Regulation of Hepatitis B Virus (HBV) Expression

Abstract

Disclosed herein are chimeric antisense compounds and methods for reducing the expression of HBV mRNA, DNA and protein. Such methods, compounds and compositions are useful for treating, preventing or ameliorating diseases, disorders or conditions associated with HBV.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 298,092, filed January 10, 2022, the contents of which are incorporated herein in their entirety. [Background technology]

[0002] Hepatitis B is a viral disease transmitted parenterally through contaminated materials, such as blood and blood products, contaminated needles, sexual contact, and vertical transmission from infected or carrier mothers to their children. The World Health Organization estimates that over 2 billion people are infected worldwide, with approximately 4 million acute cases and 1 million deaths per year, and 350–400 million chronic carriers (World Health Organization: Geographic Prevalence of Hepatitis B Prevalence, 2004. http: / / www.who.int / vaccines-surveillance / graphics / htmls / hepbprev.htm).

[0003] The HBV virus is a hepatotropic, double-stranded virus that infects only humans and nonhuman primates. Viral replication occurs primarily in the liver, with smaller replication rates in the kidneys, pancreas, bone marrow, and spleen (Hepatitis B virus biology. Microbiol Mol Biol Rev. 64:2000;51-68). Viral and immune markers are detectable in the blood, and characteristic antigen-antibody patterns develop over time. The first detectable viral marker is HBsAg, followed by hepatitis B e antigen (HBeAg) and HBV DNA. While titers may be high during the incubation period, HIV DNA and HBeAg levels begin to decline at the onset of disease and may become undetectable at the peak of clinical disease (Hepatitis B virus infection—natural history and clinical consequences. N Engl J Med., 350:2004;1118-1129). HBeAg is a viral marker detectable in the blood that correlates with active viral replication, i.e., high viral load, and infectivity (Hepatitis B e antigen—the dangerous end game of hepatitis BN Engl J Med.347:2002;208-210). The presence of anti-HBsAb and anti-HBcAb (IgG) suggests recovery and immunity in previously infected individuals.

[0004] Current treatment recommendations for chronic HBV infection by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) include interferon alfa (IFNα), pegylated interferon alfa-2a (Peg-IFN2a), entecavir, and tenofovir. The nucleoside and nucleotide therapies, entecavir and tenofovir, have been successful in reducing viral load, but the HBeAg seroconversion and HBsAg clearance rates are much lower than those achieved with IFNa therapy. Other similar treatments, including lamivudine (3TC), telbivudine (LdT), and adefovir, are also used, but resistance to nucleoside / nucleotide therapies is common and their therapeutic efficacy is limited.

[0005] Therefore, there is a need in the art for the discovery and development of new antiviral therapies. Furthermore, there is a need for new anti-HBV therapies that can increase the rate of seroconversion to HBeAg and HBsAg. Recent clinical studies have shown a correlation between seroconversion and reductions in HBeAg (Fried et al. (2008) Hepatology 47:428) and reductions in HBsAg (Moucari et al. (2009) Hepatology 49:1151). Because high levels of antigen are thought to induce immune tolerance, reducing antigen levels may enable immune control of HBV infection. Current nucleoside therapies for HBV can dramatically reduce HBV serum levels but have little effect on HBeAg and HBsAg levels.

[0006] Antisense technology has emerged as an effective means of reducing the expression of specific gene products and may prove uniquely useful in many therapeutic and diagnostic applications. Antisense therapy differs from nucleoside therapy in that it directly targets HBV antigen transcription and can reduce serum HBeAg and HBsAg levels. Because multiple overlapping transcripts are produced during HBV infection, a single antisense oligomer may reduce both HBeAg and HBsAg, as well as HBV DNA. Therefore, antisense technology has emerged as an effective means of reducing the expression of specific gene products and may prove uniquely useful in many therapeutic, diagnostic, and research applications for HBV regulation.

[0007] HBV antisense oligonucleotides have been developed that have a single gap segment immediately adjacent to the 5' wing segment and the 3' wing segment (WO2012 / 145697). However, many of the HBV antisense oligonucleotides with this structure have shown minimal efficacy in reducing serum HBsAg levels and / or have safety concerns in patients with chronic hepatitis B. Therefore, there is a need in the art for improved HBV antisense oligonucleotides. Summary of the Invention

[0008] The present disclosure extends this principle to HBV antisense oligonucleotides by providing improved modified oligonucleotide structures, which use one or more separators to provide a more segmented gap structure.

[0009] The present disclosure is based, at least in part, on the discovery that a discontinuous gap segment adjacent to the 5' wing segment and the 3' wing segment in a modified oligonucleotide results in improved activity (e.g., reduced serum HBsAg or HBeAg levels) over conventional antisense oligonucleotides with a continuous gap. One or more separator segments positioned directly between the gap segments can improve the activity of an antisense oligonucleotide, a finding that clearly contradicts current teachings in the art that suggest separator segments may result in decreased activity. The inventors unexpectedly discovered that placing a separator segment at a specific position in a full-length antisense oligonucleotide improves activity, while separator segments at other positions result in decreased activity or no change in activity. Furthermore, they discovered that only certain types of nucleoside modifications in the separator segment result in improved activity, while other types of nucleoside modifications in the separator segment result in decreased activity or no change in activity. Without being bound by theory, specific combinations of nucleoside modifications and positions of the separator segment within the full-length antisense oligonucleotide improve RNAse H endonuclease binding and activity without compromising complementarity with the HBV target sequence.

[0010] The present disclosure is based, at least in part, on the discovery that certain nucleoside modifications in the 5' and 3' wing segments reduce in vivo toxicity (e.g., lower ALT levels, lower surrogate values ​​for hepatotoxicity, or lower CC30 (the cytotoxic concentration that reduces cell viability by 30%)). The inventors unexpectedly found that certain types of modified nucleosides in the 5' wing segment result in increased in vivo toxicity, while certain types of modified nucleosides in the 3' wing segment reduce in vivo toxicity. Furthermore, the location of modified nucleosides within the 5' and 3' wing segments also contributes to in vivo toxicity. Without being bound by theory, the specific combination of nucleoside modifications and their placement within the 5' and 3' wing segments contributes to the complementarity of the full-length antisense oligonucleotide to the HBV target sequence. Combinations of certain types of modifications at certain positions improve complementarity, while other combinations of certain types of modifications and positions decrease complementarity, resulting in increased off-target binding and ultimately toxicity.

[0011] Provided herein are methods, compounds and compositions for regulating the expression of HBV mRNA and protein.In certain embodiments, the compound useful for regulating the expression of HBV mRNA and protein is an antisense compound.In certain embodiments, the antisense compound is an antisense oligonucleotide.

[0012] In certain embodiments, modulation may occur in a cell or tissue. In certain embodiments, the cell or tissue is a cell or tissue in an animal. In certain embodiments, the animal is a human. In certain embodiments, HBV mRNA levels are reduced. In certain embodiments, HBV DNA levels are reduced. In certain embodiments, HBV protein levels are reduced. In certain embodiments, HBV antigen levels are reduced. In certain embodiments, HBV s-antigen (HBsAg) levels are reduced. In certain embodiments, HBV e-antigen (HBeAg) levels are reduced. Such reduction may occur in a time-dependent manner or in a dose-dependent manner.

[0013] Also provided are methods, compounds, and compositions useful for preventing, treating, and ameliorating diseases, disorders, and conditions. In certain embodiments, the HBV-related diseases, disorders, and conditions are liver diseases. In certain embodiments, such liver diseases, disorders, and conditions include jaundice, liver cancer, liver inflammation, liver fibrosis, inflammation, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, and liver disease-associated transplantation. In certain embodiments, the HBV-related diseases, disorders, and conditions are hyperproliferative diseases, disorders, and conditions. In certain embodiments, such hyperproliferative diseases, disorders, and conditions include cancer and related malignancies and metastases. In certain embodiments, such cancers include liver cancer and hepatocellular carcinoma (HCC).

[0014] These diseases, disorders, and conditions may have one or more common risk factors, causes, or outcomes. Specific risk factors and causes for the development of liver disease or hyperproliferative diseases include aging, tobacco use, exposure to sunlight and ionizing radiation, contact with certain chemicals, infection with certain viruses and bacteria, certain hormone therapy, a family history of cancer, alcohol use, and certain lifestyle choices, including poor diet, lack of physical activity, and / or being overweight. Specific symptoms and outcomes associated with the development of liver disease or hyperproliferative diseases include, but are not limited to, flu-like illness, weakness, pain, headache, fever, loss of appetite, diarrhea, jaundice, nausea and vomiting, pain in the liver region of the body, clay-colored or gray stools, generalized itching, and dark urine.

[0015] In certain embodiments, the method of treatment comprises administering to an individual in need thereof an HBV antisense compound. In certain embodiments, the method of treatment comprises administering to an individual in need thereof an HBV antisense oligonucleotide. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 provides a description of the nucleoside modifications at each position of the sequence shown in Figure 2. The "Example" column describes each type of nucleoside modification.

[0017] [Figure 2-1] Figure 2 shows a table of exemplary modified oligonucleotides of the present disclosure. The modifications at each position of the modified oligonucleotide sequence are read using the legend in Figure 1. AUS1010-AUS1714 (SEQ ID NO: 11-SEQ ID NO: 666) represent modified oligonucleotide sequences. AUS1233 (SEQ ID NO: 10), also referred to as AUS1138, is the reference modified oligonucleotide sequence. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above. [Figure 2-6] Same as above. [Figure 2-7] Same as above. [Figure 2-8] Same as above. [Figure 2-9] Same as above. [Figure 2-10] Same as above. [Figure 2-11] Same as above.

[0018] [Figure 3] FIG. 3 shows a schematic diagram of the solid phase synthesis process used to generate modified oligonucleotides.

[0019] [Figure 4] FIG. 4 shows a schematic diagram of the dosage regimen of modified oligonucleotides and the sampling schedule for HBsAg levels in HBV Tg mice.

[0020] [Figure 5] FIG. 5 shows a schematic diagram of the dosing regimen of modified oligonucleotides and the sampling schedule for HBsAg levels in C57BL / 6 Tg mice.

[0021] [Figure 6] FIG. 6 shows a schematic diagram of the dosing regimen of modified oligonucleotides and the sampling schedule for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in C57BL / 6 Tg mice.

[0022] [Figure 7] FIG. 7 shows a schematic diagram of the dosing regimen of modified oligonucleotides and the sampling schedule for HBsAg levels in pAAV-1.2HBV-GTA HDI-HBV mice.

[0023] [Figure 8]FIG. 8 shows a schematic diagram of the dosing regimen of modified oligonucleotides and the sampling schedule for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in C57BL / 6 Tg mice.

[0024] [Figure 9] FIG. 9 shows a schematic diagram of an exemplary modified oligonucleotide dosing regimen and sampling schedule for HBsAg levels in pcDNA3.1-preS2-GTD HDI-HBV mice.

[0025] [Figure 10] FIG. 10 shows a schematic diagram of an exemplary modified oligonucleotide dosing regimen and sampling schedule for HBsAg levels in pcDNA3.1-preS2-GTA HDI-HBV mice.

[0026] [Figure 11] FIG. 11 shows a schematic diagram of an exemplary modified oligonucleotide dosing regimen and sampling schedule for HBsAg levels in GTA HBV Tg mice.

[0027] [Figure 12] FIG. 12 shows a schematic diagram of an exemplary modified oligonucleotide dosing regimen and sampling schedule for HBsAg levels in GTD AAV-HBV Tg mice.

[0028] [Figure 13] FIG. 13 shows a schematic diagram of an exemplary modified oligonucleotide dosing regimen and sampling schedule for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in C57BL / 6 Tg mice.

[0029] [Figure 14]Figure 14 depicts Table 15.1 showing serum HBsAg levels in GT-C 1.0 HBV transgenic mice after subcutaneous treatment with modified oligonucleotides AUS1233, AUS1683, AUS1684, AUS1685, AUS1220, AUS1322, AUS1323, or AUS1324 at 40 mg / kg on days 3, 7, 10, 14, 21, and 28. Mean values ​​are expressed in log10.

[0030] [Figure 15] Figure 15 depicts Table 15.2 showing serum levels of HBsAg in GT-C 1.0 HBV transgenic mice after subcutaneous treatment with modified oligonucleotides AUS1169, AUS1171, AUS1170, AUS1168, or AUS1322 at 40 mg / kg on days 3, 7, 10, 14, 21, and 28. Mean values ​​are expressed in log10.

[0031] [Figure 16] 16 depicts Table 15.3 showing serum HBsAg levels in GT-C 1.0 HBV transgenic mice after subcutaneous treatment with modified oligonucleotides AUS1434, AUS1435, AUS1440, AUS1436, AUS1437, AUS1438, AUS1439, AUS1441, AUS1422, AUS1423, AUS1424, AUS1425, AUS1426, AUS1427, AUS1428, AUS1429, AUS1430, AUS1431, AUS1432, or AUS1433 at 30 mg / kg on days 3, 7, 10, 14, and 21. Mean values ​​are expressed in log 10.

[0032] [Figure 17] Figure 17 depicts Table 15.4 showing serum levels of HBsAg in GT-C 1.0 HBV transgenic mice after subcutaneous treatment with modified oligonucleotides AUS1463, AUS1466, AUS1476, AUS1472, AUS1489, or AUS1459 at 30 mg / kg on days 3, 7, 10, 14, and 21. Mean values ​​are expressed in log10.

