucleotide sequence: 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29) [In the sequence, 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.] Comprising.

[0012] In some embodiments, the antisense strand and the sense strand are (a) 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (b) 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (c) 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (d) 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (e) 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (f) 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); or (g) 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29): [In the sequences, 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-glycol nucleic acid (GNA); and s is a phosphorothioate linkage.] It comprises a modified nucleotide sequence represented by

[0013] In some embodiments, at least one strand of the dsRNA substance comprises a 3'-overhang (protrusion) of at least one nucleotide. In some embodiments, at least one strand of the dsRNA substance comprises a 3'-overhang of two nucleotides.

[0014] In some embodiments, the double-stranded region of the dsRNA substance is a pair 19-21 nucleotides in length.

[0015] In some embodiments, each strand of the dsRNA substance independently has 19-23 nucleotides. In some embodiments, each strand of the dsRNA substance independently has 19-21 nucleotides.

[0016] In some embodiments, the dsRNA substance further comprises a ligand. In some embodiments, the ligand is conjugated to the 3'-end of the sense strand of the dsRNA substance. In some embodiments, the ligand is an N-acetylgalactosamine (GalNAc) derivative. In certain embodiments, the ligand has the formula:

Chemical formula

[0017] In some embodiments, the dsRNA substance has the formula:

Chemical formula

[0018] In some embodiments, the antisense strand is 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16), 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18), 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20), 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23), 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24), 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25), or 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28): [In the sequence, 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-glycol nucleic acid (GNA); and s is a phosphorothioate linkage.] It consists of a modified nucleotide sequence represented by

[0019] In some embodiments, the present specification provides dsRNA substances of a sense strand and an antisense strand consisting of the following modified nucleotide sequences: (a) 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (b) 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (c) 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (d) 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (e) 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); (f) 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29); or (g) 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28) and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29): In the array, 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-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and The 3'-end of the sense strand is conjugated to an N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol (L96) ligand.

[0020] In other aspects, the present specification provides cells containing the dsRNA substances disclosed herein.

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

[0022] This specification also provides a method for inhibiting hepatitis B virus (HBV) gene expression in a cell, which comprises contacting the cell with the dsRNA substance or pharmaceutical composition disclosed in the present invention to inhibit HBV gene expression in the cell. In some embodiments, the cell is within a subject. In some embodiments, the subject is a human. In some embodiments, the subject is suffering from an HBV-related disease. In some embodiments, the cell is in vitro. In some embodiments, the HBV gene expression is inhibited to at least 80%, 90%, 95% or 98%, or below the detection level of the assay.

[0023] This specification provides a method for inhibiting the replication of hepatitis B virus (HBV) in a cell, which comprises contacting the cell with the dsRNA substance or pharmaceutical composition disclosed herein to inhibit the replication of HBV in the cell. In some embodiments, the cell is within a subject. In some embodiments, the subject is a human. In certain embodiments, the subject is suffering 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 to at least 80%, 90%, 95% or 98%, or below the detection level of the assay.

[0024] Also provided herein is a method for reducing hepatitis B virus (HBV) antigen levels in a subject infected with HBV, the method comprising administering to the subject a therapeutically effective amount of a dsRNA substance or pharmaceutical composition disclosed herein to reduce the subject's HBV antigen levels. 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 is reduced in serum by at least 1 log10, at least 2 log10, at least 3 log10, or at least 4 log10, or to less than the detection level of the assay.

[0025] Also provided herein is 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 substance or pharmaceutical composition disclosed herein to reduce the subject's HBV viral load. 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 is reduced in serum by at least 1 log10, at least 2 log10, at least 3 log10, or at least 4 log10, or to less than the detection level of the assay.

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

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

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

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

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

[0031] In some embodiments of the above-described method, the method further comprises administering to the subject one or more additional therapeutic substances. The additional therapeutic substances include, for example, antiviral agents, reverse transcriptase inhibitors, immunostimulants, therapeutic vaccines, virus entry inhibitors, oligonucleotides that inhibit the secretion or release of HbsAg, capsid inhibitors, covalent closed circular (ccc) HBV DNA inhibitors, and any combination of the foregoing, but are not limited thereto. In some embodiments, the additional therapeutic substance is a reverse transcriptase inhibitor.

[0032] In some embodiments, more than one additional therapeutic substance is administered, and the additional therapeutic substances are a reverse transcriptase inhibitor and an immunostimulant. Examples of reverse transcriptase inhibitors include, but are not limited to, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, and AGX-1009. Examples of immunostimulants 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.

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

[0034] The present disclosure provides for the use of a dsRNA substance or pharmaceutical composition disclosed herein for performing any of the above methods.

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

[0036] This specification provides a kit comprising the dsRNA substances or pharmaceutical compositions disclosed herein, optionally comprising instructions for use for carrying out the methods described herein.

Brief Description of the Drawings

[0037]

Figure 1

[0038]

Figure 2

[0039]

Figure 3

[0040]

Figure 4

[0041]

Figure 5A

[0042]

Figure 5B

[0043] The dsRNA substances described herein are designed to target regions conserved across at least eight known genotypes of HBV within the HBV genome. Further, the dsRNA substances herein are designed to inhibit the expression of more than one HBV gene, thereby inhibiting all stages of the HBV life cycle, such as replication, assembly, viral secretion, and secretion of subviral antigens. Specifically, since polycistronic overlapping RNAs are generated when the HBV genome is transcribed, in some embodiments, dsRNA substances that target a single HBV gene will significantly inhibit the expression of most or all HBV transcripts. For example, since the HBV genome is transcribed into a single mRNA, the dsRNA substances herein that target the S gene will inhibit not only S gene expression but also the expression of its "downstream" polymerase gene. Further, the dsRNA substances herein are designed to inhibit HBV virus replication by targeting the HBV structural genes and the HBV X gene, thereby enabling the subject's immune system to detect and react to the presence of HBsAg and produce anti-HBV antibodies to eliminate HBV infection. Without intending to be limited to a particular theory, the combination or sub-combination of the above properties with a particular target site or particular modification of the dsRNA substances herein is thought to confer improvements in the efficacy, stability, safety, potency, and durability of the dsRNA substances herein.

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

[0045] Accordingly, this specification also provides a method of treating a subject having a disorder, such as an HBV-related disease like chronic hepatitis B virus infection (CHB), that may benefit from inhibiting or reducing the expression of the HBV gene, with a dsRNA substance composition that affects RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the HBV gene.

[0046] The dsRNA substances of this specification include an RNA strand (antisense strand) having a complementary region that is about 9 to 21 nucleotides in length, for example about 19 nucleotides in length, and that is substantially complementary to at least a portion of the mRNA transcript of the HBV gene of at least one HBV genotype. It is understood that multiple HBV genotypes exist, such that the dsRNA substances of this specification may have different degrees of complementarity to different HBV genotypes.

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

[0048] The following detailed description discloses how to make and use compositions containing dsRNA substances to inhibit the expression of the HBV gene, as well as compositions, uses, and methods for treating subjects having diseases and disorders that may benefit from inhibiting or reducing the expression of the HBV gene.

[0049] I. Definition To make this specification more readily understandable, certain terms are first defined. Further, it should be noted that whenever a value or range of values of a parameter is recited, values intermediate to and including the recited values are also intended to be part of this feature.

[0050] Unless the context requires otherwise, throughout this specification and the claims, the terms "comprise" and variations thereof, such as "comprises" and "comprising", are to be interpreted in an open, inclusive sense, i.e., "including, but not limited to". "Consisting of" is meant to exclude any other components or trace elements of substantial process steps disclosed herein. For example, a polynucleotide consists of a nucleotide sequence if it does not contain any additional nucleotides, but does not exclude the incorporation of ligands, such as targeting ligands, or modifications. The term "consisting essentially of" limits the claim to a particular material substance or process, or those that do not substantially affect the basic control of the invention claimed. For example, a pharmaceutical composition consisting essentially of the components defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers such as phosphate buffered saline, preservatives, etc. Similarly, a polynucleotide consists essentially of a nucleotide sequence if it contains additional nucleotides that contribute at least 20% of the polynucleotide length and do not substantially affect the activity of the polynucleotide (e.g., modify the activity of the polynucleotide by less than 50%). Embodiments defined by each transitional term are within the scope of the present invention.

[0051] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

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

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

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

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

[0056] As used herein, "not exceeding" or "less than" is understood, as is logical from the context, as the number from zero to the value adjacent to the phrase and the logical lower value or integer. For example, a double strand having an overhang of "not exceeding 2 nucleotides" has an overhang of 2, 1, or 0 nucleotides. When "not exceeding" is present before a series of numbers or a range, it is understood that each number within the series or range can be changed.

[0057] As used herein, a range includes both an upper limit and a lower limit.

[0058] If a conflict occurs between an array and its indicated site on a transcript or another array, the nucleotide sequences described herein shall prevail.

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

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

[0061] The HBV genome is a circular DNA that is partially double-stranded and has overlapping reading frames (for example, see Figure 1).

[0062] There are four transcripts based on size encoded by the HBV genome (which may be referred to herein as "genes" or "open reading frames"). These contain open reading frames designated 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 the pre-core protein. DNA polymerase is encoded by gene P. Gene S is the gene encoding the surface antigen (HBsAg). The HBsAg gene is a single long open reading frame containing the "start" (ATG) codons of three in-frame "initiation" codons that give rise to three different sized polypeptides (pre-S1 + pre-S2 + S, pre-S2 + S, or S) designated the large, middle, and small S antigens. The surface antigen, in addition to decorating the envelope of HBV, is also part of the subviral particles that are produced in excess compared to virion particles and serves to sequester immune tolerance and anti-HBsAg antibodies, thereby allowing infectious particles to escape immune detection. The function of the non-structural protein encoded by gene X is not fully understood but is thought to play a role in transcriptional transactivation and replication and is associated with the development of hepatocellular carcinoma.

[0063] HBV is one of the few DNA viruses that utilize reverse transcriptase in a replication process involving multiple steps including entry, uncoating, and transport of the viral genome to the nucleus. Initially, replication of the HBV genome involves the generation of an RNA intermediate, which is then reverse transcribed to produce the DNA viral genome.

[0064] When a cell is infected with HBV, the relaxed circular DNA (rcDNA) of the viral genome is transported to the cell nucleus and converted into episomal covalently closed circular DNA (cccDNA), which serves as the transcription template for viral mRNA. After transcription and nuclear export, the cytoplasmic viral pregenomic RNA (pgRNA) assembles with the HBV polymerase and capsid protein to form nucleocapsids, inside which minus-strand DNA is generated by polymerase-catalyzed reverse transcription, followed by its copying into plus-strand DNA to form progeny rcDNA genomes. The mature nucleocapsids then either are packaged into viral envelope proteins to emerge as virion particles or are shuttled to the nucleus to amplify the cccDNA reservoir via the intracellular cccDNA amplification pathway. CccDNA is an essential component of the HBV replication cycle and plays a role in the establishment of infection and viral persistence.

[0065] HBV infection produces two different particles: 1) the infectious HBV virus itself (or Dane particles), which contains a viral capsid constructed from HBcAg and is coated by an envelope consisting of a lipid membrane with the HBV surface antigen, and 2) subviral particles (or SVP), which contain small and intermediate forms of the non-infectious hepatitis B surface antigen HBsAg. For each viral particle produced, more than 10,000 SVPs are released into the blood. Thus, SVPs (and the HBsAg proteins they carry) represent the overwhelming majority of viral proteins in the blood. HBV-infected cells also secrete a soluble proteolytic product of the precore protein, called the HBV e antigen (HBeAg).

[0066] The eight genotypes of HBV are named A - H, have been sequenced, and two additional genotypes, I and J, have been proposed, each with a different geographical distribution. The virus is non-cytopathic, and virus-specific cellular immunity is the major factor determining whether the result of exposure to HBV is an acute infection with resolution of liver disease within six months or a chronic HBV infection with progressive liver injury at high frequency.

[0067] The term "HBV" includes any genotype (A - J) of HBV. The complete coding sequences of the reference sequences of the HBV genome can be found, for example, in GenBank accession numbers GI:21326584 (SEQ ID NO: 1) and GI:3582357 (SEQ ID NO: 3). The antisense sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively. The amino acid sequences of the C, X, P, and S proteins can be found, for example, in NCBI accession numbers YP_009173857.1 (C protein) (SEQ ID NO: 37); YP_009173867.1 and BAA32912.1 (X protein) (SEQ ID NO: 36 and 40); YP_009173866.1 and BAA32913.1 (P protein) (SEQ ID NO: 32 and 38); and YP_009173869.1, YP_009173870.1, YP_009173871.1, and BAA32914.1 (S protein) (SEQ ID NO: 33, 34, 35, 39). The protein and DNA sequences from HBV genotype D, strain ayw are shown in SEQ ID NO: 36 - 37. The protein and DNA sequences from the HBV gene are shown in SEQ ID NO: 38 - 39. Further examples of HBV protein and DNA sequences, or their reverse complementary sequences, are shown in SEQ ID NO: 41 - 49.

