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

Abstract

The present disclosure provides a method for treating HBV infection using siRNA targeting HBV genes. In some embodiments, the method for treating HBV involves co-administering siRNA with PEG-INFα.

Description

[0001] Statement concerning sequence listing

[0002] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is 930485_405WO_SEQUENCE_LISTING.txt. The text file is 6.5KB, created on May 6, 2020, and submitted electronically via EFS-Web. Background Art

[0003] Chronic hepatitis B virus (HBV) infection is associated with high morbidity and mortality and remains a major 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 suffer from 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). Over time, chronic HBV infection causes serious sequelae, including cirrhosis, liver failure, hepatocellular carcinoma (HCC) and death. It is estimated that nearly 800,000 people die each year from sequelae 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).

[0004] HBV prevalence varies widely, ranging from less than 2% in low-prevalence countries to greater than 8% in high-prevalence countries (Schweitzer et al., 2015). In high-prevalence countries, for example, those in sub-Saharan Africa and East Asia, transmission occurs primarily among infants and children through the perinatal and horizontal routes. In more industrialized countries, new infections are greatest among young adults, and transmission occurs primarily through injection drug use and high-risk sexual behaviors. The risk of developing chronic HBV infection depends on the age at the time of infection. Although only approximately 10% of infected adults develop chronic HBV infection, 90% of infants infected perinatally or during the first 6 months of life, and 20–60% of children infected between 6 months and 5 years of age, remain chronically infected. 25% of people infected with HBV during infancy and childhood will develop primary liver cancer or cirrhosis in adulthood.

[0005] HBV is a DNA virus that infects, replicates and is maintained 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) consists of partially double-stranded relaxed circular DNA (rcDNA) and has four open reading frames encoding seven proteins: HBcAg (HBV core antigen, 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, transcriptional regulator required for initiation of infection) (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).

[0006] In hepatocytes, rcDNA, a form of HBV nucleic acid introduced by infected virions, is converted into covalently closed circular DNA (cccDNA), which is maintained in the host cell nucleus as a free chromatin structure (Allweiss L et al., The Role of cccDNA in HBV Maintenance, Viruses 2017, 9: 156). cccDNA serves as a transcription template for all viral transcripts (Lucifora J et al., Attacking hepatitis B virus cccDNA-The holygrail to hepatitis B cure, Journal of Hepatology 2016, 64 (1): S41-S48). Pregenomic RNA (pgRNA) transcripts are reverse transcribed into new rcDNA of new virus particles, which are secreted without causing cytotoxicity. In addition to infectious virions, infected hepatocytes secrete a large number of genome-free subviral particles, which can exceed the number of secreted virions by 10,000 times (Seeger et al., 2015). The virus can also be randomly integrated into the host genome, which is a mechanism that promotes hepatocyte transformation (Levrero M et al., Mechanisms of HBV-induced hepatocellular carcinoma, Journal of Hepatology 2016, 64 (1): S84-S101). HBV is maintained in hepatocytes in the form of cccDNA and integrated DNA (intDNA).

[0007] Hepatitis B infection is characterized by serum viral markers and antibodies ( Figure 1In acute resolving infection, the virus is cleared by effective innate and adaptive immune responses, including cytotoxic T cells that cause death of infected hepatocytes and induction of B cells to 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. In contrast, chronic infection is associated with T and B cell dysfunction, mediated by multiple regulatory mechanisms, including presentation of viral epitopes on hepatocytes and 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 cccDNA retention in hepatocytes is considered a key step in the host's inability to clear the infection.

[0008] Chronic HBV infection is a dynamic process that reflects the interaction between HBV replication and the host immune response. The laboratory hallmarks of chronic HBV infection are the retention of HBsAg in the blood for more than six months and the absence of detectable anti-HBs. Chronic infection is divided into four stages based on HBV markers in the blood (HBsAg, HBeAg / anti-HBe, HBV DNA), and liver disease is based on biochemical parameters (alanine aminotransferase, "ALT") and fibrosis markers (noninvasive or based on liver biopsy) (EASL, 2017). In general, at each stage of chronic HBV infection, only a small number of patients (less than 1% / year) have cleared the disease as measured by HBsAg seroclearance.

[0009] A cure for HBV would involve complete elimination of HBV DNA or permanent transcriptional silencing of HBV DNA without the risk of relapse. Potential therapies that can eliminate or permanently silence cccDNA / intDNA have the risk of disrupting or altering human chromosomal DNA transcription.

[0010] In contrast, functional cure is defined as lifelong control of the virus. Patients with a history of acute hepatitis B who appear to be cured have an approximately 40% risk of HBV recurrence if they undergo immunosuppression. It appears that functional cure is part of the natural history of HBV infection. Potential therapies that provide functional cure may require immunomodulation. This is because chronic HBV infection causes B and T cell exhaustion, potentially due to the expression of HBV antigens (tolerogens), which can hinder the efficacy of immune modulators.

[0011] Currently, there are two main treatment options for patients with chronic HBV infection: treatment with nucleoside / nucleotide reverse transcriptase inhibitors (NRTIs) and pegylated interferon-α (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 viral particles and generally reduce serum HBV DNA to undetectable levels. However, NRTIs do not directly eliminate cccDNA, and therefore, viral proteins continue to be transcribed and translated. Therefore, expression of viral epitopes on hepatocytes, secretion of subviral particles, and immune dysfunction remain largely unaffected by NRTI therapy. As a result, this requires long-term, usually lifelong therapy (however, less than half of patients continue therapy after 5 years). NRTI therapy results in a loss of serum HBsAg at a rate of approximately 0-3% per year. Furthermore, although NRTI therapy reverses fibrosis and reduces the incidence of HCC, it does not eliminate the increased HCC risk conferred by HBV infection.

[0012] In contrast, PEG-IFN can induce long-term immune control, but only in a small percentage of patients (<10%) (Konerman MA et al., Interferon Treatment for Hepatitis B, Clinics in Liver Disease 2016, 20(4):645-665). PEG-IFN usually requires 48 weeks of therapy, and duration-dependent side effects are significant. In studies evaluating PEG-IFNα for the treatment of chronic hepatitis C infection, 12-week or 24-week regimens were associated with lower rates of serious adverse events, grade 3 adverse events, and treatment discontinuations than those observed in trials evaluating a 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. 2013, 368(20):1878-1887). =Med. 2004, 140(5):346-355; Fried MW et al., Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection, New England Journal of Medicine 2002, 347(13):975-982). The high variability of response combined with an unfavorable safety and side effect profile makes a large number of patients unsuitable or unwilling to undergo PEG-IFNα treatment.

[0013] The inability of NRTI therapy to clear the virus and the limitations of PEG-IFNα therapy highlight the clinical need for new HBV therapies that are effective, well tolerated, and do not require lifelong administration. Summary of the invention

[0014] In some aspects, the present disclosure relates to compositions and methods for treating HBV with siRNA, especially HBV02. For example, according to some embodiments, a method for treating HBV infection in a subject by administering siRNA is provided, wherein the siRNA has a sense strand comprising SEQ ID NO:5 and an antisense strand comprising SEQ ID NO:6. In some embodiments, the method of treatment further comprises administering pegylated interferon-α (PEG-INFα) to the subject. In some embodiments, PEG-INFα is administered before, simultaneously with, or after administering siRNA HBV02. In some embodiments, HBV infection is chronic. In some other embodiments, nucleoside / nucleotide reverse transcriptase inhibitors (NRTIs) are administered to the subject. In some embodiments, NRTIs are administered before, simultaneously with, or after administering HBV02. In some embodiments, NRTIs are administered 2 to 6 months before HBV02.

[0015] In some aspects, the present disclosure also provides a siRNA for treating HBV infection in a subject, wherein the siRNA is HBV02 and has a sense strand comprising SEQ ID NO: 5 and an antisense strand comprising SEQ ID NO: 6. In some additional embodiments, siRNA HBV02 is administered to a subject who also administers PEG-INFα. In some embodiments, PEG-INFα is administered before, simultaneously with, or after the administration of siRNA HBV02. In some embodiments, HBV infection is chronic. In some other embodiments, NRTI is administered to the subject. In some embodiments, NRTI is administered before, simultaneously with, or after the administration of HBV02. In some embodiments, NRTI is administered 2 to 6 months before HBV02.

[0016] In some other aspects, the present disclosure provides the use of siRNA in the manufacture of a medicament for treating HBV infection, wherein the siRNA is HBV02 and has a sense strand comprising SEQ ID NO: 5 and an antisense strand comprising SEQ ID NO: 6. In some embodiments, the use of siRNA HBV02 is used with PEG-IFNα. In some embodiments, siRNA HBV02 is used with PEG-IFNα and NRTI.

[0017] In some of the foregoing embodiments, the dosage of siRNA HBV02 is 0.8mg / kg, 1.7mg / kg, 3.3mg / kg, 6.7mg / kg, 10mg / kg or 15mg / kg. In some of the foregoing embodiments, the dosage of siRNA HBV02 is 20mg to 900mg. In some of the foregoing embodiments, the dosage of siRNA HBV02 is 20mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 400mg or 450mg. In some of the foregoing embodiments, HBV02 is administered weekly. In some of the foregoing embodiments, more than one dose of siRNA is administered. In some of the foregoing embodiments, two, three, four, five, six or more doses of siRNA are administered, each dose being 1, 2, 3 or 4 weeks apart. In some of the foregoing embodiments, six 200mg doses of siRNA are administered. In some of the foregoing embodiments, two 400mg doses of siRNA are administered. In some of the foregoing embodiments, the siRNA is administered by subcutaneous injection; for example, in some embodiments, administering siRNA HBV02 comprises administering 1, 2, or 3 subcutaneous injections per dose.

[0018] In some of the foregoing embodiments, the dose of PEG-IFNα is 50 μg, 100 μg, 150 μg, or 200 μg.

[0019] In some of the foregoing embodiments, the PEG-IFNα is administered weekly. In some of the foregoing embodiments, the PEG-IFNα is administered by subcutaneous injection.

[0020] In some of the foregoing embodiments, the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), lamivudine, adefovir, adefovir dipivoxil (adefovir dipivoxil), entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clevudine, ritonavir, dipivoxil, lobucavir, famvir, N-acetyl-cysteine ​​(NAC), PC1323, theradigm-HBV, thymosin-α, ganciclovir, besifovir (ANA-380 / LB-80380), or tenofvir-exaliades (TLX / CMX157).

[0021] In some of the foregoing embodiments, the subject is HBeAg negative. In some embodiments, the subject is HBeAg positive.

[0022] In some aspects of the present disclosure, a kit is provided, comprising: a pharmaceutical composition comprising an siRNA according to any one of the preceding embodiments and a pharmaceutically acceptable excipient; and a pharmaceutical composition comprising PEG-IFNα and a pharmaceutically acceptable excipient. The kit may also contain an NRTI and a pharmaceutically acceptable excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Characterize acute and chronic hepatitis B infection.

[0024] Figure 2 To characterize chronic hepatitis B infection. The disease is divided into 4 stages based on HBeAg status and laboratory or radiographic evidence of liver disease. The heterogeneity of the disease may be due to differences in the virus (e.g., HBV genotype, mutations), the host (e.g., immune response, age at inflection, number of infected hepatocytes), and other factors (e.g., co-infections (HDV, HCV, HIV), inter-infections, comorbidities).

[0025] Figure 3 The single ascending dose design for Part A of Example 2 is depicted. aSubjects were discharged on day 2 after completing all assessments.

[0026] Figure 4 The multiple ascending dose design for Parts B and C of Example 2 is depicted. a Additional HBsAg monitoring is indicated for subjects whose HBsAg levels decrease by >10% from the Day 1 predose level at the Week 16 visit. Visits will be conducted every 4 weeks starting at Week 20 and continuing until Week 48, or until HBsAg levels return to ≥90% of the Day 1 predose level.

[0027] FIG. 5A to FIG. 5B Depicts the cohort dosing schedule for Parts A, B, and C of Example 2, including optional cohorts and floater subjects. *If further data is needed, up to 8 subjects may be added to Part A and up to a total of 16 subjects may be added to Part B / C as an expansion of one or more existing cohorts (floater subject allocations in Part B / C do not need to be evenly distributed; the total combined n for Part B / C does not exceed 48 subjects). **The doses specified in the Part B / C schedule indicate a single HBV02 or placebo dose; subjects receive up to 2 doses total.

[0028] FIG. 6A to FIG. 6D Depicted is the cohort dosing schedule for Part D of Example 2. Fig. 6A Display the design of queue 1d; Figure 6B Display the design of queue 2d; Figure 6C Display queue 3d design; Fig.6D Displays the design of Queue 4D.

[0029] Figures 7A to 7B Describes the cohort dosing schedule for Parts A, B, C, and D of Example 2, including optional cohorts and floating subjects ( Fig. 7A dotted line on the top).

[0030] Figure 8 Depicted are the cohort dosing schedules for Parts A, B, and C of the study in Example 3.

[0031] Figures 9A to 9C The studies that generated the preliminary data in Example 3 are described. Fig. 9A Describe the study design at the time of completion of dosing for Part A Cohorts 1 to 5 (50 mg, 100 mg, 200 mg, 400 mg, 600 mg) and Part B Cohorts 1 to 2 (50 mg, 100 mg). Fig. 9B Describes the completed dosing for Part A Cohorts 1 to 5 and the withdrawals of subjects in the different cohorts. Fig. 9C Depicts the completed dosing for Part B Cohorts 1 to 2 and the withdrawals of subjects in the different cohorts.

[0032] FIG. 10A to FIG. 10B Depicted are ALT levels for subjects in Cohorts 1 to 4 of Part A of Example 3. Fig. 10A Shown are ALT levels for subjects who received 50 mg (cohort 1a) or 100 mg (cohort 2a) of HBV002. Fig. 10B ALT levels for subjects who received 200 mg (cohort 3a) or 400 mg (cohort 4a) of HBV002 are shown. One subject in the 200 mg cohort had ALT above the ULN on Day 29, which was associated with vigorous exercise and high creatinine kinase (CK: 5811 U / L). Two subjects in the 400 mg cohort had ALT values ​​above the ULN prior to dosing on Day 1; one of these subjects admitted to vigorous exercise, had high CK (20,001 U / L), and withdrew on Day 2 unrelated to adverse events, and the other subject's ALT decreased by Day 8 without intervention.

[0033] Fig.11 Depicted are ALT levels for subjects who received 50 mg (Cohort 1b) or 100 mg (Cohort 2b) of HBV002 in Part B of Example 3. One female subject in the 100 mg cohort exhibited a Grade 1 ALT elevation at Week 8.

[0034] Figures 12A to 12C Depicted are antiviral activity in Part B Cohorts 1b (50 mg) and 2b (100 mg) of Example 3 as measured by changes in HBsAg levels. Fig. 12A Shown are changes in HBsAg levels among active and placebo subjects. Fig. 12B Changes in HBsAg levels among active subjects only are shown. Fig. 12C Shown are changes in HBsAg levels (mean change in HBsAg from Day 1 after administration of HBV02) among subjects in the 50 mg (Cohort 1b) and 100 mg (Cohort 2b) cohorts.

[0035] FIG. 13A to FIG. 13E ALT levels in chronic HBV patients in Example 3 up to week 16 (n=32) are shown. Fig.13A ALT levels of all patients are shown, and the difference between the two groups is not statistically significant. Fig. 13B These results are shown in 13A (20 mg), 13C (50 mg), 13D (100 mg) and 13E (200 mg), respectively.

[0036] Fig.14Post-baseline ALT elevations occurring during treatment in healthy volunteers with normal ALT at baseline are shown corresponding to Example 3. The highest post-baseline ALT elevation occurring during treatment expressed relative to the upper limit of normal (ULN) is shown on the y-axis. The dose of HBV01 or HBV02 is shown on the x-axis. * Approximate mg / kg dose based on an average adult weight of 60 kg; fixed doses of HBV02 range from 50 to 900 mg.

[0037] FIG. 15A to FIG. 15B Shown are plasma concentration versus time curves of HBV02 (A) and AS(N-1)3'HBV02 (B) following a single subcutaneous administration in healthy volunteers corresponding to Example 3.

[0038] Fig.16 The plasma AUC of HBV02 after a single subcutaneous administration in healthy volunteers is shown corresponding to Example 3. 0-12 Dose proportionality was observed from 50 mg to 900 mg.

[0039] Fig.17 The plasma C values ​​of HBV02 after a single subcutaneous administration in healthy volunteers are shown in accordance with Example 3. max Dose proportionality was observed from 50 mg to 900 mg.

[0040] Fig.18 The plasma PK parameters of HBV02 and AS(N-1)3′HBV02 after a single SC dose in healthy volunteers in Example 3 are shown. Time parameters are presented as median (quartile [Q]1, Q3); all other data are presented as mean (coefficient of variation [CV]%). 1 / 2 ) and PK sampling schedule limitations, the terminal phase was not fully characterized; therefore, apparent clearance and t were not reported. 1 / 2 . a One volunteer who received a partial dose was excluded; b including PK from replacement volunteers; c Measurable in 3 / 6 volunteers; AUC, area under the curve; AUC 0-12 , AUC from 0 to 12 hours; AUC 最后 , AUC from the time of administration to the last measurable time point; BLQ, below the limit of quantification; C max , maximum concentration; CV, coefficient of variation; MR, metabolite:parent ratio; NC, not calculable; T max =C max Time; T 最后 , and finally the time can be measured.

[0041] Figures 19A to 19BShown are urine concentration versus time curves of HBV02 (A) and AS(N-1)3'HBV02 (B) following a single subcutaneous administration in healthy volunteers corresponding to Example 3.

[0042] Fig. 20 The plasma PK parameters of HBV02 and AS(N-1)3'HBV02 in healthy volunteers are shown in Example 3. All PK parameters are expressed as mean values ​​(CV %). a One volunteer who received a partial dose was excluded; b including PK from replacement volunteers; c AUC 0-24 For extrapolation; AUC 0-24 , AUC from 0 to 24 hours; CLR, total renal clearance; fe 0-24 , the fraction excreted between time 0 and 24 h; NC, not calculable.

[0043] Figures 21A to 21B Depicted are the antiviral activities in Parts B and C of Example 3 as measured by changes in HBsAg levels. Fig.21A Changes in HBsAg levels are shown on a logarithmic scale.

[0044] Fig. 22 Depicted are the changes from baseline in HBsAg by HBV02 dose or for placebo for Example 3. Follow-up data were available for all placebo patients up to week 16 compared to week 24 for the treatment groups.

[0045] Fig.23 Depicted are the individual maximum changes from baseline in HBsAg for Example 3. Error bars represent median (interquartile range).