[0033] [Figure 18] Figure 18 depicts Table 15.5 showing serum levels of HBsAg in GT-C 1.0 HBV transgenic mice after subcutaneous treatment with modified oligonucleotides AUS1492, AUS1495, AUS1494, AUS1493, AUS1482, or AUS1441 at 30 mg / kg on days 3, 7, 10, 14, 21, and 28. Mean values ​​are expressed in log10.

[0034] [Figure 19] Figure 19 depicts Table 15.6 showing serum HBsAg levels in GT-C 1.0 HBV transgenic mice after subcutaneous treatment with modified oligonucleotides AUS1693, AUS1694, AUS1695, AUS1696, AUS1697, AUS1698, AUS1699, or AUS1700 at 40 mg / kg on days 3, 7, 10, 14, and 21. Mean values ​​are expressed in log10.

[0035] [Figure 20] Figure 20 depicts Table 15.7 showing serum HBsAg levels in GT-C 1.0 HBV transgenic mice after treatment with modified oligonucleotides on days 3, 7, 10, 14, 21, and 28. Rows 1 and 2 show HBsAg levels after subcutaneous administration of 10 mg / kg AUS1493 or AUS1233 on day 0. Rows 3 and 4 show HBsAg levels after subcutaneous administration of 30 mg / kg AUS1493 or AUS1233 on day 0. Mean values ​​are expressed in log10.

[0036] [Figure 21] Figure 21 depicts Table 16.1 showing serum levels of HBsAg in GT-D HDI HBV mice after subcutaneous treatment with eASO compounds AUS1683, AUS1684, AUS1685, AUS1220, AUS1322, AUS1323, or AUS1324 at 60 mg / kg on days 3, 7, 10, 14, 21, and 28. Mean values ​​are expressed in log10.

[0037] [Figure 22] Figure 22 depicts Table 16.2 showing serum levels of HBsAg in GT-D HDI HBV mice after subcutaneous treatment with eASO compounds AUS1169, AUS1171, AUS1170, or AUS1168 at 60 mg / kg on days 3, 7, 10, 14, 21, and 28. Mean values ​​are expressed as log10.

[0038] [Figure 23] Figure 23 depicts Table 16.3 showing serum levels of HBsAg in GT-D HDI HBV mice after subcutaneous treatment with eASO compounds AUS1173, AUS1174, AUS1175, or AUS1176 at 60 mg / kg on days 3, 7, 10, 14, and 21. Mean values ​​are expressed in log10.

[0039] [Figure 24] Figure 24 shows the GT-D HDI following subcutaneous treatment with eASO compounds AUS1173, AUS1177, AUS1178, AUS1179, AUS1180, AUS1181, AUS1182, AUS1194, AUS1184, AUS1185, AUS1186, AUS1187, AUS1188, AUS1189, AUS1190, AUS1191, AUS1192, AUS1193, AUS1183, AUS1239, AUS1325, AUS1326, AUS1327, AUS1328, AUS1329, AUS1175, AUS1361, or AUS1362 at 60 mg / kg. Table 16.4 shows serum levels of HBsAg in HBV mice on days 3, 7, 10, and 14. Mean values ​​are expressed as log10.

[0040] [Figure 25]Figure 25 depicts Table 16.5 showing serum levels of HBsAg in GT-D HDI HBV mice after subcutaneous treatment with eASO compounds AUS1322, AUS1382, AUS1383, AUS1384, or AUS1385 at 60 mg / kg on days 3, 7, 10, 14, and 22. Mean values ​​are expressed in log10.

[0041] [Figure 26] Figure 26 represents Table 16.6 showing serum HBsAg levels in GT-D HDI HBV mice after subcutaneous treatment with eASO compounds AUS1322, AUS1388, AUS1389, AUS1390, AUS1392, AUS1393, AUS1396, AUS1403, AUS1409, AUS1411, AUS1413, AUS1414, or AUS1415 at 60 mg / kg on days 3, 7, 10, 14, and 21. Mean values ​​are expressed in log10.

[0042] [Figure 27] Figure 27 represents Table 16.7 showing serum levels of HBsAg in GT-D HDI HBV mice on days 3, 7, 10, and 14 after subcutaneous treatment with eASO compounds AUS1387, AUS1326, AUS1328, AUS1388, AUS1390, AUS1403, or AUS1360 at 60 mg / kg. Mean values ​​are expressed in log10.

[0043] [Figure 28] Figure 28 represents Table 16.8 showing serum HBsAg levels in GT-D HDI HBV mice on days 3, 7, 10, 14, and 21 after subcutaneous treatment with eASO compounds AUS1434, AUS1435, AUS1440, AUS1436, AUS1437, AUS1438, AUS1439, AUS1441, AUS1422, AUS1423, AUS1424, AUS1425, AUS1426, AUS1427, AUS1428, AUS1429, AUS1430, AUS1431, AUS1432, or AUS1433 at 40 mg / kg. Mean values ​​are expressed in log10.

[0044] [Figure 29] Figure 29 shows the eASO compounds AUS1444, AUS1458, AUS1459, AUS1460, AUS1461, AUS1462, AUS1463, AUS1464, AUS1465, AUS1466, AUS1467, AUS1468, AUS1469, AUS1470, AUS1471, AUS1472, AUS1473, AUS1474, AUS1475, AUS1476, AUS1477, AUS1478, AUS1479, AUS1480, AUS1481, AUS1482, AUS1483, AUS1484, AUS1485, AUS1486, AUS1487, AUS1488, AUS1489, AUS1490, AUS1491, AUS1492, AUS1493, AUS1494, AUS1495, AUS1496, AUS1497, AUS1498, AUS1499, AUS2000, AUS2001, AUS2002, AUS2003, AUS2004, AUS2005, AUS2006, AUS2007, AUS2008, AUS2009, AUS2010, AUS2011, AUS2012, AUS2013, AUS2014, AUS2015, AUS2016, AUS2017, AUS2018, AUS2019, AUS2020, AUS2021, AUS2022, AUS2023, AUS2024, AUS2025, AUS2026, AUS2027, AUS20 Table 16.9 shows serum HBsAg levels in GT-D HDI HBV (1.5 μg / ml pcDNA3.1 preS2 / S plasmid) mice on days 3, 7, 10, 14, and 21 after subcutaneous treatment with AUS1474, AUS1475, AUS1476, AUS1477, AUS1478, AUS1479, AUS1480, AUS1481, AUS1482, AUS1483, AUS1488, AUS1489, AUS1490, AUS1443, AUS1444, or AUS1445 at 40 mg / kg. Mean values ​​are expressed in log10.

[0045] [Figure 30] Figure 30 represents Table 16.10 showing serum HBsAg levels in GT-D HDI HBV (1.5 μg / ml pcDNA3.1 preS2 / S plasmid) mice after subcutaneous treatment with eASO compounds AUS1492, AUS1493, AUS1233, AUS1492, or AUS1493 at 15 mg / kg or 45 mg / kg on days 3, 7, 10, 14, 21, and 28. Mean values ​​are expressed in log10.

[0046] [Figure 31] Figure 31 represents Table 16.11 showing serum HBsAg levels in GT-A HDI HBV (1.5 μg / ml pcDNA3.1 preS2 / S plasmid) mice after subcutaneous treatment with eASO compounds AUS1492, AUS1493, AUS1233, AUS1492, or AUS1493 at 40 mg / kg on days 3, 7, 10, 14, 21, and 28. Mean values ​​are expressed in log10.

[0047] [Figure 32] Figure 32 represents Table 16.12 showing serum HBsAg levels in GT-A HDI HBV (1.5 μg / ml pcDNA3.1 preS2 / S plasmid) mice after subcutaneous treatment with eASO compounds AUS1441, AUS1466, AUS1472, AUS1488, or AUS1489 at 15 or 45 mg / kg on days 3, 7, 10, 14, and 21. Mean values ​​are expressed in log10.

[0048] [Figure 33] Figure 33 represents Table 16.13 showing serum HBsAg levels in GT-D HDI HBV (1.5 μg / ml pcDNA3.1 preS2 / S plasmid) mice after subcutaneous treatment with eASO compounds AUS1495, AUS1494, AUS1233, AUS1495, AUS1494, AUS1492, or AUS1441 at 15 or 45 mg / kg on days 3, 7, 10, 14, 21, and 28. Mean values ​​are expressed in log10.

[0049] [Figure 34] Figure 34 represents Table 16.14 showing serum HBsAg levels in GT-A HDI HBV (1.5 μg / ml pcDNA3.1 preS2 / S plasmid) mice at days 3, 7, 10, 14, 21, and 28 after subcutaneous treatment with eASO compounds AUS1495, AUS1494, AUS1233, AUS1495, AUS1494, AUS1492, AUS1482, or AUS1441 at 15 or 45 mg / kg. Mean values ​​are expressed in log10.

[0050] [Figure 35] Figure 35 represents Table 16.15 showing serum levels of HBsAg in GT-B HDI HBV (0.5 μg / ml pcDNA3.1 preS2 / S plasmid) mice after subcutaneous treatment with eASO compound AUS1684 at 40 mg / kg on days 3, 7, 10, 14, and 21. Mean values ​​are expressed in log10.

[0051] [Figure 36] Figure 36 represents Table 16.16 showing serum levels of HBsAg in GT-A 1.2HBV mice after subcutaneous treatment with eASO compounds AUS1492 or AUS1493 at 45 mg / kg on days 3, 7, 10, 14, 21, and 28. Mean values ​​are expressed as log10.

[0052] [Figure 37] Figure 37 represents Table 16.17 showing serum HBsAg levels in GT-A HDI HBV (1.5 μg / ml pAAV-1.2HBV plasmid) mice after subcutaneous treatment with eASO compounds AUS1396, AUS1422, AUS1423, AUS1424, AUS1425, AUS1426, AUS1427, AUS1428, AUS1429, AUS1430, AUS1431, AUS1432, or AUS1433 at 40 mg / kg on days 3, 7, 10, 14, and 21. Mean values ​​are expressed in log10.

[0053] [Figure 38] Figure 38 shows the eASO compounds AUS1444, AUS1458, AUS1459, AUS1460, AUS1461, AUS1462, AUS1463, AUS1464, AUS1465, AUS1466, AUS1467, AUS1468, AUS1469, AUS1470, AUS1471, AUS1472, and AUS14 Table 16.18 shows serum HBsAg levels on days 3, 7, 10, 14, and 21 in GT-A HDI HBV (1.5 μg / ml pAAV-1.2HBV plasmid) mice after subcutaneous treatment with 40 mg / kg of AUS1473, AUS1474, AUS1475, AUS1476, AUS1477, AUS1478, AUS1479, AUS1480, AUS1481, AUS1482, AUS1483, AUS1488, AUS1489, AUS1490, AUS1443, or AUS1434. Mean values ​​are expressed in log10.

[0054] [Figure 39]Figure 39 depicts Table 17 showing serum levels of HBsAg, HBeAg, and HBV DNA in GT-D AAV HBV (rAAV-HBV1.3-mer WT replicon) mice after subcutaneous treatment with eASO compound AUS1493 at 40 mg / kg on days 3, 7, 10, 14, 21, and 28. Mean values ​​are expressed as log10.

[0055] [Figure 40] Figure 40 shows the effect of eASO compounds AUS1434, AUS1435, AUS1440, AUS1436, AUS1437, AUS1438, AUS1439, AUS1441, AUS1178, AUS1190, AUS1188, AUS1192, AUS1422, AUS1423, AUS1424, AUS1425, AUS1426, AUS1427 on days 0, 2, and 4. , represents Table 18.1 showing serum levels of ALT in WT male C57BL / 6 mice on days 5, 7, 10, 14, 17, and 21 after subcutaneous treatment with AUS1428, AUS1429, AUS1430, AUS1431, AUS1432, AUS1433, AUS1360, AUS1401, AUS1361, AUS1362, or AUS1411 at 60 mg / kg.

[0056] [Figure 41] Figure 41 represents Table 18.2 showing serum levels of ALT in WT male C57BL / 6 mice on days 5, 7, 10, 14, 17, and 21 after subcutaneous treatment with eASO compounds AUS1443, AUS1444, AUS1445, AUS1446, AUS1447, AUS1448, AUS1449, AUS1450, AUS1452, AUS1453, AUS1454, AUS1455, AUS1456, or AUS1457 at 60 mg / kg on days 0, 2, and 4. DETAILED DESCRIPTION OF THE INVENTION

[0057] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of the invention as claimed. As used herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including" and other forms of the term, such as "includes" or "included," is not limiting. Also, unless specifically stated otherwise, terms such as "element" or "component" encompass both elements and components comprising a single unit and elements and components comprising multiple subunits.

[0058] The paragraph headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference as well as in their entirety for the portions of the documents discussed herein.

[0059] definition Unless specific definitions are provided, the technical terms used in analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as the procedures and techniques thereof, are known and commonly used in the art. Standard techniques can be used for chemical synthesis and chemical analysis. Where permitted, all patents, applications, published applications, and other publications, GENBANK accession numbers, and related sequence information available through databases such as the National Center for Biotechnology Information (NCBI), and other data referred to throughout this disclosure are incorporated by reference and in their entirety for the portions of the documents discussed herein.

[0060] Unless otherwise indicated, the following terms have the following meanings:

[0061] "2'-O-Methoxyethyl" (also 2'-MOE and 2'-O(CH2)2-OCH3) refers to an O-methoxy-ethyl modification at the 2' position of the furanose ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0062] "2'-MOE nucleoside" (also 2'-O-methoxyethyl nucleoside) means a nucleoside that includes a 2'-MOE modified sugar moiety.