[0068] Further examples of HBV mRNA sequences can be readily obtained using publicly available databases such as GenBank, UniProt, and OMIM. The international repository of hepatitis B virus strain data can be accessed from http: / / www.hpa - bioinformatics.org.uk / HepSEQ / main.php.

[0069] As used herein, the term "HBV" also means naturally occurring DNA sequence variations of the HBV genome, such as genotypes A - J and their variants.

[0070] As used herein, the term "hepatitis D virus" is used synonymously with the term "HDV" and refers to a well-known non-cytopathic liver tropic DNA virus belonging to the family Hepadnaviridae. 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 hereby incorporated by reference). Unless otherwise specified, HDV refers to all clades and variants of HDV.

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

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

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

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

[0075] As used herein, "target sequence" means a continuous portion of the nucleotide sequence of an mRNA molecule formed upon transcription of an HBV gene, including the mRNA that is the product of RNA processing of the primary transcript. In some embodiments, the target portion of the sequence is at least long enough for the dsRNA species to serve as a substrate for the cleavage of interest at or near the target portion in the nucleotide sequence of the mRNA molecule formed upon transcription of the HBV gene.

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

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

[0078] "G", "C", "A", "T", and "U" each generally mean a nucleotide containing guanine, cytosine, adenine, thymidine, and uracil as bases. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also mean a modified nucleotide, or a surrogate replacement moiety (see, e.g., Table 1) as further detailed below. One of ordinary skill in the art is well aware that guanine, cytosine, adenine, and uracil, even when replaced by other moieties, do not substantially change the base pairing of the oligonucleotide containing nucleotides with such replacement moieties. For example, without limitation, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine may be replaced, for example, by a nucleotide containing inosine in the nucleotide sequence of the dsRNA characterized herein. In another example, adenine and cytosine at any position in the oligonucleotide can be replaced by guanine and uracil, respectively, to form wobble G-U base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods characterized herein.

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

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

[0081] Generally, each strand of a dsRNA molecule can contain ribonucleotides, but as described in detail herein, each strand or both strands can also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Further, the term "dsRNA substance" as used herein can include ribonucleotides having chemical modifications; the dsRNA substance may include substantial modifications in a plurality of nucleotides. The term "modified nucleotide" as used herein independently means a nucleotide having a modified sugar moiety, a modified internucleotide linkage or a modified nucleobase. Thus, the term "modified nucleotide" encompasses substitutions, additions or removals of, for example, functional groups or atoms to the internucleoside linkage, the sugar moiety or the nucleobase. Suitable modifications for use with the substances herein include all types of modifications disclosed herein or known in the art. Any such modification used with a dsRNA substance type of molecule is encompassed by "dsRNA substance" for the purposes of this specification and the claims.

[0082] The term "inhibit" as used herein is used synonymously with "reduce", "silence", "down-regulate", "suppress" and other similar terms and includes any level of inhibition. Preferably, "inhibit" includes statistically significant or clinically significant inhibition.

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

[0084] "Inhibiting the expression of the HBV gene" includes inhibition at any level of the HBV gene or transcript, e.g., at least the HBV gene, e.g., the HBV gene S, P, X or C, or any combination thereof, e.g., partial inhibition of the expression of S, P and C. The expression of the HBV gene can be evaluated based on levels or fluctuations in levels that can vary in relation to the HBV gene, e.g., at the HBV mRNA level or the HBV protein level, or the HBV cccDNA level. This level can be evaluated, for example, in individual cells or cell populations, including samples derived from a subject, and, for example, the level can be monitored in serum. Inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, e.g., a baseline level before administration, or a level determined from a similar subject or population average from an appropriate control subject, cell or sample that has been untreated, or treated with a control (e.g., a buffer-only control or an inactive agent control).

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

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

[0087] Inhibition of HBV gene expression may be manifested by a reduction in the number of RNAs expressed by a first cell or cell population (such cells may be present, for example, in a sample derived from a subject), where the HBV gene in the first cell or cell population is transcribed and the first cell or cell population is treated (e.g., by contacting the cell with the dsRNA substance of the present specification or by administering the dsRNA substance of the present specification to the subject in which the cell is present or was present), resulting in inhibition of HBV gene expression compared to a second cell or cell population (control cells) that is substantially the same as the first cell or cell population but has not been so treated. In a preferred embodiment, the inhibition is evaluated by the rtPCR method provided in Example 2 of WO2016 / 077321 (incorporated herein by reference) using an in vitro assay performed in a suitably adapted cell line having a double-stranded concentration of 10 nM, and the level of mRNA in the treated cells is expressed as a percentage of the level of mRNA in the control cells using the following formula.

Number

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

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

[0090] Control cells or cell populations that can be used to evaluate the inhibition of HBV gene expression include cells or cell populations that have not been contacted with the dsRNA substances of the present specification. For example, the control cells or cell populations can be derived from individual subjects (e.g., human or animal subjects) before the subject is treated with the dsRNA substance. In alternative embodiments, the levels may be compared to appropriate control samples, e.g., known population control samples of individuals.

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

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

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

[0094] Alternative methods for measuring the expression level of the HBV gene in a sample include, for example, RT-PCR (the experimental embodiment described in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained 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., U.S. Patent No. 5,854,033), or any other nucleic acid amplification method, including, for example, the step of nucleic acid amplification of mRNA in the sample or reverse transcriptase (to prepare cDNA), followed by detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are particularly useful for the detection of nucleic acid molecules when such molecules are present in very small numbers. In certain embodiments herein, the level of expression of the HBV gene is measured by quantitative fluorescent RT-PCR (i.e., the TaqMan™ system), for example, using the methods provided herein.

[0095] The expression level of HBV RNA can be monitored using a membrane blot (such as those used for Northern, dot, etc. hybridization analysis), or using a microwell, sample tube, gel, bead, fiber (or any solid support containing bound nucleic acid). For teachings related to such methods, see U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Measuring the HBV expression level may also include using a nucleic acid probe in solution.

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

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

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

[0099] When the two substantially complementary strands of the dsRNA are contained in separate RNA molecules, those molecules need not be covalently linked, but may be covalently linked. When the two strands are covalently linked by means other than a continuous strand of nucleotides between the 3' end of one strand and the 5' end of the other strand that form the double-stranded structure, that linking structure is referred to as a "linker". The two RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus the number of overhangs present in the double-strand. In addition to the double-stranded structure, the dsRNA substance can include one or more nucleotide overhangs.

[0100] As used herein, the term "nucleotide overhang" means at least one unpaired nucleotide that protrudes from the double-stranded structure of a double-stranded dsRNA molecule. For example, a nucleotide overhang exists when the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand or vice versa. The dsRNA can include an overhang of at least one nucleotide, or the overhang can include two nucleotides. The nucleotide overhang can include or consist of nucleotide / nucleoside analogs that include deoxynucleotides / nucleosides. The overhang can be present in the sense strand, the antisense strand, or a combination of both. Further, the nucleotides of the overhang can be present at the 5' end, 3' end, or both ends of either the antisense or sense strand of the dsRNA. In a preferred embodiment, the nucleotide overhang is present at the 3' end of the antisense strand.

[0101] By "blunt" or "blunt end" is meant that there are no unpaired nucleotides at the ends of the double-stranded dsRNA molecule, i.e., there is no nucleotide overhang. A "blunt end" dsRNA molecule is double-stranded over its entire length, i.e., a dsRNA in which there is no nucleotide overhang at either end of the molecule. In some embodiments, the dsRNA molecules herein include dsRNA molecules having a nucleotide overhang at one end (i.e., a dsRNA molecule having one overhang and one blunt end) or having nucleotide overhangs at both ends.

[0102] The term "antisense strand" or "guide strand" means a strand of a dsRNA substance that contains a region substantially complementary to a target sequence, such as HBV mRNA. As used herein, the term "complementary region" means a region on the antisense strand that is substantially complementary to a sequence as defined herein, such as a target sequence, such as an HBV nucleotide sequence. If the complementary region is not completely complementary to the target sequence, mismatches may be present in the internal or terminal regions of the molecule. Generally, most acceptable mismatches are within 5, 4, 3, 2, or 1 nucleotide of the terminal regions, such as the 5' and / or 3' termini of the dsRNA substance. In some embodiments, the dsRNA substances herein contain nucleotide mismatches in the antisense strand. In some embodiments, the dsRNA substances herein contain nucleotide mismatches in the sense strand. In some embodiments, the nucleotide mismatches are within 5, 4, 3, 2, or 1 nucleotide from the 3' terminus of the dsRNA substance, for example. In some embodiments, the nucleotide mismatches are at the 3' terminal nucleotide of the dsRNA substance, for example.

[0103] As used herein, the term "sense strand" or "passenger strand" means a strand of a dsRNA substance that contains a region substantially complementary to the region of the antisense strand as defined herein.

[0104] As used herein, the term "complementary", unless otherwise specified, when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, as understood by one of ordinary skill in the art, means that an oligonucleotide or polynucleotide containing the first nucleotide sequence is capable of hybridizing to an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions to form a double-stranded structure. Such conditions can be, for example, stringent conditions, where stringent conditions can include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, 12-16 hours at 50 °C or 70 °C, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press"). Other conditions can be applied, such as physiologically relevant conditions that can occur within an organism. One of ordinary skill in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate application of the hybridized nucleotides.

[0105] Within the dsRNA molecule, for example, the complementary sequences within the dsRNAs described herein include base pairs over the full length of one or both nucleotide sequences of an oligonucleotide or polynucleotide containing a first nucleotide sequence to an oligonucleotide or polynucleotide containing a second nucleotide sequence. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences are either fully complementary or, while retaining the ability to hybridize under conditions optimal for their ultimate application, e.g., inhibition of gene expression via the RISC pathway, when hybridization is performed to a duplex of up to 21 base pairs, they may form one or more, but generally no more than 4, 3, or 2 mismatched base pairs. However, if two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs shall not be considered mismatches with respect to complementarity determination. For example, a dsRNA containing one 21-nucleotide-long oligonucleotide and another 23-nucleotide-long oligonucleotide can be referred to as "fully complementary" for the purposes described herein if the longer oligonucleotide contains a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide.

[0106] As used herein, "complementary" sequences may further include base pairs formed from non-Watson-Crick base pairs or non-natural and modified nucleotides, or may be formed entirely from such base pairs, so long as the above requirements related to their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogstein-type base pairs.

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

[0108] As used herein, a polynucleotide that is "substantially complementary to at least a portion" of a messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest (e.g., an mRNA encoding an HBV gene). For example, a polynucleotide is complementary to at least a portion of an HBV mRNA if its sequence is substantially complementary to a continuous portion of the mRNA encoding the HBV gene.

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

[0110] In some embodiments, the dsRNA substances herein include a sense strand that is substantially complementary to an antisense polynucleotide, and then the antisense polynucleotide is complementary to the target HBV sequence, where the sense strand polynucleotide is at least about 80% complementary over the equivalent region and the full length of the nucleotide sequence of SEQ ID NO: 2, for example, at least 85%, 90% or 95% complementary, and includes a continuous nucleotide sequence that is complementary.

[0111] In some embodiments, the dsRNA substance includes an antisense strand that is substantially complementary to the target HBV sequence, and is at least 80% complementary over the equivalent region and the full length of the nucleotide sequence of any one of the sense nucleotide sequences in Table 2, for example, at least 85%, 90% or 95% complementary, and includes a continuous nucleotide sequence that is complementary.

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

[0113] In some embodiments, the majority of the nucleotides of each strand are ribonucleotides, but as detailed herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Further, the dsRNA substance may include ribonucleotides having chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications used in the dsRNA substance are encompassed by "dsRNA substance" for the purposes of this specification and the claims.

