Compositions and methods for treating hepatitis b virus (HBV) infection

Abstract

To provide new HBV therapies that are effective, well tolerated, and do not require lifelong administration.SOLUTION: The present invention provides a method for treating HBV infection using an siRNA that targets an HBV gene. In one embodiment, the method for treating HBV includes co-administration of the siRNA and PEG-INFα, and may further include administration of a nucleoside / nucleotide reverse transcriptase inhibitor (NRTI).SELECTED DRAWING: Figure 22

Description

Technical Field

[0001] Description of Sequence Listing The sequence listing accompanying this application is submitted in text format instead of in writing and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is 930485_405WO_SEQUENCE_LISTING.txt. The text file is 6.5 KB, was created on May 6, 2020, and was submitted electronically via EFS-Web.

Background Art

[0002] Background Chronic hepatitis B virus (HBV) infection is associated with significant morbidity and mortality and remains an important global public health problem (Trepo C., A brief history of hepatitis milestones, Liver International 2014, 34(1):29-37). According to the World Health Organization (WHO), an estimated 257 million people worldwide are living with chronic HBV infection (WHO, 2017; Schweitzer A, et al., Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013, The Lancet 2015, 387(10003):1546-1555). Chronic HBV infection can lead to serious complications over time, including cirrhosis, liver failure, hepatocellular carcinoma (HCC) and death. It is estimated that nearly 800,000 people die each year from complications associated with chronic HBV infection (Stanaway JD, et al., The global burden of viral hepatitis from 1990 to 2013: findings from the Global Burden of Disease Study 2013, The Lancet 2016, 388(10049):1081-1088).

[0003] The prevalence of HBV varies geographically, ranging from less than 2% in low-prevalence areas to over 8% in high-prevalence countries (Schweitzer et al., 2015). In high-prevalence countries such as sub-Saharan Africa and East Asia, it is mainly transmitted to infants and children through the perinatal and horizontal routes. Furthermore, in more industrialized countries, new infections are highest among young adults, and transmission is mainly through injection drug use and high-risk sexual behavior. The risk of developing chronic HBV infection depends on the age at the time of infection. Only about 10% of adults infected develop chronic HBV infection, while 90% of infants infected perinatally or within 6 months after birth and 20 - 60% of children infected between 6 months and 5 years remain chronically infected. 25 percent of those who acquire HBV between infancy and childhood develop primary liver cancer or cirrhosis during adulthood.

[0004] HBV is a DNA virus that infects, replicates, and persists in human hepatocytes (Protzer U, et al., Living in the liver: hepatic infections, Nature Reviews Immunology 201, 12: 201 - 213). The small viral genome (3.2 kb) is a partially double-stranded, relaxed circular DNA (rcDNA) and has four open reading frames encoding seven proteins: HBcAg (HBV core antigen, the viral capsid protein), HBeAg (hepatitis B e-antigen), HBV Pol / RT (polymerase, reverse transcriptase), PreS1 / PreS2 / HBsAg (large, medium, and small surface envelope glycoproteins), and HBx (HBV x antigen, a regulator of transcription required for infection initiation) (Seeger C, et al., Molecular biology of hepatitis B virus infection, Virology, 2015, 479 - 480:672 - 686; Tong S, et al., Overview of viral replication and genetic variability, Journal of Hepatology, 2016, 64(1):S4 - S16).

[0005] In hepatocytes, the rcDNA, which is the form of HBV nucleic acid introduced by the infecting virion, is converted into covalently closed circular DNA (cccDNA), which persists as an episomal chromatin structure in the nucleus of the host cell (Allweiss L, et al., The Role of cccDNA in HBV Maintenance, Viruses 2017, 9: 156). The cccDNA serves as a transcription template for all viral transcripts (Lucifora J, et al., Attacking hepatitis B virus cccDNA - The holy grail to hepatitis B cure, Journal of Hepatology 2016, 64(1): S41 - S48). The pregenomic RNA (pgRNA) transcript is reverse transcribed into new rcDNA for new virions, which is secreted without causing cytotoxicity. In addition to infectious virions, infected hepatocytes secrete large amounts of genome-free subviral particles that can exceed 10,000-fold the number of secreted virions (Seeger et al., 2015). Random integration of the virus into the host genome can also occur and is a mechanism contributing to hepatocyte transformation (Levrero M, et al., Mechanisms of HBV-induced hepatocellular carcinoma, Journal of Hepatology 2016, 64(1): S84 - S101). HBV persists in hepatocytes in the form of cccDNA and integrated DNA (intDNA).

[0006] Hepatitis B infection is characterized by serological viral markers and antibodies (Figure 1). In acute resolving infection, the virus is eliminated by effective innate and adaptive immune responses, including the induction of cytotoxic T cells that kill infected hepatocytes and B cells that produce neutralizing antibodies that prevent viral spread (Bertoletti A, 2016, Adaptive immunity in HBV infection, Journal of Hepatology 2016, 64(1): S71 - S83; Maini MK, et al., The role of innate immunity in the immunopathology and treatment of HBV infection, Journal of Hepatology 2016, 64(1): S60-S70; Li Y, et al., Genome-wide association study identifies 8p21.3 associated with persistent hepatitis B virus infection among Chinese, Nature Communications 2016, 7:11664). In contrast, chronic infection is associated with T and B cell dysfunction, involving multiple regulatory mechanisms, including the presentation of viral epitopes to hepatocytes and the secretion of subviral particles (Bertoletti et al., 2016; Maini et al., 2016; Burton AR, et al., Dysfunctional surface antigen specific memory B cells accumulate in chronic hepatitis B infection, EASL International Liver Congress, Paris, France 2018). Therefore, the continued expression and secretion of viral proteins due to the persistence of cccDNA in hepatocytes is considered an important step that makes it impossible for the host to eliminate the infection.

[0007] Chronic HBV infection is a dynamic process that reflects the interaction between HBV replication and the host immune response. The laboratory hallmark of chronic HBV infection is the persistence of HBsAg in the blood for more than 6 months and the absence of detectable anti-HBs. Chronic infection can be classified into four stages based on blood HBV markers (HBsAg, HBeAg / anti-HBe, HBV DNA), liver disease based on biochemical parameters (alanine aminotransferase, "ALT"), and fibrosis markers (either non-invasive or based on liver biopsy) (EASL, 2017). Overall, across the various phases of chronic HBV infection, only a small minority of patients (< 1% / year) can be cured of the disease as determined by HBsAg seroclearance.

[0008] Eliminative cure of HBV involves complete eradication of HBV DNA or sustained transcriptional silencing of HBV DNA without the risk of recurrence. Treatments that may be able to eliminate or sustainably silence cccDNA / intDNA carry the risk of damaging human chromosomal DNA or altering transcription.

[0009] In contrast, functional cure is defined as lifelong management of the virus. Patients with a history of acute hepatitis B who appear to be cured have a recurrence risk of more than about 40% when immunosuppressed. In that sense, functional cure is part of the natural course of HBV infection. Treatments that may lead to functional cure may require immunomodulation. This is probably because chronic HBV infection induces B cell and T cell exhaustion due to the expression of HBV antigens (tolerogens), which may inhibit the effectiveness of immunomodulators.

[0010] Currently, there are two main treatment options for patients with chronic HBV infection: nucleoside / nucleotide reverse transcriptase inhibitors (NRTIs) and pegylated interferon-alpha (PEG-IFNα) (Liang TJ, et al., Present and Future Therapies of Hepatitis B: From Discovery to Cure, Hepatology 2015, 62(6):1893-1908). NRTIs inhibit the production of infectious virions and often reduce serum HBV DNA to undetectable levels. However, NRTIs do not directly eliminate cccDNA; therefore, viral protein transcription and translation continue. Consequently, the expression of viral epitopes in hepatocytes, the secretion of subviral particles, and immune dysfunction largely remain unaffected by NRTI treatment. As a result, long-term, often lifelong treatment is required (however, less than half of patients continue treatment after 5 years). NRTI treatment causes serum HBsAg loss at a rate of approximately 0 to 3% per year. Additionally, NRTI treatment restores fibrosis and reduces the incidence of HCC but does not eliminate the increased risk of HCC associated with HBV infection.

[0011] In contrast, PEG-IFN provides long-term immunological management, but only for a small proportion (<10%) of patients (Konerman MA, et al., Interferon Treatment for Hepatitis B, Clinics in Liver Disease 2016, 20(4): 645-665). PEG-IFN generally requires 48 weeks of treatment and has significant treatment-dependent side effects. In trials evaluating PEG-IFNα in the treatment of chronic hepatitis C infection, 12-week or 24-week regimens had lower rates of severe adverse events, grade 3 adverse events, and treatment discontinuation than trials evaluating the 48-week regimen (Lawitz E, et al., Sofosbuvir for previously untreated chronic hepatitis C infection, N Engl J Med. 2013, 368(20): 1878-1887); Hadziyannis SJ, et al., Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose, Ann Intern Med. 2004, 140(5): 346-355; Fried MW, et al., Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection, N Engl J Med. 2002, 347(13): 975-982). The high variability of response combined with unfavorable safety and side effect profiles renders a significant number of patients ineligible for or averse to receiving PEG-IFNα treatment.

Summary of the Invention

Problems to be Solved by the Invention

[0012] Due to the inability of NRTI treatment to eradicate the virus and the limitations of PEG-IFNα treatment, there is a strong clinical need for new HBV treatments that are effective, well-tolerated, and do not require lifelong administration.

Means for Solving the Problem

[0013] Overview In one aspect, the present invention relates to compositions and methods for treating HBV with siRNA, particularly HBV02. For example, according to certain embodiments, a method for treating HBV infection in a subject by administering siRNA, wherein the siRNA has an antisense strand comprising SEQ ID NO: 5 and an antisense strand comprising SEQ ID NO: 6, is provided. In certain embodiments, the treatment method further comprises administering pegylated interferon-alpha (PEG-INFα) to the subject. In certain embodiments, PEG-INFα is administered before, simultaneously with, or after siRNA HBV02 is administered. In certain embodiments, the HBV infection is chronic. In a further embodiment, the subject is administered a nucleoside / nucleotide reverse transcriptase inhibitor (NRTI). In certain embodiments, the NRTI is administered before, simultaneously with, or after HBV02 is administered. In certain embodiments, the NRTI is administered 2 to 6 months before HBV02.

[0014] In one aspect, the invention also provides an siRNA for use in treating HBV infection in a subject, wherein the siRNA is HBV02 and has an antisense strand comprising SEQ ID NO: 5 and an antisense strand comprising SEQ ID NO: 6. In a further embodiment, siRNA HBV02 is administered to a subject to whom PEG-IFNα is also administered. In one embodiment, PEG-IFNα is administered before, simultaneously with, or after siRNA HBV02 is administered. In one embodiment, the HBV infection is chronic. In a further embodiment, the subject is administered an NRTI. In one embodiment, the NRTI is administered before, simultaneously with, or after HBV02 is administered. In one embodiment, the NRTI is administered 2 to 6 months before HBV02.

[0015] In a further aspect, the invention provides the use of an siRNA in the manufacture of a medicament for treating HBV infection, wherein the siRNA is HBV02 and has an antisense strand comprising SEQ ID NO: 5 and an antisense strand comprising SEQ ID NO: 6. In one embodiment, the use of siRNA HBV02 is for use in combination with PEG-IFNα. In one embodiment, siRNA HBV02 is for use in combination with PEG-IFNα and an NRTI.

[0016] In some of the above embodiments, the dosage of siRNA HBV02 is 0.8 mg / kg, 1.7 mg / kg, 3.3 mg / kg, 6.7 mg / kg, 10 mg / kg or 15 mg / kg. In some of the above embodiments, the dosage of siRNA HBV02 is 20 mg to 900 mg. In some of the above embodiments, the dosage of siRNA HBV02 is 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg or 450 mg. In some of the above embodiments, HBV02 is administered weekly. In some of the above embodiments, the siRNA is administered more than once. In some of the above embodiments, the siRNA is administered 2, 3, 4, 5, 6 times or more, and each administration is separated by 1 week, 2 weeks, 3 weeks or 4 weeks. In some of the above embodiments, 6 doses of 200 mg of siRNA are administered. In some of the above embodiments, 2 doses of 400 mg of siRNA are administered. In some of the above embodiments, the siRNA is administered by subcutaneous injection; for example, in certain embodiments, the administration of siRNA HBV02 includes 1, 2 or 3 subcutaneous injections per administration.

[0017] In some of the above embodiments, the dosage of PEG-IFNα is 50 μg, 100 μg, 150 μg or 200 μg. In some of the above embodiments, PEG-IFNα is administered weekly. In some of the above embodiments, PEG-IFNα is administered by subcutaneous injection.

[0018] In some of the above embodiments, the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), lamivudine, adefovir, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clevudine, ritonavir, dipivoxil, lobucavir, famciclovir, N-acetyl-cysteine (NAC), PC1323, theradigm-HBV, thymosin-alpha, ganciclovir, besifovir (ANA-380 / LB-80380) or tenofvir-exaliades (TLX / CMX157).

[0019] In some of the above embodiments, the subject is HBeAg-negative. In certain embodiments, the subject is HBeAg-positive.

[0020] In one aspect of the invention, there is provided a kit comprising a pharmaceutical composition comprising any of the siRNAs of the above embodiments and a pharmaceutically acceptable additive; and a pharmaceutical composition comprising PEG-IFNα and a pharmaceutically acceptable additive. The kit may also include an NRTI and a pharmaceutically acceptable additive. BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0045] Detailed Description The present invention provides methods, compositions, and kits for use in the treatment of hepatitis B virus (HBV) infection, in which small interfering RNA (siRNA) molecules targeting HBV are administered. In certain embodiments, the siRNA molecules are administered in combination with peginterferon-2α (PEG-IFNα) therapy or to a subject who is receiving or scheduled to receive PEG-IFN-α therapy. In certain embodiments, the methods, compositions, and kits disclosed herein are used for the treatment of chronic HBV infection.

[0046] I. Glossary Before showing the present invention in more detail, it may be helpful to understand the definitions of certain terms used herein. Further definitions are provided throughout the present disclosure.

[0047] As used herein, the term "about" means ±20% of the indicated range, value, or structure, unless otherwise specified.

[0048] The term "comprising" means the presence of the recited characteristics, integers, steps, or elements as shown in the claims, but does not exclude the presence of one or more additional characteristics, integers, steps, elements, or groups thereof. The term "consisting essentially of" limits the claims to the specified materials or steps and those that do not materially affect the basic and novel features of the present invention.

[0049] It should be understood that the singular expressions used herein refer to "one or more" of the recited elements. The recitation of alternatives (e.g., "or") is to be understood to mean either one or the other of the alternatives, both, or any combination thereof, and may be used synonymously with "and / or". The terms "comprising" and "having" as used herein are used synonymously, and this term and its variants are intended to be construed as non-limiting.

[0050] The term "substantially" does not exclude "completely"; for example, a composition that is "substantially free of" Y may not be completely free of Y. If necessary, the term "substantially" may be omitted from the definitions provided herein.

[0051] As used herein, the term "disease" is generally synonymous with the terms "disorder" and "condition" (in a medical context), and all of these are generally used interchangeably in that they reflect an abnormal condition or a part of the body where normal function is impaired, in a human or animal body. A "disease" is manifested by generally recognized signs and symptoms and reduces the lifespan or quality of life of a human or animal.

[0052] As used herein, the terms "peptide", "polypeptide", and "protein" and variants of these terms refer to molecules, particularly proteins, including peptides, oligopeptides, polypeptides, or fusion proteins, each containing at least two amino acids linked to each other by normal peptide bonds or modified peptide bonds such as those in isosteric peptides. For example, a peptide, polypeptide, or protein can consist of amino acids selected from the 20 amino acids defined by the genetic code, linked to each other by normal peptide bonds ("classical" polypeptides). A peptide, polypeptide, or protein can consist of L-amino acids and / or D-amino acids. In particular, the terms "peptide", "polypeptide", and "protein" also include "peptidomimetics" defined as peptide analogs that contain non-peptide structural elements capable of mimicking or antagonizing the biological actions of native parent peptides. Peptidomimetics lack classical peptide features such as peptide bonds that are susceptible to enzymatic cleavage. In particular, a peptide, polypeptide, or protein may additionally contain amino acids other than the 20 amino acids defined by the genetic code in addition to these amino acids or may consist of amino acids other than the 20 amino acids defined by the genetic code. In particular, the peptides, polypeptides, or proteins of the present invention can likewise consist of amino acids modified by natural or chemical processes such as post-translational maturation processes well known to those skilled in the art. Such modifications are described in sufficient detail in the literature. These modifications can occur anywhere in the polypeptide: in the peptide backbone, amino acid chain, or carboxy or amino terminus. In particular, a peptide or polypeptide can be branched after ubiquitination or can be cyclized with or without branching. This type of modification can be the result of natural or synthetic post-translational processes well known to those skilled in the art. The terms "peptide", "polypeptide", or "protein" in the present invention particularly also include modified peptides, polypeptides, and proteins.For example, peptide, polypeptide or protein modifications can include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, senoylation, sulfation, amino acid addition such as arginylation or ubiquitination. These modifications are described in sufficient detail in the literature (Proteins Structure and Molecular Properties, 2nd Ed., T.E. Creighton, New York (1993); Post-translational Covalent Modifications of Proteins, B.C. Johnson, Ed., Academic Press, New York (1983); Seifter, et al., Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182:626-46 (1990); and Rattan, et al., Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci 663:48-62 (1992)). Accordingly, the terms "peptide", "polypeptide" and "protein" include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins and the like.

[0053] As used herein, the term “(poly)peptide” includes a single chain of amino acid monomers linked by peptide bonds as described above. As used herein, the term “protein” includes one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (poly)peptides, i.e., one or more chains of amino acid monomers linked by peptide bonds as described above. In a specific embodiment, the protein of the present invention includes 1, 2, 3, or 4 polypeptides.