[0063] "2'-substituted nucleoside" means a nucleoside that includes a substituent other than H or OH at the 2'-position of the furanosyl ring. In certain embodiments, 2'-substituted nucleosides include nucleosides with bicyclic sugar modifications.

[0064] "3' target site" refers to the nucleotide of a target nucleic acid that is complementary to the 3' terminal nucleotide of a particular antisense compound.

[0065] "5' target site" refers to the nucleotide of a target nucleic acid that is complementary to the 5' terminal nucleotide of a particular antisense compound.

[0066] "5-methylcytosine" means a cytosine modified by the addition of a methyl group at position 5. 5-methylcytosine is a modified nucleobase.

[0067] "About" means within ±7% of a value. For example, a statement that "the compound affected at least about 70% HBV inhibition" suggests that HBV levels were inhibited within the range of 63% to 77%.

[0068] By "acceptable safety profile" is meant a pattern of side effects that is within clinically acceptable limits.

[0069] "Active pharmaceutical ingredient" refers to a substance in a pharmaceutical composition that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeted to HBV is an active pharmaceutical ingredient.

[0070] "Active target region" means a target region targeted by one or more active antisense compounds. "Active antisense compound" means an antisense compound that reduces target nucleic acid levels or protein levels.

[0071] "Acute hepatitis B infection" occurs when a person exposed to the hepatitis B virus begins to develop signs and symptoms of viral hepatitis. This period, called the incubation period, averages 90 days but can last as long as 45 days or as long as 6 months. For most people, the infection causes mild to moderate discomfort and resolves on its own as the body's immune response successfully combats the virus. However, acute HBV infection can result in very serious problems, such as fulminant liver failure, especially in people with compromised immune systems, such as those with AIDS, undergoing chemotherapy, or taking immunosuppressants or steroids.

[0072] "Administered simultaneously" refers to co-administration of two agents in any manner such that the pharmacological effects of both are experienced by the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not occur simultaneously. The effects need only overlap in time, not be coextensive.

[0073] "Administering" means providing a drug to an individual, and includes, but is not limited to, administration by a medical professional and self-administration.

[0074] "Agent" refers to an active substance that can provide a therapeutic benefit when administered to an animal. "First agent" refers to a therapeutic compound described herein. For example, a first agent may be an antisense oligonucleotide that targets HBV. "Second agent" refers to a second therapeutic compound described herein (e.g., a second antisense oligonucleotide that targets HBV) and / or a non-HBV therapeutic compound.

[0075] "Amelioration" refers to a decrease in at least one indicator of the severity of a condition or disease. Indicators of severity can be determined by subjective or objective measures known to those skilled in the art.

[0076] "Animal" refers to humans or non-human animals, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

[0077] "Antibody" refers to a molecule characterized by reacting specifically with an antigen in some way, where the antibody and antigen are each defined by the other. An antibody may refer to a complete antibody molecule or to a subunit of the antibody molecule, e.g., heavy chains, light chains, F ab Area, and F c It may also refer to any fragment or region thereof, such as a region.

[0078] "Antisense activity" means any detectable or measurable activity resulting from hybridization of an antisense compound with its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.

[0079] " Antisense compound " refers to the oligomeric compound that can be hybridized with target nucleic acid through hydrogen bond.Examples of antisense compound include single-stranded compound and double-stranded compound, such as antisense oligonucleotide, siRNA, shRNA, snoRNA, miRNA and satellite repeat.

[0080] "Antisense inhibition" refers to a reduction in target nucleic acid levels in the presence of an antisense compound complementary to the target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.

[0081] "Antisense mechanisms" are all those mechanisms involved in the hybridization of a compound with a target nucleic acid, the result or effect of which is either the destruction of the target or the occupancy of the target, with the concomitant stalling of cellular machinery involved, for example, in transcription or splicing.

[0082] "Antisense oligonucleotide" means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.

[0083] "Area under the curve" or "AUC" is the integral of the concentration of a drug in plasma as a function of time. The AUC is, for example, the time until the drug is no longer detectable (AUC 0-t ), the area under the curve from time 0 to estimated infinity (AUC 0-+ ), or over a specific clipped time window, e.g., 24 hours after administration (AUC 0-24 ) can be determined for the entire time for which data is available.

[0084] "Base complementarity" refers to the capacity of the nucleobases of an antisense oligonucleotide to form precise base pairs (i.e., hybridization) with corresponding nucleobases in a target nucleic acid, mediated by Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds between the corresponding nucleobases.

[0085] "Bicyclic sugar" means a furanose ring modified by bridging two non-geminal carbon atoms. A bicyclic sugar is a modified sugar.

[0086] "Body weight" refers to the total body weight of an animal, including all tissues, including adipose tissue.

[0087] "Cap structure" or "terminal cap moiety" means a chemical modification incorporated at either end of an antisense compound.

[0088] "cEt" or "constrained ethyl" means a bicyclic sugar moiety containing a bridge connecting the 4'-carbon and the 2'-carbon, where the bridge has the following formula: 4'-CH(CH3)-O-2'.

[0089] "Constrained ethyl nucleoside" (or cEt nucleoside) means a nucleoside that includes a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge.

[0090] A "chemically distinct region" refers to a region of an antisense compound that is chemically distinct in some way from another region of the same antisense compound. For example, a region having 2'-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2'-O-methoxyethyl modifications.

[0091] By "chimeric antisense compound" is meant an antisense compound having at least two chemically distinct regions, each region having multiple subunits.

[0092] "Chronic hepatitis B infection" occurs when a person initially suffers from an acute infection and subsequently is unable to fight the infection. Whether the disease becomes chronic or completely resolves depends primarily on the age of the infected individual. Approximately 90% of infants infected at birth progress to chronic disease. However, as individuals age, the risk of chronic infection decreases, with 20%-50% of children and less than 10% of older children or adults progressing from acute to chronic infection. While chronic HBV infection is the primary therapeutic focus of embodiments of the present invention, the ASO compositions of the present invention can also treat HBV-associated conditions, such as inflammation, fibrosis, cirrhosis, liver cancer, and serum hepatitis.

[0093] "Co-administration" means administering two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition or in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered via the same or different routes of administration. Co-administration includes concurrent or sequential administration.

[0094] "Complementarity" means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

[0095] "Comply" means that an individual adheres to the recommended treatment.

[0096] It will be understood that the terms "comprise," "comrises," and "comprising" imply the inclusion of the stated step(s), element(s), or group(s) of steps or elements, but not the exclusion of any other step(s), element(s), or group(s) of steps or elements.

[0097] "Contiguous nucleobases" means nucleobases immediately adjacent to each other.

[0098] "Cure" means a method or process that restores health or a prescribed treatment for an illness.

[0099] "Deoxyribonucleotide" means a nucleotide having a hydrogen at the 2' position of the sugar moiety of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.

[0100] "Designing" or "designed for" refers to the process of designing oligomeric compounds that specifically hybridize with a selected nucleic acid molecule.

[0101] "Diluent" means an ingredient in a composition that is not pharmacologically active, but is pharmaceutically necessary or desirable. For example, in an injectable formulation, the diluent may be a liquid, such as saline.

[0102] "Dosage unit" means the form in which a pharmaceutical agent is provided, such as, for example, a pill, tablet, or other dosage unit known in the art.

[0103] "Dose" refers to a specific amount of a pharmaceutical agent provided in a single administration or provided over a specific period of time. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume that is not easily accommodated in a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reactions in individuals. In other embodiments, a pharmaceutical agent is administered by infusion or continuously over an extended period of time. A dose may be described as the amount of pharmaceutical agent per hour, day, week, or month.

[0104] A "dosing regimen" is a combination of doses designed to achieve one or more desired effects.

[0105] "Duration" means the period of time during which an activity or event continues. In certain embodiments, the duration of treatment is the period during which doses of a pharmaceutical agent are administered.

[0106] In the context of modulating an activity or treating or preventing a condition, an "effective amount" means the administration to a subject in need of such modulation, treatment, or prevention of that amount of active ingredient, in a single dose or as part of a series, effective to modulate that effect, or to treat or prevent, or ameliorate that condition. The effective amount will vary depending on the health and physical condition of the subject being treated, the taxonomic group of the subject being treated, the formulation of the composition, evaluation of the medical condition, and other relevant factors.

[0107] "Efficacy" means the ability to produce a desired effect.

[0108] "Expression" includes all processes by which genetically encoded information is converted into structures present and operating in a cell, including, but not limited to, the products of transcription and translation.

[0109] The term "fragment" as applied to a polynucleotide refers to a nucleotide sequence that is shorter in length compared to a reference nucleic acid or nucleotide sequence and will be understood to comprise, consist essentially of, and / or consist of a nucleotide sequence of contiguous nucleotides that is identical or nearly identical (e.g., 60%, 70%, 80%, 90%, 92%, 95%, 98%, or 99% identical) to the reference nucleic acid or nucleotide sequence. Such nucleic acid fragments according to the invention may, where appropriate, be included within a larger polynucleotide of which they are a component. In some embodiments, such fragments may contain, consist essentially of, and / or consist of an oligonucleotide having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more contiguous nucleotides of a nucleic acid or nucleotide sequence according to the invention.

[0110] "Fully complementary" or "100% complementary" means that each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, the first nucleic acid is an antisense compound and the target nucleic acid is the second nucleic acid.

[0111] A "fully modified motif" refers to an antisense compound that comprises a contiguous sequence of nucleosides, where essentially each nucleoside is a sugar-modified nucleoside having a uniform modification.

[0112] "Gapmer" refers to a chimeric antisense compound in which an internal region having multiple nucleosides that supports RNase H cleavage is positioned between external regions having one or more nucleosides, where the nucleosides comprising the internal region can be chemically distinct from the nucleosides comprising the external regions. The internal region can also be referred to as the "gap," and each of the 5' and 3' external regions can also be referred to as the "wings."

[0113] A "gap" is an internal segment of a chimeric antisense compound that comprises one or more linked deoxynucleosides and is located between the 5' wing (W1) and the 3' wing (W2). The gap may also be referred to as a "gap," "gap region," or "gap segment."

[0114] "HBV" refers to mammalian hepatitis B virus, including human hepatitis B virus. The term encompasses hepatitis B virus, particularly geographic genotypes of human hepatitis B virus, as well as variant strains of geographic genotypes of hepatitis B virus.

[0115] By "HBV antigen" is meant any hepatitis B virus antigen or protein, including, for example, core proteins such as "hepatitis B core antigen" or "HBcAG," and "hepatitis B E antigen" or "HBeAG," and envelope proteins such as "HBV surface antigen" or "HBsAg" or "HBsAG."

[0116] "Hepatitis B E antigen" or "HBeAg" or "HBeAG" is a secreted, non-particulate form of the HBV core protein. The HBV antigens HBeAg and HBcAg share a common primary amino acid sequence and are therefore cross-reactive at the T cell level. HBeAg is not required for viral assembly or replication, but studies suggest that it may be required for the establishment of chronic infection. Neonatal infection with HBeAg-negative mutants often results in fulminant acute infection rather than chronic HBV infection (Terezawa et al. (1991) Pediatr. Res. 29:5). In contrast, infection of young woodchucks with WHeAg-negative mutants resulted in a much lower rate of chronic WHV infection (Cote et al. (2000) Hepatology 31:190). HBeAg may function as a tolerogen by inactivating core-specific T cells through deletion or clonal anergy (Milich et al (1998) J. Immunol. 160:8102). Upon antiviral therapy and HBeAg seroconversion, a decrease in HBV viral load and antigen correlates positively with a decrease in T cell expression of the inhibitory receptor programmed death-1 (PD-1, also known as PDCD1), a negative regulator of activated T cells (Evans et al (2008) Hepatology 48:759).

[0117] By "HBV mRNA" is meant any messenger RNA expressed by the Hepatitis B virus.

[0118] "HBV nucleic acid" or "HBV DNA" refers to any nucleic acid encoding HBV. For example, in certain embodiments, HBV nucleic acid includes, but is not limited to, any viral DNA sequence encoding the HBV genome or a portion thereof, any RNA sequence transcribed from viral DNA, including any mRNA sequence encoding an HBV protein.

[0119] By "HBV protein" is meant any protein secreted by the hepatitis B virus. The term encompasses various HBV antigens, including core proteins such as, for example, "hepatitis E antigen," "HBeAg," or "HBeAG," and envelope proteins such as, for example, "HBV surface antigen" or "HBsAg."

[0120] "HBV surface antigen" or "HBsAg" or "HBsAG" is an envelope protein of infectious HBV virions, but is also secreted as non-infectious particles, and serum levels of HBsAg are 1000 times higher than those of HBV virions. Serum levels of HBsAg in infected humans or animals can be as high as 1000 g / mL (Kann and Gehrlich (1998) Topley & Wilson's Microbiology and Microbial Infections, 9 th ed.745). In acute HBV infection, the half-life of HBsAg in serum, or serum t %The time to maturity is 8.3 days (Chulanov et al. (2003) J. Med. Virol. 69:313). Internalization of HBsAg by myeloid dendritic cells inhibits the upregulation of costimulatory molecules (i.e., B7) and inhibits their ability to stimulate T cells (den Brouw et al. (2008) Immunology 126:280). Dendritic cells from chronically infected patients exhibit defects in the expression of costimulatory molecules, IL-12 secretion, and T cell stimulation in the presence of HBsAg (Zheng et al. (2004) J. Viral Hepatitis 11:217). HBsAg-specific CD8+ cells from CHB patients exhibit altered tetramer binding. These CD8+ cells are not anergic but may possess TCR topologies that confer partial tolerance or ignorance (Reignat et al. (2002) J. Exp. Med. 195:1089). Furthermore, a >1-log decline in serum HBsAg at week 24 is highly predictive of sustained virological response (SVR, defined as undetectable HBV DNA by PCR 1 year after treatment) during Peg-IFNa2a treatment (92%) (Moucari et al (2009) Hepatology 49:1151).