[0114] As used herein, the term "lower" in the context of HBV gene expression or HBV protein production in a subject, or the level of a disease marker or symptom, means a statistically significant reduction in such level. The reduction can be, for example, at least 80%, at least 85%, at least 90%, at least 95%, or less than the level of detection of the detection method, or a level approaching or reaching the normal level (which may or may not be zero). In the monitoring of HBV infection, the log10 scale is generally used to describe the level of antigenemia (e.g., serum HBsAg level) or viremia (serum HBV DNA level). A 1 log10 reduction is understood to be a 90% reduction (remaining 10%), a 2 log10 reduction is a 99% reduction (remaining 1%), and so on. In certain embodiments, the disease marker is reduced to less than the level of detection. In certain embodiments, the method comprises a clinically relevant inhibition of HBV expression, as demonstrated by a clinically relevant outcome, for example, after treating a control with a substance that reduces HBV expression. In some embodiments, at least partial inhibition of HBV gene expression is evaluated by a reduction in the number of HBV mRNAs isolatable from a first cell or cell population (wherein the HBV gene in the cell or cell population is transcribed, or the cell is treated such that the first cell or cell population is substantially the same as a second cell or cell population (control cell) that has not been so treated, but the expression of the HBV gene is inhibited as compared to the control cell).

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

[0116] As another example, the normal level of ALT is generally 7 - 55 units per liter (U / L), with the upper limit of the normal level being 55 U / L. The ALT of a subject with a pre-treatment ALT level of 100 U / L (exceeding the upper limit of the normal level by 45 U / L) and a post-treatment ALT level of 75 U / L (a reduction of 25 U / L) would be reduced towards the normal value by 55% (25 / 45 × 100%). As used herein, when a disease is associated with an increase in symptom values, "normal" is considered to be the upper limit of the normal level. When a disease is associated with a reduction in symptom values, "normal" is considered to be the lower limit of the normal level.

[0117] As used herein, the phrase "contacting a cell with a dsRNA substance", e.g., contacting with dsRNA, includes contacting the cell by any possible means. Contacting a cell with a dsRNA substance includes contacting the cell with the dsRNA substance in vitro or in vivo. The contact may be made directly or indirectly. Thus, for example, the dsRNA substance may be physically contacted with the cell by the individual performing the method, or the dsRNA substance may be placed in a situation that allows or subsequently causes it to contact the cell.

[0118] Contacting a cell in vitro can be accomplished, for example, by incubating the cell with the dsRNA substance. Contacting a cell in vivo can be accomplished, for example, by injecting the dsRNA substance into or near the tissue where the cell is located, or by injecting the dsRNA substance into another region, such as the bloodstream or subcutaneous cavity, such that the substance reaches the tissue where the contacted cell is located. For example, the dsRNA substance may contain or be coupled to a ligand, such as GalNAc3, that directs the dsRNA substance to the site of interest, such as the liver. Combinations of in vitro and in vivo contact methods are also possible. For example, the cell may be contacted with the dsRNA substance in vitro and subsequently transplanted into a subject.

[0119] In some embodiments, contacting a cell with a dsRNA substance includes "introducing the dsRNA substance into the cell" or "delivering the dsRNA substance into the cell" by promoting or effecting uptake or absorption into the cell. Absorption or uptake of the dsRNA substance can occur via passive diffusion or an active cellular process, or by an adjunct or device. Introduction of the dsRNA substance into the cell may be performed in vitro or in vivo. For example, for in vivo introduction, the dsRNA substance may be injected into a tissue site or administered systemically. In vitro introduction into the cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or are known in the art.

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

[0121] As used herein, the term "treating" or "treatment" refers to, but is not limited to, 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 HBV DNA in serum; the presence of serum or liver HBV antigens, such as HBsAg or HBeAg; elevated ALT; elevated AST (normal range can generally be considered about 10 - 34 U / L); absence or low levels of anti-HBV antibodies, liver injury; cirrhosis; delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; hepatic fibrosis; end-stage liver disease; hepatocellular carcinoma; serum sickness-like syndrome; eating disorder; nausea; vomiting, mild fever; myalgia; easy fatigability; taste and smell disorders (aversion to food and tobacco); right upper abdominal pain and epigastric pain (intermittent, mild to moderate); hepatic encephalopathy; drowsiness; disrupted sleep pattern; confusion; coma; ascites; gastrointestinal bleeding; coagulation abnormalities; jaundice; hepatomegaly (slightly enlarged, soft liver); splenomegaly; palmar erythema; spider angiomas; muscle wasting; spider telangiectasia; vasculitis; variceal bleeding; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (caput medusa); ALT level higher than AST level; elevated levels of gamma-glutamyl transpeptidase (GGT) (normal range can generally be considered about 8 - 65 U / L) and alkaline phosphatase (ALP) (normal range is generally considered about 44 - 147 IU / L (international units per liter) and does not exceed 3 times the ULN); slightly low albumin level; elevated serum iron level; leukopenia (i.e., granulocytopenia); lymphocytosis; increased erythrocyte sedimentation rate (ESR); shortened erythrocyte lifespan; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); presence of serum or liver HBsAg, HBeAg, hepatitis B core antibody (anti-HBc) immunoglobulin M (IgM); presence of hepatitis B surface antibody (anti-HBs), hepatitis B e antibody (anti-HBe) or HBV DNA; increased bilirubin level; hyperglobulinemia; presence of tissue-nonspecific antibodies such as anti-smooth muscle antibody (ASMA) or antinuclear antibody (ANA) (10 - 20%); presence of tissue-specific antibodies such as antibodies against the thyroid (10 - 20%); elevated rheumatoid factor (RF) level; reduced platelet count and white blood cell count; accompanying inflammation with lobular, degenerative and reparative hepatocyte changes;And means a beneficial or desired result, including primarily the reduction or amelioration of centrilobular necrosis, whether detectable or undetectable. The likelihood of developing hepatic fibrosis is reduced, for example, when an individual having one or more risk factors for hepatic fibrosis, such as chronic hepatitis B infection, does not develop hepatic fibrosis or develops hepatic fibrosis at a lower severity compared to a population of individuals having the same risk factors and not receiving treatment as described herein. The failure of a disease, disorder or condition to develop, or the development of signs or symptoms associated with such disease, disorder or condition being reduced (e.g., in a clinically appropriate amount), or the signs or symptoms exhibiting a delay (e.g., of days, weeks, months or years) is considered effective prevention. "Treatment" may also mean prolonging survival compared to expected survival in the absence of treatment. Prevention may require administration more than once.;

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

[0123] As used herein, the terms "hepatitis B virus-related disease" or "HBV-related disease" are diseases or disorders caused by or associated with HBV infection or replication. The term "HBV-related disease" includes diseases, disorders or conditions that would benefit from a reduction in HBV gene expression or replication. Non-limiting examples of HBV-related diseases include, for example, hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

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

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

[0126] In some embodiments, the HBV-related disease is acute hepatitis B. Acute hepatitis B includes inflammation of the liver that lasts less than 6 months. Typical symptoms of acute hepatitis B are fatigue, anorexia, nausea, and vomiting. Extremely high aminotransferase values (>1000 U / L) and hyperbilirubinemia are often observed. Severe cases of acute hepatitis B can rapidly progress to acute liver failure characterized by insufficient liver synthetic function. This is often defined as a prothrombin time (PT) of 16 seconds or an international normalized ratio (INR) of 1.5 in the absence of pre-existing liver disease. Acute hepatitis B can progress to chronic hepatitis B.

[0127] In some embodiments, the HBV-related disease is chronic hepatitis. Chronic hepatitis B (CHB) includes liver inflammation that persists for more than six months. Subjects with CHB are HBsAg positive and have either high viremia (≥104 HBV-DNA copies / ml of blood) or low viremia (<103 HBV-DNA copies / ml of blood). In some embodiments, the subject has been infected with HBV for at least 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 have immune tolerance or non-active chronic infection without evidence of active disease, and these are also asymptomatic. Patients with chronic active hepatitis may have symptoms similar to those of acute hepatitis, especially during the replication state. Subjects with chronic hepatitis B disease may have active chronic infection with necroinflammatory liver disease, or increased hepatocyte turnover in the absence of detectable necroinflammation, or non-active chronic infection without any evidence of active disease, and these are also asymptomatic. The persistence of HBV infection in CHB subjects is due to ccc HBV DNA. In some embodiments, subjects with CHB are HBeAg positive. In another embodiment, subjects with CHB are HBeAg negative. Subjects with CHB have serum HBV DNA levels of less than 105, and persistent elevations of transaminases, such as ALT, AST, and γ-glutamyltransferase. Subjects with CHB may have a liver biopsy score of less than 4 (e.g., necroinflammatory score).

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

[0129] Subjects having HBV infection, such as CHB, may develop liver fibrosis. Thus, in some embodiments, the HBV-related disease is liver fibrosis. Histologically, liver fibrosis or cirrhosis is defined as a diffuse liver lesion characterized by fibrosis (excessive fibrous connective tissue) and structural changes from normal liver structure to abnormal nodules.

[0130] Subjects having HBV infection, such as CHB, may develop end-stage liver disease. Thus, in some embodiments, the HBV-related disease is end-stage liver disease. For example, liver fibrosis can progress to the point where the body can no longer compensate for, for example, reduced liver function, resulting in, for example, mental and neurological symptoms and liver failure.

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

[0132] "HDV-related disorder" or "delta hepatitis virus-related disorder" is a disease or disorder associated with the expression of HDV. Exemplary HDV-related disorders include hepatitis B virus infection, acute hepatitis B, acute delta hepatitis; acute fulminant delta hepatitis; chronic delta hepatitis; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

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

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

[0135] As used herein, the term "sample" refers to a sample of a similar fluid, cell, or tissue isolated from a subject, as well as a sample of a fluid, cell, or tissue present within a subject. Adoption Examples of biological fluids include blood, serum, and serous fluid, plasma, lymph, urine, saliva, etc. Tissue samples can include samples from tissues, organs, or localized regions. For example, samples can be derived from specific organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples can be derived from the liver (e.g., the whole liver or a specific part of the liver, or a specific type of cell in the liver, such as hepatocytes). In a preferred embodiment, "sample from subject" refers to plasma or serum obtained from blood taken from a subject. In a further embodiment, "sample from subject" refers to liver tissue (or a subcomponent thereof) or blood tissue (or a subcomponent thereof, such as serum) from a subject.

[0136] II. dsRNA substance This specification provides dsRNA substances that inhibit the expression of one or more HBV genes. In some embodiments, the dsRNA substance comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of HBV genes in cells, such as cells in the body of a mammal, such as a human, having an HBV-related disease, such as chronic hepatitis B. The dsRNA substance comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during the expression of the HBV gene. The complementary region is about 19-21 nucleotides in length. When contacted with cells expressing the HBV gene, the dsRNA substance inhibits the expression of the HBV gene by at least 80% when assayed by, for example, a method based on PCR or branched DNA (bDNA), or a protein-based method such as immunofluorescence analysis using, for example, Western blot or flow cytometry techniques. In a preferred embodiment, the percentage of inhibition is measured using the real-time PCR method provided in Example 2, using the cell line provided therein together with the dsRNA substance used at a concentration of 10 nM in transfection.

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

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

[0139] Similarly, the complementary region of the target sequence is 19 to 23 nucleotides in length, for example, 19 - 23, 19 - 22, 19 - 21, 19 - 20, 20 - 23, 20 - 22, 20 - 21, 21 - 22, 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, the complementary region is 21 nucleotides in length. As those skilled in the art will also recognize, the region of the RNA targeted for cleavage is, in most cases, part of a larger RNA molecule (often an mRNA molecule). Where relevant, a "part" of an mRNA target is a continuous sequence of the mRNA target that is long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0140] Those skilled in the art will also recognize that the double-stranded region is the primary functional part of the dsRNA, for example, a double-stranded region of about 19 - 21 base pairs, for example, 19 - 21, 19 - 20, 20 - 21, 19, 20, or 21 base pairs. In some embodiments, the double-stranded region is 19 base pairs.

[0141] The dsRNA described herein may further include one or more single-stranded nucleotide overhangs, for example, 1 or 2 nucleotides. The nucleotide overhangs may include or consist of nucleotide / nucleoside analogs including deoxynucleotides / nucleosides. The overhangs may be on the sense strand, the antisense strand, or a combination of either. Further, the nucleotides of the overhang may be present on the 5' end, 3' end, or both ends of one or both of the antisense or sense strands of the dsRNA. In some embodiments, the dsRNA includes a 2-nucleotide overhang on the 3' end of the antisense strand.

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

[0143] The dsRNA substance compounds herein can be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Next, the component strands are annealed. The individual strands of the dsRNA substance compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that oligonucleotide strands containing unnatural or modified nucleotides can be readily prepared. The single-stranded oligonucleotides herein can be prepared using solution-phase or solid-phase organic synthesis or both.