[0046] Fig.24 Shown are individual changes in HBsAg from baseline at Week 24 for Example 3. Error bars represent median (interquartile range). DETAILED DESCRIPTION

[0047] The present disclosure provides methods, compositions and kits for treating hepatitis B virus (HBV) infection, wherein small interfering RNA (siRNA) molecules targeting HBV are administered. In some embodiments, the siRNA molecules are administered together with pegylated interferon-2α (PEG-IFNα) therapy, or administered to subjects who have received or will receive PEG-IFN-α therapy. In some embodiments, the methods, compositions and kits disclosed herein are used to treat chronic HBV infection.

[0048] I. Glossary

[0049] Before setting forth the present disclosure in more detail, providing definitions of certain terms to be used herein may aid in understanding the present disclosure. Additional definitions are set forth throughout the present disclosure.

[0050] In this specification, unless otherwise indicated, the term "about" means ±20% of the indicated range, value or structure.

[0051] The term "comprising" means the presence of the stated features, integers, steps or components as mentioned in the claims, but it does not exclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The term "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not substantially affect the basic and novel characteristics of the claimed invention.

[0052] It should be understood that, as used herein, the term "a" or "an" means "one or more" of the listed components. The use of alternative terms (e.g., "or") should be understood to mean any one, two, or any combination of the alternatives, and can be used synonymously with "and / or". As used herein, the terms "including" and "having" are used synonymously, and the terms and their variations are intended to be understood as non-limiting.

[0053] The word "substantially" does not exclude "completely"; for example, a composition that is "substantially free" of Y may be completely free of Y. If desired, the word "substantially" may be omitted from the definitions provided herein.

[0054] As used herein, the term "disease" is intended to be generally synonymous with and used interchangeably with the terms "disorder" and "condition" (as in medical conditions), wherein all terms reflect an abnormal condition of the human or animal body or a part of its impaired normal function. A "disease" is typically manifested by discernible signs and symptoms and causes a person or animal to have a shortened life span or reduced quality of life.

[0055] As used herein, the terms "peptide", "polypeptide" and "protein" and variations of these terms refer to molecules, especially peptides, oligopeptides, polypeptides or proteins including fusion proteins, which respectively contain at least two amino acids joined to each other by a normal peptide bond or, for example, by a modified peptide bond in the case of an isosteric peptide. For example, a peptide, polypeptide or protein can be composed of amino acids selected from the 20 amino acids defined by the genetic code, which are connected to each other by a normal peptide bond ("classical" polypeptide). A peptide, polypeptide or protein can be composed of L-amino acids and / or D-amino acids. Specifically, the terms "peptide", "polypeptide" and "protein" also include "peptide mimetics", which are defined as peptide analogs containing non-peptide structural elements that can simulate or antagonize one or more biological effects of a natural parent peptide. Peptide mimetics do not have classical peptide features, such as peptide bonds that are easily enzymatically cleaved. Specifically, a peptide, polypeptide or protein may also contain additional amino acids in addition to the 20 amino acids defined by the genetic code, or it may be composed of amino acids in addition to the 20 amino acids defined by the genetic code. Specifically, the peptides, polypeptides or proteins in the context of the present disclosure may equally be composed of amino acids modified by natural processes or chemical processes such as post-translational maturation processes well known to those skilled in the art. The modifications are fully described in detail in the literature. These modifications may occur at any position in the polypeptide: in the peptide backbone, in the amino acid chain or even at the carboxyl or amino terminus. Specifically, the peptide or polypeptide may be branched after ubiquitination, or may be cyclic with or without branches. This type of modification may be the result of natural or synthetic post-translational processes well known to those skilled in the art. Specifically, in the context of the present disclosure, the terms "peptide", "polypeptide" or "protein" also include modified peptides, polypeptides and proteins. For example, peptide, polypeptide or protein modifications may include acetylation, acylation, ADP-ribosylation, amidation, covalent immobilization of nucleotides or nucleotide derivatives, covalent immobilization of lipids or lipid derivatives, covalent immobilization of phosphatidylinositols, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, seneloylation, sulfatation, amino acid addition such as arginylation, or ubiquitination.These modifications are fully described in the literature (Proteins Structure and Molecular Properties, 2nd ed., TECreighton, New York (1993); Post-translational Covalent Modifications of Proteins, BC 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). Thus, the terms "peptide", "polypeptide" and "protein" include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, and the like.

[0056] As used herein, a "(poly)peptide" comprises a single chain of amino acid monomers linked by peptide bonds as explained above. As used herein, a "protein" comprises one or more, such as 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 explained above. In a specific embodiment, a protein according to the present disclosure comprises 1, 2, 3 or 4 polypeptides.

[0057] As used herein, the term "recombinant" (eg, recombinant protein, recombinant nucleic acid, etc.) refers to any molecule (protein, nucleic acid, siRNA, etc.) that is prepared, expressed, produced, or isolated by recombinant means and does not occur in nature.

[0058] 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. Nucleic acid molecules can be single-stranded or double-stranded. In a specific embodiment, the nucleic acid molecule is a double-stranded RNA molecule.

[0059] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be identical in DNA content due to intentional or unintentional mutations. Variant progeny having the same function or biological activity as screened for in the originally transformed cell are included.

[0060] As used herein, the term "sequence variant" refers to any sequence with one or more changes compared to a reference sequence, wherein the reference sequence is any sequence listed in the sequence list, i.e., any one of SEQ ID NO: 1 to SEQ ID NO: 6. Therefore, the term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. For sequence variants in the context of nucleotide sequences, the reference sequence is also a nucleotide sequence, and for sequence variants in the context of amino acid sequences, the reference sequence is also an amino acid sequence. As used herein, a "sequence variant" is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a reference sequence. Unless otherwise specified, sequence identity is usually calculated over the full length of a reference sequence (i.e., a sequence described in the present application). The percentage of identity as referred to herein can be determined, for example, using BLAST, using the default parameters [Blosum 62 matrix; gap open penalty = 11 and gap extension penalty = 1] specified by the National Center for Biotechnology Information (NCBI; http: / / www.ncbi.nlm.nih.gov / ).

[0061] "Sequence variants" in the context of nucleic acid (nucleotide) sequences have a changed sequence, wherein one or more 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 standard single-letter names (A, C, G or T). Due to the degeneracy of the genetic code, "sequence variants" of nucleotide sequences may or may not cause changes in the corresponding reference amino acid sequence, i.e., produce or not produce amino acid "sequence variants". In certain embodiments, nucleotide sequence variants are variants that do not produce amino acid sequence variants (i.e., silent mutations). However, nucleotide sequence variants that produce "non-silent" mutations are also within the scope, especially those nucleotide sequence variants that produce amino acid sequences that are 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. "Sequence variants" in the context of amino acid sequences have a changed sequence, wherein one or more amino acids are deleted, substituted or inserted compared to the reference amino acid sequence. As a result of the changes, the sequence variant has 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, the variant sequence has no more than 10 changes, i.e., any combination of deletions, insertions or substitutions, per 100 amino acids in the reference sequence, thereby being "at least 90% identical" to the reference sequence.

[0062] Although it is possible to have non-conservative amino acid substitutions, in certain embodiments, the substitutions are conservative amino acid substitutions, in which the substituted amino acid has similar structural or chemical properties as the corresponding amino acid in the reference sequence. For example, conservative amino acid substitutions involve the substitution of one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine, and isoleucine, with another; one hydroxyl-containing amino acid, such as serine and threonine, with another; one acidic residue, such as glutamic acid or aspartic acid, with another; one amide-containing residue, such as asparagine and glutamine, with another; one aromatic residue, such as phenylalanine and tyrosine, with another; one basic residue, such as lysine, arginine, and histidine, with another; and one smaller amino acid, such as alanine, serine, threonine, methionine, and glycine, with another.

[0063] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as internal insertions of single or multiple amino acid residues. Examples of terminal insertions include fusing the N- or C-terminus of an amino acid sequence to a reporter molecule or enzyme.

[0064] Unless otherwise specified, the changes in the sequence variants do not necessarily eliminate the functionality of the corresponding reference sequence, such as in the case of the present invention, the functionality of siRNA reducing HBV protein expression. It can be determined by using computer programs known in the art to guide which nucleotides and amino acid residues can be replaced, inserted or deleted without eliminating the functionality.

[0065] As used herein, a nucleic acid sequence or amino acid sequence "derived from" a specified nucleic acid, peptide, polypeptide or protein refers to the source of the nucleic acid, peptide, polypeptide or protein. In some embodiments, a nucleic acid sequence or amino acid sequence derived from a specific sequence has an amino acid sequence substantially identical to the sequence or a portion thereof from which it is derived, wherein "substantially identical" includes sequence variants as defined above. In certain embodiments, a nucleic acid sequence or amino acid sequence derived from a specific peptide or protein is derived from a corresponding domain in a specific peptide or protein. Wherein, "corresponding" refers in particular to the same functionality. For example, an "extracellular domain" corresponds to another "extracellular domain" (of another protein), or a "transmembrane domain" corresponds to another "transmembrane domain" (of another protein). Thus, a person of ordinary skill in the art can identify the "corresponding" portion of a peptide, protein and nucleic acid. Similarly, a sequence "derived from" another sequence can usually be identified in the sequence by a person of ordinary skill in the art as having another sequence origin.

[0066] In some embodiments, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be the same as the starting nucleic acid, peptide, polypeptide or protein (from which it is derived). However, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may also have one or more mutations relative to the starting nucleic acid, peptide, polypeptide or protein (from which it is derived), and in particular, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be a functional sequence variant as described above of the starting nucleic acid, peptide, polypeptide or protein (from which it is derived). For example, in a peptide / protein, one or more amino acid residues may be substituted by other amino acid residues, or one or more amino acid residues may be inserted or deleted.

[0067] As used herein, the term "mutation" refers to a change in a nucleic acid sequence and / or an amino acid sequence compared to a reference sequence, such as a corresponding genomic sequence. For example, a mutation compared to a genomic sequence can be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation induced by, for example, an enzyme, a chemical, or radiation, or a mutation obtained by site-directed mutagenesis (a molecular biological method for producing specific and intentional changes in nucleic acid sequences and / or amino acid sequences). Therefore, the term "mutation (mutation / mutating)" should be understood to also include, for example, physically generating mutations in a nucleic acid sequence or in 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. In order to achieve mutations in an amino acid sequence, mutations can be introduced into the nucleotide sequence encoding the amino acid sequence so as to express (recombinant) mutant polypeptides. Mutations can be achieved, for example, by changing the codons of a nucleic acid molecule encoding one amino acid to produce codons encoding a different amino acid, such as by site-directed mutagenesis, or by synthesizing sequence variants, such as by knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide, and by designing the synthesis of a nucleic acid molecule comprising a nucleotide sequence encoding a variant of the polypeptide, without mutating one or more nucleotides of the nucleic acid molecule.

[0068] As used herein, the term "coding sequence" is intended to refer to a polynucleotide molecule that encodes the amino acid sequence of a protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon.

[0069] As used herein, the term "expression" refers to any step involved in the production of a polypeptide including transcription, post-transcriptional modification, translation, post-translational modification, secretion, and the like.

[0070] Doses are usually expressed relative to body weight. Thus, a dose expressed as [g, mg or other unit] / kg (or g, mg, etc.) usually means "per kg (or g, mg, etc.) body weight" [g, mg or other unit], even if the term "body weight" is not explicitly mentioned.

[0071] As used herein, "hepatitis B virus", which can be used interchangeably with the term "HBV", refers to a well-known non-cell-degenerating hepadnavirus belonging to the Hepadnaviridae family. The HBV genome is a partially double-stranded circular DNA with four overlapping reading frames (which may be referred to herein as "genes", "open reading frames" or "transcripts"): C, X, P and S. The core protein is encoded by gene C (HBcAg). Hepatitis B e antigen (HBeAg) is produced by proteolytic processing of pre-core (pre-C) protein. DNA polymerase is encoded by gene P. Gene S is a gene encoding surface antigen (HBsAg). The HBsAg gene is a long open reading frame containing three in-frame "start" (ATG) codons, thereby producing three different sizes of polypeptides, called large, medium and small S antigens: pre-S1+pre-S2+S, pre-S2+S or S. In addition to being located at the envelope of HBV, surface antigens are also part of subviral particles that are produced in large excess compared to viral particles and play a role in immune tolerance and sequestering anti-HBsAg antibodies, allowing infectious particles to evade immune detection. The function of the nonstructural protein encoded by gene X is not fully understood, but it plays a role in transcriptional transactivation and replication and is associated with the development of liver cancer.

[0072] Nine HBV genotypes named A to I have been determined, and an additional genotype J has been proposed, each with a unique geographic 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 to J) of HBV. The complete coding sequence of the reference sequence of the HBV genome can be found in, for example, GenBank accession numbers GI: 21326584 and GI: 3582357. The amino acid sequences of 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). Additional examples of HBV messenger RNA (mRNA) sequences can be obtained using publicly available databases, such as GenBank, UniProt and OMIM. The International Repository for Hepatitis B Virus Strain Data is available at http: / / www.hpa-bioinformatics.org.uk / HepSEQ / main.php. As used herein, the term "HBV" also refers to naturally occurring DNA sequence variations of the HBV genome, ie, genotype AJ and variants thereof.

[0073] siRNA mediates the targeted cleavage of RNA transcripts through RNA-induced silencing complex (RISC) pathways, thereby achieving inhibition of gene expression. This process is commonly referred to as "RNA interference" (RNAi). Without wishing to be bound by a particular theory, the long double-stranded RNA (dsRNA) introduced into plant and invertebrate cells is decomposed into siRNA by a type III endonuclease called Dicer (Sharp et al., "Genes and Development (Genes Dev.)" 15:485 (2001)). Dicer, a ribonuclease-III-like enzyme, processes dsRNA into siRNAs of 19 to 23 base pairs with characteristic two-base 3' overhangs (Bernstein et al., "Nature (Nature)" 2001,409:363). siRNA is subsequently incorporated into RISC, where one or more helicases unfold the siRNA double helix so that the complementary antisense strand can guide target recognition (Nykanen et al., 2001, "Cell (Cell)" 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al., Genes & Development 2001, 15:188).

[0074] The terms "silencing", "inhibiting the expression of...", "downregulating the expression of...", "suppressing the expression of...", etc., in the context of HBV genes, refer herein to at least a partial reduction in HBV gene expression, which is embodied as follows: the amount of HBV mRNA that can be isolated from or detected in a first cell or cell group (control cells) that is substantially identical to the first cell or cell group in which the HBV gene is transcribed and has been treated with an inhibitor of HBV gene expression so that the expression of the HBV gene is inhibited is reduced compared to a second cell or cell group (control cells) that has not been treated in this way and is substantially identical to the first cell or cell group in which the HBV gene is transcribed. The degree of inhibition can be measured, for example, as the difference between the degree of mRNA expression in the control cells and the degree of mRNA expression in the treated cells. Alternatively, the degree of inhibition can be given as a parameter functionally associated with HBV gene expression, such as the amount of protein encoded by the HBV gene, or a reduction in the number of cells that display a certain phenotype, such as an HBV-infected phenotype. In principle, HBV gene silencing can be determined by any cell expressing the HBV gene, such as a cell infected with HBV or a cell engineered to express the HBV gene, and by any appropriate analysis.

[0075] The level of HBV RNA expressed by a cell or a group of cells or the level of circulating HBV RNA can be determined using any method known in the art for evaluating mRNA expression, such as the rtPCR method provided in Example 2 of the international application with publication number WO 2016 / 077321A1 and the U.S. patent application with application number US2017 / 0349900A1, which is incorporated herein by reference. In some embodiments, the expression level of the HBV gene in the sample (e.g., total HBV RNA, HBV transcripts, such as HBV 3.5kb transcripts) is determined by detecting a transcribed polynucleotide or a portion thereof, such as RNA of the HBV gene. RNA can be extracted using RNA extraction techniques, including, for example, using acid phenol / guanidine isothiocyanate extracts (RNAzol B; Biogenesis), RNeasy RNA preparation kits Or PAXgene (PreAnalytix, Switzerland) is extracted from cells. Typical analysis forms using ribonucleic acid hybridization include nuclear in vivo analysis (nuclear run-on assay), RT-PCR, RNA enzyme protection analysis (Melton DA 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 using the method described in the international application with publication number WO 2012 / 177906A1 and the U.S. patent application with application number US2014 / 0275211A1, which is incorporated herein by reference.

[0076] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of an HBV gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for RNAi-guided cleavage at or near the portion. For example, the target sequence will typically be 9 to 36 nucleotides in length, for example, 15 to 30 nucleotides in length, including all subranges therebetween. As a non-limiting example, the target sequence may be 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19 -26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides or 21-22 nucleotides.

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

[0078] As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence associated with a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising the second nucleotide sequence and form a double helical structure under certain conditions, as understood by the skilled person. The conditions may, for example, be stringent conditions, wherein stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12 to 16 hours, followed by washing. Other conditions may be applicable, such as physiologically relevant conditions that may be encountered inside an organism. The skilled person will be able to determine the set of conditions that are most suitable for the complementarity test of the two sequences based on the ultimate application of the hybridizing nucleotides.

[0079] As described herein, the complementary sequence in siRNA includes an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence, and base pairing on the full length of one or two nucleotide sequences. The sequences may be referred to herein as "completely complementary" relative to each other. However, when the first sequence is referred to herein as "substantially complementary" relative to the second sequence, the two sequences may be completely complementary, or they may form one or more when hybridizing to produce a double helix of up to 30 base pairs, but generally no more than 5, 4, 3 or 2 mismatched base pairs, while remaining in its final application, such as the ability to hybridize under the conditions most relevant to inhibiting gene expression by the RISC path. However, in the case where two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, the overhangs should not be considered as mismatches in terms of determining complementarity. For example, an siRNA comprising an oligonucleotide having a length of 21 nucleotides and another oligonucleotide having a length of 23 nucleotides, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that are completely complementary to the shorter oligonucleotide, but for purposes described herein, the oligonucleotide may still be referred to as "completely complementary".

[0080] As used herein, "complementary" sequences may also include non-Watson-Crick base pairs and / or base pairs formed by non-natural and modified nucleotides, or may be formed entirely by them, as long as the above requirements regarding their hybridization ability are met. The non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

[0081] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used with respect 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 will be understood from the context of their use.

[0082] As used herein, a polynucleotide that is "substantially complementary" to at least a portion of an mRNA refers to a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding an HBV protein). For example, if a polynucleotide is substantially complementary to an uninterrupted portion of an HBV mRNA, then the sequence is complementary to at least a portion of the HBV mRNA.