[0121] "Hepatitis B-associated condition" or "HPV-associated condition" means any disease, biological state, medical condition, or event that is exacerbated by, caused by, associated with, linked to, or traceable to infection, exposure, or disease with hepatitis B. The term hepatitis B-associated condition includes chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, transplant-associated liver disease, and conditions with symptoms that may include any or all of the following: flu-like illness, weakness, pain, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain in the liver region of the body, thick or gray stools, generalized itching, and dark urine when coupled with hepatitis B virus, a positive test for the presence of hepatitis B virus antigen, or a positive test for antibodies specific for hepatitis B virus antigen.

[0122] "Hybridization" refers to the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.

[0123] As used herein, "IC50" or "IC 50 The term "IC value" refers to the concentration of an agent at which cell viability is halved. 50 is a measure of the effectiveness of a drug in inhibiting a biological process.

[0124] "Identifying an animal infected with HBV" means identifying an animal diagnosed with HBV or identifying an animal with any symptoms of HBV infection, including, but not limited to, chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, transplant-associated liver disease, and conditions with symptoms which may include any or all of the following: means: when combined with a positive test for the presence of hepatitis B virus, hepatitis B virus antigen, or a positive test for antibodies specific for hepatitis B virus antigen; when combined with a positive test for the presence of hepatitis B virus, hepatitis B virus antigen, or a positive test for antibodies specific for hepatitis B virus antigen, flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain in the liver area of ​​the body, thick or gray stools, generalized itching, and dark urine.

[0125] "Immediately adjacent" means that there are no intervening elements between the immediately adjacent elements.

[0126] "Individual" means a human or non-human animal selected for treatment or therapy.

[0127] "Individual compliance" means adherence by an individual to a recommended or prescribed treatment.

[0128] Terms such as "induce," "inhibit," "potentiate," "elevate," "increase," and "decrease" generally refer to a quantitative difference between two states. These terms may refer to a statistically significant difference between two states. For example, an "amount effective to inhibit HBV activity or expression" means that the level of HBV activity or expression in a treated sample is quantitatively different from, and may be statistically significant, the level of HBV activity or expression in an untreated cell. These terms apply, for example, to levels of expression and levels of activity. As used herein, the terms "inhibit" or "reduce" or grammatical variations thereof refer to a decrease or decline in a particular level or activity by at least about 5%, about 10%, about 15%, about 25%, about 35%, about 40%, about 50%, about 60%, about 75%, about 80%, about 90%, about 95%, or more. In some embodiments, inhibition or reduction results in little or essentially no detectable activity (at most, an insignificant amount, eg, less than about 10% or even less than 5%).

[0129] "Inhibiting HBV" means that the level or expression of HBV mRNA, DNA, and / or protein is reduced. In certain embodiments, HBV is inhibited in the presence of an antisense compound targeting HBV, including an antisense oligonucleotide targeting HBV, compared to the expression level of HBV mRNA, DNA, and / or protein in the absence of the HBV antisense compound, including an antisense oligonucleotide.

[0130] "Inhibiting expression or activity" refers to a reduction or blocking of expression or activity, and does not necessarily indicate a complete abolition of expression or activity.

[0131] "Injection site reaction" means inflammation or abnormal redness of the skin at the site of an injection in an individual.

[0132] "Internucleoside linkage" refers to the chemical bond between nucleosides.

[0133] "Intraperitoneal administration" means administration via infusion or injection into the peritoneal cavity.

[0134] "Intravenous administration" means administration into a vein.

[0135] An "extended" antisense oligonucleotide is an oligonucleotide which has one or more additional nucleosides compared to the antisense oligonucleotides disclosed herein.

[0136] "Linked deoxynucleosides" means deoxyribonucleobases (A, G, C, T, U) linked by phosphate esters to form a nucleotide.

[0137] "Linked nucleosides" means adjacent nucleosides linked together by an internucleoside bond. Examples of linked nucleosides are linked nucleotides, where the linkage includes a phosphate atom, e.g., a phosphodiester bond.

[0138] "Locked nucleic acid" or "LNA" or "LNA nucleoside" refers to a nucleic acid monomer having a bridge connecting two carbon atoms between the 4' and 2' positions of the nucleoside sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugars include, but are not limited to, A) α-L-methyleneoxy (4'-CH2-O-2') LNA, (B) β-D-methyleneoxy (4'-CH2-O-2')-LNA, (C) ethyleneoxy (4'-(CH2)2-O-2') LNA, (D) aminooxy (4'-CH2-O- N(R)-2')LNA, and (E)oxyamino (4'-CH2-N(R)-O-2')LNA. [ka]

[0139] As used herein, LNA compounds include, but are not limited to, compounds having at least one bridge between the 4' and 2' sugar positions, where each bridge is independently -[C(R 1 )(R 2 )]n-, -C(R 1 )=C(R 2 )-, -C(R 1 )=N-, -C(=NR 1 )-, -C(=O)-, -C(=S)-, -O-, -Si(R 1 )2-, -S(=O) x - and -N(R 1 )-, where x is 0, 1, or 2; n is 1, 2, 3, or 4; and R 1 and R 2 each independently represents H, a protecting group, a hydroxyl, C-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ 1 , N.J. I J 2 , S.J. 1 , N3, COOJ 1 , acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)-J 1 ), or sulfoxyl (S(=O)-J 1 ) and J 1 and J. 2 are independently H, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C 12 Aminoalkyl, substituted C1-C 12 It is an aminoalkyl or a protecting group.

[0140] Examples of 4'-2' bridging groups encompassed within the definition of LNA include, but are not limited to, one of the following formulas: --[C(R 1 )(R 2 )] n -, -[C(R 1 )(R 2 )] n -O-, -C(R 1 )(R 2 )-N(R 1 )-O-, or -C(R 1 )(R 2 )-ON(R 1 )-. Additionally, other bridging groups encompassed within the definition of LNA are 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-ON(R 1 )-2', and 4'-CH2-N(R 1 )-O-2′-bridge, wherein R 1 and R 2 are each independently H, a protecting group, or C1-C 12 It is alkyl.

[0141] The definition of LNA according to the present invention also includes LNAs in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH2-O-2') bridge to form a bicyclic sugar moiety. The bridge may also be a methylene (-CH2-) group connecting the 2' oxygen atom and the 4' carbon atom, for which the term methyleneoxy (4'-CH2-O-2') LNA is used. Furthermore, for bicyclic sugar moieties with an ethylene bridging group at this position, the term ethyleneoxy (4'-CH2CH2-O-2') LNA is used. α-L-methyleneoxy (4'-CH2-O-2'), isomers of methyleneoxy (4'-CH2-O-2') LNA are also encompassed within the definition of LNA used herein.

[0142] "Maximum plasma concentration" or "C max " refers to the peak concentration of drug in plasma after a dose of drug is administered to a subject. Methods for measuring drug concentrations are known to those skilled in the art and include liquid chromatography and tandem mass spectrometry, among others.

[0143] A "mismatch" or "non-complementary nucleobase" refers to the case where a nucleobase of a first nucleic acid fails to pair with the corresponding nucleobase of a second or target nucleic acid.

[0144] A "modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside linkage (ie, a phosphodiester internucleoside linkage).

[0145] "Modified nucleobase" means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. "Unmodified nucleobase" means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0146] "Modified nucleoside" means a nucleoside having, independently, a modified sugar moiety and / or a modified nucleobase.

[0147] "Modified nucleotide" means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.

[0148] "Modified oligonucleotide" means an oligonucleotide containing at least one modified internucleoside linkage, modified sugar, and / or modified nucleobase.

[0149] By "modified sugar" is meant a substitution and / or any change from a natural sugar moiety.

[0150] "Monomer" refers to a single unit of an oligomer. Monomers include, but are not limited to, natural or modified nucleosides and nucleotides.

[0151] "Motif" refers to the pattern of unmodified and modified nucleosides in an antisense compound. "Natural sugar moiety" refers to a sugar moiety present in DNA (2'-H) or RNA (2'-OH). "Natural internucleoside linkage" refers to a 3' to 5' phosphodiester linkage.

[0152] The term "non-complementary nucleobases" refers to a pair of nucleobases that do not form hydrogen bonds with each other or otherwise support hybridization.

[0153] "Nucleic acid" refers to a molecule composed of monomeric nucleotides. Nucleic acids include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNAs).

[0154] "Nucleobase" means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

[0155] "Nucleobase complementarity" refers to a nucleobase that can base pair with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to the nucleobase of an antisense compound that can base pair with the nucleobase of its target nucleic acid. For example, if the nucleobase at a specific position of an antisense compound can hydrogen bond with the nucleobase at a specific position of a target nucleic acid, the hydrogen bond position between the oligonucleotide and the target nucleic acid is considered to be complementary in this nucleobase pair.

[0156] By "nucleobase sequence" is meant the order of consecutive nucleobases, independent of any sugar, linkage, and / or nucleobase modifications.

[0157] "Nucleoside" means a nucleobase linked to a sugar. Nucleosides include deoxynucleosides, such as deoxyribonucleosides.

[0158] "Nucleoside mimics" include structures used to replace sugars or sugars and bases, but do not necessarily include structures used to replace bonds at one or more positions in an oligomeric compound, such as morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic, or tricyclic sugar mimetics, e.g., nucleoside mimics with non-furanose sugar units. Nucleotide mimics include nucleosides and structures used to replace bonds at one or more positions in an oligomeric compound, such as peptide nucleic acids or morpholinos (morpholinos linked by -N(H)-C(=O)-O- or other non-phosphodiester linkages). Sugar surrogates overlap with the somewhat broader term nucleoside mimics, but are intended to refer to replacement of only the sugar unit (furanose ring). The tetrahydropyranyl ring provided herein is an example of a sugar surrogate in which the furanose sugar group is replaced with a tetrahydropyranyl ring system. "Mimetic" refers to groups substituted for the sugar, nucleobase, and / or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, while maintaining the nucleobase for hybridization to a selected target.

[0159] "Nucleotide" means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

[0160] "Off-target effect" refers to an unwanted or adverse biological effect associated with the modulation of RNA or protein expression of a gene other than the intended target nucleic acid.

[0161] By "oligomeric compound" is meant a polymer or linked monomeric subunits capable of hybridizing to at least a region of a nucleic acid molecule.

[0162] "Oligonucleoside" refers to an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.

[0163] "Oligonucleotide" means a polymer of linked nucleosides, each of which, independently of the others, may or may not be modified.

[0164] "Parenteral administration" means administration by injection (e.g., bolus injection) or infusion. Parenteral administration includes subcutaneous, intravenous, intramuscular, intraarterial, intraperitoneal, or intracranial administration, e.g., intrathecal or intraventricular.

[0165] "Peptide" means a molecule formed by linking at least two amino acids by an amide bond. As used herein, "peptide" refers to, but is not limited to, polypeptides and proteins.

[0166] "Pharmaceutically acceptable carrier" refers to a medium or diluent that does not interfere with the structure of the oligonucleotide. Certain such carriers allow the pharmaceutical composition to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges for oral ingestion by a subject.

[0167] "Pharmaceutically acceptable derivatives" includes pharmaceutically acceptable salts, conjugates, prodrugs, or isomers of the compounds described herein.

[0168] "Pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

[0169] "Pharmaceutical product" means a substance that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeting HBV is a pharmaceutical product.

[0170] "Pharmaceutical composition" refers to a mixture of substances suitable for administration to a subject. For example, a pharmaceutical composition may comprise an antisense oligonucleotide and a sterile aqueous solution. In certain embodiments, the pharmaceutical composition exhibits activity in a free uptake assay in a specific cell line.

[0171] "Phosphorothioate linkage" means a linkage between nucleosides in which the phosphodiester bond has been modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

[0172] "Portion" refers to a predetermined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a predetermined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a predetermined number of contiguous nucleobases of an antisense compound.

[0173] "Prevention" or "preventing" refers to delaying or averting the onset or development of a condition or disease for a period of hours to days, preferably weeks to months.

[0174] A "prodrug" refers to a therapeutic agent that is prepared in an inactive form that is converted to its active form (i.e., drug) within the body or its cells by the action of endogenous enzymes or by the action of other chemicals and / or conditions.

[0175] A "prophylactically effective amount" refers to an amount of a pharmaceutical agent that provides a prophylactic or preventative benefit to an animal.

[0176] "Recommended therapy" means a treatment regimen recommended by a medical professional for the treatment, amelioration, or prevention of disease.

[0177] A "region" is defined as a portion of a target nucleic acid that has at least one distinguishable structure, function, or characteristic.

[0178] "Ribonucleotide" means a nucleotide having a hydroxyl at the 2' position of the sugar moiety of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.

[0179] "Salts" means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

[0180] A "segment" is defined as a smaller portion or subportion of a region within a target nucleic acid.

[0181] A "separator" is a segment or region that separates and is located between two gap regions in a chimeric antisense compound. A separator segment may have one or more nucleosides, where the nucleosides are chemically distinct from the nucleosides that comprise the gap. Separator segments include nucleosides that have been modified to confer properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, reduced in vivo toxicity, or resistance to degradation by nucleases in vivo. A chimeric antisense compound may contain one or more separator segments. A separator may also be referred to as a "separator," "separator region," or "separator segment." Exemplary chimeric antisense compounds of the present disclosure contain one, two, three, four, five, or six separator segments.