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

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

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

[0147] The nucleic acids that can be patented in the present invention can be synthesized or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5′-terminal modifications (phosphorylation, conjugation, inverse ligation) or 3′-terminal modifications (conjugation, DNA nucleotides, inverse ligation, etc.); base modifications, such as substitution of a base with a stable base, an unstable base, or a base that base pairs with a repertoire of spreading partners, removal of a base (abasic nucleotide), or a conjugated base; sugar modifications (e.g., at the 2′ or 4′ position) or substitution of the sugar; or backbone modifications, including modification or substitution of the phosphodiester linkage. Specific examples of dsRNA substance compounds useful in the embodiments described herein include, but are not limited to, RNAs that contain a modified backbone or do not contain a native internucleoside linkage. RNAs having a modified backbone include, in particular, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, as sometimes mentioned in the art, modified RNAs that do not have a phosphorus atom in the internucleoside backbone can also be regarded as oligonucleosides. In some embodiments, the modified dsRNA substance has a phosphorus atom in its internucleoside backbone.

[0148] Modified RNA backbones include, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methylphosphonate and other alkylphosphonates including 3'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-aminophosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, and boranophosphate having a normal 3'-5' linkage, 2'-5' linkage analogs thereof, and those having an inverted polarity in which adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Also included are various salts, mixed salts and free acid forms.

[0149] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 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. Patent No. RE39464, each of which is hereby incorporated by reference herein for the teachings related to such preparation methods.

[0150] Modified RNA backbones that do not contain phosphorus atoms within have a backbone formed by short-chain alkyl or cycloalkyl nucleoside internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl nucleoside internucleoside linkages, or one or more short-chain heteroatom or heterocyclic nucleoside internucleoside linkages. These include those having morpholino linkages (partially formed from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and those having mixed N, O, S and CH 2 and others having component parts.

[0151] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 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 herein for the teachings related to such methods.

[0152] For use in the dsRNA substances provided herein, suitable RNA mimics in which both the sugar and the internucleoside linkage, i.e., the backbone of the nucleotide unit, are replaced by novel groups are contemplated. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound that has been shown to have excellent hybridization properties is an RNA mimic called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and are attached directly or indirectly to the azanitrogen atoms of the amide portions of the backbone. 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 for the teachings relevant to such methods. Further PNA compounds suitable for use in the dsRNA substances herein are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0153] Some embodiments claimed in the present invention include RNAs having a phosphorothioate backbone and oligonucleosides having a heteroatom backbone, particularly the --CH of U.S. Patent No. 5,489,677 described above. 2 --NH--CH 2 -, --CH 2 --N(CH 3 )--O--CH 2 -- [known as the methylene(methylimino) or MMI backbone], --CH 2 --O--N(CH 3 )--CH 2 --, --CH 2 --N(CH 3 )--N(CH 3 )--CH 2 -- and --N(CH 3 )--CH 2 --CH 2--[wherein the natural phosphodiester backbone is represented as --O--P--O--CH2--] and the amide backbone of U.S. Patent No. 5,602,240 described above is included. In some embodiments, the RNA patented herein has the morpholino backbone structure of U.S. Patent No. 5,034,506 described above.

[0154] The modified RNA may also contain one or more substituted sugar moieties. The dsRNA substances patented herein can contain, 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 alkyl, alkenyl and alkynyl are substituted or unsubstituted C 1 ~C 10 alkyl or C 2 ~C 10 alkenyl and alkynyl]. Exemplary suitable modifications include O[(CH 2 ) n O] m CH 3 , O(CH 2 ). n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 wherein n and m are from 1 to about 10. In other embodiments, the dsRNA has, at the 2' position, C 1 ~C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3, SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, a group that improves the pharmacokinetic properties of the dsRNA substance, or a group that improves the pharmacodynamic properties of the dsRNA substance, and one of other substituents having similar properties. In some embodiments, the modification is 2'-methoxyethoxy (also known as 2'-O-(2-methoxyethyl) or 2'-MOE), i.e., 2'-O--CH 2 CH 2 OCH 3 )(Martin et al., Helv. Chim. Acta, 1995, 78:486 - 504), i.e., it contains an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, as described in the following examples herein, i.e., O(CH 2 ) 2 ON(CH 3 ) 2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH 2 ) 2 . Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers of these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0155] Other modifications are 2'-methoxy (2'-OCH 3 ), 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH2 ) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the dsRNA substance, particularly at the 3'-terminal nucleotide or at the 3'-position of the sugar in a 2'-5' linked dsRNA and at the 5'-position of the 5'-terminal nucleotide. The dsRNA substance may also have a sugar mimic such as a cyclobutyl moiety instead of a pentofuranosyl sugar. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which are owned by the same owner as this application. The content of each of the above patent publications is hereby incorporated by reference herein for the teachings relevant to such methods.

[0156] The dsRNA molecule may also include nucleic acid base (often simply referred to as "base" in the art) modifications or substitutions. As used herein, "unmodified" or "natural" nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include other synthetic and natural nucleic acid bases, such as deoxy-thymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azauracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 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 (daazaadenine), and 3-deazaguanine and 3-deazaadenine.Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in Concise Encyclopedia Of Polymer Science and Engineering, pages 858-859, Kroschwitz, J.L, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for enhancing the binding affinity of the oligomeric compounds claimed in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines as well as N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. The 5-methylcytosine substituent has been shown to increase nucleic acid duplex stability by 0.6-1.2 °C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and is an exemplary base substitution, even more so when combined with a 2'-O-methoxyethyl sugar modification.

[0157] Representative U.S. patents that teach some of the preparation of the above-described modified nucleobases and other modified nucleobases include 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; 5,594,121; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, but are not limited thereto, and each of these is hereby incorporated by reference herein for the teachings related to such methods.

[0158] In some embodiments, the RNA of the dsRNA agent can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a bridge of two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a bridge that links two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge links the 4'-carbon and the 2'-carbon of the sugar ring. Thus, in some embodiments, the agents herein can include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, and the ribose moiety includes an additional bridge that links the 2'-carbon and the 4'-carbon. In other words, an LNA is 4'-CH 2-O-2'-bridged bicyclic sugar moiety-containing nucleotide. This structure effectively "locks" the ribose in the 3'-endo conformational configuration. Addition of a locked nucleic acid to a dsRNA substance has been shown to increase the stability of the dsRNA substance in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O.R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides herein include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, antisense polynucleotide substances herein include one or more bicyclic nucleosides containing a 4'-2' bridge. Examples of such 4'-2' bridged bicyclic nucleosides include 4'-(CH 2 )--O-2'(LNA); 4'-(CH 2 )--S-2'; 4'-(CH 2 ) 2 --O-2'(ENA); 4'-CH(CH 3 )--O-2'(also referred to as "constrained ethyl" or "cEt") and 4’-CH(CH 2 OCH 3 )--O-2'(and its analogs; see, for example, U.S. Patent No. 7,399,845); 4'-C(CH 3 )(CH 3 )--O-2'(and its analogs; see, for example, U.S. Patent No. 8,278,283); 4'-CH 2 --N(OCH 3 )-2'(and its analogs; see, for example, U.S. Patent No. 8,278,425); 4'-CH 2 --O-N(CH 3 )-2'(see, for example, U.S. Patent Publication No. 2004 / 0171570); 4'-CH 2 --N(R)-O-2'(where R is H, C1 ~C 12 is alkyl), or a protecting group (see, e.g., U.S. Patent No. 7,427,672); 4'-CH 2 --C(H)(CH 3 )-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118 - 134); and 4'-CH 2 --C(=CH 2 )-2' (and its analogs; see, e.g., U.S. Patent No. 8,278,426), among others. Each of the above regarding the modified nucleic acids is incorporated herein by reference.

[0159] Further representative U.S. patents and U.S. patent publications that teach the preparation of immobilized nucleic acid nucleotides include U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; U.S. Patent Publication No. 2008 / 0039618; and U.S. Patent Publication No. 2009 / 0012281, among others, and are incorporated herein by reference for the teachings related to such methods.

[0160] For example, any of the above bicyclic nucleosides having one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose can be prepared (see WO99 / 14226).

[0161] The RNA of the dsRNA agent can also be modified to include one or more locked ethyl nucleotides. As used herein, "locked ethyl nucleotide" or "cEt" is 4’-CH(CH 3)-0-2’ bridged bicyclic sugar moiety-containing immobilized nucleic acid. In some embodiments, the constrained ethyl nucleotide is in the S configuration, referred to herein as "S-cEt".

[0162] The dsRNA substances herein may also include one or more "stereochemically restricted nucleotides" ("CRNs"). A CRN is a nucleotide analog having a linker that connects the C2' and C4' carbons of ribose or the C3 and -C5' carbons of ribose. CRNs fix the ribose ring into a stable conformation and enhance the hybridization affinity for mRNA. The linker has a length sufficient to position oxygen in an optimal position for stability and affinity, resulting in less puckering of the ribose ring.

[0163] Representative publications teaching some preparations of the above CRNs include, but are not limited to, US Patent Publication No. 2013 / 0190383; and PCT Publication WO2013 / 036868, which are hereby incorporated by reference herein for the teachings related to such methods.

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

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

[0166] Potentially stable modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3”-phosphate, inverted base dT (idT), and the like. The disclosure of this modification is found in PCT Publication No. WO2011 / 005861.

[0167] In certain embodiments, the dsRNA agent is modified to include one or more adenosine-glycol nucleic acids (“GNA”). The term “GNA” is a polymer similar to DNA or RNA, but in composition, a glycol nucleic acid with a different “backbone” consisting of repeating glycerol units linked by phosphodiester bonds: [Chemical formula] . The description of adenosine-GNA can be found, for example, in Zhang, et al. (JACS 127(12):4174-75 (2005).

[0168] Other modifications of the nucleotides of the dsRNA substances disclosed herein include 5'-phosphate or 5'-phosphate mimetics, such as 5'-terminal phosphate or phosphate mimetics for the antisense strand of the dsRNA substance. Suitable phosphate mimetics are disclosed in US Patent Publication No. 2012 / 0157511, which is hereby incorporated by reference herein for the teachings related to such modifications.

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

[0170] In some embodiments, the dsRNA substance comprises, consists essentially of, or consists of a modified sense strand and a modified antisense strand as shown in Table 2.

[0171] IV. dsRNA substance conjugated to a ligand The dsRNA substances herein may be conjugated with a ligand. The ligand can change the distribution, targeting or lifespan of the dsRNA substance in which it is incorporated. In a preferred embodiment, the ligand improves the affinity for a selected target, such as a molecule, cell or cell type, compartment, such as a compartment of a cell or organ, body tissue, organ or region, compared to a species of substance in which such a ligand is absent. Preferred ligands do not interfere with duplex formation in double-stranded nucleic acids.

[0172] The ligand-conjugated oligonucleotides herein can be synthesized by using oligonucleotides having pendant reactive functional groups such that they are derived by binding a linking molecule to the oligonucleotide (described below). This reactive oligonucleotide can be reacted directly with a commercially available ligand, a synthetic ligand having any of various protecting groups, or a ligand to which a linking moiety is attached.

[0173] In the ligand-conjugated oligonucleotides and ligand molecules having sequence-specific linked nucleosides herein, the oligonucleotides and oligonucleosides can be assembled by a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already having a linking moiety, ligand-nucleotide or nucleoside conjugate precursors already having a ligand molecule, or non-nucleoside ligand-containing building blocks.

[0174] When using nucleotide-conjugate precursors already having a linking moiety, typically a sequence-specific linked nucleoside is synthesized and then the ligand molecule is reacted with the linking moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides herein are synthesized by an automated synthesizer using standard phosphoramidites and non-standard phosphoramidites commonly used in oligonucleotide synthesis, along with phosphoramidites derived from ligand-nucleoside conjugates.

[0175] 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; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, which are hereby incorporated by reference herein for the teachings related to such methods.

[0176] A. Carbohydrate conjugate In preferred embodiments of the compositions and methods of the present specification, the dsRNA substance oligonucleotide further comprises a carbohydrate. The carbohydrate-conjugated dsRNA substance is advantageous for in vivo nucleic acid delivery and for compositions suitable for therapeutic use in vivo, as described herein. As used herein, "carbohydrate" means a compound that is a carbohydrate itself composed of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom, or a compound having as part of it a carbohydrate moiety composed of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom 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), as well as polysaccharides such as starch, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include sugars with C5 and above (e.g., C5, C6, C7 or C8), and disaccharides and trisaccharides include sugars having 2 or 3 monosaccharide units (e.g., C5, C6, C7 or C8).