[0054] As used herein, the term “recombinant” (e.g., recombinant protein, recombinant nucleic acid, etc.) refers to any molecule (protein, nucleic acid, siRNA, etc.) that is prepared, expressed, created, or isolated by recombinant means and does not exist in nature.

[0055] As used herein, the terms “nucleic acid”, “nucleic acid molecule” and “polynucleotide” are used interchangeably and are intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded. In a specific embodiment, the nucleic acid molecule is a double-stranded RNA molecule.

[0056] As used herein, the terms “cell”, “cell line” and “cell culture” are used interchangeably and all such names include progeny. Thus, the terms “transformant” and “transformed cell” include the culture derived therefrom, regardless of the number of generations and the primary subject cell. It should be understood that all progeny may not have exactly the same DNA content due to deliberate or accidental mutations. Variant progeny having the same function or biological activity as screened in the original transformed cell are included.

[0057] As used herein, the term "array variant" refers to any array having one or more modifications as compared to a control array, where the control array is any of the arrays listed in the sequence listing, i.e., any of SEQ ID NOs: 1 to 6. Thus, the term "array variant" includes nucleotide sequence variants and amino acid sequence variants. For nucleotide sequence array variants, the control array is also a nucleotide sequence, while for amino acid sequence array variants, the control array is also an amino acid sequence. The "array variant" as used herein is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the control array. Sequence identity is usually calculated with respect to the full length of the control array (i.e., the sequences cited in this application), unless otherwise specified. The percentage identity referred to herein can be determined, for example, using BLAST with the default parameters specified by NCBI (the National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ) [Blosum 62 matrix; gap open penalty = 11 and gap extension penalty = 1].

[0058] A "sequence variant" of a nucleic acid (nucleotide) sequence has an altered sequence in which one or more of the nucleotides in the reference sequence are deleted or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by the standard single-letter notation (A, C, G, or T). Due to the degeneracy of the genetic code, a "sequence variant" of a nucleotide sequence may or may not result in a change in the corresponding amino acid sequence, i.e., an amino acid "sequence variant". In certain embodiments, a nucleotide sequence variant is a variant that does not result in an amino acid sequence variant (i.e., a silent mutation). However, nucleotide sequence variants that lead to "non-silent" mutations, particularly those that result in an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the reference amino acid sequence, are also within the scope. An "sequence variant" of an amino acid sequence has an altered sequence in which one or more amino acids are deleted, substituted or inserted as compared to the reference amino acid sequence. As a result of the modification, such sequence variants have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the reference amino acid sequence. For example, a variant sequence having more than 10 modifications per 100 amino acids of the reference sequence, i.e., any combination of deletions, insertions or substitutions, is "at least 90% identical" to the reference sequence.

[0059] Non-conservative amino acid substitutions are possible, although in certain embodiments the substitutions are conservative amino acid substitutions where the substituted amino acid has a structural or chemical property similar to the corresponding amino acid in the control sequence. By way of example, conservative amino acid substitutions include substitution of one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine and isoleucine, for another; substitution of one hydroxyl-containing amino acid, such as serine and threonine, for another; substitution of one acidic residue, such as glutamic acid or aspartic acid, for another; replacement of one amide-containing residue, such as asparagine and glutamine, for another; replacement of one aromatic residue, such as phenylalanine and tyrosine, for another; replacement of one basic residue, such as lysine, arginine and histidine, for another; and replacement of one small amino acid, such as alanine, serine, threonine, methionine and glycine, for another.

[0060] Amino acid insertions include amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from 1 residue to 100 or more residues as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include fusions to reporter molecules or enzymes at the N-terminus or C-terminus of an amino acid sequence.

[0061] Unless otherwise specified, modifications to the sequence variants do not abolish the functionality of each control sequence, e.g., in the case of the present invention, the functionality of the siRNA to reduce HBV protein expression. Guidance as to which nucleotide and amino acid residues can be substituted, inserted or deleted without abolishing such functionality can be found using computer programs known in the art.

[0062] As used herein, a nucleic acid sequence or amino acid sequence "derived from" a specified nucleic acid, peptide, polypeptide or protein refers to the origin of the nucleic acid, peptide, polypeptide or protein. In certain embodiments, a nucleic acid sequence or amino acid sequence derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof from which it is derived, where "essentially identical" includes the sequence variants defined above. In certain embodiments, a nucleic acid sequence or amino acid sequence derived from a particular peptide or protein is derived from the corresponding domain of the particular peptide or protein. Thereby, "corresponding" refers to the same functionality in particular. For example, an "extracellular domain" corresponds to another "extracellular domain" or a "transmembrane domain" corresponds to another "transmembrane domain" (of another protein). The "corresponding" portions of peptides, proteins and nucleic acids are thus identifiable to those of ordinary skill in the art. Similarly, a sequence "derived from" another sequence is generally identifiable to those of ordinary skill in the art as long as the sequence has its origin in the sequence.

[0063] In certain embodiments, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein can be identical to the starting nucleic acid, peptide, polypeptide or protein (from which it is derived). However, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein can have one or more mutations compared to the starting nucleic acid, peptide, polypeptide or protein (from which it is derived), and in particular, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein can be a functional sequence variant of the starting nucleic acid, peptide, polypeptide or protein as described above. For example, in a peptide / protein, one or more amino acid residues can be substituted with other amino acid residues or one or more insertions or deletions of amino acid residues can occur.

[0064] As used herein, the term "mutation" relates to a change in a nucleic acid sequence and / or an amino acid sequence as compared to a control sequence, e.g., a corresponding genomic sequence. For example, as compared to a genomic sequence, a mutation can be, e.g., a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutant or a site-directed mutagenesis (a molecular biology method that creates specific and intentional changes in a nucleic acid sequence and / or an amino acid sequence) induced by an enzyme, a chemical or radiation. Thus, the term "mutation" or "mutate" is understood to include, e.g., physically creating a mutation in a nucleic acid sequence or an amino acid sequence. Mutations include substitutions, deletions and insertions of one or more nucleotides or amino acids and inversions of several consecutive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, the mutation can be introduced into the nucleotide sequence encoding the amino acid sequence in order to express a (recombinant) mutant polypeptide. A mutation can be achieved, e.g., by site-directed mutagenesis, e.g., by modifying the codon of a nucleic acid molecule encoding a certain amino acid to result in a codon encoding a different amino acid or, e.g., by knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide and designing the synthesis of a nucleic acid molecule of a nucleotide sequence encoding a variant of the polypeptide without mutating one or more nucleotides of the nucleic acid molecule, by synthesis of a sequence variant.

[0065] As used herein, the term "coding sequence" refers to a polynucleotide molecule that encodes the amino acid sequence of a protein product. The boundaries of a coding sequence are generally determined by an open reading frame that usually begins with an ATG start codon.

[0066] As used herein, the term "expression" refers to all stages associated with the production of a polypeptide, including transcription, post-translational modification, translation, post-translational modification, secretion, etc.

[0067] Dosages are often expressed in relation to body weight. Thus, a dosage expressed as [g, mg or other unit] / kg (or g, mg, etc.) usually refers to [g, mg or other unit] “ / kg (or g, mg, etc.) body weight” even if the term “body weight” is not explicitly stated.

[0068] As used herein, the term “hepatitis B virus” is used interchangeably with the term “HBV” and refers to a well-known non-cytopathic, liver tropic DNA virus belonging to the Hepadnaviridae family. The HBV genome is a partially double-stranded, circular DNA with four overlapping reading frames, C, X, P, and S (referred to herein as “genes,” “open reading frames,” or “transcripts”). The core protein is encoded by gene C (HBcAg). The hepatitis B e antigen (HBeAg) is produced by proteolytic processing of the precore (preC) 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 three in-frame “start” (ATG) codons that give rise to three different sized polypeptides designated as the large, middle, and small S antigens, preS1+preS2+S, preS2+S, or S. The surface antigen, in addition to modification of the HBV envelope, is produced in large excess compared to virion particles and is part of the subviral particles that play a role in immune tolerance and sequestration of 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 has a role in transcriptional transactivation and replication and is associated with the progression of hepatocellular carcinoma.

[0069] Nine genotypes of HBV, named A-I, have been determined, and an additional genotype J has been proposed, each having a different geographical distribution (Velkov S, et al., The Global Hepatitis B Virus Genotype Distribution Approximated from Available Genotyping Data, Genes 2018, 9(10):495). The term "HBV" includes any of the genotypes (A-J) of HBV. The complete coding sequences of the reference sequences of the HBV genome can be found, for example, in GenBank Accession Nos. GI:21326584 and GI:3582357. 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); YP_009173867.1 and BAA32912.1 (X protein); YP_009173866.1 and BAA32913.1 (P protein); and YP_009173869.1, YP_009173870.1, YP_009173871.1, and BAA32914.1 (S protein). Further examples of HBV messenger RNA (mRNA) sequences are available using publicly available databases such as GenBank, UniProt, and OMIM. The International Repository for Hepatitis B Virus Strain Data is accessible at http: / / www.hpa-bioinformatics.org.uk / HepSEQ / main.php. The term "HBV" as used herein also refers to naturally occurring DNA sequence variants of the HBV genome, namely genotypes A-J and their variants.

[0070] siRNA mediates the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, thereby inhibiting gene expression. This process is often referred to as "RNA interference" (RNAi). Without wishing to be bound by a particular theory, long double-stranded RNA (dsRNA) introduced into plant and invertebrate cells is broken down into siRNA by a type III endonuclease known as Dicer (Sharp, et al., Genes Dev. 15:485 (2001)). Dicer, a ribonuclease-III-like enzyme, processes dsRNA into 19-23 base pair siRNAs with characteristic 2-base 3' overhangs (Bernstein, et al., Nature 2001, 409:363). The siRNAs are then incorporated into RISC, where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., 2001, Cell 107:309). Binding to the appropriate target mRNA causes one or more endonucleases within RISC to cleave the target and induce silencing (Elbashir, et al., Genes Dev. 2001, 15:188).

[0071] As far as the terms "silence", "inhibition of expression", "downregulation of expression", "repression of expression", etc. are concerned with respect to the HBV gene, here, the amount of HBV mRNA that can be isolated or detected from a first cell or cell population that has been treated with an inhibitor of HBV gene expression such that the HBV gene is transcribed and the expression of the HBV gene is inhibited, which is substantially the same as that of the first cell or cell population, but is manifested by a decrease when compared to a second cell or cell population (control cells) that has not been so treated, refers to at least a partial decrease in the expression of the HBV gene. The degree of inhibition can be measured, for example, as the difference in the degree of mRNA expression in the treated cells minus the degree of mRNA expression in the control cells. Alternatively, the degree of inhibition can be indicated in terms of a parameter that is functionally related to HBV gene expression, for example, the amount of protein encoded by the HBV gene or a decrease in the number of cells exhibiting a particular phenotype, for example, the HBV infection phenotype. In principle, HBV gene silencing can be determined by any appropriate assay in any cell that expresses the HBV gene, for example, HBV-infected cells or cells engineered to express the HBV gene.

[0072] The HBV RNA level or the circulating HBV RNA level expressed by a cell or cell population can be determined by any method known in the art for evaluating mRNA expression, such as the rtPCR method provided in Example 2 of International Application Publication WO2016 / 077321A1 and US Patent Application US2017 / 0349900A1, which are incorporated herein by reference. In certain embodiments, the expression level of the HBV gene in a sample (e.g., total HBV RNA, HBV transcript, e.g., HBV 3.5 kb transcript) is determined by detection of 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 / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen®), or those using PAXgene (PreAnalytix, Switzerland). Exemplary assay formats that utilize ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton D A, et al., Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter, Nuc. Acids Res. 1984, 12:7035-56), Northern blotting, in situ hybridization, and microarray analysis. Circulating HBV mRNA can be detected by the methods described in International Application Publication WO2012 / 177906A1 and US Patent Application US2014 / 0275211A1, which are incorporated herein by reference.

[0073] As used herein, the "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the HBV gene, including the mRNA which is the product of RNA processing of the primary transcript. The target portion of the sequence must be long enough to function as a substrate for RNAi-directed cleavage at least in that portion or in its vicinity. For example, the target sequence is generally 9 to 36 nucleotides in length, such as 15 to 30 nucleotides in length (including all sub-ranges therebetween). By way of non-limiting example, the target sequence can be 15 to 30 nucleotides, 15 to 26 nucleotides, 15 to 23 nucleotides, 15 to 22 nucleotides, 15 to 21 nucleotides, 15 to 20 nucleotides, 15 to 19 nucleotides, 15 to 18 nucleotides, 15 to 17 nucleotides, 18 to 30 nucleotides, 18 to 26 nucleotides, 18 to 23 nucleotides, 18 to 22 nucleotides, 18 to 21 nucleotides, 18 to 20 nucleotides, 19 to 30 nucleotides, 19 to 26 nucleotides, 19 to 23 nucleotides, 19 to 22 nucleotides, 19 to 21 nucleotides, 19 to 20 nucleotides, 20 to 30 nucleotides, 20 to 26 nucleotides, 20 to 25 nucleotides, 20 to 24 nucleotides, 20 to 23 nucleotides, 20 to 22 nucleotides, 20 to 21 nucleotides, 21 to 30 nucleotides, 21 to 26 nucleotides, 21 to 25 nucleotides, 21 to 24 nucleotides, 21 to 23 nucleotides or 21 to 22 nucleotides.

[0074] As used herein, the term "strand containing a sequence" refers to an oligonucleotide containing a nucleotide strand described by the sequence referred to using standard nucleotide nomenclature.

[0075] Unless otherwise indicated, as used herein, the term "complementary" when used to refer to a first nucleotide sequence relative to a second nucleotide sequence, as would be understood by one of ordinary skill in the art, refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize to an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions to form a double-stranded structure. Such conditions may be, for example, stringent conditions, where stringent conditions include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50 °C or 70 °C for 12-16 hours, followed by washing. Other conditions may be applied, such as physiologically appropriate conditions that may be encountered in vivo. One of ordinary skill in the art can determine the most appropriate conditions for testing the complementarity of the two sequences in the ultimate application of the hybridized nucleotides.

[0076] The complementary sequences within the siRNAs described herein include base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the full length of one or both nucleotide sequences. Such sequences may herein be referred to as “perfectly complementary” to each other. However, when a first sequence is referred to as “substantially complementary” to a second sequence herein, the two sequences may be perfectly complementary, or may form no more than 5, 4, 3, or 2 mismatched base pairs by hybridization of one or more, but generally a maximum of 30 base pairs of double-strandedness, while maintaining the ability to hybridize under the most suitable conditions for ultimate application, e.g., inhibition of gene expression via the RISC pathway. However, when two oligonucleotides are said to form one or more single-stranded overhangs by hybridization, such overhangs must not be considered mismatches with respect to determination of complementarity. For example, in an siRNA where one oligonucleotide is 21 nucleotides in length and the other oligonucleotide is 23 nucleotides in length, and the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, it may still be referred to as “perfectly complementary” for the purposes herein.

[0077] The “complementary” sequences used herein may include, or be formed entirely from, base pairs formed by non-Watson-Crick base pairs and / or non-natural and modified nucleotides, so long as the above requirements regarding hybridization are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing.

[0078] The terms “complementary,” “perfectly complementary,” and “substantially complementary” as used herein relate to base matching between the sense and antisense strands of an siRNA or between the antisense strand of an siRNA agent and a target sequence, as understood from the context of use.

[0079] The polynucleotide that is "substantially complementary" to at least a part of the mRNA used herein refers to a polynucleotide that is substantially complementary to a continuous part of the target mRNA (for example, the mRNA encoding the HBV protein). For example, if the sequence of the polynucleotide is substantially complementary to an uninterrupted part of the HBV mRNA, it is complementary to at least a part of the HBV mRNA.

[0080] As used herein, the term "siRNA" refers to an RNA interference molecule comprising an RNA molecule or molecular complex having a hybridized double-stranded region containing two anti-parallel and substantially complementary nucleic acid strands that can be referred to as having "sense" and "antisense" orientations with respect to the target RNA. The double-stranded region can be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, but generally ranges from 9 to 36 base pairs in length, for example, in the range of 15 to 30 base pairs in length. Considering double-strands of 9 to 36 base pairs, the double-strand can be of any length within this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 and, for 15 to 30 base pairs, 15 to 26 base pairs, 15 to 23 base pairs, 15 to 22 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 19 base pairs, 15 to 18 base pairs, 15 to 17 base pairs, 18 to 30 base pairs, 18 to 26 base pairs, 18 to 23 base pairs, 18 to 22 base pairs, 18 to 21 base pairs, 18 to 20 base pairs, 19 to 30 base pairs, 19 to 26 base pairs, 19 to 23 base pairs, 19 to 22 base pairs, 19 to 21 base pairs, 19 to 20 base pairs, 20 to 30 base pairs, 20 to 26 base pairs, 20 to 25 base pairs, 20 to 24 base pairs, 20 to 23 base pairs, 20 to 22 base pairs, 20 to 21 base pairs, 21 to 30 base pairs, 21 to 26 base pairs, 21 to 25 base pairs, 21 to 24 base pairs, 21 to 23 base pairs and 21 to 22 base pairs, including, but not limited to, any sub-range therebetween. siRNAs produced in cells by processing with Dicer and similar enzymes generally range from 19 to 22 base pairs in length.

[0081] One strand of the double-stranded region of the siRNA contains a sequence that is substantially complementary to a region of the target RNA. The two strands forming the double-stranded structure can form a single RNA molecule having at least one self-complementary region or can be formed from two or more other RNA molecules. When the double-stranded region is formed from two strands of a single molecule, the molecule can have a single strand of nucleotides (herein referred to as a "hairpin loop") between the 3' end of one strand forming the double-stranded structure and the 5' end of the other strand, whereby the double-stranded regions are separated. The hairpin loop can contain at least one unpaired nucleotide; in certain embodiments, the hairpin loop can contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. When the two substantially complementary strands of the siRNA constitute separate RNA molecules, these molecules can be covalently linked, although not necessarily. When the two strands are covalently linked by means other than a hairpin loop, the linking structure is referred to as a "linker".