[0083] As used herein, the term "siRNA" refers to an RNA interference molecule, which includes a complex of an RNA molecule or a molecule having a hybrid double helix region comprising two antiparallel and substantially complementary nucleic acid strands, which will be referred to as having "sense" and "antisense" orientations relative to the target RNA. The double helix region can have any length that allows the specific degradation of the desired target RNA by the RISC pathway, but typically the length will be in the range of 9 to 36 base pairs, for example, in the range of 15 to 30 base pairs. Contemplating duplexes of 9 to 36 base pairs, the duplex can be 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 any subranges therebetween, including, but not limited to, 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18- 26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs and 21-22 base pairs. siRNAs generated in cells by processing with Dicer and similar enzymes typically range from 19 to 22 base pairs in length.

[0084] One strand of the double helix region of siRNA contains a sequence that is substantially complementary to the region of the target RNA. The two strands forming the double helix structure may come from a single RNA molecule having at least one self-complementary region, or may be formed by two or more different RNA molecules. In the case where the double helix region is formed by two strands of a single molecule, the molecule may have a double helix region separated by a single strand of nucleotides (referred to herein as a "hairpin loop") between the 3' end of one strand and the 5' end of the corresponding other strand forming the double helix structure. The hairpin loop may contain at least one unpaired nucleotide; in some embodiments, the hairpin loop may 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. In the case where the two substantially complementary strands of siRNA are composed of different RNA molecules, those molecules do not have to be but may be covalently linked. In the case where the two strands are covalently linked by means other than the hairpin loop, the linking structure is referred to as a "linker".

[0085] siRNA as described herein can be synthesized by standard methods known in the art, for example by using an automated DNA synthesizer as commercially available from, for example, Biosearch, Applied Biosystems.

[0086] The term "antisense strand" or "guide strand" refers to a strand of the siRNA that includes a region that is substantially complementary to a target sequence. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence, such as a target sequence as defined herein. In the case where the complementary region is not completely complementary to the target sequence, the mismatch may be in the interior or terminal regions of the molecule. In general, the most tolerable mismatch is in the terminal region, such as within 5, 4, 3 or 2 nucleotides of the 5' and / or 3' ends.

[0087] As used herein, the term "sense strand" or "passenger strand" refers to the strand of a siRNA that includes a region that is substantially complementary to a region of the antisense strand, as that term is defined herein.

[0088] The term "RNA molecule" or "ribonucleic acid molecule" not only encompasses RNA molecules as expressed or found in nature, but also encompasses analogs and derivatives of RNA comprising one or more ribonucleoside / ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, "ribonucleoside" includes nucleoside bases and ribose, and "ribonucleotide" is a ribonucleoside with one, two or three phosphate moieties. However, as used herein, the terms "ribonucleoside" and "ribonucleotide" can be considered as synonyms. RNA can be modified in a core base structure or in a ribose phosphorylation backbone structure, such as described in more detail below. However, the siRNA molecule comprising a ribonucleoside analog or derivative maintains the ability to form a duplex. As non-limiting examples, RNA molecules can also include at least one modified ribonucleoside, including but not limited to 2'-O-methyl modified nucleosides, nucleosides comprising 5' thiophosphate groups, terminal nucleosides connected to cholesterol derivatives or dodecanoic acid bisdecylamide groups, locked nucleosides, abasic nucleosides, 2'-deoxy-2'-fluorine modified nucleosides, 2'-amino modified nucleosides, 2'-alkyl modified nucleosides, morpholino nucleosides, nucleosides comprising aminophosphorothioate or non-natural bases, or any combination thereof. In another example, RNA molecules can include at least two modified ribonucleosides, 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, up to the total length of siRNA molecules. For each of a plurality of modified ribonucleosides described in RNA molecules, modification need not be identical. In some embodiments, modified ribonucleosides include deoxyribonucleosides. For example, an siRNA may comprise one or more deoxynucleosides, including, for example, one or more deoxynucleoside overhangs or one or more deoxynucleosides within a double-stranded portion of an siRNA. However, as used herein, the term "siRNA" does not include a complete DNA molecule.

[0089] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide extending from the double helix structure of the siRNA. For example, when the 3' end of one strand of the siRNA extends beyond the 5' end of the other strand or vice versa, there is a nucleotide overhang. The siRNA may include an overhang of at least one nucleotide; or the overhang may include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang may include or consist of: nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. One or more overhangs may be on the sense strand, the antisense strand, or any combination thereof. In addition, one or more nucleotides of the overhang may be present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the siRNA.

[0090] As used herein with respect to siRNA, the term "blunt" or "blunt end" means that there are no unpaired nucleotides or nucleotide analogs at a given end of the siRNA, i.e., no nucleotide overhang. One or both ends of the siRNA may be blunt-ended. In the case where both ends of the siRNA are blunt-ended, the siRNA is said to be "blunt-ended." A "blunt-ended" siRNA is a siRNA that has blunt ends at both ends, i.e., does not have a nucleotide overhang at either end of the molecule. Most typically, the molecule will be double-stranded over its entire length.

[0091] II. siRNA targeting HBV

[0092] The present disclosure provides a treatment method, which involves administering siRNA targeting HBV and related compositions and kits. In some embodiments, the siRNA targeting HBV is HBV02. HBV02 is a synthetic chemically modified siRNA targeting HBV RNA with a covalently linked tri-antenna N-acetyl-galactosamine (GalNAc) ligand that allows specific hepatocyte uptake. HBV02 targets the HBV genome region common to all HBV viral transcripts and has pharmacological activity against HBV genotypes A to J. In preclinical models, HBV02 has been shown to inhibit viral replication, translation and secretion of HBsAg, and can provide a functional cure for chronic HBV infection. A siRNA can have a variety of antiviral effects, including degradation of pgRNA, thereby inhibiting viral replication, and degradation of all viral mRNA transcripts, thereby preventing the expression of viral proteins. This can restore a functional immune response to HBV alone or in combination with other therapies. The ability of HBV02 to reduce non-infectious subviral particles containing HBsAg also distinguishes it from currently available treatments.

[0093] HBV02 targets and inhibits the expression of mRNA encoded by the HBV genome according to the NCBI reference sequence NC_003977.2 (GenBank accession number GI: 21326584) (SEQ ID NO: 1). More specifically, HBV02 targets mRNA encoded by a portion of the HBV genome comprising the sequence GTGTGCACTTCGCTTCAC (SEQ ID NO: 2), which corresponds to nucleotides 1579 to 1597 of SEQ ID NO: 1. Because transcription of the HBV genome produces polycistronic, overlapping RNAs, HBV02 causes significant inhibition of the expression of most or all HBV transcripts.

[0094] HBV02 has a sense strand comprising 5'-GUGUGCACUUCGCUUCACA -3' (SEQ ID NO:3) and an antisense strand comprising 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO:4), wherein the nucleotides include 2'-fluoro (2'F) and 2'-O-methoxy (2'OMe) ribose modifications, phosphorothioate backbone modifications, glycol nucleic acid (GNA) modifications, and is conjugated to a triantennary N-acetyl-galactosamine (GalNAc) ligand at the 3' end of the sense strand to facilitate delivery to hepatocytes through the asialoglycoprotein receptor (ASGPR). Including the modifications, the sense strand of HBV02 comprises 5′-gsusguGfcAfCfUfucgcuucacaL96-3′ (SEQ ID NO: 5) and the antisense strand comprises 5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′ (SEQ ID NO: 6), wherein the modifications are abbreviated as shown in Table 1.

[0095] Table 1. Nucleotide monomer abbreviations used in the representation of modified nucleic acid sequences. It should be understood that unless otherwise indicated, When present in an oligonucleotide, these monomers are linked to each other via 5′-3′-phosphodiester bonds.

[0096]

[0097]

[0098] In some embodiments, the siRNA used in the methods, compositions, or kits described herein is HBV02.

[0099] In some embodiments, the siRNA used in the methods, compositions, or kits described herein comprises a sequence variant of HBV02. In specific embodiments, the portion of one or more HBV transcripts targeted by the sequence variant of HBV02 overlaps with the portion of one or more HBV transcripts targeted by HBV02.

[0100] In some embodiments, the siRNA comprises a sense strand and an antisense strand, wherein (1) the sense strand comprises SEQ ID NO:3 or SEQ ID NO:5, or a sequence that differs from 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 nucleotides, respectively; or (2) the antisense strand comprises SEQ ID NO:4 or SEQ ID NO:6, or a sequence that differs from 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 nucleotides, respectively.

[0101] In some embodiments, a shorter duplex having one of the sequences of SEQ ID NO:5 or SEQ ID NO:6 minus only a few nucleotides on one or both ends is used. Thus, the present invention encompasses a partial sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from one or both of SEQ ID NO:5 and SEQ ID NO:6, and an siRNA having the ability to inhibit HBV gene expression differs by no more than 5, 10, 15, 20, 25 or 30% inhibition from an siRNA comprising the complete sequence. In some embodiments, an siRNA having a blunt end at one or both ends formed by removing nucleotides from one or both ends of HBV02 is provided.

[0102] In some embodiments, siRNA as described herein may contain one or more mismatches with the target sequence. In some embodiments, siRNA as described herein contains no more than 3 mismatches. In some embodiments, if the antisense strand of siRNA contains a mismatch with the target sequence, the mismatch region is not located at the center of the complementary region. In a specific embodiment, if the antisense strand contains a mismatch with the target sequence, the mismatch is limited to the last 5 nucleotides of the 5' end or 3' end of the complementary region. For example, for a siRNA chain of 23 nucleotides complementary to the region of the HBV gene, the RNA chain may not contain any mismatch in the central 13 nucleotides. Methods described herein or methods known in the art can be used to determine whether the siRNA containing a mismatch with the target sequence is effective in suppressing the expression of the HBV gene.

[0103] In some embodiments, the siRNA used in the methods, compositions and kits described herein comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand and the second oligonucleotide is described as the corresponding antisense strand of the sense strand. As described elsewhere herein and as known in the art, the complementary sequence of the siRNA can also be included as a self-complementary region of a single nucleic acid molecule relative to being located on different oligonucleotides.

[0104] In some embodiments, the methods, compositions and kits described herein use a single-stranded antisense RNA molecule comprising an antisense strand of HBV02 or a sequence variant thereof. The antisense RNA molecule may have 15 to 30 nucleotides complementary to the target. For example, the antisense RNA molecule may have a sequence of at least 15, 16, 17, 18, 19, 20, 21 or more consecutive nucleotides from SEQ ID NO: 6.

[0105] In some embodiments, the siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 5 and the antisense strand comprises SEQ ID NO: 6, and further comprises additional nucleotides, modifications, or conjugates as described herein. For example, in some embodiments, the siRNA may include modifications other than those shown in SEQ ID NO: 5 and 6. The modifications may be produced using methods recognized in the art, such as those described in Current protocols in nucleic acid chemistry, Beaucage SL et al. (eds.), John Wiley & Sons, New York, NY, USA, which are incorporated herein by reference. Examples of the modifications are described in more detail below.

[0106] a. Modified siRNA

[0107] Modifications disclosed herein include, for example, (a) sugar modifications (e.g., at the 2' position or the 4' position) or replacement of sugars; (b) backbone modifications, including modification or replacement of phosphodiester bonds; (c) base modifications, such as replacement of bases with stabilized bases, destabilized bases, or bases that are base-paired with an extended partner library, removal of bases (abasic nucleotides) or conjugated bases; and (d) terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, reverse linkage, etc.), 3' terminal modifications (conjugation, DNA nucleotides, reverse linkage, etc.). Some specific examples of modifications that can be incorporated into the siRNA of the present application are shown in Table 1.

[0108] Modifications include substituted sugar moieties. The siRNA provided herein may include one of the following at the 2′ position: OH; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkenyl, or N-alkenyl; O-alkynyl, S-alkynyl, or N-alkynyl; or O-alkyl-O-alkyl; wherein alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n C3)]2, wherein n and m are 1 to about 10. In some other embodiments, the siRNA comprises one of the following at the 2' position: C1 to C10 Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of siRNA or group for improving the pharmacodynamic properties of siRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., "Swiss Chemical Journal (Helv.Chim.Acta)" 1995, 78: 486-504), i.e., alkoxy-alkoxy. Another exemplary modification is 2'-dimethylaminooxyethoxy, ie, O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2 * -O-dimethylaminoethoxyethyl or 2 * -DMAEOE), i.e., 2 * -O-CH2-O-CH2-N(CH2)2. Other exemplary modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications may also occur at other positions on the RNA of the siRNA, particularly at the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked siRNAs and at the 5' position of the 5' terminal nucleotide. Modifications may also include sugar mimetics, such as cyclobutyl moieties, in place of the pentafuroyl sugar.

[0109] Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, 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 incorporated herein by reference for its teachings relating to methods for making such modifications.

[0110] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters; methyl and other alkylphosphonates, including 3'-alkylenephosphonates and chiral phosphonates; phosphites; phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates; thiocarbonylphosphoramidates; thiocarbonylalkylphosphonates, thiocarbonylalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5'-linked analogs of these esters, and those with reverse polarity, wherein adjacent nucleoside unit pairs are linked with 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0111] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, 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; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,316; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587,361; No. 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, No. 608,035; No. 6,683,167; No. 6,858,715; No. 6,867,294; No. 6,878,805; No. 7,015,315; No. 7,041,816; No. 7,273,933; No. 7,321,029; and U.S. Pat. No. RE39464; each of which is incorporated herein by reference for its teachings relating to methods of making such modifications.

[0112] RNA with modified main chain especially includes those without phosphorus atom in the main chain. For the purpose of this specification, and as sometimes mentioned in the art, modified RNA without phosphorus atom in its internucleoside main chain can also be regarded as oligonucleoside. The modified RNA main chain without phosphorus atom has a main chain formed by: short chain alkyl or cycloalkyl nucleoside interbond, mixed heteroatom and alkyl or cycloalkyl nucleoside interbond or one or more short chain heteroatom or heterocyclic nucleoside interbond. These main chains include main chains with morpholino bond (partially formed by sugar part of nucleoside); siloxane main chain; sulfide, sulfoxide and sulfone main chain; formyl and thioformyl main chain; methylene formyl and thioformyl main chain; olefin-containing main chain; aminosulfonate main chain; methylene imino and methylene hydrazine main chain; sulfonate and sulfonamide main chain; amide main chain; and other main chains with mixed N, O, S and CH2 constituent parts.

[0113] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, 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; 5,491,687; 5,511,679; 5,643,678; 5,650,679; 5,664,671; 5,675,683; 5,692,710; 5,701,711; 5,713,712; 5,714,713; 5,715,714; 5,716,715 No. 5,419,677; each of which is incorporated herein by reference for its teachings relating to methods of making such modifications.

[0114] In some embodiments, the sugar and internucleoside bonds of the nucleotide unit, that is, the main chain are replaced by novel groups. The base unit is retained to hybridize with an appropriate nucleic acid target compound. One such oligomeric compound, i.e., an RNA mimetic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, especially an aminoethylglycine backbone. The nucleobase is retained and directly or indirectly bound to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, No. 5,539,082; No. 5,714,331; and No. 5,719,262; each of which is incorporated herein by reference. Further teachings of PNA compounds can be found in, for example, Nielsen et al. (Science, 254: 1497-1500 (1991)).

[0115] Some embodiments provided in the technology described herein include RNA with a thiophosphate backbone and oligonucleosides with a heteroatom backbone, and the heteroatom backbone is particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [referred to as a methylene (methylimino) or MMI backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [wherein the natural phosphodiester backbone is represented as -OPO-CH2-] of U.S. Pat. No. 5,489,677 and the amide backbone of U.S. Pat. No. 5,602,240. In some embodiments, the RNA provided herein has a morpholino backbone structure of U.S. Pat. No. 5,034,506.

[0116] Modifications of the siRNA disclosed herein may also include modifications or substitutions of nucleobases (commonly referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 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 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Other nucleobases include those disclosed in U.S. Pat. No. 3,687,808, Modified Nucleosides in Biochemistry, Biotechnology and Medicine (Herdewijn P, ed., Wiley-VCH, 2008); The Concise Encyclopedia Of Polymer Science And Engineering (pp. 858-859, Kroschwitz JL, ed., John Wiley & Sons, 1990), Englisch et al. (Angewandte Chemie, International Edition, 30, 613, 1991), and Sanghvi YS (Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke ST and Lebleu B, eds., CRC Press, 1993). Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds provided in the technology described herein. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C (Sanghvi YS et al., eds., dsRNA Research and Applications, Boca Raton CRC Press, pp. 276-278, 1993) and are exemplary base substitutions, even more specifically when combined with 2′-O-methoxyethyl sugar modifications.

[0117] Representative U.S. patents that teach the preparation of some of the modified nucleobases mentioned above, as well as 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 incorporated herein by reference for its teachings relating to methods of making such modifications.

[0118] siRNA can also be modified to include one or more adenosine-glycol nucleic acids (GNAs). Adenosine-GNAs are described, for example, in Zhang et al. (JACS 2005, 127(12):4174-75), which is incorporated herein by reference for its teachings related to methods for preparing GNA modifications.

[0119] The RNA of siRNA can also be modified to include one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with modified ribose moieties, wherein the ribose moiety contains an additional bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose into a 3'-endo conformation. Adding locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen J et al., Nucleic Acids Research 2005, 33(1):439-47; Mook OR et al., Mol Cancer Therapy 2007, 6(3):833-43; Grunweller A et al., Nucleic Acids Research 2003, 31(12):3185-93).

[0120] Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845; each of which is incorporated herein by reference for its teachings related to methods of making such modifications.

[0121] In some embodiments, the siRNA includes modifications involving chemical attachment to the RNA of one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the siRNA. Such moieties include, but are not limited to, lipid moieties, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA 1989, 86:6553-56), bile acid (Manoharan et al., Biorg. Med. Chem. Let. 1990, 4:1053-60), thioethers, such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci. 1992, 660:306-9); Manoharan et al., Biorg. Med. Chem. Let. 1993, 3:2765-70), thiocholesterol (Oberhauser et al., Nucleic Acids Res. 1992, 20:533-38), aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J. Chem. 1993, 660:306-9); Manoharan et al., Ann. NY Acad. Sci. 1993, 3:2765-70). 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 1,2-di-O-hexadecyl-rac-glycero-3-phosphonic acid triethyl-ammonium (Manoharan et al., Tetrahedron Letters 1995, 36:3651-54; Shea et al., Nucleic Acids Research, 1990, 18:3777-83), a polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-73), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-54), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-37), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-37).

[0122] In some embodiments, the ligand alters the distribution, targeting or lifespan of the siRNA into which it is incorporated. In some embodiments, the ligand provides enhanced affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment such as a cell or organ compartment, a tissue, an organ or a body region) as compared to a species in which the ligand is not present. In such embodiments, the ligand will not participate in duplex pairing in a duplex nucleic acid.

[0123] 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, such as 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-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly-(2-ethylacrylic acid), N-isopropylacrylamide polymer or 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 helical peptides.