[0177] In some embodiments, the carbohydrate conjugate for use in the compositions and methods of the present specification is a monosaccharide. In some embodiments, the carbohydrate conjugate for use in the compositions and methods of the present specification is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0178] In some embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). In some embodiments, the carbohydrate comprises a plurality of N-acetylgalactosamine units, such as [Chem.] including.

[0179] Another exemplary carbohydrate conjugate that can be used in the embodiments described herein is [Chem.] [wherein when one of X or Y is an oligonucleotide, the other is hydrogen] including, but not limited to.

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

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

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

[0183] Non-limiting examples of dsRNA substance carbohydrate conjugates having linkers that can be used in the compositions and methods disclosed herein include

Chemical formula

Chemical formula

Chemical formula

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

[0185] In some embodiments, the dsRNA substances disclosed herein have the formulas (XXXI)-(XXXIV):

Chemical formula

Chem.

Chem.

Chem.

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

[0187] B. Linker In some embodiments, the dsRNA substance oligonucleotide can be conjugated with the conjugates or ligands described herein by various linkers that can or cannot be cleaved.

[0188] The term "linker" or "linking group" means an organic moiety that links two parts of a compound, for example, covalently bonds two parts of a compound. A linker typically includes a direct bond or atoms, such as oxygen or sulfur, units, such as NR8, C(O), C(O)NH, SO, SO 2 、SO 2NH, 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, wherein these may be interrupted or terminated by one or more methylenes being O, S, S(O), SO 2 including those interrupted or terminated by N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic (wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic). In some embodiments, the linker is about 1 to 24, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18, 7 to 17, 8 to 17, 6 to 16, 7 to 16 or 8 to 16 atoms.

[0189] The cleavable linking group is sufficiently stable outside the cell but is cleaved upon entering the target cell, releasing the two portions held together by the linker. In some embodiments, the cleavable linking group is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or more, or at least about 100-fold faster, within the target cell or under a first reference condition (e.g., selected to mimic or represent intracellular conditions), in the subject's blood, or under a second reference condition (e.g., selected to mimic or represent conditions found in blood or serum).

[0190] The cleavable linking group is sensitive to a cleaving agent, e.g., the presence of pH, redox potential, or a degradable molecule. Generally, the cleaving agent is more prevalent inside the cell as compared to in serum or blood, or is found at a higher level or activity. Examples of such degrading agents include, for example, redox agents selected for or having substrate specificity for a particular substrate, including oxidizing or reducing enzymes present inside the cell, or reducing agents such as mercaptans that can decompose a redox-cleavable linking group by reduction; esterases; endosomes, or substances that can form an acidic environment, e.g., a substance that brings about a pH of 5 or less; enzymes, peptidases (which may be substrate-specific), and phosphatases that can hydrolyze or decompose an acid-cleavable linking group by acting as a general acid.

[0191] Cleavable linking groups, such as disulfide bonds, can be sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, in the range of about 7.1 - 7.3. Endosomes have a more acidic pH in the range of 5.5 - 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers will release the cationic lipid from the ligand inside the cell, or release it into a desired compartment of the cell, by having a cleavable linking group that is cleaved at a preferred pH.

[0192] The linker may contain a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the target cell. For example, a liver-targeting ligand can be linked to a cationic lipid via a linker containing an ester group. Since hepatocytes are rich in esterases, this linker will be cleaved more efficiently within hepatocytes compared to within cell types that are not rich in esterases. Other cell types rich in esterases include cells of the lung, renal cortex, and testis.

[0193] Linkers containing peptide bonds can be used when targeting peptidase-rich cell types such as hepatocytes and synoviocytes.

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

[0195] i. Redox-cleavable linking group In some embodiments, the cleavable linking group is a redox-cleavable linking group that is cleaved after reduction or oxidation. Examples of reductively cleavable linking groups include disulfide linking groups (-S-S-). Attention may be paid to the methods described herein to determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a particular dsRNA moiety and a particular targeting agent. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art, which mimics the rate of cleavage that can be observed in cells, such as target cells. A candidate can also be evaluated under conditions selected to mimic blood or serum conditions. In some embodiments, the candidate compound is cleaved by up to about 10% in blood. In some embodiments, a useful candidate compound is at least about 2-fold, 4-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or about 100-fold faster degraded intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of a candidate compound is determined using a standard enzyme kinetics assay under conditions selected to mimic the intracellular medium and can be compared to conditions selected to mimic the extracellular medium.

[0196] ii. Phosphate-based cleavable linking group In some embodiments, the cleavable linker comprises a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by an agent that decomposes or hydrolyzes phosphate groups. Examples of agents that cleave phosphate groups intracellularly include enzymes such as intracellular phosphatases. Examples of phosphate-based linking groups include -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)(Rk)-S-. In some embodiments, the phosphate-based linking group is -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S- or -O-P(S)(H)-S-. In some embodiments, the phosphate-based linking group is -O-P(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0197] iii. Acid-cleavable linking group In some embodiments, the cleavable linker is an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In some embodiments, the acid-cleavable linking group is cleaved in an acidic environment at a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or less), or by an agent such as an enzyme that can act as a general acid. Inside cells, certain low-pH organelles such as 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, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). In some embodiments, the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using a method similar to the method described above.

[0198] iv. Ester-based linking group In some embodiments, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved by enzymes such as intracellular esterases and amylases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups are represented by the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using a method similar to the method described above.

[0199] v. Peptide-based cleavage group 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 within 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 contain an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynylene. The peptide bond is a special type of amide bond formed between amino acids to produce peptides and proteins. The peptide-based cleaving group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to produce peptides and proteins and does not include all amide functional groups. The peptide-based cleavable linking group is represented by the general formula -NHCHRAC(O)NHCHRBC(O)-, wherein RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

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

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

[0202] For systemic administration of dsRNA substances for the treatment of diseases, the RNA may be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of the RNA, pharmaceutical carrier or pharmaceutical excipient can enable targeting of the dsRNA substance composition to the target tissue and can also avoid unwanted off-target effects. The dsRNA substance molecule can be modified by chemical conjugation, for example, by the carbohydrate conjugates described above.

[0203] VI. Pharmaceutical composition This specification also includes pharmaceutical compositions and formulations containing the dsRNA substances of this specification. In some embodiments, provided herein are pharmaceutical compositions containing the dsRNA substances described herein and pharmaceutically acceptable carriers. The pharmaceutical composition containing the dsRNA substance is useful for treating diseases or disorders related to the expression or activity of the HBV gene. Such pharmaceutical compositions are formulated based on the delivery mode. One example is a composition formulated for systemic administration via parenteral delivery, such as subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. In certain embodiments, provided herein are compositions formulated for intra-arterial (e.g., liver), intratumoral, intradermal, intravitreal injection, topical ocular, ophthalmic (eye drops), spray, topical ocular or other topical routes, suppositories, or oral administration. In a preferred embodiment, the composition is administered subcutaneously.

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

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

[0206] The repeated dosing regimen may include the periodic administration of a therapeutic amount of the dsRNA substance, such as once a month, once every other month, or once every three months. In a preferred embodiment, the dsRNA substance is administered at a frequency not exceeding once a month. After the initial treatment regimen, the treatment may be administered at a lower frequency.

[0207] The pharmaceutical composition can be administered for an indefinite period to a subject having, for example, HBV infection, such as one or more signs or symptoms of a detectable HBV antigen or HBV DNA including HBV cccDNA. In some embodiments, treatment with the dsRNA substance is performed discretely or for a defined period to provide a functional cure.

[0208] Those skilled in the art will recognize that specific factors, including but not limited to the disease or severity of the disease, previous treatments, the overall health or age of the subject, and other diseases present, can affect the dosage and duration required to effectively treat the subject. Further, treatment of a subject with a therapeutically effective amount of the composition can include a single treatment or a series of treatments. Effective dosages and in vivo half-lives for the individual dsRNA substances encompassed herein can be estimated using conventional methodologies or based on in vivo testing using appropriate animal models described elsewhere herein.

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

[0210] B. Other components The compositions of the present specification may further contain other auxiliary components conventionally found in pharmaceutical compositions, at their established usage levels in the art. Thus, for example, the composition can contain additional compatible pharmaceutically active substances, such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional substances useful for physically formulating the various dosage forms of the compositions of the present specification, such as preservatives, antioxidants and stabilizers. However, such substances, when added, should not unduly interfere with the biological activity of the components of the compositions of the present specification. The formulation may be sterilized and, if necessary, may be mixed with auxiliaries that do not interact detrimentally with the nucleic acids of the formulation, such as preservatives, stabilizers, wetting agents, emulsifying agents, salts that affect osmotic pressure, or buffers, etc.

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

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

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

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

[0215] In addition to those administrations described above, the dsRNA substances discussed herein may be administered in combination with other known agents effective in the treatment of HBV infection. In any case, the administering physician can adjust the dosage and timing of administration of the dsRNA substance based on the observed results using standard measures of effectiveness known in the art or described herein.

[0216] VII. Method This specification also provides a method for inhibiting the expression of HBV in a cell. The method includes contacting the cell with an amount of a dsRNA substance effective to inhibit the expression of HBV in the cell, such as a double-stranded dsRNA substance, to inhibit the expression of HBV in the cell.

[0217] The contacting of the cell with the dsRNA substance, such as a double-stranded dsRNA substance, may be performed in vitro or in vivo. Contacting the cell with the dsRNA substance in vivo includes contacting a cell or cell population within a subject, such as a human subject, with the dsRNA substance. A combination of in vitro and in vivo methods of contacting the cell is also possible. As discussed above, the contacting of the cell can be direct or indirect. Further, the contacting of the cell may be through a target ligand that includes any ligand described herein or known in the art. In a preferred embodiment, the target ligand is either a carbohydrate moiety, such as a GalNAc3 ligand, or another ligand that directs the dsRNA substance to a meaningful site.

[0218] In some embodiments of the methods herein, the dsRNA substance is administered to a subject and delivered to a specific site within the subject. Inhibition of the expression of the HBV gene is evaluated using measurement, or variation, of the level of HBV mRNA or HBV protein in a sample obtained from a body fluid or tissue from a specific site within the subject. In a preferred embodiment, the site is selected from the liver and blood. The site may also be a sub-section, or a sub-group, of a cell or body fluid prepared from any one of the above sites.

[0219] In some embodiments, the methods disclosed herein are useful for treating subjects having HBV infection, e.g., subjects who would benefit from a reduction in 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 a subject infected with HBV. In another aspect, the present disclosure provides a method for reducing the level of an HBV antigen, e.g., HBsAg or HBeAg, in a subject infected with HBV. In another aspect, the present disclosure provides a method for reducing the viral load of HBV in a subject infected with HBV. The present disclosure also provides a method for reducing the level of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in a subject infected with HBV (although a transient increase in ALT or AST may be associated with viral clearance). In one aspect, the present disclosure provides a method for increasing the level of anti-HBV antibodies in a subject infected with HBV. In another aspect, the present disclosure provides a method for treating a subject having HBV infection. In one aspect, the present disclosure provides a method for treating a subject having an HBV-related disease, e.g., hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; or hepatocellular carcinoma. Further, since hepatitis D virus infection depends on an essential helper function provided by HBV with respect to transmission, and a subject having HBV infection may also have hepatitis D virus infection, in some embodiments, the treatment methods described herein are also useful for treating subjects having hepatitis D virus infection or an HDV-related disorder, e.g., hepatitis B virus infection, chronic hepatitis B virus infection (CHB), chronic hepatitis B infection (CHB), cirrhosis, liver failure, and hepatocellular carcinoma (HCC). In some embodiments, the treatment methods (and uses) herein involve administering to a subject, e.g., a human, a pharmaceutical composition comprising a dsRNA substance herein that targets an HBV gene, or a therapeutically effective amount of a dsRNA substance herein that targets an HBV gene.