[0082] The siRNAs described herein can be synthesized by standard methods known in the art, for example, by use of an automated DNA synthesizer, commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0083] The term "antisense strand" or "guide strand" refers to a strand of the siRNA that contains a region that is substantially complementary to the target sequence. The term "complementary region" as used herein refers to a region of the antisense strand that is substantially complementary to a sequence as defined herein, for example, the target sequence. When the complementary region is not completely complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are within 5, 4, 3 or 2 nucleotides of the terminal regions, for example, the 5' and / or 3' termini.

[0084] As used herein, the term "sense strand" or "passenger strand" refers to the strand of an siRNA that includes a region that is substantially complementary (as defined herein) to a region of the antisense strand.

[0085] The term "RNA molecule" or "ribonucleic acid molecule" includes not only naturally occurring or found RNA molecules, but also analogs and derivatives of RNA that include one or more ribonucleotide / ribonucleoside analogs or derivatives described herein or known in the art. Strictly speaking, a "ribonucleoside" is a nucleoside base and a ribose sugar, and a "ribonucleotide" is a ribonucleoside having one, two or three phosphate moieties. However, for the purposes of this disclosure, the terms "ribonucleoside" and "ribonucleotide" may be considered equivalent. RNA can be modified, for example, in the nucleobase structure or the ribose-phosphate backbone structure, as further detailed below. However, siRNA molecules containing ribonucleoside analogs or derivatives maintain the ability to form duplexes. By way of non-limiting example, an RNA molecule may also include at least one modified ribonucleoside including, but not limited to, 2'-O-methyl modified nucleosides, nucleosides containing 5' phosphorothioate groups, terminal nucleosides conjugated to cholesteryl derivatives or didodecylamid groups of dodecanoic acid, locked nucleosides, abasic nucleosides, 2'-deoxy-2'-fluoro modified nucleosides, 2'-amino-modified nucleosides, 2'-alkyl-modified nucleosides, morpholino nucleosides, phosphoramidates or non-natural base-containing nucleosides or any combination thereof. In other examples, an RNA molecule may include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more modified ribonucleosides up to the full length of the siRNA molecule. The modifications need not be the same for each of such multiple modified ribonucleosides in an RNA molecule. In certain embodiments, the modified ribonucleosides include deoxyribonucleosides. For example, an siRNA may include one or more deoxynucleosides, such as, for example, deoxynucleotide overhangs or one or more deoxynucleosides within the duplex portion of the siRNA. However, the term "siRNA" as used herein does not include complete DNA molecules.

[0086] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of an siRNA. For example, a nucleotide overhang exists when the 3' end of one strand of the siRNA extends beyond the 5' end of the other strand or vice versa. An siRNA can include an overhang of at least one nucleotide; alternatively, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more nucleotides. The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs including deoxynucleotides / nucleosides. The overhang can be on the sense strand, the antisense strand or any combination thereof. Further, the nucleotides of the overhang can be present at the 5' end, 3' end or both ends of the antisense or sense strand of the siRNA.

[0087] As used herein in connection with siRNA, the term "blunt" or "blunt-ended" means that there are no unpaired nucleotides or nucleotide analogs at a given end of the siRNA, i.e., there is no nucleotide overhang. One or both ends of the siRNA can be blunt. When both ends of the siRNA are blunt, the siRNA is "blunt-ended". A "blunt-ended" siRNA is an siRNA that is blunt at both ends, i.e., has no nucleotide overhangs at either end of the molecule. Most often such molecules are double-stranded over their entire length.

[0088] II. siRNA targeting HBV The present invention provides methods of treatment related to the administration of HBV-targeting siRNAs and related compositions and kits. In certain embodiments, the HBV-targeting siRNA is HBV02. HBV02 is a synthetic, chemically modified siRNA targeting HBV RNA with a covalently attached, branched trisaccharide N-acetyl-galactosamine (GalNAc) ligand that enables specific uptake by hepatocytes. HBV02 targets a region of the HBV genome common to all HBV viral transcripts and is pharmacologically active against HBV genotypes A - J. In preclinical models, HBV02 has been shown to inhibit viral replication, translation, and HBsAg secretion and may provide a functional cure of chronic HBV infection. One siRNA may have multiple antiviral effects, including pgRNA degradation and thereby blocking viral replication and degradation of all viral mRNA transcripts and thereby blocking viral protein expression. This may result in the restoration of a functional immune response against HBV, either alone or in combination with other therapies. The ability of HBV02 to reduce HBsAg-containing non-infectious subviral particles also differentiates it from currently available treatments.

[0089] HBV02 targets and inhibits the expression of the mRNA encoded by the HBV genome of NCBI Reference Sequence NC_003977.2 (GenBank Accession No. GI:21326584) (SEQ ID NO:1). More specifically, HBV02 targets the mRNA encoded by a portion of the HBV genome that includes the sequence GTGTGCACTTCGCTTCAC (SEQ ID NO:2) corresponding to nucleotides 1579 - 1597 of SEQ ID NO:1. Because transcription of the HBV genome results in polycistronic, overlapping RNAs, HBV02 results in significant inhibition of the expression of most or all of the HBV transcripts.

[0090] HBV02 has a sense strand containing 5’-GUGUGCACUUCGCUUCACA-3’ (SEQ ID NO: 3) and an antisense strand containing 5’-UGUGAAGCGAAGUGCACACUU-3’ (SEQ ID NO: 4), where the nucleotides contain 2’-fluoro (2’F) and 2’-O-methoxy (2’OMe) ribose sugar modifications, phosphorothioate backbone modifications, glycol nucleic acid (GNA) modifications, and conjugates with a tris-branched N-acetyl-galactosamine (GalNAc) ligand at the 3’ end of the sense strand to facilitate delivery to hepatocytes via the asialoglycoprotein receptor (ASGPR). By including the modifications, the sense strand of HBV02 contains 5’-gsusguGfcAfCfUfucgcuucacaL96-3’ (SEQ ID NO: 5), and the antisense strand contains 5’-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3’ (SEQ ID NO: 6), where the abbreviations for the modifications are as shown in Table 1.

[0091]

Table 1

[0092] In certain embodiments, the siRNA used in the methods, compositions, or kits described herein is HBV02.

[0093] In certain embodiments, the siRNA used in the methods, compositions, or kits described herein comprises sequence variants of HBV02. In specific embodiments, the portion of the HBV transcript targeted by the sequence variant of HBV02 overlaps with the portion of the HBV transcript targeted by HBV02.

[0094] In certain embodiments, the siRNA comprises a sense strand and an antisense strand, where (1) the sense strand comprises a sequence that differs from SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 3 or SEQ ID NO: 5 by no more than 4, no more than 3, no more than 2, or no more than 1; or (2) the antisense strand comprises a sequence that differs from SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 4 or SEQ ID NO: 6 by no more than 4, no more than 3, no more than 2, or no more than 1.

[0095] In certain embodiments, short double-strands having any of the sequences of SEQ ID NO: 5 or SEQ ID NO: 6 with only a few nucleotides reduced from one or both ends are used. Thus, siRNAs having a subsequence of at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of one or both of SEQ ID NO: 5 and SEQ ID NO: 6 and having a difference in the ability to inhibit HBV gene expression of no more than 5%, 10%, 15%, 20%, 25%, or 30% from the siRNA containing the full sequence are contemplated herein. In certain embodiments, siRNAs having blunt ends formed by nucleotide removal from one or both ends of one or both ends of HBV02 are provided at one or both ends.

[0096] In certain embodiments, the siRNAs described herein may contain one or more mismatches to the target sequence. In certain embodiments, the siRNAs described herein contain no more than 3 mismatches. In certain embodiments, if the antisense strand of the siRNA contains a mismatch to the target sequence, the region of the mismatch is not located in the center of the complementary region. In a specific embodiment, if the antisense strand contains a mismatch to the target sequence, the mismatch is limited to within the last 5 nucleotides from the 5' or 3' end of the complementary region. For example, for a 23-nucleotide iRNA strand that is complementary to a region of the HBV gene, the RNA strand may not contain any mismatches within the central 13 nucleotides. Using the methods described herein or methods known in the art, it can be determined whether siRNAs containing mismatches to the target sequence are effective in inhibiting HBV gene expression.

[0097] In certain embodiments, the siRNAs used in the methods, compositions and kits described herein comprise two oligonucleotides, one oligonucleotide being referred to as the sense strand and the second oligonucleotide being referred to as the corresponding antisense strand of the sense strand. As described elsewhere herein and as is known in the art, the complementary sequences of siRNAs can also be included as self-complementary regions of a single nucleic acid molecule, as opposed to separate oligonucleotides.

[0098] In certain embodiments, single-stranded antisense RNA molecules comprising the antisense strand of HBV02 or sequence variants thereof are used in the methods, compositions and kits described herein. The antisense RNA molecules can have 15-30 nucleotide complementarity to the target. For example, the antisense RNA molecule can have a sequence of at least 15, 16, 17, 18, 19, 20, 21 or more consecutive nucleotides from SEQ ID NO: 6.

[0099] In certain embodiments, the siRNA comprises a sense strand and an antisense strand, where the sense strand comprises SEQ ID NO: 5 and the antisense strand comprises SEQ ID NO: 6, further comprising additional nucleotides, modifications or conjugates described herein. For example, in certain embodiments, the siRNA can comprise additional modifications in addition to those shown in SEQ ID NOs: 5 and 6. Such modifications can be made using established methods in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage SL, et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Examples of such modifications are further detailed below.

[0100] a. Modified siRNA The modifications disclosed herein include, for example, (a) sugar modifications (e.g., at the 2' or 4' position) or replacement of the sugar; (b) backbone modifications, including modification or replacement of the phosphodiester bond; (c) base modifications, such as replacement with a stabilized base, destabilized base, or base with an expanded repertoire of base pair partners, removal of the base (abasic nucleotide), or conjugated base; and (d) terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, inverse linkage, etc.), 3' terminal modifications (conjugation, DNA nucleotides, inverse linkage, etc.). Some specific examples of modifications that can be incorporated into the siRNA of the present application are shown in Table 1.

[0101] The modification includes a substituted sugar moiety. Characteristic siRNAs herein can include at the 2' position one of the following: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl; where the alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 alkyl or C2-C 10 alkenyl and alkynyl. Examples of suitable modifications include O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, where n and m are 1 to about 10. In certain other embodiments, the siRNA includes at the 2' position one of the following: C1-C 10It includes lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, a group that improves the pharmacokinetic properties of siRNA or a group that improves the pharmacodynamic properties of siRNA, and other substituents having similar properties. In certain embodiments, the modification is 2'-methoxyethoxy (also known as 2'-O-CH2CH2OCH3, 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin, et al., Helv. Chim. Acta 1995, 78:486-504), i.e., it includes an alkoxy-alkoxy group. Examples of other modifications include 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group also known as 2'-DMAOE and 2'-dimethylaminoethoxyethoxy (in the art also known as 2 * -O-dimethylaminoethoxyethyl or 2 * -DMAEOE), i.e., 2 * -O-CH2-O-CH2-N(CH2)2. Examples of other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of siRNA, particularly at the 3'-position of the sugar of the 3'-terminal nucleotide or the 2'-5' linked iRNA and at the 5'-position of the 5'-terminal nucleotide. The modification may also include a sugar mimic such as a cyclobutyl moiety instead of the pentofuranosyl sugar.

[0102] Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. 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, each of which is hereby incorporated by reference herein for its teachings regarding the preparation of such modifications.

[0103] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates having a normal 3'-5' linkage, their 2'-5' linkage analogs, and those having reverse polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms.

[0104] Representative U.S. patents that teach the preparation of such 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 RE39464, each of which is hereby incorporated by reference herein for its teachings regarding the preparation of such modifications.

[0105] RNAs having a modified backbone include, among others, those having no phosphorus atoms in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs having no phosphorus atoms in the internucleoside backbone may also be considered oligonucleosides. Modified RNA backbones that do not contain a phosphorus atom have a backbone formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed from the sugar portion of some nucleosides); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having a mixed N, O, S and CH2 component.

[0106] Representative U.S. patents that teach the preparation of such oligonucleosides include, but are not limited to, U.S. Pat. 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 its teachings regarding methods of preparing such modifications.

[0107] In one embodiment, both the sugar of the nucleotide unit and the internucleoside linkage, i.e., the backbone, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. Such oligomeric compounds, RNA mimics, which exhibit excellent hybridization properties, are referred to as peptide nucleic acids (PNAs). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are maintained 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. Patents 5,539,082, 5,714,331, and 5,719,262, each of which is hereby incorporated by reference. Further teachings of PNA compounds can be found, for example, in Nielsen, et al. (Science, 254:1497-1500 (1991)).

[0108] One embodiment characteristic of the technology described herein includes RNAs having phosphorothioate backbones and oligonucleosides having heteroatom backbones, particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as methylene(methylimino) or MMI backbone], -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- of U.S. Patent 5,489,677 [wherein the native phosphodiester backbone is represented as -O-P-O-CH2-] and the amide backbone of U.S. Patent 5,602,240. In one embodiment, the RNAs characteristic herein have the morpholino backbone structure of U.S. Patent 5,034,506.

[0109] The modifications of siRNA disclosed herein may also include nucleobase (often simply referred to as "base" herein) modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 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 and 3-deazaguanine and 3-deazaadenine and other synthetic and natural nucleobases such as those.Additional nucleobases include those disclosed in U.S. Patent 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine (Herdewijn P, ed., Wiley-VCH, 2008); those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering (pages 858-859, Kroschwitz JL, ed., John Wiley & Sons, 1990), those disclosed in Englisch et al. (Angewandte Chemie, International Edition, 30, 613, 1991) and those disclosed in Sanghvi YS (Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke ST and Lebleu B, ed., CRC Press, 1993). Certain of these nucleobases are particularly useful for increasing the binding affinity of oligomeric compounds characteristic of the technologies described herein. 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. 5-Methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6 - 1.2 °C (Sanghvi YS, et al., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, pp. 276-278, 1993), an example of a base substitution that is even more so when combined with, in particular, 2'-O-methoxyethyl sugar modifications.

[0110] Representative U.S. patents that teach the preparation of the above-described modified nucleobases as well as some of the other modified nucleobases include, but are not limited to, 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, each of which is hereby incorporated by reference herein for its teachings regarding the preparation of such modifications.

[0111] siRNA can also be modified to include one or more adenosine-glycol nucleic acids (GNAs). Descriptions of adenosine-GNAs can be found, for example, in Zhang, et al. (JACS 2005, 127(12):4174-75), which is hereby incorporated by reference herein for its teachings related to the preparation of GNA modifications.

[0112] The RNA of siRNA can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety that includes an extra bridge connecting the 2' and 4' carbons of the ribose moiety. This structure efficiently "locks" the ribose into the conformation of the 3'-end structure. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen J, et al., Nucleic Acids Research 2005, 33(l):439-47; Mook OR, et al., Mol Cane Ther 2007, 6(3):833-43; Grunweller A, et al., Nucleic Acids Research 2003, 31(12):3185-93).

[0113] Exemplary U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Pat. Nos. 6,268,490, 6,670,461, 6,794,499, 6,998,484, 7,053,207, 7,084,125, and 7,399,845, each of which is hereby incorporated by reference herein for its teachings regarding methods of preparing such modifications.

[0114] In certain embodiments, the siRNA includes modifications in which a chemical bond to one or more ligands, moieties or conjugates that increase the activity, cellular distribution or cellular uptake of the siRNA into RNA is involved. Such moieties include lipid moieties such as cholesterol moieties (Letsinger, et al., Proc. Natl. Acid. Sci. USA 1989, 86:6553-56), cholic acid (Manoharan, et al., Biorg. Med. Chem. Let. 1990, 4:1053-60), thioethers such as beryl-S-tritylthiol (Manoharan, et al., Ann. N.Y. Acad. Sci. 1992, 660:306-9; Manoharan, et al., Biorg. Med. Chem. Let. 1993, 3:2765-70), thiocolesterol (Oberhauser, et al., Nucl. Acids Res. 1992, 20:533-38), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras, et al., EMBO J 1991, 10:1111-18; Kabanov, et al., FEBS Lett. 1990, 259:327-30; Svinarchuk, et al., Biochimie 1993, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium l,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan, et al., Tetrahedron Lett. 1995, 36:3651-54; Shea, et al., Nucl. Acids Res. 1990, 18:3777-83), polyamines or polyethylene glycol chains (Manoharan, et al., Nucleosides & Nucleotides 1995, 14:969-73) or adamantane acetic acid (Manoharan, et al., Tetrahedron Lett. 1995, 36:3651-54), palmitoyl moieties (Mishra, et al., Biochim. Biophys.(Acta 1995, 1264:229-37) or octadecylamine or hexylamino-carbonyloxy cholesterol moieties (Crooke, et al., J. Pharmacol. Exp. Ther. 1996, 277:923-37), but are not limited thereto.

[0115] In certain embodiments, the ligand alters the distribution, targeting, or lifespan of the incorporated siRNA. In certain embodiments, the ligand provides enhanced affinity for a selected target, e.g., a molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as compared to a species lacking such a ligand. In such embodiments, the ligand does not participate in duplex formation of the double-stranded nucleic acid.

[0116] The ligand can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. The ligand can also be a recombinant or synthetic molecule such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acids such as polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactic-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, and alpha helix peptides.

[0117] The ligand may also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, such as an antibody, that binds to a specific cell type such as a hepatocyte. Targeting groups can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands are dyes, intercalators (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine), peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphates, aminos, mercaptos, PEGs (e.g., PEG-40K), MPEG, [MPEG]2, polyamines, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamins, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu complex of tetraazamacrocyclic molecule), dinitrophenyl, HRP, and AP. 3+ Complex), dinitrophenyl, HRP and AP are included.