[0124] 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 specified cell type, such as a liver cell. The targeting group may be thyrotropin, melanocyte stimulating hormone, a lectin, a glycoprotein, surfactant protein A, a mucin carbohydrate, a multivalent lactose, a multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, a multivalent fucose, a glycosylated polyamino acid, a multivalent galactose, transferrin, a bisphosphonate, a polyglutamate, a polyaspartate, a lipid, cholesterol, a steroid, a bile acid, a folic acid, vitamin B12, vitamin A, biotin or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (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, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-0 (hexadecyl) glycerol, geranyloxyhexyl, hexadecyl glycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, 03-(oleoyl) pyroxyl cholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine), peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate esters, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino groups, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl groups, HRP and AP.

[0125] The ligand can be a protein, such as a glycoprotein, or a peptide, such as a molecule with a specific affinity for a coligand, or an antibody, such as an antibody that binds to a specified cell type, such as a hepatocyte. The ligand can also include hormones and hormone receptors. It can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose and multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.

[0126] The ligand can be a substance, such as a drug, that can increase the uptake of siRNA into a cell, for example by disrupting the cytoskeleton of the cell, for example by disrupting the microtubules, microfilaments and / or intermediate filaments of the cell. The drug can be, for example, paclitaxel, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine or myoservin.

[0127] In some embodiments, the ligand is a moiety, such as a vitamin, that is absorbed by a target cell, such as a liver cell. Exemplary vitamins include vitamin A, vitamin E, and vitamin K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients absorbed by a target cell, such as a liver cell. HSA and low-density lipoprotein (LDL) are also included.

[0128] In some embodiments, the ligand connected to the siRNA as described herein serves as a pharmacokinetic (PK) regulator. As used herein, "PK regulator" refers to a pharmacokinetic regulator. PK regulators include lipophiles, cholic acid, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK regulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen (naproxen), ibuprofen (ibuprofen), vitamin E, biotin, etc. Oligonucleotides comprising multiple thiophosphate bonds are also known to bind to serum proteins, so short oligonucleotides comprising multiple thiophosphate bonds in the main chain, such as oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases are also suitable for use as ligands (e.g., described as PK regulators) herein. In addition, aptamers in conjunction with serum components (e.g., serum proteins) are also suitable for use as PK regulators in embodiments described herein.

[0129] (i) Lipid conjugates. In some embodiments, the ligand or conjugate is a lipid or lipid-based molecule. The lipid or lipid-based ligand can (a) increase the conjugate's resistance to degradation, (b) increase targeting or transport into target cells or cell membranes, and / or (c) can be used to modulate binding to serum proteins, such as HSA. The lipid or lipid-based molecule can bind to serum proteins, such as human serum albumin (HSA). HSA binding ligands allow the conjugate to be distributed to target tissues, such as non-renal target tissues of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, neproxin or aspirin can be used.

[0130] Lipid-based ligands can be used to inhibit, e.g., control the binding of the conjugate to the target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA will be less likely to be targeted to the kidneys and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds less strongly to HSA can be used to target the conjugate to the kidneys.

[0131] In some embodiments, the lipid-based ligand binds to HSA. The lipid-based ligand can bind to HSA with sufficient affinity so that the conjugate will be distributed to non-renal tissues. In certain specific embodiments, the HSA-ligand binding is reversible.

[0132] In some embodiments, the lipid-based ligand binds HSA weakly or not at all, such that the conjugate will distribute to the kidney.Other moieties that target renal cells may also be used in place of the lipid-based ligand or in addition.

[0133] (ii) Cell penetrating peptides and agents. In another aspect, the ligand is a cell penetrating agent, such as a helical cell penetrating agent. In some embodiments, the agent is amphoteric. Exemplary agents are peptides, such as tat peptides or antennapedia peptides. If the agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. In some embodiments, the helical agent is an alpha-helical agent. In certain specific embodiments, the helical agent has a lipophilic and a lipophobic phase.

[0134] "Cell penetrating peptides" are capable of penetrating cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell penetrating peptides can be, for example, alpha-helical linear peptides (e.g., LL-37 or CeropinPI), disulfide bond-containing peptides (e.g., alpha-defensins, beta-defensins or bovine antimicrobial peptides), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin).

[0135] The ligand can be a peptide or a peptide mimetic. A peptide mimetic (also referred to herein as an oligopeptide mimetic) is a molecule that can be folded into a defined three-dimensional structure similar to a natural peptide. The connection of a peptide and a peptide mimetic to siRNA can affect the pharmacokinetic distribution of RNAi, for example, by enhancing cell recognition and absorption. The peptide or peptide mimetic portion can be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids long.

[0136] The peptide or peptide mimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide portion can be a dendrimer peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide portion can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 7). RFGF analogs containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 8) can also be targeting moieties. The peptide moiety can be a "delivery" peptide that can carry 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 be able to act as delivery peptides. The peptide or peptide mimetic can be encoded by a random DNA sequence, such as a peptide identified from a phage display library, or an one-bead-one-cell (OBOC) combinatorial library (Lam et al., Nature 1991, 354: 82-84).

[0137] The cell penetrating peptide may also include a nuclear localization signal (NLS). For example, the cell penetrating peptide may be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucleic Acids Research 1993, 31: 2717-24).

[0138] (iii) Carbohydrate conjugates. In some embodiments, the siRNA oligonucleotides described herein further comprise a carbohydrate conjugate. Carbohydrate conjugates may be advantageous for in vivo delivery of nucleic acids, and compositions suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that is itself a carbohydrate 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 a compound having a carbohydrate moiety consisting of one or more monosaccharide units each having at least six carbon atoms (which may be linear, branched, or cyclic) and oxygen, nitrogen, or sulfur atoms bonded to each carbon atom as part of it. 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 C5 and above (in some embodiments, C5 to C8) sugars; and disaccharides and trisaccharides include sugars having two or three monosaccharide units (in some embodiments, C5 to C8).

[0139] In some embodiments, the carbohydrate conjugate is selected from the group consisting of:

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to:

[0146]

[0147] (Formula XXII), wherein when one of X or Y is an oligonucleotide, the other is hydrogen.

[0148] In some embodiments, the carbohydrate conjugate further comprises another ligand, such as, but not limited to, a PK modulator, an endosomolytic ligand, or a cell penetrating peptide.

[0149] (iv) Linkers. In some embodiments, the conjugates described herein can be linked to siRNA oligonucleotides using various linkers that can be cleavable or non-cleavable.

[0150] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound. The linker typically comprises a direct bond, or an atom such as oxygen or sulfur; a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH; or a chain of atoms such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroaryl Arylalkyl, 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 methylene groups may be interrupted by 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 groups; wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In certain embodiments, the linker is between 1 and 24 atoms, between 4 and 24 atoms, between 6 and 18 atoms, between 8 and 18 atoms, or between 8 and 16 atoms.

[0151] A cleavable linking group is a linking group that is sufficiently stable outside of a cell, but which cleaves after entering a target cell to release the two parts of the linker held together. In certain embodiments, the cleavable linking group cleaves at least 10 times, or at least 100 times faster in a target cell or under a first reference condition (which can, for example, be selected to simulate or represent intracellular conditions) than in the blood of a subject or under a second reference condition (which can, for example, be selected to simulate or represent conditions present in blood or serum).

[0152] The cleavable linking group is sensitive to cleavage agents, such as pH, redox potential or the presence of degradation molecules. In general, cleavage agents are more common inside cells than in serum or blood, or are found to be higher in level or activity. Examples of the degradation agent include: redox agents selected for specific substrates or without substrate specificity, including, for example, oxidases or reductases or reductants present in cells, such as thiols, which can degrade the redox cleavable linking group by reduction; esterases; endosomes or agents that can produce an acidic environment, such as those that make the pH five or lower; enzymes, peptidases (which can be substrate-specific) and phosphatases that can hydrolyze or degrade acid cleavable linking groups by acting as universal acids. Cleavable linkage groups, such as disulfide bonds, can be sensitive to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, in the range of about 7.1 to 7.3. Endosomes have a relatively 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 will have a cleavable linking group that is cleaved at a specific pH, thereby releasing the cationic lipid from the ligand inside the cell, or into a desired cellular compartment.

[0153] The linker can include a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker can depend on the targeted cell. For example, a liver targeting ligand can be linked to a cationic lipid via a linker that includes an ester group. Liver cells are rich in esterases, and thus the linker will be more efficiently cleaved in liver cells 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.

[0154] When targeting cell types rich in peptidases, such as hepatocytes and synoviocytes, linkers containing peptide bonds can be used.

[0155] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of the candidate linking group to crack by testing degradation agents (or conditions). It may also be necessary to test the ability of the candidate cleavable linking group to resist the cracking in blood or when contacting with other non-target tissues. Therefore, the relative cracking susceptibility between the first and second conditions can be determined, wherein the first condition is selected to indicate the cracking in the target cell, and the second condition is selected to indicate the cracking in other tissues or biofluids, such as blood or serum. Evaluation can be carried out in a cell-free system, cell, cell culture, organ or tissue culture or whole animal. It can be useful to perform an initial evaluation under cell-free or culture conditions and to confirm by further evaluation in whole animals. In some embodiments, compared with blood or serum (or under in vitro conditions selected to simulate extracellular conditions), the cracking of useful candidate compounds in cells (or under in vitro conditions selected to simulate intracellular conditions) is at least 2, at least 4, at least 10 or at least 100 times faster.

[0156] A class of cleavable linking groups is a redox cleavable linking group that is cleaved under reduction or oxidation. An example of a reduction cleavable linking group is a disulfide linking group (-SS-). In order to determine whether a candidate cleavable linking group is a suitable "reduction cleavable linking group", or whether it is suitable for use with a specific RNAi part and a specific targeting agent, it can be found in the method described herein. For example, the candidate can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art, and the reagents simulate the cleavage rate observed in cells, for example, target cells. The candidate can also be evaluated under conditions selected to simulate blood or serum conditions. In some embodiments, the candidate compound is cleaved up to 10% in blood. In certain embodiments, compared with blood (or under in vitro conditions selected to simulate extracellular conditions), the degradation of useful candidate compounds in cells (or under in vitro conditions selected to simulate intracellular conditions) is at least 2, at least 4, at least 10 or at least 100 times faster. The rate of cleavage of a candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0157] Phosphate-based cleavable linking groups are cleaved by factors that degrade or hydrolyze the phosphate group. Examples of factors that cleave the phosphate group in a cell are enzymes in the cell, such as phosphatases. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. In certain embodiments, the phosphate-based linking group is selected from: -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. In a specific embodiment, the phosphate linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0158] Acid cleavable linking groups are linking groups that are cleaved under acidic conditions. In some embodiments, the acid cleavable linking group is in an acidic environment of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower), or by a reagent that can act as a universal acid, such as an enzyme cleavage. In cells, specific low pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid cleavable linking groups. Examples of acid cleavable linking groups include, but are not limited to, esters of hydrazones, esters, and amino acids. Acid cleavable groups may have the general formula -C=N-, C(O)O, or -OC(O). In some embodiments, the carbon (alkoxy) connected to the oxygen of the ester is an aryl, a substituted alkyl, or a tertiary alkyl, such as a dimethylpentyl or tert-butyl group. These candidates can be evaluated using methods similar to those described above.

[0159] Ester-based cleavable linking groups are cleaved by enzymes in cells, such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene and alkynylene. Ester-cleavable linking groups have the general formula -C (O) O- or -OC (O) -. These candidates can be evaluated using methods similar to the above-mentioned method.

[0160] The cleavable linking group based on peptide is by enzyme in cell, such as peptidase and protease cleavage.The cleavable linking group based on peptide is formed between amino acid to produce the peptide bond of oligopeptide (for example, dipeptide, tripeptide etc.) and polypeptide.The cleavable group based on peptide does not include amide group (-C (O) NH-).Amide group can be formed between any alkylene, alkenylene or alkynylene.Peptide bond is the amide bond of special type formed between amino acid to produce peptide and protein.The cleavable group based on peptide is generally limited to the peptide bond (i.e. amide bond) formed between amino acid to produce peptide and protein, and does not include whole amide functional group.The cleavable linking group based on peptide has general formula-NHCHRAC (O) NHCHRBC (O)-, wherein RA and RB are the R groups of two adjacent amino acids.These candidates can be evaluated using a method similar to the above method.

[0161] Representative carbohydrate conjugates with linkers include, but are not limited to:

[0162]

[0163]

[0164] wherein when one of X or Y is an oligonucleotide, the other is hydrogen.

[0165] In certain embodiments of the compositions and methods, the ligand is one or more "N-acetylgalactosamine" (GalNAc) derivatives connected by a bivalent or trivalent branched linker. For example, in some embodiments, siRNA is conjugated to a GalNAc ligand, as shown in the following schematic diagram:

[0166]

[0167] Wherein X is O or S.

[0168] In some embodiments, the combination therapy comprises an siRNA conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any one of Formulas (XXXI)-(XXXIV):

[0169]

[0170] in:

[0171] q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C independently represent 0-20 at each occurrence, and wherein the repeating units may be the same or different;

[0172] P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C 、T 2A 、T 2B 、T 3A 、T 3B 、T 4A 、T 4B 、T 4A 、T 5B and T 5C is, independently at each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O;

[0173] Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B and Q 5C is independently absent at each occurrence, alkylene or substituted alkylene, wherein one or more methylene groups may be interrupted or terminated by one or more of the following: O, S, S(O), SO2, N(R N ), C(R')=C(R"),C≡C or C(O);

[0174] R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B and R 5C Each occurrence is independently absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, or a heterocyclic group;

[0175] L2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; that is, independently at each occurrence is a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide or a polysaccharide; and R a Is H or an amino acid side chain. The trivalent conjugated GalNAc derivative can be particularly used together with siRNA to inhibit the expression of target genes, and the derivative is as shown in formula (XXXV):

[0176] (Formula XXXV)

[0177]

[0178] Where L 5A , L 5B and L 5C Represents a monosaccharide, such as a GalNAc derivative.

[0179] Examples of suitable divalent and trivalent branched linking groups for conjugating GalNAc derivatives include, but are not limited to, the structures described above as Formula I, Formula VI, Formula X, Formula IX, and Formula XII.

[0180] Representative U.S. patents that teach the preparation of RNA conjugates include Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,51 No. 2,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,9 No. 63; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,371,241, No. 5,391,723; No. 5,416,203, No. 5,451,463; No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,565,552; No. 5,567,810 ; U.S. Patent Nos. 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 incorporated herein by reference for its teachings relating to the preparation method.

[0181] In some cases, the RNA of the siRNA can be modified by non-ligand groups. Many non-ligand molecules have been conjugated to siRNA in order to enhance the activity, cellular distribution or cellular uptake of the siRNA, and procedures for performing such conjugations can be found in the scientific literature. The non-ligand moiety includes a lipid moiety, 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., Bioorganic and Medicinal Chemistry Letters 4:1053 (1994)), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Annals of the New York Academy of Sciences 660:306 (1992); Manoharan et al., Bioorganic and Medicinal Chemistry Letters 3:2765 (1993)), thiocholesterol (Oberhauser et al., Nucleic Acids Res. 20:533 (1992)), an aliphatic chain, such as dodecanediol or an undecyl residue (Saison-Behmoaras et al., EMBO J. 10:111 (1991); Kabanov et al., FEBS Lett. 259:327 (1990); Svinarchuk et al., Biochemistry 75:49 (1993)), phospholipids, such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonic acid triethylammonium (Manoharan et al., Tetrahedron Lett. 36:3651 (1995); Shea et al., Nucleic Acids Res. 18:3777 (1990)), A polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides 14:969 (1995)), or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett. 36:3651 (1995)), a palmityl moiety (Mishra et al., Biochimica et Biophysica Sinica 1264:229 (1995)), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., Journal of Pharmacology & Exp. Therapeutics 277:923 (1996)).

[0182] Typical conjugation protocols involve synthesizing RNA with an amino linker at one or more positions of the sequence. The amino group then reacts with the molecule to be conjugated using an appropriate coupling or activating reagent. The conjugation reaction can be performed in the solution phase while the RNA is still bound to a solid support or after RNA cleavage. Purification of the RNA conjugate by HPLC generally yields a pure conjugate.

[0183] b. Pharmaceutical compositions and delivery of siRNA

[0184] In some embodiments, a pharmaceutical composition containing siRNA as described herein and a pharmaceutically acceptable carrier or excipient is provided. The pharmaceutical composition containing siRNA can be used to treat HBV infection. The pharmaceutical composition is formulated based on the delivery mode. For example, the composition can be formulated for delivery by parenteral, for example, by subcutaneous (SC) delivery for systemic administration.

[0185] A "pharmaceutically acceptable carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more agents, such as nucleic acids, to an animal. Excipients can be liquid or solid and are selected with regard to the intended mode of administration so as to provide the desired body, consistency, etc., when combined with the agent (e.g., nucleic acid) and other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers or excipients include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone, hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

[0186] The siRNA compositions can also be prepared using pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not react harmfully with nucleic acids. Suitable pharmaceutically acceptable carriers for the preparations delivered non-parenterally include, but are not limited to, water, saline solutions, alcohol, polyethylene glycol, gelatin, lactose, straight-chain starch, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinyl pyrrolidone, etc.

[0187] The formulation for topical administration of nucleic acid may include aseptic and non-sterile aqueous solutions, common solvents, such as non-aqueous solutions in alcohol, or nucleic acid solutions / melts in liquid or solid oil matrices. The solution may also contain buffer, diluent and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not react harmfully with nucleic acid may be used.

[0188] In some embodiments, administration of the pharmaceutical compositions and formulations described herein can be topical (e.g., by a transdermal patch), pulmonary (e.g., by inhalation or insufflation of a powder or mist, including by a nebulizer); intratracheal; intranasal; epidermal and transdermal; oral; or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, and intramuscular injection or infusion; subdermal administration (e.g., by an implant device); or intracranial administration (e.g., by intraparenchymal, intrathecal, or intraventricular administration).

[0189] In some embodiments, the pharmaceutical composition comprises a sterile solution of HBV02 formulated in water for subcutaneous injection. In some 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.

[0190] In some embodiments, the pharmaceutical composition containing siRNA as described herein is administered with a dosage sufficient to suppress the expression of HBV genes. In some embodiments, the dosage of siRNA is in the range of 0.001 to 200.0mg per kilogram of recipient body weight per day, or in the range of 1 to 50mg per kilogram of body weight per day. For example, siRNA can be administered with 0.01mg / kg, 0.05mg / kg, 0.5mg / kg, 1mg / kg, 1.5mg / kg, 2mg / kg, 3mg / kg, 10mg / kg, 20mg / kg, 30mg / kg, 40mg / kg or 50mg / kg / single dose. The pharmaceutical composition can be administered once daily, or it can be administered as two, three or more sub-doses at appropriate intervals in one day, or even using continuous infusion or delivered by controlled release formulations. In this case, the siRNA contained in each sub-dosage must be correspondingly smaller, so as to reach a total daily dose. The dosage unit can also be compounded to deliver within several days, for example using conventional sustained release formulations, which provide sustained release of siRNA within a few days. Sustained release formulations are well known in the art and are particularly useful for delivering agents at specific locations, such as can be used with agents of the technology described herein. In the described embodiment, the dosage unit contains a daily dose of a corresponding multiple.