[0220] In one aspect, the present specification provides a method for preventing at least one symptom in a subject having HBV infection, such as 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; an increase in ALT; an increase in AST; the absence or low levels of anti-HBV antibodies; liver injury; cirrhosis; delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; hepatic fibrosis; end-stage liver disease; hepatocellular carcinoma; serum sickness-like syndrome; eating disorder; nausea; vomiting, mild fever; myalgia; easy fatigability; taste and olfactory disorders (aversion to food and tobacco); right upper abdominal pain and epigastric pain (intermittent, mild to moderate); hepatic encephalopathy; drowsiness; disrupted sleep pattern; confusion; coma; ascites; gastrointestinal bleeding; coagulation abnormalities; jaundice; hepatomegaly (a slightly enlarged and soft liver); splenomegaly; palmar erythema; spider nevus; muscle wasting; spider angioma; vasculitis; aneurysm bleeding; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (caput medusa); an ALT level higher than the AST level; an increase not exceeding three times the ULN of gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) levels; a slightly low albumin level; an increase in serum iron level; leukopenia (i.e., granulocytopenia); lymphocytosis; an increase in erythrocyte sedimentation rate (ESR); shortened erythrocyte lifespan; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); the presence of serum or liver HBsAg, HBeAg, hepatitis B core antibody (anti-HBc) immunoglobulin M (IgM); hepatitis B surface antibody (anti-HBs), hepatitis B e antibody (anti-HBe) or HBV DNA; an increase in bilirubin level; hyperglobulinemia; the presence of tissue-nonspecific antibodies, such as anti-smooth muscle antibody (ASMA) or antinuclear antibody (ANA) (10 - 20%); the presence of tissue-specific antibodies, such as antibodies against the thyroid (10 - 20%); an increase in the level of rheumatoid factor (RF); a reduction in the number of platelets and white blood cells; lobular, degenerative and reparative hepatocyte changes, and accompanying inflammation; or mainly centrilobular necrosis, whether detectable or undetectable.This method involves administering to a subject the dsRNA substance of the present invention, such as dsRNA, or a pharmaceutical composition comprising a therapeutically effective amount of the dsRNA substance, thereby preventing at least one symptom in a subject having a disorder that would benefit from a reduction in HBV gene expression, such as a subject having HBV infection or a subject having both HBV and HDV infections.

[0221] In another aspect, the present disclosure provides for the use of a therapeutically effective amount of a dsRNA substance of the present disclosure for treating a subject who can benefit from a reduction or inhibition of HBV gene expression, such as a subject having HBV infection or a subject having both HBV and HDV infections.

[0222] In a further aspect, the present disclosure provides for the use of a dsRNA substance of the present disclosure that targets the HBV gene, such as dsRNA, or a pharmaceutical composition comprising a dsRNA substance that targets the HBV gene, in the manufacture of a therapeutic medicament for treating a subject who can benefit from a reduction in HBV gene expression or HBV replication, such as a subject having HBV infection or a subject having both HBV and HDV infections, and a subject having a disorder that would benefit from a reduction in HBV gene expression, such as an HBV-related disease.

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

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

[0225] In some embodiments, when a dsRNA substance is administered to a subject, the dsRNA substance-targeted HBV is administered to a subject having HBV infection, having both HBV and HDV infections, or having an HBV-related disease, for example, to reduce or normalize the expression of one or more HBV genes, the HBV cccDNA level, the HBV antigen level, the HBV viral load level, ALT or AST by at least 80%, 85%, 90%, 95%, 98% or towards the standard in the cells, tissues, blood, other tissues or body fluids of the subject.

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

[0227] Administration of dsRNA by the methods and uses described herein results in a reduction in the severity, signs, symptoms or markers of such diseases or disorders in patients having HBV infection or both HBV and HDV infections, or HBV-related diseases. "Reduction" in this context means a clinically significant reduction in such levels. The reduction can be, for example, at least 80%, 85%, 90%, 95% or 98%, or below the detection level.

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

[0229] The effectiveness of a treatment can be evaluated, for example, by measuring disease progression, disease remission, symptom severity, pain reduction, quality of life, the dosage of a drug required to sustain the treatment effect, the level of a disease marker, or any other measurable parameter appropriate for a given disease being treated. Monitoring the effectiveness of a treatment by measuring any one of such parameters, or any combination of parameters, is within the ability of a person of ordinary skill in the art. For example, the effectiveness of the treatment of CHB can be evaluated, for example, by regular monitoring of viral load and transaminase levels. Comparison of an initial reading with a later reading provides an indication to the physician as to whether the treatment is effective. Monitoring the effectiveness of a treatment by measuring any one of such parameters, or any combination of parameters, is within the ability of a person of ordinary skill in the art. In connection with the administration of a dsRNA agent or its pharmaceutical composition that targets HBV, "effective against" an HBV-related disease means that, by administration in a clinically appropriate manner, there is an improvement in symptoms, a cure, a reduction in the disease, an extension of lifespan, an improvement in quality of life, or other effects generally recognized as favorable by physicians knowledgeable in the treatment of HBV infection or HBV-related diseases and related causative agents, resulting in a beneficial effect for at least a statistically significant proportion of patients.

[0230] The treatment effect is evident when there is a clinically significant improvement in one or more parameters of the disease state, or when there is no development of a worsening or a symptom that was originally expected. As an example, a favorable change in at least 50%, preferably at least 70% or more, of the measurable parameters of a disease can indicate an effective treatment. The effectiveness of a given dsRNA agent drug or its pharmaceutical formulation can also be determined, as is known in the art, using an experimental animal model for a given disease. When using an experimental animal model, the effectiveness of a treatment is demonstrated when a statistically significant reduction in signs or symptoms is observed.

[0231] Administration of the dsRNA substance can, for example, reduce the presence of serum or liver HBV cccDNA, serum or liver HBV antigens, such as HBsAg or HBeAg; or the normalized ALT level or AST level, to at least 70%, 75%, 80%, 85%, 90%, 95%, or to less than the level of detection of the assay, towards or up to the upper limit of the normal value of the clinical test value.

[0232] Administration of the dsRNA substance can, for example, make detectable the presence of serum or liver anti-HBV antibodies, such as anti-HBsAg antibodies, in the cells, tissues, blood or other compartments of a patient, or increase it by at least 80%, 85%, 90%, 95% or more, or make the antibody detectable if none was detected before treatment.

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

[0234] Subjects who benefit from the reduction or inhibition of HBV gene expression are those having an HBV infection or an HBV-related disease or disorder as described herein.

[0235] Treatment of subjects who benefit from the reduction or inhibition of HBV gene expression includes therapeutic and prophylactic treatments

[0236] The present specification further provides methods and uses of a dsRNA substance or a pharmaceutical composition thereof for treating subjects who benefit from the reduction or inhibition of HBV gene expression, such as subjects having an HBV-related disease, in combination with other pharmaceuticals or other therapies, such as known pharmaceuticals or known therapies, for example those currently used for the treatment of these disorders.

[0237] For example, in some embodiments, a dsRNA substance that targets one or more HBV genes is administered, for example, in combination with a substance useful for the treatment of HBV-related diseases described herein. For example, additional therapeutic agents and therapies suitable for treating a subject who may benefit from a reduction in HBV expression, such as a subject having an HBV-related disease, include dsRNA substances that target different portions of the HBV genome, antiviral agents, a nucleotide analog, a nucleoside analog, a reverse transcriptase inhibitor (e.g., tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine, clevudine, ritonavir, dipivoxil, lobucavir, famciclovir, FTC, N-acetyl-cysteine (NAC), PC1323, theradigm-HBV, thymosin-alpha, and ganciclovir), an immunostimulant (e.g., pegylated interferon alpha2a (PEG-IFN-α2a), interferon alpha-2b, recombinant human interleukin-7, and toll-like receptor 7 (TLR7) agonist), a therapeutic vaccine (e.g., GS-4774, DV-601, and TG1050), a virus entry inhibitor (e.g., Myrcludex), an oligonucleotide that inhibits the secretion or release of HbsAg (e.g., REP9AC), a capsid inhibitor (e.g., Bay41-4109 and NVR-1221), a cccDNA inhibitor (e.g., IHVR-25), or other therapeutic substances or procedures for treating HBV-related diseases, such as liver transplantation or chemotherapy, or any combination of the foregoing.

[0238] The subject to which the dsRNA substance of the present specification is administered is further administered with one or more other therapeutic agents that function by a non-RNAi mechanism and are useful for the treatment of HBV infection. Exemplary therapies that can be used in combination with the therapies of the present specification include, for example, immune modulators that stimulate the immune system by enhancing T cell helper activity, maturation of B lymphocytes, inhibition of T-cell suppressors, and HLA class I expression. Suitable immune modulators include interferons having various properties including antiviral effects, immunomodulatory effects, and antiproliferative effects.

[0239] For example, current treatment for chronic hepatitis B is interferon therapy, which is administered to subjects having confirmed HBV infection for at least 6 months, elevated liver enzymes (AST and ALT), and a virus that is actively replicating in their blood (HBeAg or HBV DNA positive test). Interferon-α therapy produces a long-term sustained remission of the disease, with normalization of liver enzymes and disappearance of three markers of active infection (HBeAg, HBV DNA, and HBsAg), in approximately 35% of patients with chronic hepatitis B. Subjects with acute HBV infection, end-stage cirrhosis, or other serious medical problems are generally not treated with interferon.

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

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

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

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

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

[0245] Cells suitable for treatment using the methods disclosed herein can be any cells that express the HBV gene, such as cells infected with HBV, cells containing an expression vector comprising the HBV genome or a portion of the HBV gene, or transgenic mice that express the HBV gene. Cells suitable for use in the methods and uses disclosed herein can be mammalian cells, such as primate cells (human cells or non-human primate cells, such as monkey cells or chimpanzee cells, etc.) or non-primate cells (mouse cells, rat cells, or other mammalian cells). In certain embodiments, the cells are cells that can be infected with HBV. In certain embodiments, the cells are human cells, such as human hepatocytes.

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

[0247] HBV replication can be inhibited in cells to at least 80%, 85%, 90%, 95% or more, for example, to levels below the detection level of the assay.

[0248] The in vivo methods and uses disclosed herein can include administering to a subject a composition containing a dsRNA substance comprising a nucleotide sequence complementary to at least a portion of the RNA transcript of the HBV gene of the mammal being treated. When the subject being treated is a human, the composition can be administered by any means known in the art, including but not limited to subcutaneous administration, intravenous administration or intramuscular administration. In some embodiments, the composition is administered by subcutaneous injection. In some embodiments, the dsRNA substance is formulated to be administered as a single injection of the total dose. In some embodiments, the present disclosure provides compositions formulated for intra-arterial (e.g., liver), intratumoral, intradermal, intravitreal injection, topical ocular, eye (eye drops), spray, topical ocular or other topical routes, suppository, or oral administration.

[0249] In one aspect, the present specification also provides a method for inhibiting the expression of the HBV gene in a mammal, such as a human. The present specification also provides a composition comprising a dsRNA substance targeting the HBV gene in mammalian cells for use in inhibiting the expression of the HBV gene in a mammal. In another embodiment, the present specification provides the use of a dsRNA substance targeting the HBV gene in mammalian cells in the manufacture of a therapeutic medicament for inhibiting the expression of the HBV gene in a mammal.

[0250] The method and use comprise administering to a mammal, such as a human, a composition comprising a dsRNA substance targeting the HBV gene in mammalian cells and maintaining the mammal for a time sufficient to obtain degradation of the HBV gene mRNA transcript, thereby inhibiting the expression of the HBV gene in the mammal.

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

[0252] In some embodiments, in vivo verification of RISC-mediated cleavage of the target after administration of the dsRNA substance is performed by 5'-RACE or a modification of a protocol known in the art (Lasham A et al., (2010) Nucleic Acid Res., 38 (3) p-e19)(Zimmermann et al. (2006) Nature 441: 111-4).

[0253] The present invention is further illustrated by the following examples, which should not be construed as limiting.

Example

[0254] Example 1 Synthesis of the DSRNA Substance Supplier of reagents When the supplier of a reagent is not specifically indicated herein, such a reagent can be obtained from any molecular biology reagent supplier to meet the quality / purity standards for molecular biology applications.

[0255] Design of dsRNA substance As described in WO / 2016 / 077321, the selection of dsRNA designs targeting HBV was based on two fundamental factors: a) efficacy, and b) the desire to use it, for substances that almost completely match a large number of published HBV sequences of all known genotypes (A - H), and substances that cover more than 90% of their fragments. The coordinates for selecting the RNA substances were determined relative to the NCBI HBV reference genome sequence NC_003977.1 (GenBank accession number GI:21326584 (SEQ ID NO:1)). A first set of RNA substances containing structure - activity modifications, including various 2'-O - methyl and 2'-fluoro substitution patterns, centered around two adjacent regions of the HBV genome encoding the surface antigen (HbSAg) and the HBV polymerase, were designed, synthesized, and screened in vitro. Also, a second set of substances targeting positions 1581 - 1599 of SEQ ID NO:1, which are further regions in the HBV genome, specifically the regions encoding HbSAg, polymerase, and the X gene, were designed, synthesized, and screened in vitro. The selected sequences were further chemically modified and tested. These double - strand designs are described in WO2016 / 077321 (the entire content of which is incorporated herein by reference), and detailed lists of the unmodified HBV sense - strand and antisense - strand nucleotide sequences are in Tables 3, 6, 12, 22, and 25 of WO2016 / 077321, and detailed lists of the modified HBV sense - strand and antisense - strand nucleotide sequences are described in Tables 4, 7, 13, 23, and 26 of WO2016 / 077321. The results from the screening assays performed with those substances are also described there.