[0182] "Seroconversion" is defined as absence of serum HBeAg plus presence of serum HBeAb if HBeAg was being monitored as the determinant of seroconversion, or absence of serum HBsAg if HBsAg was being monitored as the determinant of seroconversion, as determined by the currently available detection limits of commercially available ELISA systems.

[0183] "Shortened" or "truncated" versions of the antisense oligonucleotides taught herein have one, two, or more nucleosides missing.

[0184] "Side effects" refer to physiological responses resulting from treatment other than the desired effect. In certain embodiments, side effects include, but are not limited to, injection site reactions, liver function test abnormalities, renal function abnormalities, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and myopathy. For example, an increase in serum aminotransferase levels may indicate hepatotoxicity or liver function abnormalities. For example, an increase in bilirubin may indicate hepatotoxicity or liver function abnormalities.

[0185] As used herein, "significant" means a measurable or observable, e.g., significant result. For example, a significant improvement or a significant reduction generally refers to a measurable or observable result, e.g., a measurable or observable improvement or reduction.

[0186] As used herein, a "site" is defined as a unique nucleobase position within a target nucleic acid.

[0187] By "slowing progression" is meant a reduction in the development of the disease.

[0188] "Specifically hybridizable" refers to an antisense compound that has a sufficient degree of complementarity between the antisense oligonucleotide and the target nucleic acid to induce the desired effect under conditions where specific binding is desired, i.e., physiological conditions in the case of in vivo assays and therapeutic treatments, while having minimal or no effect on non-target nucleic acids. "Stringent hybridization conditions" or "stringent conditions" refer to conditions under which an oligomeric compound hybridizes to its target sequence but weakly hybridizes to other sequences.

[0189] As used herein, "statistically significant" means a measurable or observable parameter that is unlikely to occur by chance.

[0190] "Subcutaneous administration" refers to administration just below the skin. "Subject" means a human or non-human animal selected for treatment or therapy.

[0191] "Target" refers to a protein whose modulation is desired.

[0192] "Target gene" refers to a gene that encodes a target.

[0193] "Targeting" refers to the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

[0194] "Target nucleic acid," "target RNA," "target RNA transcript," and "nucleic acid target" all refer to a nucleic acid capable of being targeted by antisense compounds.

[0195] "Target region" means a portion of a target nucleic acid to which one or more antisense compounds are targeted.

[0196] "Target segment" refers to the nucleotide sequence of a target nucleic acid to which an antisense compound is targeted. "5' target site" refers to the 5'-terminal nucleotide of a target segment. "3' target site" refers to the 3'-terminal nucleotide of a target segment.

[0197] "Therapeutically effective amount" means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.

[0198] "Treatment" refers to administering a composition to alter or ameliorate a disease or condition.

[0199] "Unmodified" nucleobase means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0200] "Unmodified nucleotide" means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, the unmodified nucleotide is an RNA nucleotide (i.e., a 3-D-ribonucleoside) or a DNA nucleotide (i.e., a 3-D-deoxyribonucleoside).

[0201] An "effective target segment" is defined as at least an eight nucleobase portion (ie, eight consecutive nucleobases) of a target region to which an active oligomeric compound is targeted.

[0202] "Wings" are terminal segments of a chimeric antisense oligonucleotide that have been modified to confer properties to the oligonucleotide, such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, reduced in vivo toxicity, or resistance to degradation by nucleases in vivo. Wings may also be referred to as "wings," "wing regions," or "wing segments." As used herein, a wing comprises at least two linked nucleosides, a subset of which may comprise one or more deoxynucleosides, although the entire wing is not composed exclusively of deoxynucleosides.

[0203] The chimeric antisense compounds of the present disclosure comprise a 5' wing segment (W1) located at the 5' end of the chimeric antisense oligonucleotide, wherein the residue at the 3' end of W1 cannot be a deoxynucleoside.The chimeric antisense compounds of the present disclosure also comprise a 3' wing segment (W2) located at the 3' end of the chimeric antisense oligonucleotide, wherein the residue at the 5' end of W2 cannot be a deoxynucleoside.

[0204] The 5' wing (W1) begins at the 5' end of the chimeric antisense oligonucleotide, extends in the 5' to 3' direction, and ends at the first nucleoside that is directly linked to a deoxynucleoside of the first gap rather than a deoxynucleoside, thus representing the 3' end of W1 and the 5' end of the first gap (G1).

[0205] The 3' wing (W2) begins at the 3' end of the chimeric antisense oligonucleotide, extends in the 3' to 5' direction, and ends at the first nucleoside that is directly linked to a deoxynucleoside of the gap rather than a deoxynucleoside, thus representing the 3' end of the final gap and the 5' end of W2.

[0206] Exemplary Chimeric Antisense Oligonucleotides The present disclosure provides at least the following exemplary chimeric antisense oligonucleotides:

[0207] In one embodiment, a modified oligonucleotide is provided, the modified oligonucleotide comprising, in the 5' to 3' direction: 5'W1-G1-S1-G2-W2 3' (Formula I), During the ceremony, W1 is the 5' wing segment; W2 is the 3' wing segment; G1 is the first gap segment, S1 is a first separator segment; G2 is the second gap segment, - is an internucleoside linkage, and At least one nucleoside of the oligonucleotide is modified.

[0208] In some embodiments, the modified oligonucleotide has, in the 5' to 3' direction: 5'W1-G1-S1-G2-S2-G3-W2 3' (Formula II), During the ceremony, S2 is a second separator segment, and G3 is the third gap segment.

[0209] In some embodiments, the modified oligonucleotide has, in the 5' to 3' direction: 5'W1-G1-S1-G2-S2-G3-S3-G4-W2 3' (Formula III), During the ceremony, S3 is the third separator segment, and G4 is the fourth gap segment.

[0210] In some embodiments, the modified oligonucleotide has, in the 5' to 3' direction: 5'W1-G1-S1-G2-S2-G3-S3-G4-S4-G5-W2 3' (Formula IV), During the ceremony, S4 is the fourth separator segment, and G5 is the fifth gap segment.

[0211] In some embodiments, the modified oligonucleotide has, in the 5' to 3' direction:

[0212] 5'W1-G1-S1-G2-S2-G3-S3-G4-S4-G5-S5-G6-W2 3' (Formula V), During the ceremony, S5 is the fifth separator segment, and G6 is the sixth gap segment.

[0213] In some embodiments, the modified oligonucleotide has, in the 5' to 3' direction: 5'W1-G1-S1-G2-S2-G3-S3-G4-S4-G5-S5-G6-S6-G7-W2 3' (Formula VI), During the ceremony, S6 is the sixth separator segment, and G7 is the seventh gap segment.

[0214] In some embodiments, W1 contains 2 to 25 linked nucleosides. In some embodiments, W1 contains one or more linked deoxynucleosides.

[0215] In some embodiments, W2 contains 2 to 35 linked nucleosides. In some embodiments, W2 contains one or more linked deoxynucleosides.

[0216] In some embodiments, any one or more of G1, G2, G3, G4, G5, G6 and / or G7 comprises 1 to 10 linked deoxynucleosides.

[0217] In some embodiments, any one or more of S1, S2, S3, S4, S5, and / or S6 comprises 1, 2, 3, 4, or 5 linked nucleosides.

[0218] In some embodiments, the modified oligonucleotide is 18 to 50 nucleobases in length. In some embodiments, the modified oligonucleotide is at least 20 nucleobases in length. In some embodiments, the modified oligonucleotide is 20 nucleobases in length.

[0219] In some embodiments, the modified oligonucleotide is of Formula I, wherein W1 comprises 4 to 6 linked nucleosides, G1 comprises 1 to 6 linked deoxynucleosides, S1 comprises 1 linked nucleoside, G2 comprises 1 to 6 linked deoxynucleosides, and W2 comprises 4 to 6 linked nucleosides.

[0220] In some embodiments, the modified oligonucleotide is of Formula I, wherein W1 comprises 4 to 6 linked nucleosides, G1 comprises 5 linked deoxynucleosides, S1 comprises 1 linked nucleoside, G2 comprises 5 linked deoxynucleosides, and W2 comprises 4 to 6 linked nucleosides.

[0221] In some embodiments, the modified oligonucleotide is of Formula I, wherein W1 comprises 4 linked nucleosides, G1 comprises 5 linked deoxynucleosides, S1 comprises 1 linked nucleoside, G2 comprises 5 linked deoxynucleosides, and W2 comprises 5 linked nucleosides.

[0222] In some embodiments, the modified oligonucleotide is of Formula I and the length of G1-S1-G2 is between 8 and 12 nucleobases in length.

[0223] The following embodiments relate to any of Formulas I-VI: In some embodiments, G1, G2, G3, G4, G5, G6, and G7 may contain nucleosides containing 2'-deoxynucleoside modifications. In some embodiments, any one or more of G1, G2, G3, G4, G5, G6, and G7 contain nucleosides containing a 2'-deoxy 5-methylcytidine sugar modification. In some embodiments, S1, S2, S3, S4, S5, and / or S6 contain nucleosides containing a 2'-O-methoxyethyl sugar modification. In some embodiments, S1, S2, S3, S4, S5, and / or S6 contain nucleosides containing 5-methylcytidine. In some embodiments, S1, S2, S3, S4, S5, and / or S6 contain nucleosides containing a 2'-O-methyl sugar modification. In some embodiments, S1, S2, S3, S4, S5, and / or S6 comprise nucleosides comprising a 2'-OH sugar modification. In some embodiments, S1, S2, S3, S4, S5, and / or S6 comprise nucleosides comprising a 2'-fluoro sugar modification. In some embodiments, S1, S2, S3, S4, S5, and / or S6 comprise nucleosides comprising a 2'-fluoro-arabinonucleic acid (2'-fluoro-ANA) sugar modification. In some embodiments, S1, S2, S3, S4, S5, and / or S6 comprise glycol nucleic acid (GNA). In some embodiments, S1, S2, S3, S4, S5, and / or S6 comprise LNA. In some embodiments, W1 comprises a nucleoside comprising a 2'-deoxy sugar modification (e.g., the 2'-deoxy sugar modification is at position 2 and / or 5 of a sequence corresponding to SEQ ID NO: 2). In some embodiments, W1 contains a nucleoside containing a 2'-O-methoxyethyl sugar modification (e.g., a 2'-O-methoxyethyl sugar modification at positions 1, 2, 3, 4, and / or 5 of a sequence corresponding to SEQ ID NO: 2). In some embodiments, W1 contains a 2'-O-methoxyethyl 5-methylcytidine at position 2 of a sequence corresponding to SEQ ID NO: 2. In some embodiments, W1 contains a nucleoside containing a 2'-O-methyl sugar modification (e.g., a 2'-O-methyl sugar modification at positions 1, 2, 3, 4, and / or 5 of a sequence corresponding to SEQ ID NO: 2).In some embodiments, W1 contains a 2'-O-methyl 5-methylcytidine at position 2 of the sequence corresponding to SEQ ID NO:2. In some embodiments, W1 contains a nucleoside containing a 2'-fluoro sugar modification. In some embodiments, W1 contains a nucleoside containing a 2'-fluoro-arabinonucleic acid (2'-fluoro-ANA) modification. In some embodiments, W1 contains a glycol nucleic acid (GNA) (e.g., a GNA at position 2 of the sequence corresponding to SEQ ID NO:2). In some embodiments, W1 contains a modified nucleoside, wherein the modified nucleoside is a locked nucleic acid (LNA) (e.g., an LNA at position 1 and / or 3 of the sequence corresponding to SEQ ID NO:2). In some embodiments, W2 contains a nucleoside containing a 2'-deoxy sugar modification (e.g., a 2'-deoxy sugar modification at position 15 and / or 16 of the sequence corresponding to SEQ ID NO:2). In some embodiments, W2 contains a nucleoside containing a 2'-O-methoxyethyl sugar modification (e.g., the 2'-O-methoxyethyl sugar modification is at position 15, 16, 17, 18, 19, and / or 20 of the sequence corresponding to SEQ ID NO: 2). In some embodiments, W2 contains a nucleoside containing a 2'-O-methyl sugar modification (e.g., the 2'-O-methyl sugar modification is at position 15, 16, 17, 18, 19, and / or 20 of the sequence corresponding to SEQ ID NO:2). In some embodiments, W2 contains a nucleoside containing a 2'-fluoro sugar modification. In some embodiments, W2 contains a nucleoside containing a 2'-fluoro-arabinonucleic acid (2'-fluoro-ANA) modification. In some embodiments, W2 contains a modified nucleoside, wherein the modified nucleoside is a glycol nucleic acid (GNA). In some embodiments, W2 contains a modified nucleoside, wherein the modified nucleoside is a locked nucleic acid (LNA) (e.g., the LNA is at position 16, 17, 18, 19, and / or 20 of the sequence corresponding to SEQ ID NO:2).

[0224] Exemplary Target Sequences and Exemplary Modified Oligonucleotides Certain embodiments provide methods, compounds, and compositions for inhibiting HBV mRNA expression.

[0225] Certain embodiments provide antisense compounds that target HBV nucleic acid sequences. Exemplary HBV nucleic acid sequences include, but are not limited to, those shown in Table 1. [Table 1]

[0226] In certain embodiments, the HBV nucleic acid is the sequence set forth in GENBANK Accession No. U95551.1 (incorporated herein as SEQ ID NO:3). In some embodiments, the antisense compound targets the sequence listed in SEQ ID NO:3, or a portion thereof. In some embodiments, the antisense compound targets the sequence from positions 1583 to 1602 of SEQ ID NO:3.