[0191] In some embodiments, a pharmaceutical composition comprising an siRNA targeting HBV described herein (eg, HBV02) contains a dose of 0.8 mg / kg, 1.7 mg / kg, 3.3 mg / kg, 6.7 mg / kg, or 15 mg / kg of the siRNA.

[0192] In some embodiments, a pharmaceutical composition comprising a siRNA described herein (e.g., HBV02) contains 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 of siRNA.

[0193] In some embodiments, a pharmaceutical composition comprising an siRNA described herein (eg, HBV02) contains a dose of 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 450 mg of siRNA.

[0194] In some embodiments, a pharmaceutical composition comprising an siRNA described herein (eg, HBV02) contains a 200 mg dose of the siRNA.

[0195] III. Treatments and additional therapeutic agents

[0196] The present disclosure provides methods for treating HBV infection with the siRNA described herein. In some embodiments, a method for treating HBV is provided, comprising administering HBV02 to a subject.

[0197] In some embodiments of the foregoing methods, the method further comprises administering pegylated interferon-α (PEG-IFNα) to the subject.

[0198] In some other embodiments of the aforementioned method, the method further comprises administering a nucleoside / nucleotide reverse transcriptase inhibitor (NRTI) to the subject. In some embodiments, the NRTI is administered sequentially before, simultaneously with, or after the administration of HBV02.

[0199] In some embodiments, a method for treating HBV is provided, comprising administering HBV02 and PEG-IFNα to a subject. In some embodiments, PEG-IFNα is administered before, simultaneously with, or after the administration of HBV02.

[0200] In some embodiments, a method of treating HBV is provided, comprising administering HBV02 and PEG-IFNα to a subject, wherein the subject has previously been administered NRTI. In some embodiments, PEG-IFNα is administered simultaneously with HBV02 or sequentially after administration of HBV02.

[0201] In some embodiments, a method of treating HBV is provided comprising administering HBV02, wherein the subject has been previously administered PEG-IFNα and has been previously administered NRTI.

[0202] In any of the aforementioned methods, the HBV infection may be a chronic HBV infection.

[0203] As used herein, "nucleoside / nucleotide reverse transcriptase inhibitors" or "nucleoside(acid) reverse transcriptase inhibitors" (NRTIs) refer to inhibitors of DNA replication that are structurally similar to nucleotides or nucleosides and specifically inhibit HBV cccDNA replication by inhibiting the action of HBV polymerase, and do not significantly inhibit host (e.g., human) DNA replication. The inhibitors include tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), lamivudine, adefovir, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clavudine, ritonavir, dipivoxil, lobcavir, panvir, N-acetyl-cysteine ​​(NAC), PC1323, theradigm-HBV, thymosin-α, ganciclovir, besifovir (ANA-380 / LB-80380) and tenofvir-exaliades (TLX / CMX157). In some embodiments, the NRTI is entecavir (ETV). In some embodiments, the NRTI is tenofovir. In some embodiments, the NRTI is lamivudine. In some embodiments, the NRTI is adefovir or adefovir dipivoxil.

[0204] As used herein, "subject" is an animal, such as a mammal, including any mammal that can be infected with HBV, such as a primate (such as a human, a non-human primate, such as a monkey or a chimpanzee), or an animal considered as an acceptable clinical model of HBV infection, a HBV-AAV mouse model (see, e.g., Yang et al., Cell and Mol Immunol 11:71 (2014)) or a HBV 1.3xfs transgenic mouse model (Guidotti et al., J. Virol. 69:6158 (1995)). In some embodiments, the subject is infected with hepatitis B virus (HBV). In some embodiments, the subject is a human, such as a human with HBV infection, particularly chronic hepatitis B virus infection.

[0205] As used herein, the terms "treating" and "treatment" refer to beneficial or desired results, including but not limited to alleviating or ameliorating one or more signs or symptoms associated with undesirable HBV gene expression or HBV replication, for example, 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 to 34 U / L), absence or low levels of anti-HBV antibodies; liver damage; cirrhosis; hepatitis D; acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma; serum sickness syndrome; anorexia; nausea; vomiting; mild fever; myalgia; easy fatigability sexual; taste and smell disturbances (food and cigarette aversions); or right upper quadrant and epigastric pain (intermittent, mild to moderate); hepatic encephalopathy; somnolence; disturbed sleep patterns; confusion; coma; ascites; gastrointestinal bleeding; coagulopathy; jaundice; hepatomegaly (mildly enlarged, soft liver); splenomegaly; palmar erythema; spider angiomas; muscular atrophy; spider angiomas; vasculitis; varicose bleeding; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (abdominal collateral vein) (caput medusa); ALT level higher than AST level; elevated gamma-glutamyl transpeptidase (GGT) (normal range is generally considered to be about 8 to 65 U / L) and alkaline phosphatase (ALP) levels (normal range is generally considered to be about 44 to 147 IU / L (international units per liter), not more than 3 times the ULN); slightly low albumin level; elevated serum iron level; leukopenia (ie, granulocytopenia); lymphocytosis; increased erythrocyte sedimentation rate (ESR); shortened red blood cell survival; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); serum or liver HBsAg, HBeAg, hepatitis B core antibody (anti-HBc) immunoglobulin M (IgM); hepatitis B surface antibody (anti-HBs), hepatitis B e antibody (anti-HBe), or HBV The presence of DNA; increased bilirubin levels; hyperglobulinemia; the presence of tissue nonspecific antibodies, such as anti-smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA) (10 to 20%); the presence of tissue-specific antibodies, such as antibodies against the thyroid gland (10 to 20%); elevated rheumatoid factor (RF) levels; low platelet and white blood cell counts; lobular and degenerative and regenerative hepatocellular changes and concomitant inflammation; and marked centrilobular necrosis, whether detectable or undetectable.For example, when an individual with one or more risk factors for liver fibrosis, such as chronic hepatitis B infection, does not suffer from liver fibrosis, or suffers from liver fibrosis to a lesser degree relative to a cohort with the same risk factors and not receiving treatment as described herein, the likelihood of suffering from liver fibrosis is reduced. "Treatment" can also mean an extension of survival compared to the expected survival in the absence of treatment.

[0206] As used herein, the terms "preventing" and "prevention" refer to not developing a disease, disorder, or condition, or reducing the development of signs or symptoms associated with the disease, disorder, or condition (e.g., a clinically relevant amount), or delaying the development of signs or symptoms (e.g., a delay of days, weeks, months, or years). Prevention may require administration of more than one dose.

[0207] In some embodiments, the treatment of HBV infection causes a "functional cure" of hepatitis B. As used herein, functional cure is understood as the clearance of circulating HBsAg, and may be accompanied by a state in which HBsAg antibodies become detectable using clinically relevant analyses. For example, detectable antibodies may include a signal higher than 10mIU / ml as measured by chemiluminescent micron particle immunoassay (CMIA) or any other immunoassay. Functional cure does not require the removal of all replicative forms of HBV (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 HBV are often observed to remain for decades, indicating that functional cure rather than complete cure occurs. Without being bound by a specific mechanism, the immune system may be able to control HBV under conditions where functional cure has been achieved. Functional cure permits interruption of any treatment for HBV infection. However, it should be understood that a "functional cure" of HBV infection may not be sufficient to prevent or treat a disease or condition caused by HBV infection, such as liver fibrosis, HCC, or cirrhosis. In some specific embodiments, "functional cure" can refer to a sustained reduction in serum HBsAg, e.g., <1 IU / mL, for at least 3 months, at least 6 months, or at least one year after initiation of a treatment regimen or completion of a treatment regimen. The official endpoint accepted by the U.S. Food and Drug Administration or FDA for demonstrating functional cure of HBV is the absence of detectable HBsAg in the blood six months after the end of therapy, defined as less than 0.05 international units / milliliter or IU / ml, and HBV DNA below the limit of quantification.

[0208] As used herein, the term "hepatitis B virus-related disease" or "HBV-related disease" is a disease or condition caused by or associated with HBV infection or replication. The term "HBV-related disease" includes diseases, disorders or conditions that benefit from reduced HBV gene expression or replication. Non-limiting examples of HBV-related diseases include, for example, hepatitis D virus infection, hepatitis D, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0209] In some embodiments, the HBV-related disease is chronic hepatitis. Chronic hepatitis B is defined by one of the following criteria: (1) positive serum HBsAg, HBV DNA, or HBeAg at two times at least 6 months apart (any combination of these tests performed 6 months apart is acceptable); or (2) negative HBV core antigen immunoglobulin M (IgM) antibody (IgM anti-HBc) and a positive result for one of the following tests: HBsAg, HBeAg, or HBV DNA (see Figure 2 Chronic HBV usually involves inflammation of the liver that lasts for more than six months. Subjects with chronic HBV are HBsAg positive and have high viremia (≥10 4 HBV-DNA copies / ml blood) or low viremia (<10 3 HBV-DNA copies / ml blood). In certain embodiments, the subject has been infected with HBV for at least five years. In certain embodiments, the subject has been infected with HBV for at least ten years. In certain embodiments, the subject was infected with HBV at birth. Subjects with chronic hepatitis B disease can be immune tolerant, or have an inactive chronic infection without any signs of active disease, and the subject is also asymptomatic. Patients with chronic active hepatitis, especially during the replicative state, may have symptoms similar to those of acute hepatitis. Subjects with chronic hepatitis B disease may have active chronic infection accompanied by necroinflammatory liver disease, have increased hepatocyte turnover in the absence of detectable necroinflammation, or have an inactive chronic infection without any signs of active disease, and the subject is also asymptomatic. The persistence of HBV infection in chronic HBV subjects is a result of ccHBV DNA.

[0210] HBeAg status indicates multiple differences between subjects (Table 2). HBeAg status can affect the response to different therapies, and approximately one-third of HBV patients are HBeAg positive.

[0211] Table 2: HBeAg status.

[0212] HBeAg positive HBeAg negative age Younger Older Approximate mean HBsAg level <![CDATA[10 4 -10 5 IU / mL]]> <![CDATA[10 3 IU / mL]]> Transcriptional activity cccDNA>intDNA intDNA>cccDNA HBV-specific immune profile Less immune damage Severe immune damage

[0213] In some embodiments, the subject with chronic HBV is HBeAg positive. In some other embodiments, the subject with chronic HBV is HBeAg negative. The subject with chronic HBV has less than 10 5 Serum HBV DNA levels and persistently elevated transaminases, such as ALT, AST, and γ-glutamyl transferase. Subjects with chronic HBV may have a liver biopsy score (e.g., necroinflammatory score) of less than 4.

[0214] In some embodiments, the HBV-related disease is acute fulminant hepatitis B. Subjects with acute fulminant hepatitis B have symptoms of acute hepatitis and the additional symptoms of confusion or coma (due to the liver's failure to detoxify chemicals) and bruising or bleeding (due to lack of clotting factors).

[0215] Subjects with HBV infection, such as chronic HBV, may suffer from liver fibrosis. Thus, in some embodiments, the HBV-related disease is liver fibrosis. Liver fibrosis or cirrhosis is histologically defined as a diffuse liver process characterized by fibrosis (excess fibrous connective tissue) and transformation of normal liver architecture into structurally abnormal nodules.

[0216] Subjects with HBV infection, such as chronic HBV, may suffer from end-stage liver disease. Thus, in some embodiments, the HBV-related disease is end-stage liver disease. For example, liver fibrosis may progress to the point where the body may no longer be able to compensate for the reduced liver function caused by, for example, liver fibrosis (i.e., decompensated liver), and produce, for example, psychiatric and neurological symptoms and liver failure.

[0217] Subjects with HBV infection, such as chronic HBV, may suffer from hepatocellular carcinoma (HCC), also known as malignant hepatoma. Therefore, in some embodiments, the HBV-related disease is HCC. HCC usually develops in subjects with chronic HBV and can be fibrolamellar, pseudoductal (adenoid proliferation), pleomorphic (giant cell) or clear cell type.

[0218] In some embodiments of the methods and purposes described herein, siRNA, PEG-IFN α or both of a therapeutically effective amount are administered to a subject. As used herein, "therapeutically effective amount" means the amount including an activating agent, which, when administered to a subject to treat a subject with HBV infection or HBV-related disease, is sufficient to achieve the treatment of the disease (e.g., by reducing or maintaining one or more symptoms of an existing disease or disease). "Therapeutically effective amount" may vary depending on the activating agent, its mode of administration, disease and its severity and the medical history, age, body weight, family history, gene composition, the stage of the pathological process mediated by HBV gene expression, the type of previous or concomitant treatment (if present) and other individual characteristics of the subject to be treated. The therapeutically effective amount may need to be administered more than one dosage.

[0219] "Therapeutically effective amount" also includes the amount of active agent that produces some desired effect at a reasonable benefit / risk ratio applicable to any treatment. The therapeutic agent (e.g., siRNA, PEG-IFNα) used in the methods of the present disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to the treatment.

[0220] As used herein, the term "sample" includes a collection of similar fluids, cells or tissues separated from a subject and fluids, cells or tissues present in a subject. Examples of biological fluids include blood, serum and serosal fluid, plasma, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs or local areas. For example, a sample may be derived from a specific organ, a part of an organ or a fluid or cell in those organs. In some embodiments, a sample may be derived from liver (such as whole liver or certain sections of liver or certain types of cells in liver, such as hepatocytes). In some embodiments, "sample derived from a subject" refers to blood or plasma or serum obtained from blood extracted from a subject. In other embodiments, "sample derived from a subject" refers to liver tissue (or its subcomponents) or blood tissue (or its subcomponents, such as serum) derived from a subject.

[0221] Some embodiments of the present disclosure provide a method for treating chronic HBV infection or HBV-related diseases in a subject in need thereof, comprising: administering to the subject an siRNA, wherein the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and a sense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 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-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol. In some embodiments of the method, the method further comprises administering pegylated interferon-α (PEG-IFNα) to the subject. In some embodiments, siRNA and PEG-IFNα are administered to a subject in the same period. In some embodiments, siRNA is administered to a subject for a period of time before PEG-IFNα is administered to a subject. In some embodiments, PEG-IFNα is administered to a subject for a period of time before siRNA is administered to a subject. In some embodiments, PEG-IFNα has been administered to a subject before siRNA is administered. In some embodiments, PEG-IFNα is administered to a subject during the same period of time during which siRNA is administered to a subject. In some embodiments, PEG-IFNα is subsequently administered after siRNA is administered to a subject.

[0222] In some embodiments of the aforementioned method, the method further includes administering NRTI to the subject. In some embodiments of the aforementioned method, prior to administering siRNA, NRTI has been administered to the subject to whom siRNA is administered. In some embodiments, prior to administering siRNA, NRTI has been administered to the subject for at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months. In some embodiments, prior to administering siRNA, NRTI has been administered to the subject for at least 2 months. In some embodiments, prior to administering siRNA, NRTI has been administered to the subject for at least 6 months. In some embodiments, NRTI is administered to the subject during the same period of time that siRNA is administered to the subject. In some embodiments of the method, NRTI is subsequently administered after siRNA is administered to the subject.

[0223] Some embodiments of the present disclosure provide an siRNA for treating chronic HBV infection in a subject, wherein the siRNA has a sense strand comprising 5′-gsusguGfcAfCfUfucgcuucacaL96-3′ (SEQ ID NO: 5) and a base pair 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-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol. In some embodiments of the siRNA for use, PEG-IFNα is also administered to the subject. In some embodiments, the siRNA and PEG-IFNα are administered to the subject during the same period of time. In some embodiments, siRNA is administered to the subject for a period of time before PEG-IFNα is administered to the subject. In some embodiments, PEG-IFNα is administered to the subject for a period of time before siRNA is administered to the subject. In some embodiments, PEG-IFNα has been administered to the subject before siRNA is administered. In some embodiments, PEG-IFNα is administered to the subject during the same period of time during which siRNA is administered to the subject. In some embodiments, PEG-IFNα is subsequently administered to the subject. In any of the aforementioned siRNAs for use, NRTI may also be administered to the subject or NRTI has been previously administered to the subject. In some embodiments, NRTI has been administered to the subject before siRNA is administered. In some embodiments, NRTI has been administered to the subject before siRNA is administered for at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months. In some embodiments, NRTI has been administered to the subject before siRNA is administered for at least 2 months. In some embodiments, NRTI has been administered to the subject before siRNA is administered for at least 6 months. In some embodiments, NRTI is administered to the subject during the same period of time during which siRNA is administered to the subject. In some embodiments, the subject is subsequently administered an NRTI.

[0224] Some embodiments of the present disclosure provide for use of siRNA in the manufacture of a medicament for treating chronic HBV infection, wherein the siRNA has a sense strand comprising 5′-gsusguGfcAfCfUfucgcuucacaL96-3′ (SEQ ID NO: 5) and a nucleotide strand comprising 5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′ (SEQ ID NO: 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-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0225] Some embodiments of the present disclosure provide the use of siRNA and PEG-IFNα in the manufacture of a medicament for treating chronic HBV infection, wherein the siRNA has a sense strand comprising 5′-gsusguGfcAfCfUfucgcuucacaL96-3′ (SEQ ID NO: 5) and a nucleotide strand comprising 5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′ (SEQ ID NO: 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-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0226] Some embodiments of the present disclosure provide for use of siRNA, PEG-IFNα and NRTI in the manufacture of a medicament for treating chronic HBV infection, wherein the siRNA has a sense strand comprising 5′-gsusguGfcAfCfUfucgcuucacaL96-3′ (SEQ ID NO: 5) and a sense strand comprising 5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′ (SEQ ID NO: 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-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0227] In some embodiments of the aforementioned methods, compositions for use, or uses, 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. In some embodiments of the aforementioned methods, compositions for use, or uses, the dose 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 some embodiments of the aforementioned methods, compositions for use, or uses, the dose of siRNA is 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 450 mg. In some embodiments of the aforementioned methods, compositions for use, or uses, the dose of siRNA is 200 mg. In some embodiments of the aforementioned methods, compositions for use, or uses, the dose of siRNA is at least 200 mg.

[0228] In some embodiments of the foregoing methods, compositions for use, or uses, the siRNA is administered weekly.

[0229] In some embodiments of the aforementioned methods, compositions for use, or uses, more than one dose of siRNA is administered. For example, in some embodiments, two doses of siRNA are administered, wherein the second dose is administered 2, 3, or 4 weeks after the first dose. In some specific embodiments, two doses of siRNA are administered, wherein the second dose is administered 4 weeks after the first dose.