[0256] These studies have identified that double-stranded AD-66810 has an antisense strand with a modified nucleotide sequence: 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 13), and a sense strand with a modified nucleotide sequence: 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29) [in the sequence, 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 here, the N-acetylgalactosamine moiety N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyproline (also known as Hyp-(GalNAc-alkyl)3 or referred to herein as L96) is covalently attached to the 3' end of the sense strand].

[0257] By conducting further studies described herein, a potent and specific dsRNA agent molecule was identified based on the previously identified sequence AD-66810 that targets the X transcript of hepatitis B virus (HBV; U95551). To achieve this goal, a series of chemically modified dsRNA agents were designed, synthesized, and tested for their in vitro activity using the Dual-Luc reporter basic assay and the HepG2.2.15 cell line. All compounds were conjugated to a ternary N-acetylgalactosamine (GalNac) ligand (L96) covalently attached to the 3' end of their sense strand.

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

[0259] After completion of solid-phase synthesis, the oligoribonucleotides were cleaved from the solid support and deprotected at 60 °C for 20 min using 200 μL of methylamine water-soluble reagent in a sealed 96-deep well plate. At the end of the cleavage and deprotection steps, the synthesis plate was allowed to reach room temperature and precipitated by adding 1 mL of an acetonitrile:ethanol mixture (9:1). The plate was cooled at -80 °C for 2 h and the supernatant was carefully decanted using a multi-channel pipette. The oligonucleotide pellet was resuspended in 20 mM NaOAc buffer and desalted using a 5 mL HiTrapTM size-exclusion column (GE HealthcareTM) on an AKTA purification system equipped with an A905 autosampler and a Frac 950 fraction collector. The desalted samples were collected into 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 measured by IEX chromatography.

[0260] Single-stranded annealing was performed with a Tecan liquid handling robot. Equimolar mixtures of sense and antisense single strands were combined and annealed in a 96-well plate. After combining the complementary single strands, the 96-well plate was tightly sealed, heated in an oven at 100 °C for 10 min, and allowed to slowly reach room temperature over 2 - 3 h. The concentration of each double strand was normalized to 1 μM with 1X PBS.

[0261] Abbreviations for the modified nucleotide monomers disclosed herein are shown in Table 1. Table 2 shows the AD-66810 and HBV dsRNA substances synthesized using the above method.

[0262] Table 1. Abbreviations of nucleotide monomers used for modified nucleic acid sequence representation Unless otherwise specified, it will be understood that these monomers are linked to each other by 5'-3'-phosphodiester bonds when present in an oligonucleotide.

Table 1-1

Table 1-2

[0263]

Table 2

[0264] Example 2 In Vitro Screening of DSRNA Substance Double Strands Dual-Glo® Luciferase Assay: Cos7 cells (ATCC®, Manassas, VA) were grown in DMEM (ATCC) supplemented with 10% FBS at 37 °C in an atmosphere of 5% CO 2 until almost confluent and then detached from the plate by trypsin treatment. 5 μl of RNA double strand per well and 5 μl of psiCHECK2-HBV (or psiCHECK2-HBV genotype A, C, E or F) plasmid were added together with 5 μl of Opti-MEM® plus 0.1 μl of Lipofectime TM RNAiMax (Invitrogen TM , Carlsbad CA. cat# 13778-150) to perform transfection of Cos7 cells with double-stranded substances and psiCHECK2-HBV (an expression vector containing a nucleotide sequence encoding a portion of the matched wild-type HBV sequence; SEQ ID NO: 5), or double-stranded substances expressing the nucleotide sequences of HBV genotypes A, C, E, or F (SEQ ID NO: 6, 7, 8 or 9 respectively) and psiCHECK2 vector, and incubated at room temperature for 15 minutes. Next, this mixture was added to the cells resuspended in 35 μl of fresh complete medium. The transfected cells were incubated in an atmosphere of 50% CO 2 .

[0265] After transfection with the dsRNA substance and the psiCHECK2 plasmid, Firefly (transfection control) and Renilla (fused to the HBV target sequence) luciferase were measured 28 hours later. First, the medium was removed from the cells. Next, Firefly luciferase activity was measured by adding 20 μl of Dual-Glo® luciferase reagent equal to the medium volume to each well and mixing. After incubating this mixture at room temperature for 30 minutes, luminescence (500 nm) was measured with a Spectramax® (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 20 μl of room temperature Dual-Glo® Stop & Glo® reagent to each well, incubating the plate for 10 - 15 minutes, and then measuring luminescence again to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® reagent stops the Firefly luciferase signal and maintains the luminescence of the Renilla luciferase reaction. The dsRNA substance activity was determined by normalizing the Renilla (HBV) signal to the Firefly (control) signal within each well. Next, the magnitude of the dsRNA substance activity was evaluated compared to cells transfected with the same vector but not treated with the dsRNA substance or treated with a non-targeting dsRNA substance. All transfections were performed with n = 2 or more.

[0266] The results of these assays were provided in Table 3 using the agents described in Table 2.

[0267] HepG2.2.15 and PLC in vitro screening: HepG2.2.15 and PLC (human hepatoma cells; ATCC® CRL-8024) cells were cultured in DMEM or EMEM medium (ATCC) supplemented with 10% FBS (ATCC) in 5% CO 2After growing at 37°C in an atmosphere until almost confluent, it was detached from the plate by trypsin treatment. 5 μl of double-stranded dsRNA substance per well, 14.8 μl of Opti-MEM® plus 0.2 μl of Lipofectamine TM RNAiMax (Invitrogen TM , Carlsbad CA. cat # 13778-150) were added together to perform reverse transfection and incubated at room temperature for 15 minutes. Next, 80 μl of antibiotic-free complete growth medium containing 2x104 HepG2.2.15 or PLC cells was added. After incubating the cells for 24, 48, and 72 hours, RNA purification was performed.

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

[0269] Total RNA isolation using MagMAX-96 Total RNA Isolation Kit (Applied Biosystem, Forer City CA, part # AM1830): After collecting the cells and dissolving them in 140 μl of lysis / binding solution, they were mixed at 850 rpm for 1 minute using an Eppendorf TM Thermomixer (the mixing speed was the same throughout the process). 20 μl of the electromagnetic bead and lysis / binding enhancer mixture was added to the cell lysate and mixed for 5 minutes. The electromagnetic beads were captured using a magnetic stand and the supernatant was removed without disturbing the beads. After removing the supernatant, the electromagnetic beads were washed with washing solution 1 (to which isopropanol was added) and mixed for 1 minute. The beads were captured again and the supernatant was removed. Next, the beads were washed with 150 μl of washing solution 2 (to which ethanol was added), captured, and the supernatant was removed. Next, 50 μl of DNase mixture (MagMax turbo DNase buffer and Turbo DNase) was added to the beads and they were mixed for 10 - 15 minutes. After mixing, 100 μl of RNA rebinding solution was added and mixed for 3 minutes. The supernatant was removed, the electromagnetic beads were washed again with 150 μl of washing solution 2, mixed for 1 minute, and the supernatant was completely removed. After mixing the electromagnetic beads for 2 minutes to dry them, the RNA was eluted with 50 μl of water.

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

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

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

[0273] Example 3 Transfection of GalNAc-conjugated HBV-targeting DSRNA Substances in the DUAL-LUC System Table 3 shows the silencing of HBV mRNA after transfection with each conjugate dsRNA substance. Each dsRNA substance was tested by performing transfection at dsRNA substance concentrations of 50 nM, 10 nM, and 0.1 nM in Cos7 cells. Based on the screening results, 7 out of 20 dsRNA substances showed efficacy equivalent to that of the AD-66810 parent.

Table 3

[0274] Example 4 Transfection of GALNAC-conjugated HBV-targeted DSRNA Substances in HepG2.2.15 Cells Table 4 shows the silencing of HBV mRNA (PORF1 and SORF2 mRNA determined using forward and reverse primers and TaqMan probe) in HepG2.2.15 cells after transfection with each conjugate dsRNA substance. Overall, stronger silencing of the PORF1 and SORF2 viral transcripts was observed in HepG2.2.15 cells compared to the silencing observed in the Dual-Luc overexpression system, and 10 dsRNA substances had efficacy equivalent to that of the parent molecule AD-66810. Similar dsRNA substance activity was observed against PORF1 and SORF2 mRNA, which was expected since the target sites of the dsRNA substances are present in both transcripts.

Table 4

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

[0276] Example 5 Pharmacological Evaluation of AD-81890 after Single Subcutaneous Injection in an Adeno-Associated HBV Mouse Model The pharmacology of AD-81890 was assayed in an adeno-associated HBV (HBV-AAV) mouse model. AAV8-HBV (SignaGen Laboratories) was diluted with 1×PBS to a final concentration of 2×10¹² GC / mL. Male C57BL / 6 mice, 6 - 8 weeks old, were intravenously injected with 2×10¹¹ GC / mouse in a fixed volume of 100 μL via the lateral tail vein.

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

Table 5

[0278] After blood collection, the blood was allowed to clot for 30 minutes and then centrifuged at 13,000 rpm for 10 minutes at 4°C in a microcentrifuge. The serum was aspirated and stored at -20°C.

[0279] The hepatitis B virus surface antigen protein level was evaluated via ELISA (BioTang, Waltham, MA). A standard curve was generated using the HBsAg protein from US Biologics (Memphis, TN). Serum samples were diluted 1:2000 or 1:500 with 1×PBS (Gibco, Gaithersburg, MD) and evaluated using a slightly modified ELISA protocol. Briefly, 50 μL / well of the diluted serum or standard was loaded onto the plate and incubated at 37 °C for 1 hour. After this incubation, 50 μL / well of the enzyme conjugate was added to each well and the plate was incubated at 37 °C for 30 minutes. The plate was washed three 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, an additional 100 μL / well of stop solution was added and the absorbance was measured at a wavelength of 450 nm. If the calculated HBsAg level was below the lower limit of quantification (LLOQ) of the assay, the value was recorded as the LLOQ (i.e., 313 ng / mL).

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

[0281] Example 6 Pharmacological Evaluation of AD-81890 in an Adeno-Associated HBV Mouse Model after Multiple Subcutaneous Injections The pharmacology of AD-81890 was assayed in an adeno-associated HBV (HBV-AAV) mouse model. AAV8-HBV (SignaGen Laboratories) was diluted with 1×PBS to a final concentration of 2×1012 GC / mL. Male C57BL / 6 mice, 6 - 8 weeks old, were intravenously injected with 2×1011 GC / mouse via the lateral tail vein in a fixed volume of 100 μL.

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

[0283] As described in the previous example, blood samples were processed and ELISA assays were performed. The results are shown in Table 7 below. The results including standard deviation are shown in Figure 3. [Table 7]

[0284] Dose-responsive serum HBsAg levels were observed in the AAV8-HBV mouse model after administration of AD-81890. After a single administration of AD-81890 at 9 mg / kg or q2w×6 administrations at 3 mg / kg, a maximum HBsAg reduction of approximately 2.7 log10 was observed. Animals administered q2w×6 at 3 mg / kg had a total HBsAg reduction of more than 2 log10 that persisted for approximately 8 weeks.

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

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

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

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

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

Table 8

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

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

[0292] Example 8 In Vitro Analysis of AD-81890 Specificity Using RNA-SEQ in HEPG2.2.15 Cells The effects of chemical modification of dsRNA species were measured by comparing AD-66810 and AD-81890 in HBV-expressing HepG2.2.15 cell lines using transcriptome-wide changes in expression levels using RNA-Seq. The AD-66810 and AD-81890 molecules have the same nucleotide sequence but differ by the substitution of a single glycol nucleic acid (GNA) at position 6 from the 5'-end of the molecule on the antisense strand (see Table 2).