[0227] Exemplary HBV nucleic acid target sequences include, but are not limited to, those shown in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0228] In some embodiments, the HBV target comprises the sequence of SEQ ID NOs: 1, 4, 675-838. In some embodiments, the HBV target comprises the sequence of CTTGG TCATG GGCCA TCAG (SEQ ID NO: 1). In some embodiments, the HBV target comprises the sequence of GCACT TCGCT TCACC TCTGC (SEQ ID NO: 4).

[0229] In certain embodiments, the compounds provided herein comprise modified oligonucleotides. In certain embodiments, the compounds comprise modified oligonucleotides and conjugates described herein. In certain embodiments, the modified oligonucleotides are pharmaceutically acceptable derivatives.

[0230] In certain embodiments, the HBV target comprises the sequence set forth in SEQ ID NO: 3, or a portion thereof, or a variant thereof. In certain embodiments, the modified oligonucleotide is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% complementary to an HBV nucleic acid.

[0231] In certain embodiments, the HBV target comprises the sequence set forth in SEQ ID NO: 1. In certain embodiments, the modified oligonucleotide is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% complementary to SEQ ID NO: 1.

[0232] In certain embodiments, the HBV target comprises the sequence set forth in SEQ ID NO: 4. In certain embodiments, the modified oligonucleotide is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% complementary to SEQ ID NO: 4.

[0233] In some embodiments, the compound or composition comprises a modified oligonucleotide of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 linked nucleosides in length that binds to the HBV target sequence. In certain embodiments, the compound or composition comprises a modified oligonucleotide of 20 linked nucleosides that binds to the HBV target sequence.

[0234] In certain embodiments, the 20 linked nucleoside modified oligonucleotide has the nucleobase sequence GCAGA GGTGA AGCGA AGTGC (SEQ ID NO: 2). A chart showing the positions of the nucleobases in SEQ ID NO: 2 is provided below. [Table 3]

[0235] In certain embodiments, the 20 linked nucleoside modified oligonucleotide has the nucleobase sequence GTGAA GCGAA GTGCA CACGG (SEQ ID NO: 5). A chart showing the positions of the nucleobases in SEQ ID NO: 5 is provided below. [Table 4]

[0236] In certain embodiments, the compound comprises a modified oligonucleotide described herein. Examples of modified oligonucleotides include, but are not limited to, those shown in Figures 1-2. In some embodiments, the compound comprises a modified oligonucleotide comprising the sequence of any one of AUS1010-AUS1714. In some embodiments, the compound comprises a modified oligonucleotide comprising the sequence of any one of SEQ ID NOs: 11-666, or a modified oligonucleotide comprising 1, 2, 3, 4, or 5 modifications to those sequences.

[0237] In certain embodiments, the compound comprises a modified oligonucleotide comprising any one of the sequences A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z, AA, AB, AC, AD, AE, AF, AG, AH, AI, AJ, AK, AL, AM, AN, AO, AP, AQ, AR, AS, AT, AU, AV, AW, or AX. Table 3 shows the correspondence between the alphabetic identifier, AUS identifier, and SEQ ID NO: corresponding to the modified oligonucleotide sequence. [Table 5-1] [Table 5-2]

[0238] Characteristics of modified oligonucleotides In certain embodiments, a compound comprises a modified oligonucleotide of 20 linked nucleosides consisting of: a first gap segment consisting of the linked nucleosides, a second gap segment consisting of the linked nucleosides, a separator segment consisting of nucleosides linked to the first gap segment and the second gap segment, a 5' wing segment consisting of the linked nucleosides, and a 3' wing segment consisting of the linked nucleosides, wherein the first gap segment, separator segment, and second gap segment are disposed between the 5' wing segment and the 3' wing segment, the first gap segment is attached to the 5' wing segment, the second gap segment is attached to the 3' wing segment, and the first gap segment, separator segment, and second gap segment collectively consist of 8, 9, 10, 11, or 12 linked nucleosides.

[0239] In certain embodiments, the compound comprises a modified oligonucleotide of 20 linked nucleosides consisting of: a first gap segment consisting of linked nucleosides, a second gap segment consisting of linked nucleosides, a separator segment consisting of linked nucleosides between the first gap segment and the second gap segment (the first gap segment, separator segment, and second gap segment collectively consist of 8 or 9 or 10 or 11 or 12 nucleotides), a 5' wing segment consisting of linked nucleosides, and a 3' wing segment consisting of linked nucleosides. In this case, the first gap segment, second gap segment, and separator segment are disposed between the 5' wing segment and the 3' wing segment, the first gap segment is attached to the 5' wing segment and the second gap segment is attached to the 3' wing segment, the first gap segment is comprised of linked nucleosides comprising a 2'-deoxy sugar, the second gap segment is comprised of linked nucleosides comprising a 2'-deoxy sugar, the separator segment is comprised of a 2'-O(CH2)2-OCH3 sugar or a single nucleoside comprising a 2'-OCH3 sugar, and the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 1, and the modified oligonucleotide has the nucleobase sequence of SEQ ID NO: 2 (GCAGA GGTGA AGCGA AGTGC).

[0240] In certain embodiments, the compound comprises a modified oligonucleotide of 20 linked nucleosides consisting of: a first gap segment consisting of linked nucleosides, a second gap segment consisting of linked nucleosides, a separator segment consisting of linked nucleosides between the first gap segment and the second gap segment (the first gap segment, separator segment, and second gap segment collectively consist of 8 or 9 or 10 or 11 or 12 nucleotides), a 5' wing segment consisting of linked nucleosides, and a 3' wing segment consisting of linked nucleosides. and wherein the first gap segment, the second gap segment, and the separator segment are disposed between the 5' wing segment and the 3' wing segment, the first gap segment is attached to the 5' wing segment and the second gap segment is attached to the 3' wing segment, the first gap segment is comprised of linked nucleosides comprising a 2'-deoxy sugar, the second gap segment is comprised of linked nucleosides comprising a 2'-deoxy sugar, the separator segment is comprised of a nucleoside comprising a 2'-O(CH2)2-OCH3 sugar or a 2'-OCH3 sugar, at least one of the nucleosides in the 5' wing segment comprises a 2'-O(CH2)2-OCH3 sugar and at least one of the nucleosides in the 3' wing segment comprises a 2'-O(CH2)2-OCH3 sugar, and the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 1, and the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 2 (GCAGA GGTGA It has the nucleic acid base sequence AGCGA AGTGC).

[0241] A. 5' wing segment, overview In certain embodiments, at least one of the nucleosides in the 5' wing segment contains a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, each nucleoside in the 5' wing segment contains a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 5' wing segment contains a nucleoside having a 2'-deoxy sugar. In certain embodiments, the 5' wing segment contains a nucleoside having a 2'-deoxy sugar, and the other nucleoside in the 5' wing segment contains a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the nucleoside at position 2 of SEQ ID NO:2 contains a 2'-deoxy sugar. In certain embodiments, the nucleoside at position 3 of SEQ ID NO:2 contains a 2'-deoxy sugar. In certain embodiments, the nucleoside at position 4 of SEQ ID NO:2 contains a 2'-deoxy sugar. In certain embodiments, the 5' wing segment comprises two nucleosides, each having a 2'-deoxy sugar. In certain embodiments, the nucleosides at positions 2 and 3 of SEQ ID NO:2 comprise a 2'-deoxy sugar. In certain embodiments, the nucleosides at positions 2 and 4 of SEQ ID NO:2 comprise a 2'-deoxy sugar. In certain embodiments, the nucleosides at positions 3 and 4 of SEQ ID NO:2 comprise a 2'-deoxy sugar. In certain embodiments, the nucleosides at positions 2 and 5 of SEQ ID NO:2 comprise a 2'-deoxy sugar.

[0242] In certain embodiments, one, two, three, or four of the nucleosides in the 5' wing segment comprise a sugar modification described herein. In certain embodiments, one, two, three, or four of the nucleosides in the 5' wing segment comprise a bicyclic sugar. In certain embodiments, one, two, three, or four of the nucleosides in the 5' wing segment comprise a constrained ethyl sugar.

[0243] In certain embodiments, one, two, three, or four of the nucleosides in the 5' wing segment comprise a locked nucleic acid. In certain embodiments, one, two, three, or four of the nucleosides in the 5' wing segment comprise a 4'-CH2-O-2' sugar. In certain embodiments, two of the nucleosides in the 5' wing segment comprise a locked nucleic acid. In certain embodiments, two of the nucleosides in the 5' wing segment comprise a 4'-CH2-O-2' sugar. In certain embodiments, the nucleosides at positions 1 and 3 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the nucleosides at positions 1 and 3 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0244] B1. 5' wing segment with 4 linked nucleosides In certain embodiments, a 5' wing segment is comprised of four linked nucleosides. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, wherein each of the four linked nucleosides contains a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, wherein three of the four linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, wherein two of the four linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 5' wing segment consists of four linked nucleosides, wherein one of the four linked nucleosides contains a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0245] B2. 5' wing segment with four linked nucleosides, 2'-deoxy combination In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, wherein one of the linked nucleosides contains a nucleoside having a 2'-deoxy sugar. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, wherein two of the linked nucleosides contain a nucleoside having a 2'-deoxy sugar. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, wherein one of the four linked nucleosides contains a nucleoside having a 2'-deoxy sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, where one of the four linked nucleosides contains a nucleoside having a 2'-deoxy sugar, one of the four linked nucleosides contains a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, where one of the four linked nucleosides contains a nucleoside having a 2'-deoxy sugar, two of the four linked nucleosides contain a locked nucleic acid, and the other linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0246] In certain embodiments, a 5' wing segment consists of four linked nucleosides, where the nucleoside at position 2 of SEQ ID NO:2 comprises a 2'-deoxy sugar. In certain embodiments, a 5' wing segment consists of four linked nucleosides, where the nucleoside at position 2 of SEQ ID NO:2 comprises a 2'-deoxy sugar, and the other three linked nucleosides each comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment consists of four linked nucleosides, where the nucleoside at position 3 of SEQ ID NO:2 comprises a 2'-deoxy sugar. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, where the nucleoside at position 3 of SEQ ID NO:2 comprises a 2'-deoxy sugar and the other three linked nucleosides each comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, where the nucleoside at position 4 of SEQ ID NO:2 comprises a 2'-deoxy sugar. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, where the nucleoside at position 4 of SEQ ID NO:2 comprises a 2'-deoxy sugar and the other three linked nucleosides each comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0247] In certain embodiments, a 5' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 2 and 3 of SEQ ID NO: 2 comprise a 2'-deoxy sugar. In certain embodiments, a 5' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 2 and 3 of SEQ ID NO: 2 comprise a 2'-deoxy sugar and the other two linked nucleosides each comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 2 and 4 of SEQ ID NO: 2 comprise a 2'-deoxy sugar. In certain embodiments, a 5' wing segment consists of four linked nucleosides, where the nucleosides at positions 2 and 4 of SEQ ID NO: 2 comprise a 2'-deoxy sugar, and the other two linked nucleosides each comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment consists of four linked nucleosides, where the nucleosides at positions 3 and 4 of SEQ ID NO: 2 comprise a 2'-deoxy sugar. In certain embodiments, a 5' wing segment consists of four linked nucleosides, where the nucleosides at positions 3 and 4 of SEQ ID NO: 2 comprise a 2'-deoxy sugar, and the other two linked nucleosides each comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0248] B3. 5' wing segment with four linked nucleosides, 2'-LNA combination In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, wherein one of the four linked nucleosides comprises a locked nucleic acid. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, wherein two of the four linked nucleosides comprise a locked nucleic acid. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, wherein one of the four linked nucleosides comprises a 4'-CH2-O-2' sugar. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, wherein two of the four linked nucleosides comprise a 4'-CH2-O-2' sugar.