[0230] In some embodiments of the foregoing methods, two, three, four, five, six or more doses of siRNA are administered. For example, in some embodiments, two 400 mg doses of siRNA are administered to the subject. In some embodiments, six 200 mg doses of siRNA are administered to the subject.

[0231] In some embodiments of the methods, compositions for use, or uses described herein, the method comprises:

[0232] (a) administering to the subject two or more doses of at least 200 mg of siRNA having a sense strand comprising 5′-gsusguGfcAfCfUfucgcuucacaL96-3′ (SEQ ID NO: 5) and a sense strand comprising 5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′ (SEQ ID NO: 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-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol; and

[0233] (b) administering a nucleoside / nucleotide reverse transcriptase inhibitor (NRTI) to the subject;

[0234] The subject is HBeAg negative or HBeAg positive.

[0235] In some embodiments, the method further comprises administering PEG-IFNα to the subject.

[0236] In some embodiments of the aforementioned methods, compositions for use, or uses, the siRNA is administered by subcutaneous injection. In some embodiments, the siRNA comprises 1, 2, or 3 subcutaneous injections per dose.

[0237] In some embodiments of the aforementioned methods, compositions for use, or uses, the dose of PEG-IFNα is 50 μg, 100 μg, 150 μg, or 200 μg. In some embodiments, the dose of PEG-IFNα is 180 μg.

[0238] In some embodiments of the foregoing methods, compositions for use, or uses, the PEG-IFNα is administered weekly.

[0239] In some embodiments of the aforementioned methods, compositions for use, or uses, the PEG-IFNα is administered by subcutaneous injection.

[0240] In some embodiments of the aforementioned methods, compositions for use, or uses, the NRTI may be tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), lamivudine, adefovir, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clavudine, ritonavir, dipivoxil, lobcavir, panvir, N-acetyl-cysteine ​​(NAC), PC1323, theradigm-HBV, thymosin-α, ganciclovir, besifovir (ANA-380 / LB-80380) or tenofvir-exaliades (TLX / CMX157). In some embodiments, the NRTI is entecavir (ETV). In some embodiments, the NRTI is tenofovir. In some embodiments, the NRTI is lamivudine. In some embodiments, the NRTI is adefovir or adefovir dipivoxil.

[0241] In some embodiments of the aforementioned methods, compositions for use, or uses, the subject is HBeAg negative. In some embodiments, the subject is HBeAg positive.

[0242] The siRNA may be present in the same pharmaceutical composition as the other active agents, or the active agents may be present in different pharmaceutical compositions. The different pharmaceutical compositions may be administered in combination / simultaneously or at different times or in different locations (e.g., different parts of the body).

[0243] IV. HBV therapy kit

[0244] Also provided herein is a kit comprising components of HBV therapy. The kit may include siRNA (e.g., HBV02) and optionally one or two of the following: (a) PEG-IFNα and (b) NRTI (e.g., entecavir, tenofovir, lamivudine or adefovir or adefovir dipivoxil). The kit may further include instructions for preparing and / or administering components of HBV combination therapy.

[0245] Some embodiments of the present disclosure provide a kit comprising: a pharmaceutical composition comprising the siRNA according to any one of the preceding claims and a pharmaceutically acceptable excipient; and a pharmaceutical composition comprising PEG-IFNα and a pharmaceutically acceptable excipient. In some embodiments, the kit further comprises an NRTI and a pharmaceutically acceptable excipient.

[0246] Example

[0247] Example 1

[0248] Treatment of chronic HBV infection with HBV02

[0249] The safety, tolerability, pharmacokinetics (PK) and antiviral activity of HBV02 were evaluated in a Phase 1 / 2 randomized, double-blind, placebo-controlled clinical study. The study consisted of three parts. Part A was a single ascending dose design in healthy volunteers. Parts B and C were multiple ascending dose designs in subjects with chronic HBV who were undergoing nucleoside (acid) reverse transcriptase inhibitor (NRTI) therapy. Subjects in Part B were HBeAg negative; subjects in Part C were HBeAg positive. HBeAg positivity reflects high levels of active viral replication in human liver cells.

[0250] In Part A, a single dose of HBV02 is administered to healthy adult subjects. Each dose can consist of up to 2 subcutaneous (SC) injections based on the specified dose level. Part A includes four dose level cohorts: 50 mg, 100 mg, 200 mg, and 400 mg. Two sentinel subjects are randomized 1:1 to HBV02 or placebo. Sentinel subjects are given the drug at the same time and monitored for 24 hours; if the investigator does not find safety issues, the remaining subjects in the same cohort are given the drug. The remaining subjects will be randomly divided into HBV02 or placebo 5:1. Two optional cohorts (including sentinel dosing) can be added to Part A in the same stratification method, up to a maximum dose of 900 mg. In addition to the optional cohorts, a total of 8 "floater" subjects can be added to expand any cohort in Part A. "Floating" subjects will be added in increments of 4 and randomly divided into HBV02 or placebo 3:1. The dose escalation plan for Part A is shown in Table 3. The single ascending dose design for Part A was Figure 3 shown.

[0251] Table 3. Dose escalation plan for Part A

[0252] queue Dosage based on body weight (mg / kg) <![CDATA[Fixed dose a (mg)]]> Dose escalation factor 1a 0.8 50 - 2a 1.7 100 2.0 times 3a 3.3 200 2.0 times 4a 6.7 400 2.0 times Optional: 5a and 6a Up to 15 Up to 900 Up to 2.25 times

[0253] a Based on an average adult weight of 60 kg

[0254] Data from Part A were reviewed prior to initiation of dose level cohorts in subjects with chronic HBV infection. The population dosing strategy for Part B / C of this study was staggered; 2 dose levels in Part A (1a: 50 mg and 2a: 100 mg) were completed and data were reviewed prior to initiation of the starting dose in Part B (1b: 50 mg). Part C was initiated concurrently with the Part C starting dose (3c: 200 mg) and the equivalent Part B dose level cohort (3b: 200 mg).

[0255] Subjects in Part B were non-cirrhotic adult subjects with HBeAg-negative chronic HBV infection who had been on NRTI therapy for ≥6 months and had serum HBV DNA levels <90 IU / mL. Screening included a non-invasive assessment of liver fibrosis, such as a FibroScan evaluation, to exclude the presence of fibrosis or cirrhosis, unless the subject had results from a FibroScan evaluation performed within 6 months prior to screening or a liver biopsy performed within 1 year prior to screening that confirmed the absence of Metavir F3 fibrosis or F4 cirrhosis.

[0256] Two doses of HBV02 were administered to the subjects 4 weeks apart. Each dose can be composed of up to 2 SC injections based on the specified dose level. Part B includes three dose level cohorts of 50 mg, 100 mg and 200 mg, so that the cumulative doses received by the subjects in Part B are 100 mg, 200 mg and 400 mg. Each cohort is randomly divided into HBV02 or placebo 3:1. In Part B, two optional cohorts can be added by 1.5 times according to the same stratification method, up to a maximum of 450 mg / dose (900 mg cumulative dose). In addition to the optional cohorts, a total of 16 "floating" subjects can be added to expand any cohort in Part B. "Floating" subjects will be added in increments of 4 and randomly divided into HBV02 or placebo 3:1. After accumulating and reviewing all available safety data, including the 4th week laboratory and clinical data of the last available healthy volunteer subject in the 100 mg cohort (cohort 2a), cohort 1b is started. The dose escalation plan for Parts B and C is shown in Table 4. The multiple ascending dose design of Part B / C is as follows Figure 4 shown.

[0257] Subjects in Part C are non-cirrhotic adult subjects with HBeAg-positive chronic HBV infection and have been on NRTI therapy for ≥6 months, and serum HBV DNA levels are <90 IU / mL. In order to exclude the presence of fibrosis or cirrhosis, screening includes non-invasive assessment of liver fibrosis, such as FibroScan evaluation, unless the subject has a FibroScan evaluation performed within 6 months before screening or a liver biopsy performed within 1 year before screening, which confirms the absence of Metavir F3 fibrosis or F4 cirrhosis. Two doses of HBV02 were administered to the subject 4 weeks apart. Each dose can be composed of up to 2 SC injections based on the specified dose level. In order to accommodate the expected lower prevalence of HBeAg-positive patients undergoing NRTI therapy, HBeAg-positive subjects are only planned for 1 dose level cohort (200 mg). Part C includes a dose level cohort of 200 mg, so that the cumulative dose received by the subjects in Part C is 400 mg. Cohort 3:1 randomization is HBV02 or placebo. In Part C, two optional cohorts can be added in 1.5 times the same stratification method, up to a maximum of 450 mg / dose (900 mg cumulative dose). In addition to the optional cohorts, a total of 16 "floating" subjects can be added to expand any cohort in Part C. "Floating" subjects will be added in increments of 4 and randomized 3:1 to HBV02 or placebo. After reviewing all available safety data, including clinical and laboratory data from Week 6 of Cohort 2b, the only cohort planned in Part C, Cohort 3c, was started simultaneously with Cohort 3b. Subjects in Cohort 3c received HBV02 (200 mg, administered twice at a 4-week dosing interval) at the same dose level as subjects in Cohort 3b.

[0258] Table 4. Dose escalation plan for part B / C

[0259]

[0260]

[0261] a Based on an average adult weight of 60 kg

[0262] A summary of study drug doses and administration in Part AC is shown in Tables 5 and Figure 5A and Figure 5B as shown in .

[0263] Table 5. Study Drug Dosages and Administration

[0264]

[0265] a The injection volume per site should not exceed 1.5 mL

[0266] HBV02 was supplied as a sterile solution for SC injection at a free acid concentration of 200 mg / mL. Placebo was a sterile preservative-free 0.9% solution in normal saline for SC injection.

[0267] After administration of HBV02 or placebo, any adverse effects were recorded. PK parameters of HBV02 and possible metabolites were also measured and may include plasma: maximum concentration, time to maximum concentration, area under the concentration versus time curve [until the last measurable time point and until infinity], extrapolated area percentage, apparent terminal elimination half-life, clearance and volume of distribution; urine: urinary elimination and renal clearance. The following will also be determined: maximum reduction in serum HBsAg from Day 1 until Week 16; number of subjects with serum HBsAg loss at any time point; number of subjects with persistent serum HBsAg loss for ≥6 months; number of subjects with anti-HBs seroconversion at any time point; number of subjects with HBeAg loss and / or anti-HBe seroconversion at any time point (for HBeAg-positive subjects in Part C only); assessment of the effect of HBV02 on other markers of HBV infection, including detection of serum HBcrAg, HBV RNA, and HBV DNA; and potential biomarkers of host response to infection and / or therapy, including evaluation of genetic, metabolic, and proteomic parameters. To evaluate PK parameters, blood samples were collected before dosing (≤15 minutes before dosing), and then at 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours and 48 hours after dosing; and urine samples were collected before dosing (≤15 minutes before dosing), and then collected and mixed at 0 to 4 hours, 4 to 8 hours, 8 to 12 hours, 12 to 24 hours, 48 ​​hours and 1 week after dosing. For subjects in Part B or C, blood samples may be collected at one or more of the following time points for measurement of HBsAg, anti-HBs, HBeAg, anti-HBe, HBV DNA, HBV RNA or HBcrAg: screening (28 days to 1 day before dosing), day 1 (dosing), day 2 (after dosing), weekly during the dosing phase, weekly for 4 weeks after dosing, 12 weeks after dosing, 16 weeks after dosing, 20 weeks after dosing, and 24 weeks after dosing.

[0268] Fasting was not required for study procedures.

[0269] Example 2

[0270] HBV02 alone or in combination with PEG-IFNα for the treatment of chronic HBV

[0271] The safety, tolerability, pharmacokinetics and antiviral activity of HBV02 alone or in combination with PEG-IFNα were evaluated in a Phase 1 / 2 clinical study. The study consisted of four parts. Part AC was a randomized, double-blind, placebo-controlled clinical study of subcutaneous administration of HBV02 in healthy adult subjects or non-cirrhotic adult subjects with chronic HBV infection undergoing NRTI therapy. Part A was a single ascending dose design in healthy volunteers. Parts B and C were multiple ascending dose designs in non-cirrhotic subjects with chronic HBV undergoing NRTI therapy. Subjects in Part B were HBeAg-negative; subjects in Part C were HBeAg-positive. HBeAg positivity reflects a high level of active viral replication in human liver cells. Part D was a randomized, open-label Phase 2 study of HBV02 alone or in combination with PEG-IFNα in non-cirrhotic adult subjects with chronic HBV undergoing NRTI therapy; Part D included HBeAg-positive and HBeAg-negative subjects.

[0272] In Part A, a single dose of HBV02 is administered to healthy adult subjects. Each dose may consist of up to 3 subcutaneous (SC) injections based on the specified dose level. Part A includes four dose level cohorts: 50 mg, 100 mg, 200 mg, and 400 mg. Two sentinel subjects are randomized 1:1 to HBV02 or placebo. Sentinel subjects are dosed at the same time and monitored for 24 hours; if the investigator finds no safety issues, the remaining subjects in the same cohort are dosed. The remaining subjects will be randomized 5:1 to HBV02 or placebo. Two optional cohorts (including sentinel dosing) can be added to Part A in the same stratified manner, with a maximum dose of up to 900 mg. In addition to the optional cohorts, a total of 8 "floating" subjects can be added to expand any cohort in Part A. "Floating" subjects will be added in increments of 4 and randomized 3:1 to HBV02 or placebo. The single ascending dose design for Part A is as follows: Figure 3 shown.

[0273] Subjects in Part B are non-cirrhotic adult subjects with HBeAg-negative chronic HBV infection who have been on NRTI therapy for ≥6 months and have serum HBV DNA levels <90 IU / mL. To exclude the presence of fibrosis or cirrhosis, screening includes non-invasive assessment of liver fibrosis, such as FibroScan evaluation. Two doses of HBV02 are administered to subjects 4 weeks apart. Each dose can consist of up to 2 SC injections based on the specified dose level. Part B includes three dose level cohorts of 50 mg, 100 mg and 200 mg, so that the cumulative doses received by subjects in Part B are 100 mg, 200 mg and 400 mg. Each cohort is randomly divided into HBV02 or placebo in a 3:1 ratio. In order to accommodate the expected lower prevalence of HBeAg-positive patients undergoing NRTI therapy, only 1 dose level cohort (200 mg) is planned for HBeAg-positive subjects. Two optional cohorts can be added to Part B in the same stratification method, up to a maximum of 450 mg / dose (900 mg cumulative dose). In addition to the optional cohorts, a total of 16 "floating" subjects can be added to expand any cohort in Part B. "Floating" subjects will be added in increments of 4 and randomized 3:1 to HBV02 or placebo. Cohort 1b will be initiated after cumulative review of all available safety data, including week 4 laboratory and clinical data from the last available healthy volunteer subject in the 100 mg cohort (cohort 2a). The dose escalation plan for Parts B and C is shown in Table 5. The multiple ascending dose design for Parts B / C is shown in Table 5. Figure 4 shown.

[0274] Subjects in Part C are non-cirrhotic adult subjects with HBeAg-positive chronic HBV infection, and have been on NRTI therapy for ≥6 months, and serum HBV DNA levels are <90 IU / mL. Two doses of HBV02 are administered to subjects 4 weeks apart. Each dose can be composed of up to 2 SC injections based on the specified dose level. Part C includes a dose level cohort of 200 mg, so that the cumulative dose received by the subjects in Part C is 400 mg. The cohorts are randomly divided into HBV02 or placebo 3:1. Two optional cohorts can be added to Part C by the same stratification method, up to a maximum of 450 mg / dose (900 mg cumulative dose). In addition to the optional cohorts, a total of 16 "floating" subjects can be added to expand any cohort in Part C. "Floating" subjects will be added in increments of 4 and randomly divided into HBV02 or placebo 3:1.

[0275] A summary of study drug doses and administration in Part AC is shown in Tables 5 and Figure 5A and Figure 5B shown.

[0276] The subjects in Part D are non-cirrhotic adult subjects with HBeAg-positive or HBeAg-negative chronic HBV infection, and have been undergoing NRTI therapy for ≥2 months, and serum HBV DNA levels <90IU / mL and serum HBsAg levels>50IU / mL. Based on the safety and tolerability of HBV02 in Part AC and the analysis of the antiviral activity of HBV02 in Part B and C, the dose level and dose number of HBV02 in Part D are determined. The dose level in Part D does not exceed the highest dose level evaluated in Parts B and C, and the number of doses will be up to 6 doses (e.g., between 3 and 6 doses) administered every 4 weeks. Subjects are randomly grouped into one of the queues 1d, 2d, 3d, and 4d (optional) (e.g., a total of 100 subjects, 25 subjects / cohort). In the queue 1d, up to 6 doses (e.g., 3 to 6 doses) of HBV02 are administered to subjects at a frequency of every 4 weeks. Each subject receives a dose of HBV02 on day 1, week 4, and week 8, and may receive additional doses at weeks 12, 16, and 20. In cohort 2d, subjects are administered up to 6 times (e.g., 3 to 6 doses) of HBV02 4 weeks apart, and 24 weekly doses of PEG-IFNα are administered starting on day 1 (ie, each administration is 1 week apart). Each subject receives a dose of HBV02 on day 1, week 4, and week 8, and may receive additional doses at weeks 12, 16, and 20. In cohort 3d, subjects are administered up to 6 times (e.g., 3 to 6 doses) of HBV02 4 weeks apart, and 12 weekly doses of PEG-IFNα are administered starting on week 12 (ie, each administration is 1 week apart). Each subject receives a dose of HBV02 on day 1, week 4, and week 8, and may receive additional doses at weeks 12, 16, and 20. In cohort 4d, subjects were administered 3 doses of HBV02 4 weeks apart, and 12 weekly doses of PEG-IFNα were administered starting on day 1 (ie, each administration was 1 week apart). Each subject received a dose of HBV02 on day 1, week 4, and week 8. The dose of PEG-INFα administered to subjects in cohorts 2d, 3d, and 4d was 180 μg, administered by SC injection. Figures 6A-6D Schematic diagram illustrating the study design for Part D. The medication administration schedule for Cohort 4d is shown in Table 6.

[0277] Table 6.

[0278] Cohort 4d study drug administration schedule (D1 = Day 1, W1 = Week 1, etc.)

[0279] D1 W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 HBV02 X X X <![CDATA[PEG-INFα a ]]> X X X X X X X X X X X X

[0280] aSubjects who discontinued PEG-IFNα treatment due to PEG-IFNα-related adverse reactions could continue to receive HBV02 treatment.