[0118] A ligand can be an antibody that has specific affinity for a protein, such as a glycoprotein or a peptide, such as a co-ligand, or an antibody that binds to a specific cell type, such as a hepatocyte. A ligand can also include hormones and hormone receptors. Non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, and polyvalent fucose can also be included. A ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

[0119] A ligand can be a substance, such as a drug, that can increase the uptake of siRNA into cells by, for example, disrupting the cytoskeleton of the cells, such as by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cells. Drugs can be, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, inadanocin, or myoservin.

[0120] In certain embodiments, the ligand is a moiety that is taken up by a target cell, such as a hepatocyte, such as a vitamin. Examples of vitamins include vitamin A, E, and K. Other examples of vitamins include vitamins B, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by a target cell, such as a hepatocyte. HSA and low density lipoprotein (LDL) are also included.

[0121] In certain embodiments, the ligand conjugated to the siRNA described herein acts as a pharmacokinetic (PK) modulator. As used herein, "PK modulator" refers to a pharmacokinetic modulator. PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Examples of PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacyl glycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamins, biotin, and the like. Oligonucleotides containing multiple phosphorothioate linkages are also known to bind to serum proteins, and thus short oligonucleotides containing multiple phosphorothioate linkages in the backbone, such as oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, are also suitable as ligands (e.g., PK modulating ligands) for the technology described herein. Additionally, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

[0122] (i) Lipid conjugates. In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Lipid or lipid-based ligands can be used to (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport to target cells or cell membranes, and / or (c) modulate binding to serum proteins, such as HSA. Such lipid or lipid-based molecules can bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the conjugate to target tissues, such as non-renal target tissues of the body. For example, the target tissue can be the liver, including hepatocytes of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used.

[0123] The binding of the conjugate to the target tissue can be inhibited, e.g., controlled, using a lipid-based ligand. For example, the stronger the binding of a lipid or lipid-based ligand to HSA, the lower the likelihood of targeting the kidney and, therefore, the lower the likelihood of elimination from the body. Lipids or lipid-based ligands with less strong binding to HSA can be used to target the conjugate to the kidney.

[0124] In certain embodiments, the lipid-based ligand binds to HSA. The lipid-based ligand can bind to HSA with sufficient affinity for the conjugate to be distributed to non-renal tissues. In certain specific embodiments, the HSA-ligand binding is reversible.

[0125] In certain embodiments, the lipid-based ligand binds weakly or not at all to HSA such that the conjugate is distributed to the kidney. Other moieties that target renal cells can also be used instead of or in addition to the lipid-based ligand.

[0126] (ii) Cell-penetrating peptides and agents. In other embodiments, the ligand is a cell-penetrating agent such as a helical cell-penetrating agent. In certain embodiments, the agent is amphiphilic. Examples of agents are peptides such as tat or antennapedia. If the agent is a peptide, it can be modified to include peptidomimetics, reverse isomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. In certain embodiments, the helical agent is an alpha-helical agent. In certain specific embodiments, the helical agent has a lipophilic and a hydrophobic phase.

[0127] "Cell-penetrating peptides" can penetrate cells, such as microbial cells like bacterial or fungal cells or mammalian cells like human cells. Microbial cell-penetrating peptides can be, for example, alpha-helical linear peptides (e.g., LL-37 or Ceropin PI), disulfide bond-containing peptides (e.g., alpha-defensin, beta-defensin or bacteriocin) or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin).

[0128] The ligand can be a peptide or a peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule that can fold into a defined three-dimensional structure similar to that of a natural peptide. The binding of peptides and peptidomimetics to siRNA can affect the pharmacokinetic distribution of RNAi, such as by enhancing cell recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in length.

[0129] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphiphilic peptide or a hydrophobic peptide (e.g., consisting mainly of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, a constrained peptide or a cross-linked peptide. In other alternatives, the peptide moiety can contain a hydrophobic membrane translocation sequence (MTS). An example of a hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 7). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 8)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide capable of transporting large polar molecules including peptides, oligonucleotides and proteins across the cell membrane. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 9)) and the Drosophila antennapedia protein (RQIKIWFQNRRMKWK (SEQ ID NO: 10)) have been found to function as delivery peptides. The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage display library or a one-bead one-compound (OBOC) combinatorial library (Lam, et al., Nature 1991, 354:82-84).

[0130] The cell-penetrating peptide can also contain a nuclear localization signal (NLS). For example, the cell-penetrating peptide can be a bipartite amphiphilic peptide such as MPG derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni, et al., Nucl. Acids Res. 1993, 31:2717-24).

[0131] (iii) Carbohydrate conjugate. In certain embodiments, the siRNA oligonucleotides described herein further comprise a carbohydrate conjugate. Carbohydrate conjugates can be advantageous for compositions suitable for in vivo delivery and in vivo therapeutic use of nucleic acids. As used herein, "carbohydrate" is a compound that is a carbohydrate itself consisting of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched or cyclic) and oxygen, nitrogen or sulfur atoms bonded to each carbon atom; or, in part, a compound having a carbohydrate moiety consisting of one or more monosaccharide units each having at least 6 carbon atoms (which may be linear, branched or cyclic) and oxygen, nitrogen or sulfur atoms bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides and oligosaccharides containing about 4 to 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include sugars having C5 or more (in certain embodiments, C5 - C8); and disaccharides and trisaccharides include sugars having 2 or 3 monosaccharide units (in certain embodiments, C5 - C8).

[0132] In certain embodiments, the carbohydrate conjugate is selected from the group consisting of.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0133] Other representative carbohydrate conjugates used in the embodiments described herein are

Chemical formula

[0134] In certain embodiments, the carbohydrate conjugate further comprises other ligands including, but not limited to, a PK modulator, an endosomolytic ligand or a cell penetrating peptide.

[0135] (iv) Linker. In certain embodiments, the conjugates described herein can be attached to the siRNA oligonucleotide with a variety of linkers that can be cleavable or non-cleavable.

[0136] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound. Linkers generally contain an atomic chain such as a direct bond or an atom such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or 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 and alkynylheteroaryl (wherein one or more methylenes may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or substituted or unsubstituted heterocyclic), but are not limited thereto, where R8 is hydrogen, acyl, aliphatic or substituted aliphatic).In certain embodiments, the linker is 1 to 24 atoms, 4 to 24 atoms, 6 to 18 atoms, 8 to 18 atoms, or 8 to 16 atoms.

[0137] The cleavable linking group is sufficiently stable extracellularly, but once it enters the target cell, it cleaves to release the two moieties that the linker holds together. In certain embodiments, the cleavable linking group cleaves at least 10-fold or at least 100-fold faster in the target cell or under a first reference condition (e.g., selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (e.g., selected to mimic or represent conditions found in blood or serum).

[0138] The cleavable linking group is sensitive to cleavage factors, such as pH, redox potential, or the presence of degradative molecules. Generally, the cleavage factors are found predominantly or at higher levels or activities intracellularly than in serum or blood. Examples of such degradative factors include redox agents selected for or without substrate specificity, such as mercaptans present in cells that can cleave redox-cleavable linking groups upon reduction, including oxidative or reductive enzymes or reducing agents; esterases; endosomes or agents that can create an acidic environment resulting in a pH of 5 or less; enzymes, peptidases (which can be substrate-specific), and phosphatases that can hydrolyze or cleave acid-cleavable linking groups by acting as general acids. Cleavable linking groups, such as disulfide bonds, can be sensitive to pH. The pH of human serum is 7.4, whereas the average intracellular pH is slightly lower, in the range of about 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have cleavable linking groups that cleave at a specific pH, thereby releasing a cationic lipid from a ligand intracellularly or releasing it to a desired compartment of the cell.

[0139] The linker may include a cleavable linking group that is cleavable by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the target cell. For example, a liver targeting ligand may be attached to a cationic lipid via a linker that includes an ester group. Hepatocytes are rich in esterases, and thus the linker is cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include cells of the lung, renal cortex, and testis.

[0140] Linkers containing peptide bonds can be used when targeting cell types rich in peptidases, such as hepatocytes and synoviocytes.

[0141] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test the ability of a candidate cleavable linking group to withstand cleavage when contacted with blood or other non-target tissues. Thus, the relative sensitivity to cleavage between a first condition and a second condition can be determined, where the first is selected to be an indicator of cleavage in the target cell and the second is selected to be an indicator of cleavage in other tissues or body fluids, such as blood or serum. The evaluation can be performed in a cell-free system, cells, cell culture, organ or tissue culture, or whole animals. It can be useful to perform an initial evaluation under cell-free or culture conditions and confirm further evaluation in whole animals. In certain embodiments, a useful candidate compound is cleaved at least 2-fold, at least 4-fold, at least 10-fold, or at least 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0142] A group having a cleavable linking group is a redox-cleavable linking group that is cleaved by reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-S-S-). The methods described herein can be emulated to determine whether a cleavable linking group candidate is a suitable "reductively cleavable linking group" or is suitable for use, for example, with a particular RNAi 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 that mimic the cleavage rate observed in cells, e.g., target cells. A candidate can also be evaluated under conditions selected to mimic blood or serum conditions. In certain embodiments, a candidate compound is cleaved up to 10% in blood. In certain embodiments, a useful candidate compound is cleaved at least 2-fold, at least 4-fold, at least 10-fold, or at least 100-fold faster in cells (or in vitro conditions selected to mimic intracellular conditions) compared to blood (or in vitro conditions selected to mimic extracellular conditions). The cleavage rate of a candidate compound can be determined by using a standard enzyme kinetics assay under conditions selected to mimic the intracellular medium and comparing it to conditions selected to mimic the extracellular medium.

[0143] Phosphate-based cleavable linking groups are cleaved by agents that decompose or hydrolyze the phosphate group. Examples of agents that cleave phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are -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 certain embodiments, the phosphate-based linking group is selected from -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-Ρ(O)(Η)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S- and -O-P(S)(H)-S-. In a specific embodiment, the phosphate linking group is -O-P(O)(OH)-O-. These candidates can be evaluated using methods analogous to those described above.

[0144] An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In certain embodiments, the acid-cleavable linking group is decomposed in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less) or by an agent such as an enzyme that can act as a general acid. In cells, specific low-pH organelles such as endosomes and lysosomes can provide the 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. The acid-cleavable group may have the general formula -C=N-, C(O)O or -OC(O). In certain embodiments, the carbon (alkoxy group) bonded to the oxygen of the ester is an aryl group such as dimethylpentyl or t-butyl, a substituted alkyl group or a tertiary alkyl group. These can be evaluated using methods analogous to those described above.

[0145] Ester-based cleavable linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene groups, alkenylene groups, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These can be evaluated using methods analogous to those described above.

[0146] Peptide-based cleavable linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids, giving rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not contain an amide group. The amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids, giving rise to peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) between amino acids that give rise to peptides and proteins and do not contain a complete amide functional group. Peptide-based cleavable linking groups generally have the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These can be evaluated using methods analogous to those described above.

[0147] Representative carbohydrate conjugates with linkers include, but are not limited to, the following.

Chemical formula

Chemical formula

[0148] In certain embodiments of the compositions and methods, the ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives conjugated via a divalent or trivalent branched linker. For example, in certain embodiments, siRNA conjugates to a GalNAc ligand as shown in the following scheme.

Chemical formula

[0149] In certain embodiments, the combination therapy is of the formulae (XXXI)-(XXXIV)

Chemical formula

Chem.

Chem.

[0150] Examples of suitable divalent and trivalent branched linker groups conjugated to GalNAc derivatives include, but are not limited to, the structures shown above as formulas I, VI, X, IX and XII.

[0151] Representative U.S. patents that teach the preparation of RNA conjugates include U.S. Pat. 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, and 5,688,941, 6,294,664, 6,320,017, 6,576,752, 6,783,931, 6,900,297, and 7,037,646, each of which is hereby incorporated herein by reference for its teachings regarding such preparation methods.

[0152] In some cases, the RNA of siRNA can be modified by non-ligand groups. A number of non-ligand molecules have been conjugated to siRNA to enhance its activity, cellular distribution or cellular uptake, and methods for performing such conjugation are available from the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol (Kubo, T., et al., Biochem. Biophys. Res. Comm. 365(1):54-61 (2007); Letsinger, et al., Proc. Natl. Acad. Sci. USA 86:6553 (1989)), cholic acid (Manoharan, et al., Bioorg. Med. Chem. Lett. 4:1053 (1994)), thioethers such as hexyl-S-tritylthiol (Manoharan, et al., Ann. N.Y. Acad. Sci. 660:306 (1992); Manoharan, et al., Bioorg. Med. Chem. Let. 3:2765 (1993)), thiocolesterol (Oberhauser, et al., Nucl. Acids Res. 20:533 (1992)), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras, et al., EMBO J. 10:111 (1991); Kabanov, et al., FEBS Lett. 259:327 (1990); Svinarchuk, et al., Biochimie 75:49 (1993)), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan, et al., Tetrahedron Lett. 36:3651 (1995); Shea, et al., Nucl. Acids Res. 18:3777 (1990)), polyamines or polyethylene glycol chains (Manoharan, et al., Nucleosides & Nucleotides 14:969 (1995)) or adamantane acetic acid (Manoharan, et al., Tetrahedron Lett. 36:3651 (1195)), palmitoyl moiety (Mishra, et al., Biochim. Biophys. Acta 1264:229 (1995)) or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke, et al., J. Pharmacol. Exp. Ther. 277:923 (1996)).

[0153] Typical conjugation protocols involve the synthesis of RNA bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using an appropriate coupling or activating agent. The conjugation reaction can be carried out on the RNA still attached to the solid support or in solution phase after cleavage of the RNA. Purification of the RNA conjugate by HPLC generally yields a pure conjugate.

[0154] b. Pharmaceutical Compositions and Delivery of siRNA In certain embodiments, pharmaceutical compositions are provided that comprise the siRNAs described herein and a pharmaceutically acceptable carrier or additive. Pharmaceutical compositions containing siRNAs can be used for the treatment of HBV infection. Such pharmaceutical compositions are formulated based on the method of delivery. For example, the composition can be formulated for systemic administration via parenteral delivery, such as subcutaneous (SC) delivery.

[0155] "Pharmaceutically acceptable carrier" or "additive" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert medium for delivering one or more agents, such as nucleic acids, to an animal. The additive can be liquid or solid and is selected in a dosage method designed with consideration to provide the desired bulk, consistency, etc. when combined with the agent (e.g., nucleic acid) and other elements of a pharmaceutical composition. Typically pharmaceutically acceptable carriers or additives include binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, hydroxypropylmethylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate), but are not limited thereto.

[0156] Organic or inorganic pharmaceutically acceptable additives that do not react detrimentally with nucleic acids and are suitable for parenteral administration can also be used in the formulation of siRNA compositions. Suitable pharmaceutically acceptable carriers for formulations used in parenteral delivery include, but are not limited to, water, saline solutions, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0157] Formulations for topical administration of nucleic acids can include solutions of the nucleic acid in sterile and non-sterile aqueous solutions, non-aqueous solutions of common solvents such as alcohol, or liquid or solid oily bases. The solutions can also include buffers, diluents and other suitable additives. Organic or inorganic pharmaceutically acceptable additives that do not react detrimentally with nucleic acids and are suitable for parenteral administration can be used.

[0158] In certain embodiments, administration of the pharmaceutical compositions and formulations described herein can be local (e.g., by transdermal patch), pulmonary (e.g., by inhalation or insufflation of a powder or aerosol, including by nebulizer), intratracheal, intranasal, epithelial and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, and intramuscular injection or infusion; subcutaneous administration (e.g., via an implant device); or intracranial administration (e.g., intracerebral, intrathecal, or intraventricular administration).

[0159] In certain embodiments, the pharmaceutical composition comprises a sterile solution of HBV02 formulated in water for subcutaneous injection. In certain embodiments, the pharmaceutical composition comprises a sterile solution of HBV02 formulated in water for subcutaneous injection at a free acid concentration of 200 mg / mL.

[0160] In certain embodiments, a pharmaceutical composition comprising the siRNA described herein is administered in a dosage sufficient to inhibit the expression of the HBV gene. In certain embodiments, the dosage of siRNA ranges from 0.001 to 200.0 mg / kg recipient body weight / day or from 1 to 50 mg / kg body weight / day. For example, the siRNA is administered at 0.01 mg / kg, 0.05 mg / kg, 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 administration. The pharmaceutical composition may be administered once daily or may be administered using divided doses of 2, 3, or more at appropriate intervals throughout the day or via continuous infusion or delivery via a controlled release formulation. In this case, the siRNA contained in each divided dose must be correspondingly smaller in order to achieve the total daily dosage. Dosage units may also be combined for delivery over several days, for example, using conventional controlled release formulations that provide for the sustained release of the siRNA over several days. Controlled release formulations are well known in the art and are particularly useful for the delivery of drugs at specific sites such that they can be used with the drugs of the technology described herein. In such embodiments, the dosage unit contains the corresponding number of unit doses.

[0161] In certain embodiments, a pharmaceutical composition comprising an HBV-targeting siRNA described herein (e.g., HBV02) comprises siRNA at a dose of 0.8 mg / kg, 1.7 mg / kg, 3.3 mg / kg, 6.7 mg / kg or 15 mg / kg.

[0162] In certain embodiments, a pharmaceutical composition comprising an siRNA described herein (e.g., HBV02) comprises siRNA at a dose of 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg or 900 mg.

[0163] In certain embodiments, a pharmaceutical composition comprising an siRNA described herein (e.g., HBV02) comprises siRNA at a dose of 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg or 450 mg.

[0164] In certain embodiments, a pharmaceutical composition comprising an siRNA described herein (e.g., HBV02) comprises siRNA at a dose of 200 mg.