[0249] In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, where one of the four linked nucleosides contains a locked nucleic acid and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, where two of the four linked nucleosides contain a locked nucleic acid and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, where one of the four linked nucleosides contains a 4'-CH2-O-2' sugar and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of four linked nucleosides, where two of the four linked nucleosides contain a 4'-CH2-O-2' sugar and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0250] In certain embodiments, the 5' wing segment consists of four linked nucleosides, and the nucleosides at positions 1 and 3 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 5' wing segment consists of four linked nucleosides, and the nucleosides at positions 1 and 3 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0251] In certain embodiments, the 5' wing segment consists of four linked nucleosides, where the nucleosides at positions 1 and 3 of SEQ ID NO:2 contain a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 5' wing segment consists of four linked nucleosides, where the nucleosides at positions 1 and 3 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0252] C1. 5' wing segment with 5 linked nucleosides In certain embodiments, a 5' wing segment consists of five linked nucleosides. In certain embodiments, a 5' wing segment consists of five linked nucleosides, wherein each of the five linked nucleosides contains a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment consists of five linked nucleosides, wherein four of the five linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment consists of five linked nucleosides, wherein three of the five linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein two of the five linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides contains a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0253] C2. 5' wing segment with five linked nucleosides, 2'-deoxy combination In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein one of the linked nucleosides contains a nucleoside having a 2'-deoxy sugar. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein two of the linked nucleosides contain a nucleoside having a 2'-deoxy sugar. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides contains a nucleoside having a 2'-deoxy sugar, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides contains a nucleoside having a 2'-deoxy sugar, one of the five linked nucleosides contains a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides contains a nucleoside having a 2'-deoxy sugar, two of the five linked nucleosides contain a locked nucleic acid, and the other two linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0254] In certain embodiments, a 5' wing segment consists of five linked nucleosides, wherein the nucleoside at position 2 of SEQ ID NO:2 comprises a 2'-deoxy sugar. In certain embodiments, a 5' wing segment consists of five linked nucleosides, wherein the nucleoside at position 2 of SEQ ID NO:2 comprises a 2'-deoxy sugar and the other four linked nucleosides each comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment consists of five linked nucleosides, wherein the nucleoside at position 3 of SEQ ID NO:2 comprises a 2'-deoxy sugar. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein the nucleoside at position 3 of SEQ ID NO:2 comprises a 2'-deoxy sugar and the other four linked nucleosides each comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein the nucleoside at position 4 of SEQ ID NO:2 comprises a 2'-deoxy sugar. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein the nucleoside at position 4 of SEQ ID NO:2 comprises a 2'-deoxy sugar and the other four linked nucleosides each comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0255] In certain embodiments, a 5' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 2 and 3 of SEQ ID NO: 2 comprise a 2'-deoxy sugar. In certain embodiments, a 5' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 2 and 3 of SEQ ID NO: 2 comprise a 2'-deoxy sugar and the other three linked nucleosides each comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 2 and 4 of SEQ ID NO: 2 comprise a 2'-deoxy sugar. In certain embodiments, a 5' wing segment consists of five linked nucleosides, where the nucleosides at positions 2 and 4 of SEQ ID NO: 2 comprise a 2'-deoxy sugar, and the other three linked nucleosides each comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment consists of five linked nucleosides, where the nucleosides at positions 3 and 4 of SEQ ID NO: 2 comprise a 2'-deoxy sugar. In certain embodiments, a 5' wing segment consists of five linked nucleosides, where the nucleosides at positions 3 and 4 of SEQ ID NO: 2 comprise a 2'-deoxy sugar, and the other three linked nucleosides each comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0256] C3. 5' wing segment with 5 linked nucleosides, 2'-LNA combination In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides comprises a locked nucleic acid. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein two of the five linked nucleosides comprise a locked nucleic acid. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides comprises a 4'-CH2-O-2' sugar. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein two of the five linked nucleosides comprise a 4'-CH2-O-2' sugar.

[0257] In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides contains a locked nucleic acid and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, wherein two of the five linked nucleosides contain a locked nucleic acid and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, where one of the five linked nucleosides contains a 4'-CH2-O-2' sugar and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 5' wing segment is comprised of five linked nucleosides, where two of the five linked nucleosides contain a 4'-CH2-O-2' sugar and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0258] In certain embodiments, the 5' wing segment consists of five linked nucleosides, and the nucleosides at positions 1 and 3 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 5' wing segment consists of five linked nucleosides, and the nucleosides at positions 1 and 3 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0259] In certain embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 1 and 3 of SEQ ID NO:2 contain a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 1 and 3 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0260] D. 3' wing segment, overview In certain embodiments, at least one of the nucleosides in the 3' wing segment contains a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, each nucleoside in the 3' wing segment contains a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment contains a nucleoside having a 2'-deoxy sugar. In certain embodiments, the 3' wing segment contains a nucleoside having a 2'-deoxy sugar, and the other nucleoside in the 3' wing segment contains a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment contains two nucleosides, each having a 2'-deoxy sugar.

[0261] In certain embodiments, one, two, three, or four of the nucleosides in the 3' wing segment comprise a sugar modification described herein. In certain embodiments, one, two, three, or four of the nucleosides in the 3' wing segment comprise a bicyclic sugar. In certain embodiments, one, two, three, or four of the nucleosides in the 3' wing segment comprise a constrained ethyl sugar.

[0262] In certain embodiments, one, two, three, or four of the nucleosides in the 3' wing segment comprise a locked nucleic acid. In certain embodiments, one, two, three, or four of the nucleosides in the 3' wing segment comprise a 4'-CH2-O-2' sugar. In certain embodiments, two of the nucleosides in the 3' wing segment comprise a locked nucleic acid. In certain embodiments, two of the nucleosides in the 3' wing segment comprise a 4'-CH2-O-2' sugar. In certain embodiments, three of the nucleosides in the 3' wing segment comprise a locked nucleic acid. In certain embodiments, three of the nucleosides in the 3' wing segment comprise a 4'-CH2-O-2' sugar.

[0263] In certain embodiments, the nucleosides at positions 16 and 18 of SEQ ID NO:2 comprise a locked nucleic acid. In certain embodiments, the nucleosides at positions 16 and 18 of SEQ ID NO:2 comprise a 4'-CH2-O-2' sugar. In certain embodiments, the nucleosides at positions 16 and 19 of SEQ ID NO:2 comprise a locked nucleic acid. In certain embodiments, the nucleosides at positions 16 and 19 of SEQ ID NO:2 comprise a 4'-CH2-O-2' sugar. In certain embodiments, the nucleosides at positions 16 and 20 of SEQ ID NO:2 comprise a locked nucleic acid. In certain embodiments, the nucleosides at positions 16 and 20 of SEQ ID NO:2 comprise a 4'-CH2-O-2' sugar. In certain embodiments, the nucleosides at positions 17 and 18 of SEQ ID NO:2 comprise a locked nucleic acid. In certain embodiments, the nucleosides at positions 17 and 18 of SEQ ID NO:2 comprise a 4'-CH2-O-2' sugar. In certain embodiments, the nucleosides at positions 17 and 19 of SEQ ID NO:2 comprise a locked nucleic acid. In certain embodiments, the nucleosides at positions 17 and 19 of SEQ ID NO:2 comprise a 4'-CH2-O-2' sugar. In certain embodiments, the nucleosides at positions 17 and 20 of SEQ ID NO:2 comprise a locked nucleic acid. In certain embodiments, the nucleosides at positions 17 and 20 of SEQ ID NO:2 comprise a 4'-CH2-O-2' sugar. In certain embodiments, the nucleosides at positions 18 and 19 of SEQ ID NO:2 comprise a locked nucleic acid. In certain embodiments, the nucleosides at positions 18 and 19 of SEQ ID NO:2 comprise a 4'-CH2-O-2' sugar. In certain embodiments, the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar. In certain embodiments, the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0264] In certain embodiments, the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar. In certain embodiments, the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0265] E1. 3' wing segment with 4 linked nucleosides In certain embodiments, a 3' wing segment is comprised of four linked nucleosides. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, wherein each of the four linked nucleosides contains a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, wherein three of the four linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, wherein two of the four linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, wherein one of the four linked nucleosides contains a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0266] E2. 3' wing segment with four linked nucleosides, 2'-deoxy combination In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, wherein one of the linked nucleosides contains a nucleoside having a 2'-deoxy sugar. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, wherein two of the linked nucleosides contain a nucleoside having a 2'-deoxy sugar. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, wherein one of the four linked nucleosides contains a nucleoside having a 2'-deoxy sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, where one of the four linked nucleosides contains a nucleoside having a 2'-deoxy sugar, one of the four linked nucleosides contains a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, where one of the four linked nucleosides contains a nucleoside having a 2'-deoxy sugar, two of the four linked nucleosides contain a locked nucleic acid, and the other linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0267] E3. 3' wing segment with four linked nucleosides, 2'-LNA combination In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, wherein one of the four linked nucleosides comprises a locked nucleic acid. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, wherein two of the four linked nucleosides comprise a locked nucleic acid. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, wherein one of the four linked nucleosides comprises a 4'-CH2-O-2' sugar. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, wherein two of the four linked nucleosides comprise a 4'-CH2-O-2' sugar.

[0268] In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, where one of the four linked nucleosides contains a locked nucleic acid and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, where two of the four linked nucleosides contain a locked nucleic acid and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, where three of the four linked nucleosides contain a locked nucleic acid and the other linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, where one of the four linked nucleosides contains a 4'-CH2-O-2' sugar and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of four linked nucleosides, where two of the four linked nucleosides contain a 4'-CH2-O-2' sugar and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, where three of the four linked nucleosides contain a 4'-CH2-O-2' sugar and the other linked nucleoside contains a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0269] In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0270] In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 16 and 18 of SEQ ID NO:2 contain a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 16 and 18 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0271] In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0272] In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 16 and 19 of SEQ ID NO:2 contain a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 16 and 19 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0273] In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0274] In certain embodiments, the 3' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 16 and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 16 and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0275] In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0276] In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 17 and 18 of SEQ ID NO:2 contain a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 17 and 18 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0277] In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0278] In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 17 and 19 of SEQ ID NO:2 contain a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 17 and 19 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0279] In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0280] In certain embodiments, the 3' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 17 and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 17 and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0281] In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0282] In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 18 and 19 of SEQ ID NO:2 contain a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 18 and 19 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0283] In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0284] In certain embodiments, the 3' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 18 and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 18 and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0285] In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0286] In certain embodiments, the 3' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 19 and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 19 and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0287] In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0288] In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 17, 18, and 19 of SEQ ID NO:2 contain locked nucleic acids, and the other linked nucleosides contain nucleosides with a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 17, 18, and 19 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other linked nucleosides contain nucleosides with a 2'-O(CH2)2-OCH3 sugar.

[0289] In certain embodiments, the 3' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0290] In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 17, 19, and 20 of SEQ ID NO:2 contain locked nucleic acids, and the other linked nucleosides contain nucleosides with 2'-O(CH2)2-OCH3 sugars. In certain embodiments, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 17, 19, and 20 of SEQ ID NO:2 contain 4'-CH2-O-2' sugars, and the other linked nucleosides contain nucleosides with 2'-O(CH2)2-OCH3 sugars.

[0291] F1. 3' wing segment with 5 linked nucleosides In certain embodiments, a 3' wing segment consists of five linked nucleosides. In certain embodiments, a 3' wing segment consists of five linked nucleosides, wherein each of the five linked nucleosides contains a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment consists of five linked nucleosides, wherein four of the five linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment consists of five linked nucleosides, wherein three of the five linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, wherein two of the five linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides contains a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0292] F2. 3' wing segment with five linked nucleosides, 2'-deoxy combination In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, wherein one of the linked nucleosides contains a nucleoside having a 2'-deoxy sugar. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, wherein two of the linked nucleosides contain a nucleoside having a 2'-deoxy sugar. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides contains a nucleoside having a 2'-deoxy sugar, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides contains a nucleoside having a 2'-deoxy sugar, one of the five linked nucleosides contains a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides contains a nucleoside having a 2'-deoxy sugar, two of the five linked nucleosides contain a locked nucleic acid, and the other two linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0293] F3. 3' wing segment with 5 linked nucleosides, 2'-LNA combination In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides comprises a locked nucleic acid. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, wherein two of the five linked nucleosides comprise a locked nucleic acid. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides comprises a 4'-CH2-O-2' sugar. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, wherein two of the five linked nucleosides comprise a 4'-CH2-O-2' sugar.

[0294] In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, wherein one of the five linked nucleosides contains a locked nucleic acid and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, wherein two of the five linked nucleosides contain a locked nucleic acid and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, where three of the five linked nucleosides contain a locked nucleic acid and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, where one of the five linked nucleosides contains a 4'-CH2-O-2' sugar and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, where two of the five linked nucleosides contain a 4'-CH2-O-2' sugar and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of five linked nucleosides, where three of the five linked nucleosides contain a 4'-CH2-O-2' sugar and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0295] In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0296] In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 16 and 18 of SEQ ID NO:2 contain a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 16 and 18 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0297] In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0298] In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 16 and 19 of SEQ ID NO:2 contain a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 16 and 19 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0299] In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0300] In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 16 and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 16 and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0301] In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0302] In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 17 and 18 of SEQ ID NO:2 contain a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 17 and 18 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0303] In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0304] In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 17 and 19 of SEQ ID NO:2 contain a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 17 and 19 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0305] In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0306] In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 17 and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 17 and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0307] In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0308] In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 18 and 19 of SEQ ID NO:2 contain a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 18 and 19 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0309] In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0310] In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 18 and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 18 and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0311] In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0312] In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 19 and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 19 and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0313] In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0314] In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 17, 18, and 19 of SEQ ID NO:2 contain a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 17, 18, and 19 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0315] In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0316] In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 17, 19, and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 17, 19, and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0317] G1. 3' wing segment with 6 linked nucleosides In certain embodiments, a 3' wing segment is comprised of six linked nucleosides. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, wherein each of the six linked nucleosides contains a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, wherein five of the six linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, wherein four of the six linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, wherein three of the six linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, wherein two of the six linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, wherein one of the six linked nucleosides contains a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0318] G2. 3' wing segment with six linked nucleosides, 2'-deoxy combinations In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, wherein one of the linked nucleosides contains a nucleoside having a 2'-deoxy sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, wherein two of the linked nucleosides contain a nucleoside having a 2'-deoxy sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, wherein one of the six linked nucleosides contains a nucleoside having a 2'-deoxy sugar, and the other five linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, where one of the six linked nucleosides contains a nucleoside having a 2'-deoxy sugar, one of the six linked nucleosides contains a locked nucleic acid, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, where one of the six linked nucleosides contains a nucleoside having a 2'-deoxy sugar, two of the five linked nucleosides contain a locked nucleic acid, and the other three linked nucleosides contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0319] G3. 3' wing segment with six linked nucleosides, 2'-LNA combination In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, wherein one of the six linked nucleosides comprises a locked nucleic acid. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, wherein two of the six linked nucleosides comprise a locked nucleic acid. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, wherein one of the six linked nucleosides comprises a 4'-CH2-O-2' sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, wherein two of the six linked nucleosides comprise a 4'-CH2-O-2' sugar.