[0281] Screening of subjects participating in Parts B / C and D included a noninvasive assessment of liver fibrosis, such as a FibroScan evaluation, to exclude the presence of cirrhosis, unless the subject had results from a FibroScan evaluation performed within 6 months prior to Screening or a liver biopsy performed within 1 year prior to Screening that confirmed the absence of Metavir F3 fibrosis or F4 cirrhosis.

[0282] HBV02 was supplied as a sterile solution for SC injection at a free acid concentration of 200 mg / mL. Placebo was a sterile preservative-free 0.9% solution in normal saline for SC injection.

[0283] After administration of HBV02 or placebo, any adverse effects were recorded. PK parameters of HBV02 and possible metabolites were also measured and may include plasma: maximum concentration, time to maximum concentration, area under the concentration versus time curve [until the last measurable time point and until infinity], extrapolated area percentage, apparent terminal elimination half-life, clearance and volume of distribution; urine: urinary elimination and renal clearance. The following will also be determined: maximum reduction in serum HBsAg from Day 1 until Week 16; number of subjects with serum HBsAg loss at any time point; number of subjects with persistent serum HBsAg loss for ≥6 months; number of subjects with anti-HBs seroconversion at any time point; number of subjects with HBeAg loss and / or anti-HBe seroconversion at any time point (for HBeAg-positive subjects in Parts C and D only); assessment of the effect of HBV02 on other markers of HBV infection, including detection of serum HBcrAg, HBV RNA, and HBV DNA; and potential biomarkers of host response to infection and / or therapy, including assessment of genetic, metabolic, and proteomic parameters.

[0284] Data from Part A were reviewed prior to initiation of dose-level cohorts in subjects with chronic HBV infection. The population dosing strategy for Part B / C of this study was staggered; 2 dose levels in Part A (1a: 50 mg and 2a: 100 mg) were completed and data reviewed prior to initiation of dosing at the starting dose in Part B (1b: 50 mg). Part C was initiated concurrently with the Part C starting dose (3c: 200 mg) and the equivalent Part B dose-level cohort (3b: 200 mg).

[0285] Fasting was not required for study procedures.

[0286] Fig. 7A and 7BThe study design of the AD part is shown.

[0287] Example 3

[0288] HBV02 alone or in combination with PEG-IFNα for the treatment of chronic HBV

[0289] The safety, tolerability, pharmacokinetics and antiviral activity of HBV02 were evaluated in a Phase 1 / 2 clinical study. The study consisted of four parts. Part AC was a randomized, double-blind, placebo-controlled clinical study of subcutaneous administration of HBV02 in healthy adult subjects or non-cirrhotic adult subjects with chronic HBV infection undergoing NRTI therapy. Part A was a single ascending dose design in healthy volunteers. Parts B and C were multiple ascending dose designs in non-cirrhotic subjects with chronic HBV undergoing NRTI therapy. Subjects in Part B were HBeAg-negative; subjects in Part C were HBeAg-positive. HBeAg positivity reflects a high level of active viral replication in human liver cells. HBeAg-positive patients are typically younger and are thought to have more complete immune function than HBeAg-negative patients, who are typically older and have experienced greater immune exhaustion. HBeAg-negative patients are also thought to have a larger amount of integrated DNA than HBeAg-positive patients. Part D is a randomized, open-label, Phase 2 study of HBV02 administered alone or in combination with PEG-IFNα in non-cirrhotic adult subjects with chronic HBV on NRTI therapy; Part D includes both HBeAg-positive and HBeAg-negative subjects.

[0290] i. Preparing Animal Dosage Studies

[0291] 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 toxicology studies and applying safety margins to those HEDs. 2) conversion factor was used to calculate the HED for animal doses. No toxicity was observed in a rat Good Laboratory Practice (GLP) study after 3 biweekly doses of HBV02 at the highest dose tested, which was 150 mg / kg, corresponding to a HED of 24 mg / kg / dose (Table 7). No toxicity was observed in a non-human primate (NHP) GLP study after 3 biweekly doses of HBV02 at the highest dose tested, which was 300 mg / kg, corresponding to a HED of 97 mg / kg / dose (Table 7). Using this approach, the suggested starting dose of 0.8 mg / kg in humans represents a 30-fold safety margin of the HED of the NOAEL expected in rats, and a 120-fold safety margin of the HED of the NOAEL expected in NHPs. Other siRNAs using the GalNAc platform exhibited meaningful liver target engagement at 1 to 15 mg / kg. Furthermore, statistically significant reductions in HBsAg were observed in a preclinical HBV mouse model at doses ranging from 1 to 9 mg / kg.

[0292] Table 7. Recommended starting dose of HBV02

[0293]

[0294] A fixed dose of HBV02 was used in clinical studies because HBV02, like other GalNAc-conjugated siRNAs, is taken up by the liver and minimally distributed to other organs and tissues. Therefore, weight-based dosing is not expected to reduce the inter-individual variability of the pharmacokinetics (PK) of HBV02 in adults, and a fixed dose has the advantage of avoiding potential dose calculation errors.

[0295] ii. Methods

[0296] The study design is shown in Figure 12.

[0297] In Part A, a single dose of HBV02 was administered to healthy adult subjects. Each dose consisted of up to 3 subcutaneous (SC) injections based on the specified 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 randomly divided into HBV02 or placebo 1:1. Sentinel subjects were administered at the same time and monitored for 24 hours; if the investigators found no safety issues, the remaining subjects in the same cohort were administered.

[0298] Subjects in Part B are non-cirrhotic adult subjects with HBeAg-negative chronic HBV infection, and have been on NRTI therapy for ≥6 months, and serum HBV DNA levels <90 IU / mL. In order to exclude the presence of fibrosis or cirrhosis, screening includes non-invasive assessment of liver fibrosis. Two doses of HBV02 (i.e., on the 1st day and the 29th day) are administered to subjects 4 weeks apart. Each dose consists of up to 2 SC injections based on the specified dose level. Six cohorts of 20 mg, 50 mg, 100 mg or 200 mg doses are included in Part B, so that the cumulative dose received by the subjects in Part B is 40 mg, 100 mg, 200 mg or 400 mg. Each cohort is randomly divided into HBV02 or placebo 3:1. After accumulating and reviewing all available safety data, including the 4th week laboratory and clinical data of the last available healthy volunteer subject in the 100 mg cohort, the 50 mg cohort of Part B is started.

[0299] Subjects in Part C are non-cirrhotic adult subjects with HBeAg-positive chronic HBV infection, and have been undergoing NRTI therapy for ≥6 months, and serum HBV DNA levels <90 IU / mL. In order to adapt to the expected lower prevalence of HBeAg-positive patients undergoing NRTI therapy, HBeAg-positive subjects only include 2 dose level cohorts (50 mg and 200 mg). Two doses of HBV02 (i.e., on the 1st day and the 29th day) are administered to subjects 4 weeks apart. Each dose is composed of up to 2 SC injections based on the specified dose level. Part C includes two dose level cohorts 50 mg and 200 mg, so that the cumulative dose received by the subjects in Part C is 100 mg or 400 mg. Cohort 3:1 is randomly divided into HBV02 or placebo.

[0300] Patients with chronic HBV who experienced a greater than 10% reduction in HBsAg from baseline serum HBsAg at Week 16 were followed up for up to 32 additional weeks.

[0301] Inclusion criteria for Parts B and C included: age 18 to 65 years; detectable serum HBsAg for ≥6 months; NRTI therapy for ≥6 months; HBsAg>150IU / mL; HBV DNA<90IU / mL; and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤2×upper limit of normal (ULN). Exclusion criteria included: significant fibrosis or cirrhosis (FibroScan>8.5kPa 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 hepatitis D virus infection; and creatinine clearance <60mL / min (Cockcroft-Gault).

[0302] The subjects in Part D are non-cirrhotic adult subjects with HBeAg-positive or HBeAg-negative chronic HBV infection, and have been undergoing NRTI therapy for ≥2 months, and serum HBV DNA levels <90IU / mL and serum HBsAg levels>50IU / mL. Based on the safety and tolerability of HBV02 in Part AC, and the analysis of the antiviral activity of HBV02 in Part B and C, the dose level and dose number of HBV02 in Part D are determined. The dose level in Part D does not exceed the highest dose level evaluated in Part B and Part C, and the number of doses will be up to 6 doses (e.g., between 3 and 6 doses) administered every 4 weeks. Subjects are randomly grouped into one of the queues 1d, 2d, 3d, and 4d (optional) (e.g., a total of 100 subjects, 25 subjects / cohort). In the queue 1d, up to 6 doses (e.g., 3 to 6 doses) of HBV02 are administered to subjects at a frequency of every 4 weeks.

[0303] Each subject receives a dose of HBV02 on day 1, week 4, and week 8, and may receive additional doses at weeks 12, 16, and 20. In cohort 2d, subjects are administered up to 6 times (e.g., 3 to 6 doses) of HBV02 4 weeks apart, and 24 weekly doses of PEG-IFNα are administered starting on day 1 (ie, each administration is 1 week apart). Each subject receives a dose of HBV02 on day 1, week 4, and week 8, and may receive additional doses at weeks 12, 16, and 20. In cohort 3d, subjects are administered up to 6 times (e.g., 3 to 6 doses) of HBV02 4 weeks apart, and 12 weekly doses of PEG-IFNα are administered starting on week 12 (ie, each administration is 1 week apart). Each subject receives a dose of HBV02 on day 1, week 4, and week 8, and may receive additional doses at weeks 12, 16, and 20. In cohort 4d, subjects were administered 3 doses of HBV02 4 weeks apart, and 12 weekly doses of PEG-IFNα were administered starting on day 1 (ie, each administration was 1 week apart). Each subject received a dose of HBV02 on day 1, week 4, and week 8. The dose of PEG-INFα administered to subjects in cohorts 2d, 3d, and 4d was 180 μg, administered by SC injection. Figures 6A-6D Schematic diagram illustrating the study design for Part D. The medication administration schedule for Cohort 4d is shown in Table 8.

[0304] Table 8.

[0305] Cohort 4d study drug administration schedule (D1 = Day 1, W1 = Week 1, etc.)

[0306] D1 W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 HBV02 X X X <![CDATA[PEG-INFα a ]]> X X X X X X X X X X X X

[0307] a Subjects who discontinued PEG-IFNα treatment due to PEG-IFNα-related adverse reactions could continue to receive HBV02 treatment.

[0308] Screening of subjects enrolled in Parts B and C included a noninvasive assessment of liver fibrosis, such as a FibroScan evaluation, to exclude the presence of cirrhosis, unless the subject had results from a FibroScan evaluation performed within 6 months prior to screening or a liver biopsy performed within 1 year prior to screening that confirmed the absence of Metavir F3 fibrosis or F4 cirrhosis. The same approach was used to exclude subjects with cirrhosis from inclusion in Part D.

[0309] HBV02 was supplied as a sterile solution for SC injection at a free acid concentration of 200 mg / mL. Placebo was a sterile preservative-free 0.9% solution in normal saline for SC injection.

[0310] After administration of HBV02 or placebo, any adverse effects were recorded.PK parameters of HBV02 and possible metabolites were also measured, including plasma: maximum concentration, time to maximum concentration, area under the concentration versus time curve [until the last measurable time point and until infinity], extrapolated area percentage, apparent terminal elimination half-life, clearance and volume of distribution; urine: urinary elimination and renal clearance. The following will also be determined: maximum reduction in serum HBsAg from Day 1 until Week 16; number of subjects with serum HBsAg loss at any time point; number of subjects with persistent serum HBsAg loss for ≥6 months; number of subjects with anti-HBs seroconversion at any time point; number of subjects with HBeAg loss and / or anti-HBe seroconversion at any time point (for HBeAg-positive subjects in Parts C and D only); assessment of the effect of HBV02 on other markers of HBV infection, including detection of serum HBcrAg, HBV RNA, and HBV DNA; and potential biomarkers of host response to infection and / or therapy, including assessment of genetic, metabolic, and proteomic parameters. To evaluate the PK parameters of subjects in Part A, blood samples were collected before dosing (≤15 minutes before dosing), and then at 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours and 48 hours after dosing; and urine samples were collected before dosing (≤15 minutes before dosing), and then collected and mixed at 0 to 4 hours, 4 to 8 hours, 8 to 12 hours, 12 to 24 hours, 48 ​​hours and 1 week after dosing. For subjects in Part B or C, blood samples were collected at one or more of the following time points for measurement of HBsAg, anti-HBs, HBeAg, anti-HBe, HBVDNA, HBV RNA or HBcrAg: screening (28 days to 1 day before dosing), day 1 (dosing), day 2 (after dosing), weekly during the dosing phase, weekly for 4 weeks after dosing, 12 weeks after dosing, 16 weeks after dosing, 20 weeks after dosing, and 24 weeks after dosing.

[0311] Data from Part A were reviewed prior to initiation of dose level cohorts in subjects with chronic HBV infection. The population dosing strategy for Part B / C of this study was staggered; 2 dose levels in Part A (50 mg and 100 mg) were completed and data reviewed prior to reinitiating dosing at the starting dose in Part B (50 mg). Part C was initiated concurrently with the Part C starting dose (200 mg) and the equivalent Part B dose level cohort (200 mg).

[0312] Fasting was not required for study procedures.

[0313] iii. Preliminary results from Parts A and B

[0314] Fig. 9A Describe the study design for Part A, Part B, and Part C at the time of completion of dosing for Part A Cohorts 1 to 5 (50 mg, 100 mg, 200 mg, 400 mg, 600 mg) and Part B Cohorts 1 to 2 (50 mg, 100 mg). Fig. 9B Describes the completed dosing for Part A Cohorts 1 to 5 and the withdrawals of subjects in the different cohorts. Fig. 9C Describe the completed dosing in Part B Cohorts 1 to 2 and the withdrawals of subjects in the different cohorts.

[0315] Preliminary demographic data for subjects included in Parts A and B are shown in Table 9 below.

[0316] Table 9: Demographics of subjects participating in Part A and Part B

[0317]

[0318]

[0319] A summary of adverse events (AEs) from the primary analysis of the completed dosing portion of Parts A and B is shown in Table 10.

[0320] Table 10 Overview of adverse events

[0321]

[0322] No subjects in Parts A and B had significant abnormalities in laboratory values, hyperbilirubinemia, or elevated INR. Some subjects in Parts A and B had abnormalities in their liver function laboratory values ​​( Fig. 10A , Fig. 10B and Fig.11 ). Two of the 41 subjects in Part A had ALT elevations prior to dosing on Day 1 (ALT was normal at screening). In Part B, 1 of 12 subjects developed Grade 1 elevations in 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) had ALT reaching the upper limit of normal on Day 29, associated with vigorous exercise and high creatinine kinase (CK: 5811 U / L). Two subjects in Cohort 4a (400 mg) had ALT above the upper limit of normal prior to dosing on Day 1. One admitted to vigorous exercise, had a high CK of 20,001 U / L, and withdrew on Day 2 without adverse events. The second subject with elevated ALT resolved his symptoms by Day 8 without intervention. As Fig.11 As shown in the data from Cohort 2b (100 mg), one female subject experienced Grade 1 ALT elevation at Week 8.

[0323] Subjects from Part B experienced reductions in HBsAg over time in the active group in Cohorts 1 and 2. Fig. 12A Depicted are changes in HBsAg in subjects receiving HBV02 or placebo in cohorts 1b (50 mg) and 2b (100 mg). Fig. 12B Depicted are changes in HBsAg for subjects who received only HBV02 in Cohorts 1b and 2b. In Cohort 4b (20 mg x 2 groups), subjects had a 0.47 log reduction 2 weeks after the first dose.

[0324] Fig. 12C Shown are mean changes in HBsAg from Day 1 to Week 4 or Week 20 (depending on cohort) following administration of HBV02 in Cohorts 1b and 2b, where three subjects with chronic HBV infection (HBeAg negative) received 50 mg HBV02 on Days 1 and 28 and six subjects received 100 mg on Day 1. In the 50 mg cohort, the mean reduction in HBsAg was 1.5 log at Week 12 after two doses. 10 or approximately a 30-fold reduction. All subjects in this cohort achieved an apparent maximum reduction in their HBsAg, ranging from 0.6 to 2.2 log 10 In the 100 mg cohort, all subjects reached Week 4, at which point a mean reduction of 0.7 log was observed after one dose. 10 Or about a six-fold reduction.

[0325] Of the 10 HBeAg-negative subjects in Part B, 7 subjects were good responders, showing a 0.29 to 0.95 log reduction in HBsAg 2 weeks after the first dose (20, 50 or 100 mg). Two of the 10 were moderate responders, showing a 0.06 to 0.21 log reduction in HBsAg 2 weeks after the first dose of 20, 50 or 100 mg. Finally, one of the 10 subjects was a "non-responder," showing a 0.16 log increase in HBsAg 2 weeks after the first dose. The possible reasons for the presence of moderate and non-responders include: dose response, pharmacokinetics, viral resistance and host factors.

[0326] HBV02 was well tolerated among subjects. In healthy volunteer subjects, a single dose ranging from 50 to 600 mg was well tolerated. In HBeAg-negative subjects, two doses ranging from 50 to 100 mg were well tolerated. HBsAg reduction was highly variable between patients, and there was a rebound 12 weeks after the last dose.

[0327] iv. Demographics and Baseline Characteristics – Parts A, B, and C

[0328] 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 inhibited and had FibroScan ≤ 8.5 kPa or Metavir F0 / F1 / F2.

[0329] Table 11.

[0330] Demographics and baseline characteristics of subjects (healthy volunteers) in Part A

[0331] SD = standard deviation.

[0332] a Including replacement volunteers

[0333] Table 12. Demographics and Baseline Characteristics of Part B Subjects (HBeAg-negative Patients)

[0334]

[0335]

[0336] SD = standard deviation.

[0337] Table 13. Demographics and Baseline Characteristics of Subjects in Part C (HBeAg-Positive Patients)

[0338]

[0339] SD = standard deviation.

[0340] v. Safety and tolerability – results from parts A, B, and C

[0341] Preliminary data were obtained from Parts A, B, and C based on 37 healthy volunteers who received HBV02; 12 healthy volunteers who received placebo; 24 patients with chronic HBV who received HBV02 as an NRTI; and 8 patients with chronic HBV who received placebo as an NRTI. HBV02 was generally well tolerated.

[0342] In healthy volunteers and chronic HBV patients, HBV02 was generally well tolerated at a single dose of up to 900 mg in healthy volunteers and at two doses of 20 mg, 50 mg, 100 mg, or 200 mg per dose in patients. No clinically significant abnormalities in alanine aminotransferase (ALT), a marker of liver inflammation, were observed at week 16 in chronic HBV patients (Parts B and C). Figures 13A-13E No clinically relevant changes or trends in grade ≥2 ALT elevations, bilirubin levels >ULN, or other laboratory parameters, vital signs, or ECGs were observed.