[0165] III. Treatment methods and additional therapeutic agents The present invention provides a method of treating HBV infection with an siRNA described herein. In certain embodiments, a method of treating HBV is provided, comprising administering HBV02 to a subject.

[0166] In certain embodiments of the above method, the method further comprises administering pegylated interferon-alpha (PEG-IFNα) to the subject.

[0167] In certain further embodiments of the above method, the method further comprises administering a nucleoside / nucleotide reverse transcriptase inhibitor (NRTI) to the subject. In certain embodiments, the NRTI is administered before, simultaneously with or after administration of HBV02.

[0168] In certain embodiments, a method of treating HBV is provided, comprising administering HBV02 and PEG-IFNα to a subject. In certain embodiments, PEG-IFNα is administered before, simultaneously with, or after the administration of HBV02.

[0169] In certain embodiments, a method of treating HBV is provided, comprising administering HBV02 and PEG-IFNα to a subject, wherein the subject has been previously administered an NRTI. In certain embodiments, PEG-IFNα is administered simultaneously with or after the administration of HBV02.

[0170] In certain embodiments, a method of treating HBV is provided, comprising administering HBV02 to a subject, wherein the subject has been previously administered PEG-IFNα and has been previously administered an NRTI.

[0171] In any of the above methods, the HBV infection can be chronic HBV infection.

[0172] As used herein, the "nucleoside / nucleotide reverse transcriptase inhibitor" or "nucleos(t)ide reverse transcriptase inhibitor" (NRTI) refers to an inhibitor of DNA replication that is structurally similar to a nucleotide or nucleoside, specifically inhibits the replication of HBV cccDNA by inhibiting the action of HBV polymerase, and does not significantly inhibit the replication of host (e.g., human) DNA. Such inhibitors include tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), lamivudine, adefovir, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clevudine, ritonavir, dipivoxil, lobucavir, famciclovir, N-acetyl-cysteine (NAC), PC1323, theradigm-HBV, thymosin-alpha, ganciclovir, besifovir (ANA-380 / LB-80380), and tenofvir-exaliades (TLX / CMX157). In certain embodiments, the NRTI is entecavir (ETV). In certain embodiments, the NRTI is tenofovir. In certain embodiments, the NRTI is lamivudine. In certain embodiments, the NRTI is adefovir or adefovir dipivoxil.

[0173] As used herein, the "subject" is any mammal that can be infected with HBV, such as primates (e.g., humans, non-human primates, such as monkeys or chimpanzees) or animals considered to be preclinical models permissive for HBV infection, HBV-AAV mouse models (e.g., Yang, et al., Cell and Mol Immunol 11:71 (2014)) or HBV 1.3xfs transgenic mouse models (Guidotti, et al., J. Virol. 69:6158 (1995)), such as mammals. In certain embodiments, the subject has a hepatitis B virus (HBV) infection. In certain embodiments, the subject is a human, such as a human having an HBV infection, particularly a chronic hepatitis B virus infection.

[0174] As used herein, the term "treating" or "treatment" refers to one or more signs or symptoms associated with unwanted HBV gene expression or HBV replication, such as the presence of serum or liver HBV cccDNA, the presence of serum HBV DNA, the presence of serum or liver HBV antigens, such as HBsAg or HBeAg, elevated ALT, elevated AST (normal range is generally considered to be 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, low-grade fever; myalgia; easy fatigability; taste and olfactory disturbances (aversion to food and tobacco); or right upper abdominal and epigastric pain (intermittent, mild to moderate); hepatic encephalopathy; drowsiness; abnormal sleep pattern; confusion; lethargy; ascites; gastrointestinal bleeding; coagulation disorder; jaundice; hepatomegaly (slightly enlarged, soft liver); splenomegaly; palmar erythema; spider nevus; muscle weakness; spider angioma; vasculitis; aneurysm bleeding; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (caput medusae); ALT level higher than AST level; elevated gamma-glutamyl transpeptidase (GGT) (normal range is generally considered to be about 8 - 65 U / L) and alkaline phosphatase (ALP) level (normal range is generally considered to be about 44 - 147 IU / L (international units / liter), not exceeding 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); 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; low platelet and white blood cell counts; lobules with inflammation associated with degenerative and regenerative stem cell changes; and beneficial or desired results including, but not limited to, reduction or improvement of predominantly centrilobular necrosis, whether detectable or not.For example, the likelihood of developing liver fibrosis, for example, in an individual with one or more risk factors for liver fibrosis, such as chronic hepatitis B infection, is reduced if the individual develops liver fibrosis at a lower severity compared to a population having the same risk factors and not receiving the treatment described herein. "Treatment" can also mean an extension of survival compared to the predicted survival period when not treated.

[0175] As used herein, the term "prevent" or "prevention" refers to not developing a disease, disorder or condition or reducing (e.g., by a clinically appropriate amount) the development of signs or symptoms associated with such disease, disorder or condition or presenting a sign delay or symptom delay (e.g., on a daily, weekly, monthly or yearly basis). Prevention may require more than one administration.

[0176] In certain embodiments, treatment of HBV infection results in a “functional cure” of hepatitis B. As used herein, functional cure is understood as the elimination of circulating HBsAg and may be accompanied by a conversion to a state where HBsAg antibodies are detectable using clinically relevant assays. For example, detectable antibodies may have a signal greater than 10 mIU / ml as measured by chemiluminescent microparticle immunoassay (CMIA) or any other immunoassay. Functional cure does not require the elimination of all forms of HBV replication (e.g., cccDNA from the liver). Anti-HBs seroconversion occurs spontaneously in about 0.2–1% of chronically infected patients each year. However, even after anti-HBs seroconversion, low levels of residual HBV are often observed for several years, indicating that a functional, rather than a complete cure has occurred. Without being bound to a particular mechanism, it is possible that the immune system can continue to monitor HBV under conditions where functional cure has been achieved. Functional cure allows for discontinuation of any treatment for HBV infection. However, it is understood that “functional cure” of HBV infection may not be sufficient for the prevention or treatment of diseases or conditions resulting from HBV infection, such as liver fibrosis, HCC, or cirrhosis. In certain specific embodiments, “functional cure” can be the persistence of reduced serum HBsAg, e.g., <1 IU / mL, for at least 3 months, at least 6 months, or at least 1 year after initiation or completion of a treatment regimen. To demonstrate functional cure of HBV, the public endpoints approved by the U.S. Food and Drug Administration or FDA are undetectable HBsAg in the blood at 6 months after treatment end, defined as <0.05 international units / milliliter or IU / ml, and HBV DNA below the lower limit of quantification.

[0177] As used herein, the term “hepatitis B virus-related disease” or “HBV-related disease” is a disease or disorder caused by or associated with HBV infection or replication. The term “HBV-related disease” includes diseases, disorders, or conditions that 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.

[0178] In one embodiment, the HBV-related disease is chronic hepatitis. Chronic hepatitis B is defined by one of the following criteria: (1) serum HBsAg, HBV DNA, or HBeAg positive on two occasions at least six months apart (any combination of these tests performed six months apart is acceptable); or (2) negative immunoglobulin M (IgM) antibody to HBV core antigen (IgM anti-HBc) and a positive result in one of the following tests: HBsAg, HBeAg, or HBV DNA (see Figure 2). Chronic HBV generally includes liver inflammation that persists for more than six months. Subjects with chronic HBV are HBsAg positive and have either high viremia (≥10 4 HBV-DNA copies / ml of blood) or low viremia (<10 3 HBV-DNA copies / ml of blood). In one embodiment, the subject has been infected with HBV for at least five years. In one embodiment, the subject has been infected with HBV for at least ten years. In one embodiment, the subject was infected with HBV at birth. Subjects with chronic hepatitis B disease may be immunotolerant or have non-active chronic infection without any evidence of active disease and are also asymptomatic. In particular, patients with chronic active hepatitis during the replication state may have symptoms similar to acute hepatitis. Subjects with chronic hepatitis B disease may have active chronic infection associated with necroinflammatory liver disease, have an increased hepatocyte turnover without detectable necroinflammation, or have non-active chronic infection without any evidence of active disease and are also asymptomatic. The persistence of HBV infection in chronic HBV subjects is a result of cccHBV DNA.

[0179] The HBeAg status represents multiple differences among subjects (Table 2). The HBeAg status can affect the response to various treatments, and approximately one-third of patients with HBV are HBeAg positive.

[0180]

Table 2

[0181] In certain embodiments, a subject having chronic HBV is HBeAg positive. In certain other embodiments, a subject having chronic HBV is HBeAg negative. A subject having chronic HBV has serum HBV DNA levels of less than 10 5 and persistent elevations of transaminases, such as ALT, AST, and gamma-glutamyl transferase. A subject having chronic HBV may have a liver biopsy score of less than 4 (e.g., necroinflammatory score).

[0182] In certain embodiments, the HBV-related disease is acute fulminant hepatitis B. A subject having acute fulminant hepatitis B has the symptoms of acute hepatitis and additional symptoms of confusion or coma (because the liver cannot detoxify chemicals) and subcutaneous bleeding or hemorrhage (because of a lack of clotting factors).

[0183] A subject having HBV infection, e.g., a subject having chronic HBV, may develop liver fibrosis. Thus, in certain embodiments, the HBV-related disease is liver fibrosis. Liver fibrosis or cirrhosis has historically been defined as a diffuse liver process characterized by fibrosis (excessive fibrous connective tissue) and the conversion of normal liver architecture into structurally abnormal nodules.

[0184] A subject having HBV infection, e.g., a subject having chronic HBV, may develop end-stage liver disease. Thus, in certain embodiments, the HBV-related disease is end-stage liver disease. For example, liver fibrosis may progress to the point where the body can no longer compensate for the results of liver fibrosis, e.g., decreased liver function (i.e., decompensated liver), which can result in, for example, mental and neurological symptoms and liver failure.

[0185] A subject having HBV infection, e.g., a subject having chronic HBV, may develop hepatocellular carcinoma (HCC), also referred to as malignant hepatoma. Thus, in certain embodiments, the HBV-related disease is HCC. HCC generally develops in subjects having chronic HBV and can be fibrolamellar, pseudoglandular (gland-like), pleomorphic (giant cell), or clear cell.

[0186] In certain embodiments of the methods and uses described herein, a therapeutically effective amount of siRNA, PEG-IFNα, or both, is administered to a subject. As used herein, "therapeutically effective amount" is intended to include an amount of an active agent that is sufficient to effect the treatment of a subject when administered to the subject for the treatment of HBV infection or an HBV-related disease (e.g., by reduction or maintenance of an existing disease or one or more symptoms of the disease). The "therapeutically effective amount" can vary depending on the active agent, its method of administration, the disease and its severity and history, age, weight, family history, genetic makeup, the stage of the pathological process mediated by HBV gene expression, if any, the type of prior or concurrent treatment, and other individual characteristics of the subject being treated. A therapeutically effective amount may require more than one administration.

[0187] "Therapeutically effective amount" also includes an amount of an active agent that produces a desired effect to some extent at a reasonable benefit / risk ratio applicable to any treatment. The therapeutic agents (e.g., siRNA, PEG-IFNα) used in the methods of the present invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio appropriate for such treatment.

[0188] As used herein, the term "sample" includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of body fluids can include blood, serum and serous fluid, plasma, lymph, urine, saliva, and the like. Tissue samples can include samples from tissue, organs, or localized regions. For example, a sample can be derived from a particular organ, a part of an organ, or the fluid or cells within these organs. In certain embodiments, a sample can be derived from the liver (e.g., the whole liver or a region of the liver or a particular type of cell of the liver, such as hepatocytes). In certain embodiments, "a sample derived from a subject" refers to blood drawn from the subject or plasma or serum obtained from the blood. In further embodiments, "a sample derived from a subject" refers to liver tissue (or a minor component thereof) or blood tissue (or a minor component thereof, e.g., serum) derived from the subject.

[0189] Certain embodiments of the present invention provide a method of treating chronic HBV infection or HBV-related diseases in a subject in need of treatment, comprising administering siRNA to the subject, wherein the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6) (wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyproline). In certain embodiments of the method, the method further comprises administering pegylated interferon-alpha (PEG-IFNα) to the subject. In certain embodiments, the siRNA and PEG-IFNα are administered to the subject over the same period. In certain embodiments, the siRNA is administered to the subject during a period prior to the administration of PEG-IFNα to the subject. In certain embodiments, PEG-IFNα is administered to the subject during a period prior to the administration of siRNA to the subject. In certain embodiments, the subject has been administered PEG-IFNα prior to the administration of siRNA. In certain embodiments, the subject is administered PEG-IFNα during the same period that the subject is being administered siRNA. In certain embodiments, the subject is administered PEG-IFNα subsequent to the administration of PEG-IFNα.

[0190] In certain embodiments of the above method, the method further comprises administering an NRTI to a subject. In certain embodiments of the above method, the subject to whom the siRNA is administered has been administered an NRTI prior to administration of the siRNA. In certain embodiments, the subject has been administered an NRTI for at least 2 months, at least 3 months, at least 4 months, at least 5 months or at least 6 months prior to administration of the siRNA. In certain embodiments, the subject has been administered an NRTI for at least 2 months prior to administration of the siRNA. In certain embodiments, the subject has been administered an NRTI for at least 6 months prior to administration of the siRNA. In certain embodiments, the subject is administered an NRTI for the same period of time that the subject is being administered the siRNA. In certain embodiments of the method, the subject is subsequently administered an NRTI after administration of the NRTI.

[0191] Certain embodiments of the present invention provide siRNAs for use in treating chronic HBV infection in a subject, wherein the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6) (wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyproline). In certain embodiments of the siRNA for use, the subject is also administered PEG-IFNα. In certain embodiments, the siRNA and PEG-IFNα are administered to the subject over the same period. In certain embodiments, the siRNA is administered to the subject during a period prior to the administration of PEG-IFNα to the subject. In certain embodiments, PEG-IFNα is administered to the subject during a period prior to the administration of siRNA to the subject. In certain embodiments, the subject has been administered PEG-IFNα prior to administration of the siRNA. In certain embodiments, the subject is administered PEG-IFNα during the same period that the subject is being administered the siRNA. In certain embodiments, the subject is administered PEG-IFNα sequentially. In any of the siRNAs for the above use, the subject may be administered an NRTI or may have been administered an NRTI previously. In certain embodiments, the subject has been administered an NRTI prior to administration of the siRNA. In certain embodiments, the subject has been administered an NRTI for at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months prior to administration of the siRNA.In certain embodiments, the subject has been administered an NRTI for at least 2 months prior to administration of the siRNA. In certain embodiments, the subject has been administered an NRTI for at least 6 months prior to administration of the siRNA. In certain embodiments, the subject is administered an NRTI for the same period of time that the subject is being administered the siRNA. In certain embodiments, the NRTI is administered sequentially.

[0192] Certain embodiments of the invention provide for the use of siRNA in the manufacture of a medicament for the treatment of chronic HBV infection, wherein the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6) (wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyproline).

[0193] Certain embodiments of the present invention provide for the use of siRNA and PEG-IFNα in the manufacture of a medicament for the treatment of chronic HBV infection, where the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6) (wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyproline).

[0194] Certain embodiments of the present invention provide for the use of siRNA, PEG-IFNα, and NRTI in the manufacture of a medicament for the treatment of chronic HBV infection, wherein the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6) (wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyproline).

[0195] In certain embodiments of the methods, compositions for use, or uses, the dosage of siRNA is 0.8 mg / kg, 1.7 mg / kg, 3.3 mg / kg, 6.7 mg / kg, or 15 mg / kg. In certain embodiments of the methods, compositions for use, or uses, the dosage of siRNA is 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, or 900 mg. In certain embodiments of the methods, compositions for use, or uses, the dosage of siRNA is 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 450 mg. In certain embodiments of the methods, compositions for use, or uses, the dosage of siRNA is 200 mg. In certain embodiments of the methods, compositions for use, or uses, the dosage of siRNA is at least 200 mg.

[0196] In the above method, composition for use or an embodiment in which it is used, the siRNA is administered weekly.

[0197] In the above method, composition for use or an embodiment in which it is used, the siRNA is administered more than once. For example, in one embodiment, the siRNA is administered twice, where the second administration is 2, 3 or 4 weeks after the first administration. In certain embodiments, the siRNA is administered twice, where the second administration is 4 weeks after the first administration.

[0198] In certain embodiments of the above method, the siRNA is administered 2, 3, 4, 5, 6 or more times. For example, in one embodiment, two 400 mg doses of siRNA are administered to the subject. In one embodiment, six 200 mg doses of siRNA are administered to the subject.

[0199] In the methods, compositions for use or embodiments in which they are used described herein, the method is (a) Administer to a subject at least two doses of an siRNA having a sense strand comprising at least 200 mg of 5’-gsusguGfcAfCfUfucgcuucacaL96-3’ (SEQ ID NO: 5) and an antisense strand comprising 5’-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3’ (SEQ ID NO: 6) (wherein a, c, g and u are 2’-O-methyladenosine-3’-phosphate, 2’-O-methylcytidine-3’-phosphate, 2’-O-methylguanosine-3’-phosphate and 2’-O-methyluridine-3’-phosphate, respectively; Af, Cf, Gf and Uf are 2’-fluoroadenosine-3’-phosphate, 2’-fluorocytidine-3’-phosphate, 2’-fluoroguanosine-3’-phosphate and 2’-fluorouridine-3’-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyproline); and (b) Administer to the subject a nucleoside / nucleotide reverse transcriptase inhibitor (NRTI); comprising, wherein the subject is HBeAg negative or HBeAg positive.

[0200] In certain embodiments, the method further comprises administering PEG-IFNα to the subject.

[0201] In the above method, composition for use or certain embodiments of the use, the siRNA is administered by subcutaneous injection. In certain embodiments, the siRNA is administered by one, two or three subcutaneous injections per dose.