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

[0294] The stocks of dsRNA substances were diluted to a concentration of 1,000 nM or 100 nM with 1X DPBS. RNAiMAX (ThermoFisher, Cat#13778150) was diluted with Opti-MEM (ThermoFisher Cat#31985062) to a concentration of 0.3 μL RNAiMAX / 10 μL Opti-MEM and incubated at room temperature for 5 minutes. After incubation, 10 μL / well was added to each well of a 96-well collagen-coated plate (BD Biocoat, Cat#356407) together with 10 μL / well of the appropriate dsRNA substance dilution, gently mixed, and incubated at room temperature for 20 minutes. 80 μL of the prepared cell suspension was added to each well so that the final cell density was 15,000 cells / well and the final dsRNA substance concentrations were 100 nM and 10 nM. The cells were incubated in an incubator at 37 °C containing 5% CO 2 for 16 - 22 hours. The cells were plated such that each experimental condition had 16 wells, and the experiment was performed twice.

[0295] RNA was isolated using the ThermoFisher RNAqueous-96 Total RNA Isolation Kit according to the protocol. Briefly, after 16 - 22 hours, the supernatant was aspirated from each well, 100 μL / well of 1× DPBS was added to wash away the remaining medium, and then aspirated. 200 μL of lysis / binding solution was added to each well in the first and fifth columns of each plate, pipetted up and down several times, and transferred to subsequent columns such that 4 wells / condition were pooled to ensure sufficient RNA recovery. This resulted in 4 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. A series of centrifugation steps (1,900 × g, 1 minute) were used to wash the samples with the provided wash buffer, treat with DNase reagent, and elute in 100 μL of nuclease-free water. The RNA concentration was measured using a NanoDrop 8000 spectrophotometer (ThermoFisher).

[0296] The RNA was further treated with TURBO DNase (Ambion). Each RNA sample (≤10 μg of RNA / sample) was mixed with 2 μL of DNase, 10 μL of 10X buffer, and nuclease-free water to a total volume of 100 μL and incubated at 37 °C for 30 minutes. As per the protocol, after DNase treatment, the RNA was further purified using the RNeasy MinElute Cleanup Kit (Qiagen). The RNA was eluted in 30 μL of nuclease-free water and the RNA concentration was measured using a NanoDrop 8000 spectrophotometer. The RNA was stored at -80 °C. Subsequently, this RNA was used for cDNA library preparation using the TruSeq Stranded Total RNA Library Preparation Kit (Illumina), and sequencing was performed on a NextSeq500 desktop sequencer (Illumina), all according to the manufacturer's instructions. The experimental repeats were performed twice.

[0297] HepG2.2.15 cells were transfected four-fold with 10 nM or 100 nM of AD-66810 or AD-81890 and cultured for 24 hours together with an untreated control group. The RNA extracted using the Purelink RNA kit (ThermoFisher) was used for cDNA library preparation using the TruSeq Stranded Total RNA Library Preparation Kit (Illumina) with Ribo-Zero Human / Mouse / Rat for rRNA removal, and sequencing was performed on a NextSeq500 desktop sequencer (Illumina), all according to the manufacturer's instructions. A total of 40 samples were pooled per NextSeq 500 / 550 High Output v2 (75 cycle) flow cell (Illumina). The experimental repeats were performed twice.

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

[0299] MA plots were used for visualization of both on-target HBV knockdown and off-target effects. Analysis of GNA chemistry to mitigate global off-target effects was limited to downregulated genes (log2 fold change <0). Upregulated genes (log2 fold change >0) were considered secondary effects. To achieve near-maximal HBV knockdown, evaluation of off-target effects was restricted to the lowest (10 nM) dose. To compare transcriptome noise, significantly downregulated (adjusted p-value threshold <0.05) genes were identified with AD-66810 and / or AD-81890. The degree of downregulation was visualized by boxplots of log2 fold change (Figure 4) as the statistical difference between AD-66810 and AD-81890 evaluated using Welch's two-sample t-test (Table 9).

Table 9

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

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

[0302] Research was conducted in PBX - mice to compare the hepatotoxicity of AD - 66810 and AD - 81890. On days 0, 21, 28, 35, and 42, mice were administered 12 mg / kg, 36 mg / kg, or 100 mg / kg of AD - 66810, AD - 81890, or PBS (control) by subcutaneous injection (n = 4 per group). General condition observation and body weight measurement were performed twice a week until day 49. Blood was collected twice a week by retro - orbital bleeding, and serum was prepared using the usual method. Terminal bleed was performed, and the animals were sacrificed by cardiac puncture and exsanguination. Post - exsanguination autopsy was performed. The liver was collected and weighed. The liver was separated and stored in RNAlater solution (Ambion), stored in formalin, and snap - frozen before paraffin embedding.

[0303] All animals maintained a body weight above 80% of the initial level throughout the study period. Furthermore, the lowest arithmetic mean value in the compound - treated groups was higher than that in the control PBS - treated group.

[0304] Plasma human serum albumin levels were monitored throughout the study period. All surviving animals maintained a plasma h - Alb concentration above 7.0 mg / ml during the survival period of the study.

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

Table 10

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

[0307] At necropsy, there were no findings specific to the test compound in any of the groups. There were no obvious changes in the relative (liver / body weight) liver weights of the animals in the compound treatment groups compared to the control group.

[0308] equivalent One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, that numerous equivalents to the specific embodiments and methods described herein exist. Such equivalents are intended to be encompassed by the following claims.

[0309] Although specific embodiments have been illustrated and described, it will be readily appreciated that various modifications can be made thereto without departing from the spirit and scope of the invention, and further embodiments can be provided by combining the various embodiments described above.

[0310] All figures, U.S. patents, U.S. patent application publication, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein or listed in the filing data sheet, including U.S. Provisional Patent Application No. 62 / 718,314, filed on August 13, 2018, are hereby incorporated by reference in their entirety herein, unless otherwise indicated. Where it is necessary to employ concepts from various patents, applications, and publications to provide further embodiments, aspects of the embodiments can be modified.

[0311] In light of the foregoing detailed description, these and other modifications can be made to the embodiments. In general, in the following claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed in the specification and the claims, but rather the claims should be construed to include all possible embodiments together with all equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present specification.

Claims

1. A double-stranded ribonucleic acid (dsRNA) material comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand being 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16), 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18), 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20), 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23), 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24), 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25), or 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28): [wherein a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; dA, dC, dG and dT are 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxythymidine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); and s is a phosphorothioate linkage. A dsRNA substance comprising a modified nucleotide sequence represented by:

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

2. The dsRNA agent of claim 1, comprising:

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

2. The dsRNA agent of claim 1, comprising a modified nucleotide sequence as set forth below:

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

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

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

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

8. 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 to 8, further comprising a ligand.

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

12. The ligand has the formula: 【Chemistry 1】 12. The dsRNA agent of claim 11, wherein

13. formula: 【Chemistry 2】 "Wherein, X is O or S" 13. The dsRNA agent of claim 12, wherein the dsRNA agent is conjugated to a ligand,

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

15. The antisense strand is 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16), 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18), 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20), 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23), 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24), 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25), or 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28): "In the sequence, a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; dA, dC, dG and dT are 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxythymidine-3'-phosphate, respectively; (Agn) is adenosine glycol nucleic acid (GNA); and s is a phosphorothioate linkage." 2. The dsRNA agent of claim 1, comprising a modified nucleotide sequence as set forth below:

16. The antisense strand and the sense strand are (a) 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); (b) 5'-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3' (SEQ ID NO: 18) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); (c) 5'-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 20) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); (d) 5'-usGfsudGadAgdCGfaaguGfcAfcacsusu-3' (SEQ ID NO: 23) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); (e) 5'-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 24) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); (f) 5'-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3' (SEQ ID NO: 25) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); or (g) 5'-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3' (SEQ ID NO: 28) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10): "In the sequence, a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; dA, dC, dG and dT are 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxythymidine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.” 2. The dsRNA agent of claim 1, comprising a modified nucleotide sequence as set forth below:

17. The antisense and sense strands were 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 16) and 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 10); "In the sequence, a, c, g and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; dA, dC, dG and dT are 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxythymidine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.” 2. The dsRNA agent of claim 1, comprising a modified nucleotide sequence as set forth below:

18. 18. The dsRNA agent of any one of claims 1 to 17, wherein the dsRNA agent inhibits expression of Hepatitis B Virus (HBV) in a cell.

19. A cell comprising a dsRNA agent according to any one of claims 1 to 18.

20. A pharmaceutical composition comprising a dsRNA agent according to any one of claims 1 to 19, and a pharmaceutical excipient.

21. 21. A method for inhibiting Hepatitis B Virus (HBV) gene expression in a cell by contacting said cell with a dsRNA agent according to any one of claims 1 to 18, or with a pharmaceutical composition according to claim 20.

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

23. 21. A method for inhibiting the replication of Hepatitis B Virus (HBV) in a cell by contacting said cell with a dsRNA agent according to any one of claims 1 to 18, or with a pharmaceutical composition according to claim 20.

24. 24. The method of claim 23, which inhibits intracellular HBV replication by at least 80%, 90%, 95% or 98%, or to below the detection level of the assay method.

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

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

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

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

29. 21. A method for reducing the level of Hepatitis B Virus (HBV) antigen in a subject infected with HBV by administering to said subject a therapeutically effective amount of a dsRNA agent according to any one of claims 1 to 18, or a pharmaceutical composition according to claim 20.

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

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

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

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

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

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

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

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

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

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

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

41. 21. A method of treating a subject having a Hepatitis B Virus (HBV) infection, comprising administering to the subject a therapeutically effective amount of a dsRNA agent of any one of claims 1 to 18, or a pharmaceutical composition of claim 20, thereby treating the subject.

42. 21. A method of treating a subject having a Hepatitis B Virus (HBV) associated disorder, comprising administering to the subject a therapeutically effective amount of a dsRNA agent of any one of claims 1 to 18, or a pharmaceutical composition of claim 20, thereby treating the subject.

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

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

45. 45. The method of any one of claims 21 to 44, wherein the dsRNA agent is administered to the subject at a dose of 0.01 mg / kg to 10 mg / kg, 0.5 mg / kg to 50 mg / kg, or 3 mg / kg to 10 mg / kg.

46. 46. ​​The method of claim 45, wherein the dsRNA agent is administered to the subject at a dose of 3 mg / kg to 10 mg / kg.

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

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

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

50. 50. The method of any one of claims 21-49, wherein the dsRNA agent is administered to the subject monthly, bimonthly, or trimonthly.

51. 50. 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. 52. The method of any one of claims 21 to 51, further comprising administering to the subject one or more additional therapeutic agents.

53. 53. The method of claim 52, wherein the additional therapeutic agent is an antiviral agent, a reverse transcriptase inhibitor, an immunostimulant, a therapeutic vaccine, a viral entry inhibitor, an oligonucleotide that inhibits secretion or release of HbsAg, a capsid inhibitor or a covalently closed circular (ccc) HBV DNA inhibitor, or any combination thereof.

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

55. 53. The method of claim 52, wherein the additional therapeutic agents are a reverse transcriptase inhibitor and an immunostimulant.

56. 56. The method of claim 54 or 55, wherein the reverse transcriptase inhibitor is tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine or AGX-1009.

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

58. A pharmaceutical composition for carrying out any method according to any one of claims 21 to 57 comprising a dsRNA agent according to any one of claims 1 to 18 and a pharmaceutical excipient.

59. A dsRNA agent according to any one of claims 1 to 18, or a pharmaceutical composition according to claim 20, for use in the treatment of Hepatitis B Virus (HBV) infection in a subject.

60. A dsRNA agent according to any one of claims 1 to 18, or a pharmaceutical composition according to claim 20, for use in the treatment of a Hepatitis B Virus (HBV)-related disorder in a subject.

61. 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. 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 negative.

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

64. 64. The dsRNA agent or pharmaceutical composition for use according to claim 63, wherein the additional therapeutic agent is an antiviral agent, a reverse transcriptase inhibitor, an immunostimulant, a therapeutic vaccine, a viral entry inhibitor, an oligonucleotide that inhibits the secretion or release of HbsAg, a capsid inhibitor or a covalently closed circular (ccc) HBV DNA inhibitor, or any combination thereof.

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

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

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

69. Use of a dsRNA agent according to any one of claims 1 to 18 or a pharmaceutical composition according to claim 20 for carrying out any method according to any one of claims 21 to 57.

70. Use of a dsRNA agent according to any one of claims 1 to 18 or a pharmaceutical composition according to claim 20 for the preparation of a medicament for carrying out any method according to any one of claims 21 to 57.

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