[0343] For Part A, there were no post-baseline ALT elevations >ULN associated with increases in bilirubin >ULN. No changes in liver function status (e.g., albumin, coagulation parameters) or clinical signs / symptoms of abnormal liver function were observed in any HBV02-treated subject. Transient ALT elevations were observed in 1 / 6 (17%) and 4 / 6 (67%) subjects after single doses of HBV02 1 and 3 mg / kg, respectively. These elevations were asymptomatic and not associated with hyperbilirubinemia. In contrast, no ALT elevations potentially related to HBV02 were observed with single doses of HBV02 ranging from 50 to 600 mg (approximately 0.8 to 10 mg / kg). In the Part A 900 mg (approximately 15 mg / kg) cohort, mild asymptomatic Grade 1 ALT elevations were observed in a subset of subjects (5 / 6 subjects with ALT elevations of 1.1 to 2.6×ULN) without associated changes in bilirubin. ALT levels of subjects in Part A, including comparisons with subjects administered HBV01 (similar siRNA without GNA modification), as Fig.14 These results demonstrate that incorporation of ESC+ technology (provided by incorporation of GNA modifications to provide enhanced stability and minimized off-target activity) reduces the propensity of siRNA to elicit ALT elevations in healthy volunteers at dose levels expected to be clinically relevant.

[0344] No dose-related trend was observed in the frequency of adverse events. Most of the adverse events reported during treatment were mild in severity, and no patient discontinued due to adverse events. The most common adverse event was headache (6 / 24, 25%). Three grade 3 adverse events were reported: upper respiratory tract infection, chest pain, and low phosphate levels in the blood, but were considered unrelated to HBV02. A single grade 3 adverse event, hypophosphatemia, was observed in patients receiving tenofovir disoproxil fumarate. Two serious adverse events or SAEs were reported in Part B. The first event, grade 2 headache, was resolved with intravenous infusions and non-opioid pain medications. This patient had additional symptoms of fever, nausea, vomiting, and dehydration, which were assessed to be consistent with viral syndrome. The second SAE, grade 4 depression, occurred within 50 days after the last dose of the drug and was assessed to be unrelated to HBV02 treatment.

[0345] A summary of adverse events that occurred during treatment is shown in Table 14.

[0346] Table 14. Summary of adverse events (AEs) that occurred during treatment

[0347]

[0348] vi. Pharmacokinetics – Results from Part A

[0349] Preliminary pharmacokinetic (PK) data from a first-in-human Phase 1 randomized, blinded, placebo-controlled dose-ranging study of HBV02 in healthy volunteers were analyzed. Plasma samples from six single ascending dose cohorts of eight subjects (6:2 active:placebo) receiving a single subcutaneous (SC) dose of HBV02 ranging from 50 to 900 mg were evaluated.

[0350] Eligibility criteria included the following: age 18 to 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.

[0351] Plasma and urine PK samples were collected centrally for 1 week. Serial plasma samples were collected within 24 hours, 48 ​​hours, and 1 week after dosing. Pooled urine samples were collected within 24 hours after dosing, and single voided urine samples were collected at 48 hours and 1 week after dosing. Concentrations of HBV02 and (N-1)3′HBV02 antisense metabolite in plasma and urine were measured using validated liquid chromatography tandem mass spectrometry (lower limit of quantification (LLOQ) 10 ng / mL in plasma and urine). PK parameters were measured using The standard noncompartmental method was estimated in V6.3.0 (Certara LP, Princeton, NJ). AS(N-1)3'HBV02, the primary circulating metabolite with the same potency as HBV02, is formed by the loss of one nucleotide from the 3' end of the antisense strand of HBV02.

[0352] Fig.15A and Fig. 15B The plasma concentrations of HBV02 and AS(N-1)3′HBV02 after a single SC administration in healthy volunteers are shown respectively versus time. HBV02 showed linear kinetics in plasma after SC injection. HBV02 was expressed in plasma with a median T of 4 to 8 hours after SC injection. max Absorption. HBV02 was undetectable in plasma after 48 hours for any subject, consistent with rapid GalNAc-mediated hepatic uptake; the median apparent elimination half-life (t 1 / 2) ranged from 2.85 to 5.71 hours. The short plasma half-life may represent a distribution half-life (see Agarwal S et al., Clin Pharmacol Ther. Jan 29, 2020, doi:10.1002 / cpt.1802). Rapid conversion of HBV02 to the (N-1)3′ metabolite, termed AS(N-1)3'HBV02, was observed. AS(N-1)3'HBV02 has a median T of 2-10 hours max , quantifiable only at doses ≥100 mg, and concentrations are generally approximately 10-fold lower than HBV02.

[0353] HBV02 plasma exposure (AUC 0-12 and C max ) appeared to increase in a dose-proportional manner up to 200 mg, and exhibited a slightly greater than dose-proportional increase at doses above 200 mg ( Fig.16 ; Fig.17 ; Table 15). After a single SC dose of 50 to 900 mg HBV02, the plasma area under the curve (AUC 最后 ) and the average maximum concentration (C max ) increased with dose, with mean exposures ranging from 786 to 74,700 ng*hr / mL and 77.8 to 6010 ng / mL, respectively. Similar trends were observed for AS(N-1)3′HBV02. These results suggest that at higher doses, transient saturation of ASGPR-mediated hepatic uptake of HBV02 results in higher circulating concentrations (see Agarwal et al., 2020, supra).

[0354] Table 15. HBV02 plasma exposure and fold changes between doses

[0355] Dosage Range Fold change <![CDATA[AUC 0-12 ]]> <![CDATA[C max ]]> 50-200mg 4 4.57 4.59 200-900mg 4.5 8.08 7.05

[0356] The inter-patient variability of HBV02 plasma PK parameters was generally low (approximately 30%).

[0357] The most prevalent active metabolite (approximately 12%), AS(N-1)3′HBV02, was as effective as HBV02. AS(N-1)3′HBV02 was detectable in plasma in 0 / 6 subjects at 50 mg, in 3 / 6 subjects at 100 mg, and in all subjects at 200, 400, 600, and 900 mg. The PK properties of the metabolite were similar to HBV02, and the AUC of AS(N-1)3′HBV02 in plasma was 1.34 ± 0.13 % (P < 0.05). 最后 and C max Value ≤ 11% of HBV02.

[0358] AUC of AS(N-1)3′HBV02 in plasma 0-12 and C max ≤ 11% of total drug-related materials.

[0359] Characteristics of plasma PK parameters of HBV02 and AS(N-1)3′HBV02 observed after a single SC dose in healthy volunteers Fig.18 shown.

[0360] The urine concentrations of HBV02 and AS(N-1)3'HBV02 versus time curves are shown in Fig.19A and 19B As shown. Lower concentrations of HBV02 and AS(N-1)3'HBV02 were detected in urine through the last measurement time point 1 week after dosing in all cohorts. When calculable, the PK properties of HBV02 in urine were consistent with those in plasma.

[0361] Summary of urine PK parameters of HBV02 and AS(N-1)3'HBV02 in healthy volunteers Fig. 20 As shown. During the first 24-hour period, approximately 17% to 46% and 2% to 7% of the administered dose (50 to 900 mg) were excreted in the urine as unchanged HBV02 and AS(N-1)3'HBV02, respectively. The urination rate of HBV02 increased with dose level within 24 hours after administration. This may be caused by the HBV02 hepatic absorption rate of ASGPR far exceeding renal elimination (see Agarwal et al., 2020, supra), and is consistent with the increase in plasma HBV02 being greater than proportional to the dose. The renal clearance of HBV02 is close to the glomerular filtration rate.

[0362] These preliminary data suggest that HBV02 exhibits favorable PK properties in healthy volunteers.

[0363] vii. Efficacy – Results from Parts B and C

[0364] Preliminary data from B and C were obtained based on 24 patients with chronic HBV receiving HBV02 as NRTI; and 8 patients with chronic HBV receiving placebo as NRTI. The initial data showed a significant reduction in HBsAg in patients at doses ranging from 20 mg to 200 mg.

[0365] The biological activity of HBV02 was evaluated by the reduction of HBsAg. Fig.21A and 21B The activity of HBV02 in the 200 mg cohorts in the HBeAg-negative part B and the HBeAg-positive part C is shown in Figure 1. The mean baseline HBsAg levels were 3.3 log for parts B and C, respectively. 10IU / mL and 3.9log 10 At week 16, the mean reduction in HBsAg in both HBeAg-negative and HBeAg-positive subjects was 1.5 log 10 , or approximately a 32-fold reduction. Following two 200 mg doses of HBV02 given four weeks apart, the observed HBsAg reduction at week 16 was 0.97 log 10 to 2.2log 10 The mean HBsAg level at week 16 was 314 IU / mL, with half of the patients achieving HBsAg values ​​<100 IU / mL and 5 / 6 achieving HBsAg values ​​<1000 IU / mL.

[0366] Changes in HBsAg from baseline to week 16 by dose Fig. 22 The percentage of patients with HBsAg levels <100 IU / mL at Week 24 was 33% for patients receiving 20 mg HBV02, 44% for patients receiving 50 mg HBV02, 50% for patients receiving 100 mg HBV02, and 50% for patients receiving 200 mg HBV02. The individual maximum changes in HBsAg from baseline are shown in Table 1. Fig.23 Similar reductions were observed in HBeAg-positive and HBeAg-negative patients. At week 24, the mean change in HBsAg observed in patients taking 20 mg, 50 mg, 100 mg, and 200 mg HBV02 was -0.76 log, respectively. 10 、-0.93log 10 、-1.23log 10 and -1.43log 10 All 6 patients who received 2 200 mg doses achieved ≥ 1.0 log 10 HBsAg decreased. Fig.24 Individual changes in HBsAg from baseline at Week 24 are shown, demonstrating dose-dependent durability of HBsAg reduction.

[0367] These results indicate that HBV02 was well tolerated and no safety signals were observed. Dose-dependent HBsAg reductions were observed in HBeAg-negative and HBeAg-positive patients over a dose range of 20 to 200 mg HBV02 (delivered in 2 doses), which persisted for at least 6 months at higher doses. Similar HBsAg reductions were observed in HBeAg-negative and HBeAg-positive patients, indicating that HBV02 can reduce HBsAg in patients regardless of the stage of their disease. All patients who received 2 200 mg doses achieved ≥ 1-log 10HBsAg decreased, and at week 24, the mean reduction in HBsAg was -1.43log 10 Overall, these results support the potential for HBV02 to serve as the basis for a limited therapeutic regimen toward a functional cure for chronic HBV infection. Specifically, the ability of HBV02 to induce a significant reduction in HBsAg after only two doses suggests that HBV02 has the potential to play an important role in a functional cure for chronic HBV.

[0368] While specific embodiments have been illustrated and described, it will be readily appreciated that the various embodiments described above may be combined to provide further embodiments, and that various changes may be made therein without departing from the spirit and scope of the invention.

[0369] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification or listed in the Application Data Sheet, including U.S. Provisional Patent Application No. 62 / 846927 filed on May 13, 2019, U.S. Provisional Patent Application No. 62 / 893646 filed on August 29, 2019, U.S. Provisional Patent Application No. 62 / 992785 filed on March 20, 2020, U.S. Provisional Patent Application No. 62 / 994177 filed on March 24, 2020, and U.S. Provisional Patent Application No. 63 / 009910 filed on April 14, 2020, are incorporated herein by reference in their entirety unless expressly stated otherwise. Aspects of the embodiments may be modified, if necessary, to employ concepts of the various patents, applications, and publications to provide further embodiments.

[0370] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the appended claims, the terms used should not be interpreted as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted to include all possible embodiments and the full scope of equivalents authorized by the claims. Therefore, the claims are not limited by the present disclosure. Sequence Listing <110> Vir Biotechnology, Inc. Pang, Phillip S. Bakardjiev, Anna Connolly, Lynn E. <120> Compositions and methods for treating hepatitis B virus (HBV) infection <130> 930485.405WO <140> PCT <141> 2020-05-12 <150> US 63 / 009,910 <151> 2020-04-14 <150> US 62 / 994,177 <151> 2020-03-24 <150> US 62 / 992,785 <151> 2020-03-20 <150> US 62 / 893,646 <151> 2019-08-29 <150> US 62 / 846,927 <151> 2019-05-13 <160> 10 <170> FastSEQ for Windows version 4.0 <210> 1 <211> 3182 <212> DNA <213> Hepatitis B virus <220> <223> Hepatitis B virus genome, NCBI reference sequence NC_003977.2 <400> 1 aattccacaa ccttccacca aactctgcaa gatcccagag tgagaggcct gtatttccct 60 gctggtggct ccagttcagg aacagtaaac cctgttctga ctactgcctc tcccttatcg 120 tcaatcttct cgaggattgg ggaccctgcg ctgaacatgg agaacatcac atcaggattc 180 ctaggacccc ttctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata 240 ccgcagagtc tagactcgtg gtggacttct ctcaattttc taggggaac taccgtgtgt 300 cttggccaaa attcgcagtc cccaacctcc aatcactcac caacctcttg tcctccaact 360 tgtcctggtt atcgctggat gtgtctgcgg cgttttatca tcttctctt catcctgctg 420 ctatgcctca tcttcttgtt ggttcttctg gactatcaag gtatgttgcc cgtttgtcct 480 ctaattccag gatcctcaac aaccagcacg ggaccatgcc ggacctgcat gactactgct 540 caaggaacct ctatgtatcc ctcctgttgc tgtaccaaac cttcggacgg aaattgcacc 600 tgtattccca tcccatcatc ctgggctttc ggaaaattcc tatgggagtg ggcctcagcc 660 cgtttctcct ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttcccc 720 actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 780 ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc 840 ctaaaaaac aaagagatgg ggttactctc taaattttat gggttatgtc attggatgtt 900 atgggtcctt gccacaagaa cacatcatac aaaaaatcaa agaatgtttt agaaaacttc 960 ctattaacag gcctattgat tggaaagtat gtcaacgaat tgtgggtctt ttgggttttg 1020 ctgccccttt tacacaatgt ggttatcctg cgttgatgcc tttgtatgca tgtattcaat 1080 ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatacctga 1140 acctttaccc cgttgcccgg caacggccag gtctgtgcca agtgtttgct gacgcaaccc 1200 ccactggctg gggcttggtc atgggccatc agcgcatgcg tggaaccttt tcggctcctc 1260 tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa 1320 acattatcgg gactgataac tctgttgtcc tatcccgcaa atatacatcg tttccatggc 1380 tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg 1440 cgctgaatcc tgcggacgac ccttctcggg gtcgcttggg actctctcgt ccccttctcc 1500 gtctgccgtt ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc 1560 cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac 1620 cgtgaacgcc caccaaatat tgcccaaggt cttacataag aggactcttg gactctcagc 1680 aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaaag actgggagga 1740 gttgggggag gagattaggt taaaggtctt tgtactagga ggctgtaggc ataaattggt 1800 ctgcgcacca gcaccatgca actttttcac ctctgcctaa tcatctcttg ttcatgtcct 1860 actgttcaag cctccaagct gtgccttggg tggctttggg gcatggacat cgacccttat 1920 aaagaatttg gagctactgt ggagttactc tcgtttttgc cttctgactt ctttccttca 1980 gtacgagatc ttctagatac cgcctcagct ctgtatcggg aagccttaga gtctcctgag 2040 cattgttcac ctcaccatac tgcactcagg caagcaattc tttgctgggg ggaactaatg 2100 actctagcta cctgggtggg tgttaatttg gaagatccag cgtctagaga cctagtagtc 2160 agttatgtca acactaatat gggcctaaag ttcaggcaac tcttgtggtt tcacatttct 2220 tgtctcactt ttggaagaga aacagttata gagtatttgg tgtctttcgg agtgtggatt 2280 cgcactcctc cagcttatag accaccaaat gcccctatcc tatcaacact tccggagact 2340 actgttgtta gacgacgagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga 2400 aggtctcaat cgccgcgtcg cagaagatct caatctcggg aatctcaatg ttagtattcc 2460 ttggactcat aaggtgggga actttactgg gctttattct tctactgtac ctgtctttaa 2520 tcctcattgg aaaacaccat cttttcctaa tatacattta caccaagaca tttcaaaaa 2580 atgtgaacag tttgtaggcc cactcacagt taatgagaaa agaagattgc aattgattat 2640 gcctgccagg tttatccaa aggttaccaa atattacca ttggataagg gtattaaacc 2700 ttattatcca gaacatctag ttaatcatta cttccaaact agacactatt tacacactct 2760 atggaaggcg ggtatattat ataagagaga aacaacacat agcgcctcat ttgtggggtc 2820 accatattct tgggaacaag atctacagca tggggcagaa tctttccacc agcaatcctc 2880 tgggattct tcccgaccac cagttggatc cagccttcag agcaaacacc gcaaatccag 2940 attgggactt caatcccaac aaggacacct ggccagacgc caacaaggta ggagctggag 3000 cattcgggct gggtttcacc ccaccgcacg gaggcctttt ggggtggagc cctcaggctc 3060 agggcatact acaaactttg ccagcaaatc cgcctcctgc ctccaccaat cgccagtcag 3120 gaaggcagcc taccccgctg tctccacctt tgagaaacac tcatcctcag gccatgcagt 3180 gg3182 <210> 2 <211> 18 <212> DNA <213> Hepatitis B virus <220> <223> Hepatitis B virus genome, nucleotides 1579-1597 of NC_003977.2 <400> 2 gtgtgcactt cgcttcac 18 <210> 3 <211> 19 <212> RNA <213> Artificial sequence <220> <223> HBV02, sense strand <400> 3 gugugcacuu cgcuucaca 19 <210> 4 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> HBV02, antisense strand <400> 4 ugugaagcga agugcacacu u 21 <210> 5 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Modified HBV02, sense strand <400> 5 gugugcacuu cgcuucaca 19 <210> 6 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Modified HBV02, antisense strand <400> 6 ugugaagcga agugcacacu u 21 <210> 7 <211> 16 <212> PRT <213> Artificial sequence <220> <223> RFGF <400> 7 Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 <210> 8 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Peptide RFGF analogs containing membrane translocation sequences <400> 8 Ala Ala Leu Leu Pro Val Leu Leu Ala Ala Pro 1 5 10 <210> 9 <211> 13 <212> PRT <213> Artificial sequence <220> <223> HIV Tat protein <400> 9 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln 1 5 10 <210> 10 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Drosophila antennapedia protein <400> 10 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys 1 5 10 15

Claims

1. A kit comprising: A pharmaceutical composition comprising siRNA and a pharmaceutically acceptable excipient, wherein (a) 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), where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate bond; and L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol; and A pharmaceutical composition comprising PEG-IFNα and a pharmaceutically acceptable excipient.

2. The kit according to claim 1, further comprising an NRTI and a pharmaceutically acceptable excipient.

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