[0202] In the above method, composition for use or certain embodiments of the use, the dose of PEG-IFNα is 50 μg, 100 μg, 150 μg or 200 μg. In certain embodiments, the dose of PEG-IFNα is 180 μg.

[0203] In the above method, composition for use or embodiment in use, PEG-IFNα is administered weekly.

[0204] In the above method, composition for use or embodiment in use, PEG-IFNα is administered by subcutaneous injection.

[0205] In the above method, composition for use or embodiment in use, the NRTI can be tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), lamivudine, adefovir, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clevudine, ritonavir, dipivoxil, lobucavir, famciclovir, N-acetyl-cysteine (NAC), PC1323, theradigm-HBV, thymosin-alpha, ganciclovir, besifovir (ANA-380 / LB-80380) or tenofvir-exaliades (TLX / CMX157). In certain embodiments, the NRTI is entecavir (ETV). In certain embodiments, the NRTI is tenofovir. In certain embodiments, the NRTI is lamivudine. In certain embodiments, the NRTI is adefovir or adefovir dipivoxil.

[0206] In the above method, composition for use or embodiment in use, the subject is HBeAg-negative. In certain embodiments, the subject is HBeAg-positive.

[0207] The siRNA may be present in the same pharmaceutical composition as the other active agent or in a different pharmaceutical composition with a different active agent. Such different pharmaceutical compositions can be administered in combination / simultaneously or at different times or at different locations (e.g., different parts of the body).

[0208] IV. Kit for HBV treatment Also provided herein is a kit comprising elements for the treatment of HBV. The kit may include siRNA (e.g., HBV02) and, optionally, one or both of (a) PEG-IFNα and (b) an NRTI (e.g., entecavir, tenofovir, lamivudine, or adefovir or adefovir dipivoxil). The kit may further include instructions for the preparation and / or administration of elements of the HBV combination therapy.

[0209] Certain embodiments of the invention provide a kit comprising a pharmaceutical composition comprising the above siRNA and a pharmaceutically acceptable additive; and a pharmaceutical composition comprising PEG-IFNα and a pharmaceutically acceptable additive. In certain embodiments, the kit further comprises an NRTI and a pharmaceutically acceptable additive.

Example

[0210] Example 1 Treatment of Chronic HBV Infection with HBV02 The safety, tolerability, pharmacokinetics (PK), and antiviral activity of HBV02 are evaluated in a Phase 1 / 2, randomized, double-blind, placebo-controlled clinical trial. The trial consists of three parts. Part A is a single-dose escalation design in healthy volunteers. Parts B and C are multiple-dose escalation designs in subjects with chronic HBV undergoing nucleoside (tide) reverse transcriptase inhibitor (NRTI) therapy. Subjects in Part B are HBeAg-negative; subjects in Part C are HBeAg-positive. HBeAg positivity reflects high-level active replication of the virus in the individual's hepatocytes.

[0211] In Part A, HBV02 is administered as a single dose to healthy adult subjects. Each administration can consist of up to two subcutaneous (SC) injections based on the assigned dose level. Part A includes four dose level cohorts of 50 mg, 100 mg, 200 mg, and 400 mg. Two sentinel subjects are randomized 1:1 to HBV02 or placebo. Administer concomitantly to the sentinel subjects and monitor for 24 hours; if the investigator has no safety concerns, administer to the remaining subjects in the same cohort. Randomize the remaining subjects 5:1 to HBV02 or placebo. Two optional cohorts in Part A following the same stratification, including sentinel dosing up to a maximum dose of 900 mg, may be added. In addition to the optional cohorts, up to eight "floater" subjects may be added to expand any cohort in Part A. Add "floater" subjects on a four-increment basis and randomize 3:1 to HBV02 or placebo. The Part A dose escalation plan is shown in Table 3. The single-dose escalation design for Part A is shown in Figure 3.

[0212]

Table 3

[0213] Review the data from Part A prior to initiating the dose level cohorts in subjects with chronic HBV infection. The cohort dosing strategies for Parts B / C of this trial are mutually adjusted; when two dose levels in Part A (1a: 50 mg and 2a: 100 mg) are completed, review the data prior to initiating dosing at the starting dose for Part B (1b: 50 mg). Initiate Part C at the starting dose for Part C (3c: 200 mg) simultaneously with the initiation of the equivalent Part B dose level cohort (3b: 200 mg).

[0214] The subjects in Part B are non-cirrhotic adult subjects who have HBeAg-negative chronic HBV infection, have received NRTI treatment for ≥6 months, and have a serum HBV DNA level <90 IU / mL. Screening includes non-invasive assessment of liver fibrosis, such as FibroScan assessment, unless there is a FibroScan assessment performed within 6 months prior to screening or a liver biopsy result performed within 1 year prior to screening to confirm that the subject does not have Metavir F3 fibrosis or F4 cirrhosis to exclude the presence of fibrosis or cirrhosis.

[0215] Administer HBV02 to the subjects twice, 4 weeks apart. Each administration may consist of up to 2 SC injections based on the assigned dose level. Part B includes 3 dose level cohorts of 50 mg, 100 mg, and 200 mg such that the cumulative doses received by the subjects in Part B are 100 mg, 200 mg, and 400 mg. Randomize each cohort 3:1 to HBV02 or placebo. Two optional cohorts in Part B may be added at 1.5-fold up to a maximum of 450 mg per administration (900 mg cumulative dose) following the same stratification. In addition to the optional cohorts, up to 16 "floater" subjects may be added to expand any of the cohorts in Part B. Add "floater" subjects in increments of 4 and randomize 3:1 to HBV02 or placebo. Start Cohort 1b after a cumulative review of all available safety data, including the 4-week test values and clinical data of the last available healthy volunteer subject in the 100 mg cohort (Cohort 2a). The dose escalation schedules for Parts B and C are shown in Table 4. The repeated dose escalation design for Parts B / C is shown in Figure 4.

[0216] The subjects in Part C are non-cirrhotic adult subjects who have HBeAg-positive chronic HBV infection, have received NRTI treatment for ≥6 months, and have a serum HBV DNA level <90 IU / mL. Screening includes a non-invasive assessment of liver fibrosis, such as FibroScan evaluation, unless there is a FibroScan evaluation performed within 6 months prior to screening or a liver biopsy result performed within 1 year prior to screening to confirm that the subject does not have Metavir F3 fibrosis or F4 cirrhosis to exclude the presence of fibrosis or cirrhosis. HBV02 is administered to the subjects twice, 4 weeks apart. Each time, it can consist of a maximum of 2 SC injections based on the assigned dose level. To accommodate the prediction of low prevalence in HBeAg-positive patients receiving NRTI treatment, only one dose level cohort (200 mg) in HBeAg-positive subjects is planned. Part C includes a 200 mg one-dose level cohort such that the cumulative dose received by the subjects in Part C is 400 mg. Randomize the cohort to HBV02 or placebo at a ratio of 3:1. Two optional cohorts in Part C may be added at 1.5-fold, up to a maximum of 450 mg per administration (900 mg cumulative dose), following the same stratification. In addition to the optional cohorts, a total of 16 "floater" subjects may be added to expand any of the cohorts in Part C. Add the "floater" subjects on a 4-increment basis and randomize them to HBV02 or placebo at a ratio of 3:1. The only planned cohort in Part C, cohort 3c, starts simultaneously with cohort 3b after review of all available safety data, including 6-week clinical data and laboratory value data from cohort 2b. Subjects in cohort 3c receive HBV02 at the same dose level as the subjects in cohort 3b (administer 200 mg twice at 4-week dosing intervals).

[0217]

Table 4

[0218] The summary of the test drug usage and dosage for Parts A - C is shown in Table 5 and Figures 5A and 5B.

Table 5

[0219] HBV02 is supplied as a sterile solution for SC injection with a free acid concentration of 200 mg / mL. The placebo is a sterile, preservative-free 0.9% normal saline solution for SC injection.

[0220] Record any adverse effects after administration of HBV02 or placebo. Also measure the PK parameters of HBV02 and possible metabolites, including for plasma: maximum concentration, time to maximum concentration, area under the concentration-time curve [up to the last measurable time point and to infinity], ratio of the extrapolated portion, apparent elimination half-life, clearance, and volume of distribution; and for urine: percentage excreted in urine and renal clearance. The following items are also determined: maximum decrease in serum HBsAg from day 1 to week 16; number of subjects who lost serum HBsAg at any time point; number of subjects who had sustained loss of serum HBsAg for more than 6 months; number of subjects who had anti-HBs seroconversion at any time point; number of subjects who had loss of HBeAg and / or anti-HBe seroconversion at any time point (for HBeAg-positive subjects only in part C); assessment of the effect of HBV02 on other markers of HBV infection, including detection of serum HBcrAg, HBV RNA, and HBV DNA; and assessment of potential biomarkers for the host response to infection and / or treatment, including genetic, metabolic, and proteomic parameters. To evaluate the PK parameters, collect blood samples before dosing (pre-dose ≤ 15 minutes), then at 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, and 48 hours post-dose; and collect urine samples before dosing (pre-dose ≤ 15 minutes), then at 0 - 4 hours, 4 - 8 hours, 8 - 12 hours, 12 - 24 hours, 48 hours, and 1 week post-dose and pool them. For subjects in part B or C, collect blood samples for measuring HBsAg, anti-HBs, HBeAg, anti-HBe, HBV DNA, HBV RNA, or HBcrAg at screening (28 - 1 days before dosing), day 1 (dosing), day 2 (post-dose), weekly during the dosing period, weekly for 4 weeks after dosing, 12 weeks after dosing, 16 weeks after dosing, 20 weeks after dosing, and 24 weeks after dosing at one or more time points.

[0221] Fasting is not required during the study.

[0222] Example 2 Treatment of chronic HBV with HBV02 alone or in combination with PEG-IFNα Evaluate the safety, tolerability, pharmacokinetics and antiviral activity of HBV02 alone or in combination with PEG-IFNα in a Phase 1 / 2 clinical trial. The trial consists of 4 parts. Parts A - C are randomized, double-blind, placebo-controlled clinical trials of HBV02 administered subcutaneously to healthy adult subjects or subjects with non-cirrhotic adult chronic HBV infection on NRTI treatment. Part A is a single-dose escalation design in healthy volunteers. Parts B and C are repeated-dose escalation designs in non-cirrhotic subjects with chronic HBV on NRTI treatment. Subjects in Part B are HBeAg-negative; subjects in Part C are HBeAg-positive. HBeAg positivity reflects high-level active replication of the virus in the individual's hepatocytes. Part D is a randomized, open-label Phase 2 trial of HBV02 administered alone or in combination with PEG-IFNα in non-cirrhotic adult subjects with chronic HBV on NRTI treatment; Part D includes HBeAg-positive and HBeAg-negative subjects.

[0223] In Part A, administer HBV02 as a single dose to healthy adult subjects. Each dose consists of up to 3 subcutaneous (SC) injections based on the assigned dose level. Part A includes 4 dose-level cohorts of 50 mg, 100 mg, 200 mg and 400 mg. Randomize 2 sentinel subjects 1:1 to HBV02 or placebo. Administer concomitantly to the sentinel subjects and monitor for 24 hours; if the investigator has no safety concerns, administer to the remaining subjects in the same cohort. Randomize the remaining subjects 5:1 to HBV02 or placebo. It is possible to add 2 optional cohorts in Part A following the same stratification, including sentinel dosing up to a maximum dose of 900 mg. In addition to the optional cohorts, it is possible to add a total of 8 "floater" subjects to expand any cohort in Part A. Add "floater" subjects on a 4-increment basis and randomize 3:1 to HBV02 or placebo. The single-dose escalation design for Part A is shown in Figure 3.

[0224] The subjects in Part B are non-cirrhotic adult subjects who have HBeAg-negative chronic HBV infection, have received NRTI treatment for ≥6 months, and have a serum HBV DNA level <90 IU / mL. To exclude the presence of fibrosis or cirrhosis, screening includes non-invasive assessment of liver fibrosis such as FibroScan evaluation. HBV02 is administered to the subjects twice, 4 weeks apart. Each time, it can consist of up to 2 SC injections based on the assigned dose level. Part B includes 3 dose level cohorts of 50 mg, 100 mg, and 200 mg such that the cumulative doses received by the subjects in Part B are 100 mg, 200 mg, and 400 mg. Each cohort is randomized to HBV02 or placebo at a ratio of 3:1. To accommodate the low prevalence prediction in HBeAg-positive patients on NRTI treatment, only 1 dose level cohort (200 mg) is planned for HBeAg-positive subjects. Two optional cohorts in Part B may be added following the same stratification up to a maximum of 450 mg per administration (900 mg cumulative dose). In addition to the optional cohorts, up to 16 "floater" subjects may be added to expand any of the cohorts in Part B. The "floater" subjects are added on a 4-increment basis and randomized to HBV02 or placebo at a ratio of 3:1. Cohort 1b is initiated after a cumulative review of all available safety data, including the 4-week test values and clinical data of the last available healthy volunteer subject in the 100 mg cohort (Cohort 2a). The dose escalation plans for Part B and C are shown in Table 5. The repeated dose escalation design for Part B / C is shown in Figure 4.

[0225] The subjects in Part C are non-cirrhotic adult subjects who have HBeAg-positive chronic HBV infection, have received NRTI treatment for ≥6 months, and have a serum HBV DNA level <90 IU / mL. HBV02 is administered to the subjects twice, 4 weeks apart. Each time, it may consist of a maximum of two SC injections based on the assigned dose level. Part C includes a 200 mg single-dose level cohort such that the cumulative dose received by the subjects in Part C is 400 mg. The cohorts are randomized 3:1 to HBV02 or placebo. Two optional cohorts in Part C may be added following the same stratification up to a maximum of 450 mg per administration (900 mg cumulative dose). In addition to the optional cohorts, up to 16 "floater" subjects may be added to expand any of the cohorts in Part C. The "floater" subjects are added on a 4-increment basis and randomized 3:1 to HBV02 or placebo.

[0226] The summary of the test article usage and dosage for Parts A - C is shown in Table 5 and Figures 5A and 5B.

[0227] The subjects in Part D are non-cirrhotic adult subjects who have HBeAg-positive or HBeAg-negative chronic HBV infection, have received NRTI treatment for ≥2 months, and have a serum HBV DNA level <90 IU / mL and a serum HBsAg level >50 IU / mL. The dose level and number of administrations of HBV02 in Part D are determined based on the analysis of the safety and tolerability of HBV02 in Parts A - C and the antiviral activity of HBV02 in Parts B and C. The dose level in Part D does not exceed the highest dose level evaluated in Parts B and C, and the number of administrations is a maximum of 6 times (e.g., 3 - 6 times) administered every 4 weeks. The subjects are randomized into one of Cohort 1d, Cohort 2d, Cohort 3d, and Cohort 4d (optional) (e.g., a total of 100 subjects, 25 subjects per cohort). In Cohort 1d, HBV02 is administered to the subjects at a frequency of every 4 weeks for a maximum of 6 times (e.g., 3 - 6 times). Each subject receives HBV02 on Day 1, Week 4, and Week 8, and may further receive it at Week 12, Week 16, and Week 20. In Cohort 2d, HBV02 is administered to the subjects at a maximum of 6 times (e.g., 3 - 6 times) at 4-week intervals, and PEG-IFNα is administered starting on Day 1 at a weekly dose for 24 weeks (i.e., each administration is 1 week apart). Each subject receives HBV02 on Day 1, Week 4, and Week 8, and may further receive it at Week 12, Week 16, and Week 20. In Cohort 3d, HBV02 is administered to the subjects at a maximum of 6 times (e.g., 3 - 6 times) at 4-week intervals, and PEG-IFNα is administered starting at Week 12 at a weekly dose for 12 weeks (i.e., each administration is 1 week apart). Each subject receives HBV02 on Day 1, Week 4, and Week 8, and may further receive it at Week 12, Week 16, and Week 20. In Cohort 4d, HBV02 is administered to the subjects 3 times at 4-week intervals, and PEG-IFNα is administered starting on Day 1 at a weekly dose for 12 weeks (i.e., each administration is 1 week apart). Each subject is administered HBV02 on Day 1, Week 4, and Week 8. The dose of PEG-INFα administered to the subjects in Cohorts 2d, 3d, and 4d is 180 μg and is administered by SC injection. Figures 6A - 6D are diagrams explaining the study design of Part D. The drug administration schedule for Cohort 4d is shown in Table 6.

[0228]

Table 6

[0229] To exclude the presence of cirrhosis, screening of subjects enrolled in Part B / C and Part D includes non-invasive assessment of liver fibrosis, such as FibroScan evaluation, unless the subject has no Metavir F3 fibrosis or F4 cirrhosis, as confirmed by FibroScan evaluation performed within 6 months before screening or liver biopsy results performed within 1 year before screening.

[0230] HBV02 is supplied as a sterile solution for SC injection with a free acid concentration of 200 mg / mL. The placebo is a sterile, preservative-free 0.9% normal saline solution for SC injection.

[0231] Record any adverse effects after administration of HBV02 or placebo. Also measure the PK parameters of HBV02 and possible metabolites, including plasma: maximum concentration, time to maximum concentration, area under the concentration-time curve [up to the last measurable time point and to infinity], ratio of the extrapolated portion, apparent elimination half-life, clearance and volume of distribution; urine: percentage excreted in urine and renal clearance. The following are also determined: maximum decrease in serum HBsAg from day 1 to week 16; number of subjects who lost serum HBsAg at any time point; number of subjects in whom serum HBsAg loss persisted for more than 6 months; number of subjects who had anti-HBs seroconversion at any time point; number of subjects who had HBeAg loss and / or anti-HBe seroconversion at any time point (only for HBeAg-positive subjects in parts C and D); assessment of the effect of HBV02 on other markers of HBV infection, including detection of serum HBcrAg, HBV RNA and HBV DNA; and assessment of potential biomarkers for host response to infection and / or treatment, including genetic, metabolic and proteomic parameters.

[0232] Review the data from part A prior to initiating the dose-level cohorts in subjects with chronic HBV infection. The cohort dosing strategies for parts B / C of this trial are staggered; the two dose levels in part A (1a: 50 mg and 2a: 100 mg) are completed and the data are reviewed prior to initiating dosing at the starting dose of part B (1b: 50 mg). Initiate part C at the starting dose of part C (3c: 200 mg) simultaneously with the initiation of the equivalent part B dose-level cohort (3b: 200 mg).

[0233] Fasting is not required during the course of the trial.

[0234] Figures 7A and 7B show the study designs for parts A - D.

[0235] Example 3 Treatment of chronic HBV with HBV02 alone or in combination with PEG-IFNα The safety, tolerability, pharmacokinetics, and antiviral activity of HBV02 were evaluated in a Phase 1 / 2 clinical trial. The trial consisted of four parts. Parts A - C were randomized, double - blind, placebo - controlled clinical trials of HBV02 administered subcutaneously to healthy adult subjects or subjects with non - cirrhotic adult chronic HBV infection on NRTI therapy. Part A was a single - dose escalating design in healthy volunteers. Parts B and C were repeated - dose escalating designs in non - cirrhotic subjects with chronic HBV on NRTI therapy. Subjects in Part B were HBeAg - negative; subjects in Part C were HBeAg - positive. HBeAg - positivity reflects high - level active replication of the virus in an individual's hepatocytes. HBeAg - positive patients are generally older and are thought to be generally younger and have more preserved immune function compared to HBeAg - negative patients who experience significant immune exhaustion. HBeAg - negative patients are also thought to have a greater amount of integrated DNA compared to HBeAg - positive patients. Part D was a randomized, open - label Phase 2 trial of HBV02 administered alone or in combination with PEG - IFNα to non - cirrhotic adult subjects with chronic HBV on NRTI therapy; Part D included HBeAg - positive and HBeAg - negative subjects.

[0236] i. Preliminary animal administration studies The doses of HBV02 used in the studies were determined by calculating the human equivalent doses (HEDs) of the no - observed - adverse - effect levels (NOAELs) in animal toxicity studies and applying a safety margin to these HEDs. Body surface area (m / kg 2)The conversion factor was used for the calculation of the HED of the animal dose. In the rat Good Laboratory Practice (GLP) test, at the maximum test dose of 150 mg / kg corresponding to an HED of 24 mg / kg / dose, no toxicity was observed after three bi-weekly administrations of HBV02 (Table 7). In the non-human primate (NHP) GLP test, at the maximum test dose of 300 mg / kg corresponding to an HED of 97 mg / kg / dose, no toxicity was observed after three bi-weekly administrations of HBV02 (Table 7). Using this method, the proposed starting dose of 0.8 mg / kg in humans represents a 30-fold safety margin of the HED of the predicted NOAEL in rats and a 120-fold safety margin of the HED of the predicted NOAEL in NHP. Other siRNAs using the GalNAc platform have shown significant liver target binding at 1 - 15 mg / kg. Furthermore, in the preclinical HBV mouse model, a statistically significant decrease in HBsAg was observed in the dose range of 1 - 9 mg / kg.

[0237]

Table 7

[0238] Similar to other GalNAc-conjugated iRNAs, HBV02 is taken up by the liver and has minimal distribution to other organs and tissues, so a fixed dose of HBV02 was used in the clinical trial. Therefore, dosing based on body weight is not predicted to reduce the inter-individual variability of the pharmacokinetics (PK) of HBV02 in adults, and the fixed dose has the advantage of avoiding the possibility of dosing calculation errors.

[0239] ii. Method The test design is shown in Figure 12.

[0240] In Part A, HBV02 was administered to healthy adult subjects as a single dose. Each dose consisted of up to three subcutaneous (SC) injections based on the assigned dose level. Six dose-level cohorts were included in Part A: 50 mg, 100 mg, 200 mg, 400 mg, 600 mg, and 900 mg. Two sentinel subjects were randomized 1:1 to HBV02 or placebo. The sentinel subjects were co-administered and monitored for 24 hours; if the investigators had no safety concerns, the remaining subjects in the same cohort were administered.

[0241] Subjects in Part B were non-cirrhotic adult subjects with HBeAg-negative chronic HBV infection who had received ≥6 months of NRTI therapy and had serum HBV DNA levels <90 IU / mL. To exclude the presence of fibrosis or cirrhosis, screening included a non-invasive assessment of hepatic fibrosis. HBV02 was administered to the subjects twice, 4 weeks apart (i.e., on Day 1 and Day 29). Each administration consisted of up to two SC injections based on the assigned dose level. Six cohorts at doses of 20 mg, 50 mg, 100 mg, or 200 mg were included in Part B such that the cumulative dose received by the subjects in Part B was 40 mg, 100 mg, 200 mg, or 400 mg. Each cohort was randomized 3:1 to BV02 or placebo. The 50 mg cohort in Part B was initiated after a cumulative review of all available safety data, including the Week 4 test values and clinical data of the last available healthy volunteer subjects in the 100 mg cohort.

[0242] The subjects in Part C were non-cirrhotic adult subjects who had HBeAg-positive chronic HBV infection, had received ≥6 months of NRTI treatment, and had a serum HBV DNA level <90 IU / mL. To be eligible for prediction of the low prevalence in HBeAg-positive patients on NRTI treatment, only 2 dose-level cohorts (50 mg and 200 mg) were included for HBeAg-positive subjects. HBV02 was administered to the subjects 2 times, 4 weeks apart (i.e., on Day 1 and Day 29). Each administration consisted of a maximum of 2 SC injections based on the assigned dose level. Part C included 2 dose-level cohorts of 50 mg and 200 mg such that the cumulative dose received by the subjects in Part C was 100 mg or 400 mg. The cohorts were randomized 3:1 to BV02 or placebo.

[0243] Patients with chronic HBV who experienced a >10% decrease from baseline serum HBsAg in HBsAg at Week 16 were followed for up to an additional 32 weeks.

[0244] The inclusion criteria for Parts B and C included age 18 - 65 years; ≥6 months of detectable serum HBsAg; ≥6 months of NRTI treatment; HBsAg >150 IU / mL; HBV DNA <90 IU / mL; and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤2 × the upper limit of normal (ULN). The exclusion criteria included significant fibrosis or cirrhosis (FibroScan >8.5 kPa at screening or Metavir F3 / F4 on liver biopsy within 1 year); bilirubin, international normalized ratio (INR), or prothrombin time >ULN; active HIV, HCV, or delta hepatitis virus infection; and creatinine clearance <60 mL / min (Cockcroft-Gault).

[0245] The subjects in Part D are non-cirrhotic adult subjects who have HBeAg-positive or HBeAg-negative chronic HBV infection, have received NRTI treatment for ≥2 months, and have serum HBV DNA levels <90 IU / mL and serum HBsAg levels >50 IU / mL. The dose level and number of administrations of HBV02 in Part D are determined based on the analysis of the safety and tolerability of HBV02 in Parts A - C and the antiviral activity of HBV02 in Parts B and C. The dose level in Part D does not exceed the highest dose level evaluated in Parts B and C, and the number of administrations is a maximum of 6 times (e.g., 3 - 6 times) administered every 4 weeks. The subjects are randomized into one of Cohort 1d, Cohort 2d, Cohort 3d, and Cohort 4d (optional) (e.g., a total of 100 subjects, 25 subjects per cohort). In Cohort 1d, HBV02 is administered to the subjects at a frequency of every 4 weeks for a maximum of 6 times (e.g., 3 - 6 times). Each subject receives HBV02 on Day 1, Week 4, and Week 8, and may further receive it at Week 12, Week 16, and Week 20. In Cohort 2d, HBV02 is administered to the subjects at a maximum of 6 times (e.g., 3 - 6 times) at 4-week intervals, and PEG-IFNα is administered starting on Day 1 at a weekly dose for 24 weeks (i.e., each administration is 1 week apart). Each subject receives HBV02 on Day 1, Week 4, and Week 8, and may further receive it at Week 12, Week 16, and Week 20. In Cohort 3d, HBV02 is administered to the subjects at a maximum of 6 times (e.g., 3 - 6 times) at 4-week intervals, and PEG-IFNα is administered starting at Week 12 at a weekly dose for 12 weeks (i.e., each administration is 1 week apart). Each subject receives HBV02 on Day 1, Week 4, and Week 8, and may further receive it at Week 12, Week 16, and Week 20. In Cohort 4d, HBV02 is administered to the subjects 3 times at 4-week intervals, and PEG-IFNα is administered starting on Day 1 at a weekly dose for 12 weeks (i.e., each administration is 1 week apart). Each subject is administered HBV02 on Day 1, Week 4, and Week 8. The dose of PEG-INFα administered to the subjects in Cohorts 2d, 3d, and 4d is 180 μg and is administered by SC injection. Figures 6A - 6D are diagrams explaining the study design of Part D. The drug administration schedule for Cohort 4d is shown in Table 8.

[0246]

Table 8

[0247] To exclude the presence of cirrhosis, the screening of subjects enrolled in Parts B and C included a non-invasive assessment of liver fibrosis, such as FibroScan evaluation, unless there was a FibroScan evaluation performed within 6 months before screening or a liver biopsy result performed within 1 year before screening that confirmed the absence of Metavir F3 fibrosis or F4 cirrhosis in the subject. The same method was used to exclude cirrhotic subjects from entering Part D.

[0248] HBV02 was supplied as a sterile solution for SC injection with a free acid concentration of 200 mg / mL. The placebo was a sterile, preservative-free 0.9% normal saline solution for SC injection.

[0249] Adverse events were recorded after HBV02 or placebo administration. PK parameters of HBV02 and possible metabolites were also measured, including in plasma: maximum concentration, time to maximum concentration, area under the concentration-time curve [up to the last measurable time point and to infinity], ratio of the extrapolated portion, apparent elimination half-life, clearance and volume of distribution; and in urine: percentage excreted in urine and renal clearance. The following were also determined: maximum decrease in serum HBsAg from Day 1 to Week 16; number of subjects who lost serum HBsAg at any time point; number of subjects in whom serum HBsAg loss persisted for more than 6 months; number of subjects who seroconverted to anti-HBs at any time point; number of subjects who lost HBeAg and / or seroconverted to anti-HBe at any time point (for HBeAg-positive subjects only in Parts C and D); assessment of the effect of HBV02 on other markers of HBV infection, including detection of serum HBcrAg, HBV RNA and HBV DNA; and assessment of potential biomarkers for host response to infection and / or treatment, including genetic, metabolic and proteomic parameters. To evaluate the PK parameters of subjects in Part A, blood samples were collected pre-dose (pre-dose ≤ 15 minutes), then at 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours and 48 hours post-dose; and urine samples were collected pre-dose (pre-dose ≤ 15 minutes), then pooled from 0 - 4 hours, 4 - 8 hours, 8 - 12 hours, 12 - 24 hours, 48 hours and 1 week post-dose. For subjects in Part B or C, blood samples for measuring HBsAg, anti-HBs, HBeAg, anti-HBe, HBV DNA, HBV RNA or HBcrAg were collected at one or more of the time points of screening (28 - 1 days before dosing), Day 1 (dosing), Day 2 (post-dose), weekly during the dosing period, weekly every 4 weeks post-dose, 12 weeks after dosing, 16 weeks after dosing, 20 weeks after dosing and 24 weeks after dosing.

[0250] Data from Part A was reviewed prior to initiation of the dose-level cohorts in subjects with chronic HBV infection. The cohort dosing strategies for Parts B / C of this trial were staggered; two dose levels in Part A (50 mg and 100 mg) were completed and the data were reviewed prior to initiation of dosing at the starting dose in Part B (50 mg). Part C was initiated at the starting dose for Part C (200 mg) simultaneously with the initiation of the equivalent Part B dose-level cohort (200 mg).

[0251] Fasting was not required during the course of the trial.

[0252] iii. Preliminary Results from Parts A and B Figure 9A shows the Part A, Part B, and Part C study designs at the time of completion of dosing in Part A cohorts 1-5 (50 mg, 100 mg, 200 mg, 400 mg, 600 mg) and Part B cohorts 1-2 (50 mg, 100 mg). Figure 9B depicts completion of dosing and subject withdrawals across different cohorts for Part A cohorts 1-5. Figure 9C depicts completion of dosing and subject withdrawals across different cohorts for Part B cohorts 1-2.

[0253] Preliminary demographic data for the subjects included in Parts A and B are shown in Table 9 below.

[0254]

Table 9

[0255] An overview of adverse events (AEs) from the preliminary analysis of the completed dosing portions of Parts A and B is shown in Table 10.

Table 10

[0256] The subjects in Parts A and B showed no significant abnormalities in test values, hyperbilirubinemia, or elevated INR. Some subjects in Parts A and B showed abnormal liver function test values (Figures 10A, 10B, and 11). Among the 41 subjects in Part A, 2 subjects had elevated ALT 1 day before dosing (normal ALT at screening). In Part B, among the 12 subjects, 1 subject showed elevated Grade 1 ALT (39 U / L, 1.1×ULN) and AST (50 U / L, 1.5×ULN) at week 8. One subject in Cohort 3a (200 mg) where ALT was at the upper limit of normal on day 29 had associated excessive exercise and high creatine kinase (CK: 5811 U / L). Two subjects in Cohort 4a (400 mg) had ALT exceeding the upper limit of normal 1 day before dosing. One subject where excessive exercise was permitted had a high CK of 20,001 U / L and discontinued on day 2 unrelated to adverse events. The second subject with elevated ALT resolved by day 8 without intervention. As shown in Figure 11, one female subject in Cohort 2b (100 mg) showed elevated Grade 1 ALT at week 8.

[0257] Subjects from Part B showed a time-dependent decrease in HBsAg in the active groups of Cohorts 1 and 2. Figure 12A depicts the changes in HBsAg in Cohorts 1b (50 mg) and 2b (100 mg) receiving HBV02 or placebo. Figure 12B depicts the changes in HBsAg in Cohorts 1b and 2b only for subjects receiving HBV02. In Cohort 4b (20 mg × 2 groups), the subjects had a 0.47 log decrease 1 week after the first dose.

[0258] Figure 12C shows the mean change in HBsAg from day 1 to week 4 or week 20 (depending on the cohort) after HBV02 administration in Cohorts 1b and 2b for 3 subjects (HBeAg-negative) with chronic HBV infection receiving 50 mg of HBV02 on day 1 and day 28 and 6 subjects receiving 100 mg on day 1 and day 28. In the 50 mg cohort, the mean decrease at week 12 after two doses was 1.5 log 10 or a decrease of approximately 30-fold. All subjects in this cohort had a decrease of 0.6 - 2.2 log 10The apparent maximum decrease in HBsAg within the range was reached. In the 100 mg cohort, all subjects reached an average decrease of 0.7 log 10 or approximately 6-fold decrease by week 4 after a single dose.

[0259] Among the 10 HBeAg-negative subjects in part B, 7 subjects were good responders, showing a 0.29 - 0.95 log decrease in HBsAg 1 week after the first dose (20 mg, 50 mg, or 100 mg). Two out of 10 subjects were moderate responders, showing a 0.06 - 0.21 log decrease in HBsAg 1 week after the first dose of 20 mg, 50 mg, or 100 mg. Finally, 1 out of 10 subjects was a "non-responder", showing a 0.16 log increase in HBsAg 1 week after the first dose. Reasons for the possible existence of moderate and non-responders include dose response, pharmacokinetics, viral resistance, and host factors.

[0260] HBV02 had good tolerance in the subjects. Single doses in the range of 50 - 600 mg had good tolerance in healthy volunteer subjects. Two doses in the range of 50 - 100 mg had good tolerance in HBeAg-negative subjects. There was high inter-patient variability in HBsAg reduction, and it rebounded 12 weeks after the last dose.

[0261] iv. Demographics and baseline characteristics - Parts A, B, and C The demographics and baseline characteristics of the subjects in parts A, B, and C are shown in Tables 11, 12, and 13, respectively. All subjects in parts B and C were NRTI-suppressed and had a FibroScan ≤ 8.5 kPa or Metavir F0 / F1 / F2.

[0262] [Table 11] SD = standard deviation. a Including alternating volunteers

[0263] [Table 12] SD = Standard Deviation.

[0264]

Table 13

[0265] v. Safety and Tolerability - Results from Parts A, B, and C Preliminary data from Parts A, B, and C were obtained based on 37 healthy volunteers who received HBV02; 12 healthy volunteers who received placebo; 24 patients with chronic HBV on NRTI who received HBV02; and 8 patients with chronic HBV on NRTI who received placebo. HBV02 was generally well tolerated.

[0266] Among healthy volunteers and patients with chronic HBV, HBV02 was generally well tolerated in healthy volunteers given as a single dose up to 900 mg and in patients given as two doses of 20 mg, 50 mg, 100 mg, or 200 mg each. Clinically significant alanine transaminase (ALT) abnormalities, which are markers of liver inflammation, were not observed up to week 16 in patients with chronic HBV (Parts B and C) (Figures 13A - 13E). Grade ≥2 ALT elevations, levels of bilirubin >ULN, or clinically relevant changes or trends in other test value parameters, vital signs, or ECG were not observed.

[0267] For Part A, the post-baseline ALT increase to ULN was not correlated with the increase in bilirubin ULN. Changes in the functional state of the liver (e.g., albumin, coagulation parameters) or clinical signs / symptoms of liver dysfunction were not observed in any HBV02 treatment subjects. Transient ALT increases were observed in 1 / 6 (17%) and 4 / 6 (67%) of the subjects after single doses of 1 mg / kg and 3 mg / kg of HBV02, respectively. These increases were asymptomatic and were not accompanied by hyperbilirubinemia. In contrast, ALT increases potentially associated with HBV02 were not observed with single doses of HBV02 in the range of 50 - 600 mg (about 0.8 - 10 mg / kg). In the Part A, 900 mg (about 15 mg / kg) cohort, mild, asymptomatic grade 1 ALT increases without bilirubin changes were seen in some of the subjects (5 / 6 of the subjects had ALT increases of 1.1 - 2.6 × ULN). The ALT levels of the subjects in Part A, including a comparison with subjects administered HBV01 (a similar siRNA without GNA modification), are shown in Figure 14. These results suggest that the incorporation of ESC+ technology (providing enhanced stability and minimized off-target activity through the incorporation of GNA modification) reduces the properties of siRNA that cause ALT increases in healthy volunteers at clinically relevant dose levels.

[0268] No dose-related trend in the frequency of adverse events was observed. Most of the adverse events that occurred under treatment were of mild severity, and no patients discontinued due to adverse events. The most common adverse event was headache (6 / 24, 25%). Three grade 3 adverse events of upper respiratory tract infection, chest pain, and low blood phosphate levels were reported but were not considered related to HBV02. One grade 3 adverse event of hypophosphatemia was observed in a patient receiving tenofovir disoproxil fumarate. No two serious adverse events or SAEs were reported in Part B. The first was a grade 2 headache, which resolved with intravenous fluids and non-opioid analgesics. This patient also had symptoms of fever, nausea, vomiting, and dehydration, which were considered consistent with a viral syndrome. The second was an SAE, grade 4 depression, which occurred more than 50 days after the last dose and was evaluated as unrelated to HBV02 treatment.

[0269] A summary of the adverse events that occurred under treatment is shown in Table 14.

[0270]

Table 14

[0271] vi. Pharmacokinetics - Results from Part A Preliminary pharmacokinetic (PK) data from a first-in-human phase 1 randomized, blinded, placebo-controlled, dose-escalation study of HBV02 in healthy volunteers were analyzed. Plasma samples were evaluated in a 6-single-dose escalation cohort of 8 subjects (6:2 active:placebo) who received a single subcutaneous (SC) dose of HBV02 in the range of 50 - 900 mg.

[0272] Eligibility criteria included age 18 - 55 years; body mass index (BMI) 18.0 - ≤32 kg / m 2 ; CLcr < 90 mL / min (Cockcroft-Gault); and no clinically significant ECG abnormalities or clinically significant chronic medical conditions.

[0273] Intensive plasma and urine PK samples were collected for 1 week. Serial plasma samples were collected over 24 hours, 48 hours and 1 week after dosing. Spot urine samples were collected over 24 hours and single void urine samples were collected 48 hours and 1 week after dosing. Concentrations of HBV02 and (N-1)3’ HBV02 antisense metabolites in plasma and urine were measured using a validated liquid chromatography tandem mass spectrometry assay (lower limit of quantification (LLOQ) of 10 ng / mL in plasma and urine). PK parameters were estimated using the standard non-compartmental method of WinNonlin®, V6.3.0 (Certara L.P., Princeton, NJ). S(N-1)3’ HBV02, a primary circulating metabolite with potency equivalent to HBV02, is formed by the loss of 1 nucleotide from the 3’ end of the antisense strand of HBV02.

[0274] Figures 15A and 15B show the plasma concentration-time profiles of HBV02 and AS(N-1)3’ HBV02, respectively, after single SC administration to healthy volunteers. HBV02 showed linear kinetics in plasma after SC injection. HBV02 was absorbed after SC injection with a median T max of 4 - 8 hours. HBV02 was undetectable in plasma at 48 hours in all subjects, consistent with rapid GalNAc-mediated hepatic uptake; the median apparent elimination half-life (t 1 / 2 ) was in the range of 2.85 - 5.71 hours. The short plasma half-life may represent the distribution half-life (Agarwal S, et al., Clin Pharmacol Ther. 2020 Jan 29, doi: 10.1002 / cpt.1802). Rapid conversion of HBV02 to its (N-1)3’ metabolite, designated AS(N-1)3’ HBV02, was observed. AS(N-1)3’ HBV02 had a median T max of 2 - 10 hours, was quantifiable only at doses ≥100 mg, and concentrations were generally approximately 10-fold lower compared to HBV02.

[0275] HBV02 plasma exposure (AUC 0-12 and C max) increased in a dose-proportional manner up to 200 mg and appeared to show a slightly greater increase than dose-proportional increase at doses above 200 mg (Figure 16; Figure 17; Table 15). After single SC administration of 50 - 900 mg of HBV02, the area under the plasma curve (AUC last ) and the mean maximum concentration (C max ) increased with dose, with mean exposures in the ranges of 786 - 74,700 ng*h / mL and 77.8 - 6010 ng / mL, respectively. A similar trend was observed with AS(N-1)3’ HBV02. These results indicate transient saturation of ASGPR-mediated hepatic uptake of HBV02 at high doses, resulting in anti-circulating concentrations (see Agarwal et al., 2020, supra).

[0276]

Table 15

[0277] Inter-patient variability in HBV02 plasma PK parameters was generally low (about 30%).

[0278] The most predominant active metabolite, AS(N-1)3’ HBV02 (about 12%), was as effective as HBV02. AS(N-1)3’ HBV02 was detectable in plasma of 0 / 6 subjects at 50 mg, 3 / 6 subjects at 100 mg, and all subjects at 200 mg, 400 mg, 600 mg, and 900 mg. The PK profile of the metabolite was similar to that of HBV02, and the AUC last value and C max value of AS(N-1)3’ HBV02 in plasma were ≤11% of those of HBV02.

[0279] The AUC 0-12 and C max of AS(N-1)3’ HBV02 in plasma were ≤11% of the total drug-related substances.

[0280] Figure 18 shows an overview of the plasma PK parameters of HBV02 and AS(N-1)3’ HBV02 observed after single SC administration to healthy volunteers.

[0281] The urine concentration-time profiles of HBV02 and AS(N-1)3’Hepatitis B virus 02 are shown in Figures 19A and 19B, respectively. Low levels of HBV02 and AS(N-1)3’ HBV02 were observed in urine up to the last measurement time point at 1 week after dosing across all cohorts. The PK profile of urinary HBV02 reflected that of plasma that could be calculated.

[0282] The summary of the urinary PK parameters of HBV02 and AS(N-1)3’ HBV02 in healthy volunteers is shown in Figure 20. In the first 24 hours, approximately 17 - 46% and 2 - 7% of the administered doses (50 - 900 mg), respectively, were excreted in urine as unchanged HBV02 and AS(N-1)3’ HBV02. Over 24 hours after dosing, the percentage of HBV02 excreted in urine increased with the dose level. This may be due to the fact that the rate of HBV02 liver uptake by ASGPR is greatly in excess of renal excretion (see Agarwal et. al, 2020, supra), reflecting a supra-proportional increase with plasma HBV02. The renal clearance of HBV02 approached the glomerular filtration rate.

[0283] These preliminary data indicate that HBV02 demonstrated favorable PK properties in healthy volunteers.

[0284] vii. Efficacy - Results of Parts B and C Preliminary data were obtained from B and C for 24 patients with chronic HBV on NRTI who received HBV02; and 8 patients with chronic HBV on NRTI who received placebo. Initial data showed a substantial decrease in HBsAg in patients at doses in the range of 20 mg to 200 mg.

[0285] The biological activity of HBV02 was evaluated by the decrease in HBsAg. The activity of HBV02 up to week 16 in the 200 mg cohort of part B (HBeAg-negative) and part C (HBeAg-positive) is shown in Figures 21A and 21B. For parts B and C, the mean baseline HBsAg levels were 3.3 log 10 IU / mL and 3.9 log 10 IU / mL, respectively. The mean decrease in HBsAg across HBeAg-negative and HBeAg-positive subjects at week 16 was 1.5 log 10 or a ~32-fold decrease. The observed decrease in HBsAg at week 16, after two 200 mg doses of HBV02 separated by 4 weeks, was in the range of 0.97 log 10 to 2.2 log 10 or a ~9- to 160-fold decrease. The mean HBsAg level at week 16 was 314 IU / mL, with half of the patients achieving an HBsAg value <100 IU / mL and 5 / 6 achieving an HBsAg value <1000 IU / mL.

[0286] The change in HBsAg from baseline to week 16 at each dose is shown in Figure 22. The percentage of patients with an HBsAg level <100 IU / mL at week 24 was 33% in patients receiving 20 mg of HBV02, 44% in patients receiving 50 mg of HBV02, 50% in patients receiving 100 mg of HBV02, and 50% in patients receiving 200 mg of HBV02. The maximum change in HBsAg from individual baselines is shown in Figure 23. Similar decreases were observed in HBeAg-positive and HBeAg-negative patients. The mean changes in HBsAg observed at week 24 in patients administered 20 mg, 50 mg, 100 mg, and 200 mg of HBV02 were -0.76 log 10 , -0.93 log 10 , -1.23 log 10 and -1.43 log 10 , respectively. All 6 patients who received two doses of 200 mg achieved a ≧1.0 log 10 decrease in HBsAg. The individual changes in HBsAg from baseline at week 24 are shown in Figure 24, demonstrating the dose-dependent persistence of HBsAg reduction.

[0287] These results showed that no safety signal was observed and HBV02 had good tolerability. Dose-dependent HBsAg reduction in both HBeAg-negative and HBeAg-positive patients was observed in the dose range of 20 - 200 mg of HBV02 (two deliveries), which was persistent for at least 6 months at high doses. Similar HBsAg reduction was observed in both HBeAg-negative and HBeAg-positive patients, indicating that HBV02 can reduce the patients' HBsAg regardless of the disease stage. All patients who received 200 mg twice achieved a ≧ -1log 10 reduction in HBsAg, and at week 24, the mean reduction in HBsAg was -1.43log 10 . Overall, these results support the potential of HBV02 as the center of a limited treatment regimen aimed at the functional cure of chronic HBV infection. In particular, the ability of HBV02 to significantly reduce HBsAg after only two administrations suggests that HBV02 may have an important role in the functional cure of chronic HBV.

[0288] Although specific embodiments have been described and set forth, it will be readily recognized that the various embodiments can be combined with additional embodiments and can be varied in various ways without departing from the spirit and scope of the invention.

[0289] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications cited herein or listed on the filing data sheet, including U.S. Provisional Patent Application 62 / 846,927 filed on May 13, 2019, U.S. Provisional Patent Application 62 / 893,646 filed on August 29, 2019, U.S. Provisional Patent Application 62 / 992,785 filed on March 20, 2020, U.S. Provisional Patent Application 62 / 994,177 filed on March 24, 2020, and U.S. Provisional Patent Application 63 / 009,910 filed on April 14, 2020, are hereby incorporated by reference in their entirety herein, unless otherwise expressly stated. The embodiments described herein can be modified, if necessary, to rely on the ideas of the various patents, applications, and publications to provide further embodiments.

[0290] In view of the foregoing detailed description, various modifications may be made to the embodiments. In general, in the appended claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed in this specification and the claims, but rather the claims should be construed to cover all possible embodiments along the full scope of equivalents to which the claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

1. 1. A method of treating chronic HBV infection in a subject in need thereof, comprising administering an siRNA, wherein the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO:5) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO:6). 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; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol).

2. The method of claim 1, further comprising administering to the subject pegylated interferon-alpha (PEG-IFNα).

3. The method of claim 2, wherein the siRNA and the PEG-IFNα are administered to the patient over the same period of time.

4. The method of claims 2-3, wherein the siRNA is administered to the subject for a period of time before PEG-IFNα is administered to the subject.

5. The method of claims 2-3, wherein PEG-IFNα is administered to the subject for a period of time before the siRNA is administered to the subject.

6. The method of claim 1, wherein the subject has been administered PEG-IFNα prior to administration of the siRNA.

7. The method of claim 1 or 6, wherein the subject is administered PEG-IFNα for the same period that the subject is administered the siRNA.

8. The method of claim 1, 6 or 7, wherein the subject is subsequently administered PEG-IFNα.

9. 9. The method of any of claims 1-8, further comprising administering to the subject a nucleoside / nucleotide reverse transcriptase inhibitor (NRTI).

10. The method of any one of claims 1 to 8, wherein the subject is administered an NRTI prior to administration of the siRNA.

11. The method of claim 10, wherein the subject has been administered an NRTI at least 2 months or at least 6 months prior to administration of the siRNA.

12. The method of any of claims 1-11, wherein the subject is administered an NRTI for the same period that the subject is administered the siRNA.

13. The method of any of claims 1 to 12, wherein the subject is subsequently administered an NRTI.

14. 1. An siRNA for use in treating chronic HBV infection in a subject, the siRNA having a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO:5) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO:6). 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; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol).

15. The siRNA for use according to claim 14, wherein the subject is also administered PEG-IFNα.

16. The siRNA for use according to claim 15, wherein the siRNA and the PEG-IFNα are administered to the patient over the same period of time.

17. The siRNA for use according to claim 15 or 16, wherein the siRNA is administered to the subject during a period before PEG-IFNα is administered to the subject.

18. The siRNA for use according to claim 15 or 16, wherein the PEG-IFNα is administered to the subject for a period of time before the siRNA is administered to the subject.

19. The siRNA for use according to claim 15 or 16, wherein the subject has been administered PEG-IFNα prior to administration of the siRNA.

20. 20. The siRNA for use according to claim 15, 16 or 19, wherein the subject is administered PEG-IFNα during the same period that the subject is administered the siRNA.

21. The siRNA for use according to claims 15-20, wherein the subject is subsequently administered PEG-IFN.

22. The siRNA for use according to any of claims 14 to 21, wherein the subject is also administered an NRTI.

23. The siRNA for use according to any one of claims 14 to 22, wherein the subject is administered an NRTI prior to administration of the siRNA.

24. The siRNA for use according to any of claims 14 to 23, wherein the subject has been administered an NRTI at least 2 months or at least 6 months prior to administration of the siRNA.

25. The siRNA for use according to any of claims 14 to 24, wherein the subject is administered an NRTI during the same period that the subject is administered the siRNA.

26. The siRNA for use according to any of claims 14 to 25, wherein the subject is subsequently administered an NRTI.

27. Use of siRNA in the manufacture of a medicament for the treatment of chronic HBV infection, wherein the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO:5) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO:6). 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; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol).

28. Use of siRNA and PEG-IFNα in the manufacture of a medicament for the treatment of chronic HBV infection, wherein the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6). 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; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol).

29. Use of siRNA, PEG-IFNα and NRTI in the manufacture of a medicament for the treatment of chronic HBV infection, wherein the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6). 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; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol).

30. The method, composition for use or use of any one of claims 1 to 29, wherein the dose of siRNA is 0.8 mg / kg, 1.7 mg / kg, 3.3 mg / kg, 6.7 mg / kg or 15 mg / kg.

31. The method, composition for use or use of any of claims 1 to 30, wherein the dose of siRNA is 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg or 450 mg.

32. 32. The method, composition for use or use of any of claims 1 to 31, wherein the siRNA is administered weekly or more than once, each administration being 2 weeks, 3 weeks or 4 weeks apart.

33. 33. The method, composition for use or use of any of claims 1 to 32, wherein the siRNA is administered 2, 3, 4, 5, 6 or more times, each administration being separated by 1 week, 2 weeks, 3 weeks or 4 weeks.

34. The method (a) administering to a subject at least 200 mg of an siRNA having a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6) at least twice; 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; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate linkage; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol; and (b) administering to the subject a nucleoside / nucleotide reverse transcriptase inhibitor (NRTI); 34. The method, composition for use or use of any one of claims 1 to 33, wherein the subject is HBeAg negative or HBeAg positive.

35. 35. The method, composition for use or use of claim 34, wherein the method further comprises administering to the subject pegylated interferon-alpha (PEG-IFNα).

36. 36. The method, composition for use or use of any of claims 1 to 35, wherein six 200 mg doses of siRNA are administered.

37. 36. The method, composition for use or use of any of claims 1 to 35, wherein two 400 mg doses of siRNA are administered.

38. The method, composition for use or use of any one of claims 1 to 37, wherein the siRNA is administered by subcutaneous injection.

39. 40. The method, composition for use or use of claim 38, wherein administration of the siRNA comprises administration by subcutaneous injection at one, two or three sites per administration.

40. The method, composition for use or use of any one of claims 2 to 39, wherein the dose of PEG-IFNα is 50 μg, 100 μg, 150 μg or 200 μg.

41. The method, composition for use or use of any of claims 2 to 13, 15 to 26 and 28 to 40, wherein PEG-IFNα is administered weekly.

42. The method, composition for use or use of any one of claims 2 to 13, 15 to 26 and 28 to 40, wherein PEG-IFNα is administered by subcutaneous injection.

43. 43. The method, composition for use, or use of any of claims 9 to 13, 22 to 26, and 29 to 42, wherein the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), lamivudine, adefovir, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clevudine, ritonavir, dipivoxil, lobucavir, famvir, N-acetyl-cysteine ​​(NAC), PC1323, theradigm-HBV, thymosin-alpha, ganciclovir, besifovir (ANA-380 / LB-80380) or tenofvir-exaliades (TLX / CMX157).

44. 44. The method, composition for use or use of claim 43, wherein the NRTI is entecavir (ETV).

45. 44. The method, composition for use or use of claim 43, wherein the NRTI is tenofovir.

46. 44. The method, composition for use or use of claim 43, wherein the NRTI is lamivudine.

47. 44. The method, composition for use or use of claim 43, wherein the NRTI is adefovir or adefovir dipivoxil.

48. 48. The method, composition for use or use of any one of claims 1 to 47, wherein the subject is HBeAg negative.

49. 48. The method, composition for use or use of any one of claims 1 to 47, wherein the subject is HBeAg positive.

50. A pharmaceutical composition comprising the siRNA according to any one of claims 1 to 49 and a pharma- ceutically acceptable excipient; and Pharmaceutical composition comprising PEG-IFNα and a pharma- ceutical acceptable excipient - Patent application Including the kit.

51. 51. The kit of claim 50, further comprising an NRTI and a pharma- ceutically acceptable excipient.

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