[0320] In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, where one of the six linked nucleosides contains a locked nucleic acid and the other five linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, where two of the six linked nucleosides contain a locked nucleic acid and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, where three of the six linked nucleosides contain a locked nucleic acid and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, where one of the six linked nucleosides contains a 4'-CH2-O-2' sugar and the other five linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, where two of the six linked nucleosides contain a 4'-CH2-O-2' sugar and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, a 3' wing segment is comprised of six linked nucleosides, where three of the six linked nucleosides contain a 4'-CH2-O-2' sugar and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0321] In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0322] In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 16 and 18 of SEQ ID NO:2 contain a locked nucleic acid, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 16 and 18 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0323] In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0324] In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 16 and 19 of SEQ ID NO:2 contain a locked nucleic acid, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 16 and 19 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0325] In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0326] In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 16 and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 16 and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0327] In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0328] In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 17 and 18 of SEQ ID NO:2 contain a locked nucleic acid, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 17 and 18 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0329] In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0330] In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 17 and 19 of SEQ ID NO:2 contain a locked nucleic acid, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 17 and 19 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0331] In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0332] In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 17 and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 17 and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0333] In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0334] In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 18 and 19 of SEQ ID NO:2 contain a locked nucleic acid, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 18 and 19 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0335] In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0336] In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 18 and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 18 and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0337] In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0338] In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 19 and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 19 and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0339] In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0340] In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 17, 18, and 19 of SEQ ID NO:2 contain a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 17, 18, and 19 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0341] In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 comprise a locked nucleic acid. In certain embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0342] In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 17, 19, and 20 of SEQ ID NO:2 contain a locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, wherein the nucleosides at positions 17, 19, and 20 of SEQ ID NO:2 contain a 4'-CH2-O-2' sugar, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0343] H-0. Gap segment In some embodiments, the modified oligonucleotide comprises or consists of at least one gap segment. In some embodiments, the modified oligonucleotide comprises or consists of at least two gap segments. In some embodiments, the modified oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 or 10 gap segments. The gap segments are sequentially designated as the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth gap segments, respectively, with the first gap segment being closest to the 5' end of the modified oligonucleotide and the last gap segment being closest to the 3' end of the modified oligonucleotide. In some embodiments, the modified oligonucleotide comprises or consists of two gap segments. In some embodiments, the modified oligonucleotide comprises or consists of three gap segments. In some embodiments, the modified oligonucleotide comprises or consists of four gap segments. In some embodiments, the modified oligonucleotide comprises or consists of five gap segments. In some embodiments, the modified oligonucleotide comprises or consists of six gap segments. In some embodiments, the modified oligonucleotide comprises or consists of seven gap segments.

[0344] In some embodiments, a gap segment contains or consists of 1 to 20 linked nucleosides. In some embodiments, a gap segment consists of 1 nucleoside. In some embodiments, a gap segment consists of 2 linked nucleosides. In some embodiments, a gap segment consists of 23 linked nucleosides. In some embodiments, a gap segment consists of 4 linked nucleosides. In some embodiments, a gap segment consists of 5 linked nucleosides. In some embodiments, a gap segment consists of 6 linked nucleosides. In some embodiments, a gap segment consists of 7 linked nucleosides. In some embodiments, a gap segment consists of 8 linked nucleosides. In some embodiments, a gap segment consists of 9 linked nucleosides. In some embodiments, a gap segment consists of 10 linked nucleosides.

[0345] H. First Gap Segment In certain embodiments, the first gap segment comprises or consists of 1, 2, 3, 4, 5, 6, 7, or 8 linked nucleosides. In certain embodiments, the first gap segment comprises or consists of 4 or 5 linked nucleosides. In certain embodiments, the first gap segment comprises or consists of 4 linked nucleosides. In certain embodiments, the first gap segment comprises or consists of 5 linked nucleosides.

[0346] In certain embodiments, the first gap segment comprises or consists of 1, 2, 3, 4, 5, 6, 7, or 8 linked nucleosides, each having a 2'-deoxy sugar. In certain embodiments, the first gap segment comprises or consists of 4 or 5 linked nucleosides, each having a 2'-deoxy sugar. In certain embodiments, the first gap segment comprises or consists of 4 linked nucleosides, each having a 2'-deoxy sugar. In certain embodiments, the first gap segment comprises or consists of 5 linked nucleosides, each having a 2'-deoxy sugar.

[0347] In certain embodiments, the first gap segments each have a 2'-deoxy sugar and are located at positions 6, 7, 8, and 9 of SEQ ID NO: 2. In certain embodiments, the first gap segments each have a 2'-deoxy sugar and are located at positions 5, 6, 7, 8, and 9 of SEQ ID NO: 2. In certain embodiments, the first gap segments each have a 2'-deoxy sugar and are located at positions 6, 7, 8, 9, and 10 of SEQ ID NO: 2.

[0348] I. Second Gap Segment In certain embodiments, the second gap segment comprises or consists of 1, 2, 3, 4, 5, 6, 7, or 8 linked nucleosides. In certain embodiments, the second gap segment comprises or consists of 4 or 5 linked nucleosides. In certain embodiments, the first gap segment comprises or consists of 4 linked nucleosides. In certain embodiments, the first gap segment comprises or consists of 5 linked nucleosides.

[0349] In certain embodiments, the second gap segment comprises or consists of 1, 2, 3, 4, 5, 6, 7, or 8 linked nucleosides, each having a 2'-deoxy sugar. In certain embodiments, the second gap segment comprises or consists of 4 linked nucleosides, each having a 2'-deoxy sugar. In certain embodiments, the second gap segment comprises or consists of 5 linked nucleosides, each having a 2'-deoxy sugar. In certain embodiments, the second gap segment comprises or consists of 6 linked nucleosides, each having a 2'-deoxy sugar.

[0350] In certain embodiments, the second gap segments each have a 2'-deoxy sugar and are located at positions 11, 12, 13, and 14 of SEQ ID NO: 2. In certain embodiments, the second gap segments each have a 2'-deoxy sugar and are located at positions 11, 12, 13, 14, and 15 of SEQ ID NO: 2. In certain embodiments, the second gap segments each have a 2'-deoxy sugar and are located at positions 11, 12, 13, 14, 15, and 16 of SEQ ID NO: 2.

[0351] J. Separator segment In some embodiments, the separator segment comprises 0, 1, 2, 3, 4, or 5 linked nucleosides. In some embodiments, the separator segment comprises 1 nucleoside. In some embodiments, the nucleoside comprises a modification. Exemplary modifications of nucleosides include, but are not limited to, 2' methoxyethyl nucleosides, 2'-O-methyl nucleosides, 2'OH nucleosides, 2' fluoro 2'-deoxynucleosides, 2'-F-arabinonucleic acid (2'-F-ANA), glycol nucleic acid (GNA), or locked nucleic acid (LNA).

[0352] In certain embodiments, the separator segment consists of a single nucleoside comprising a 2'-OCH3 sugar. In certain embodiments, the separator segment consists of a single nucleoside comprising a 2'-O(CH2)2-OCH3 sugar.

[0353] In certain embodiments, the separator segment is at position 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 of SEQ ID NO: 2. In certain embodiments, the separator segment is at position 9 or 10 or 11 of SEQ ID NO: 2. In certain embodiments, the separator segment is at position 10 of SEQ ID NO: 2.

[0354] In certain embodiments, the separator segment is at position 10 of SEQ ID NO:2 and consists of a single nucleoside comprising a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the separator segment is at position 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 of SEQ ID NO:2. In certain embodiments, the separator segment is at position 9 or 10 or 11 of SEQ ID NO:2. In certain embodiments, the separator segment is at position 10 of SEQ ID NO:2.

[0355] In certain embodiments, the separator segment consists of a single nucleoside comprising a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the separator segment consists of a single nucleoside comprising a 2'-OCH3 sugar. In certain embodiments, the separator segment is located at position 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14 of SEQ ID NO:2. In certain embodiments, the separator segment is located at position 9, or 10, or 11 of SEQ ID NO:2. In certain embodiments, the separator segment is located at position 10 of SEQ ID NO:2.

[0356] K. Oligonucleotide backbone In certain embodiments, the modified oligonucleotide is a single-stranded modified oligonucleotide. In certain embodiments, at least one internucleoside linkage is a modified internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.

[0357] L. Modified nucleobases In certain embodiments, the modified oligonucleotide comprises at least one modified nucleobase. In certain embodiments, the modified oligonucleotide comprises one, two, or three modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, the cytosines at positions 2, 13, and 20 of SEQ ID NO: 2 are each 5-methylcytosine.

[0358] M1. 5-4-1-5-5 modified oligonucleotides In certain embodiments, the modified oligonucleotide comprises a 5' wing segment of five linked nucleosides, a first gap segment of four linked nucleosides having 2'-deoxy sugars, a separator segment at position 10 of SEQ ID NO:2, a second gap segment of five linked nucleosides having 2'-deoxy sugars, and a 3' wing segment of five linked nucleosides. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosines. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises an internucleoside linkage, each of which is a phosphorothioate internucleoside linkage. In an exemplary embodiment, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosine and further comprise internucleoside linkages, each linkage being a phosphorothioate internucleoside linkage.

[0359] In certain embodiments, the modified oligonucleotide comprises a 5' wing segment described in Sections C1, C2, or C3, a first gap segment consisting of four linked nucleosides having 2'-deoxy sugars, a separator segment at position 10 of SEQ ID NO:2, a second gap segment consisting of five linked nucleosides having 2'-deoxy sugars, and a 3' wing segment described in Sections F1, F2, or F3. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosines. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises an internucleoside linkage, wherein each linkage is a phosphorothioate internucleoside linkage. In an exemplary embodiment, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosine, and further comprises an internucleoside linkage, each linkage being a phosphorothioate internucleoside linkage.

[0360] In certain embodiments, a modified oligonucleotide comprises a sequence that is A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, or Z.

[0361] M2. 4-5-1-5-5 modified oligonucleotides In certain embodiments, the modified oligonucleotide comprises a 5' wing segment of four linked nucleosides, a first gap segment of five linked nucleosides having 2'-deoxy sugars, a separator segment at position 10 of SEQ ID NO:2, a second gap segment of five linked nucleosides having 2'-deoxy sugars, and a 3' wing segment of five linked nucleosides. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosines. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises an internucleoside linkage, each of which is a phosphorothioate internucleoside linkage. In an exemplary embodiment, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosine and further comprise internucleoside linkages, each linkage being a phosphorothioate internucleoside linkage.

[0362] In certain embodiments, the modified oligonucleotide comprises a 5' wing segment described in Sections B1, B2, or B3, a first gap segment consisting of five linked nucleosides having 2'-deoxy sugars, a separator segment at position 10 of SEQ ID NO:2, a second gap segment consisting of five linked nucleosides having 2'-deoxy sugars, and a 3' wing segment described in Sections F1, F2, or F3. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosines. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises an internucleoside linkage, wherein each linkage is a phosphorothioate internucleoside linkage. In an exemplary embodiment, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosine, and further comprises an internucleoside linkage, each linkage being a phosphorothioate internucleoside linkage.

[0363] In certain embodiments, the modified oligonucleotide comprises a sequence that is AA, AB, AC, AD, AE, AF, AG, AH, AI, AJ, AK, AL, AM, AN, or AO.

[0364] Exemplary annotations of the positions and segments of AO (AUS1493 or SEQ ID NO: 456) are shown below. [Table 6]

[0365] M3. 5-4-1-6-4 modified oligonucleotides In certain embodiments, the modified oligonucleotide comprises a 5' wing segment of five linked nucleosides, a first gap segment of four linked nucleosides having 2'-deoxy sugars, a separator segment at position 10 of SEQ ID NO:2, a second gap segment of six linked nucleosides having 2'-deoxy sugars, and a 3' wing segment of four linked nucleosides. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosines. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises an internucleoside linkage, each of which is a phosphorothioate internucleoside linkage. In an exemplary embodiment, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosine and further comprise internucleoside linkages, each linkage being a phosphorothioate internucleoside linkage.

[0366] In certain embodiments, the modified oligonucleotide comprises a 5' wing segment described in Sections C1, C2, or C3, a first gap segment consisting of four linked nucleosides having 2'-deoxy sugars, a separator segment at position 10 of SEQ ID NO:2, a second gap segment consisting of six linked nucleosides having 2'-deoxy sugars, and a 3' wing segment described in Sections E1, E2, or E3. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosines. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises an internucleoside linkage, wherein each linkage is a phosphorothioate internucleoside linkage. In an exemplary embodiment, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosine, and further comprises an internucleoside linkage, each linkage being a phosphorothioate internucleoside linkage.

[0367] In certain embodiments, the modified oligonucleotide comprises a sequence that is AP, AQ, AR, or AS.

[0368] M4. 4-5-1-4-6 modified oligonucleotides In certain embodiments, the modified oligonucleotide comprises a 5' wing segment of four linked nucleosides, a first gap segment of five linked nucleosides having 2'-deoxy sugars, a separator segment at position 10 of SEQ ID NO:2, a second gap segment of four linked nucleosides having 2'-deoxy sugars, and a 3' wing segment of six linked nucleosides. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosines. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises an internucleoside linkage, each of which is a phosphorothioate internucleoside linkage. In an exemplary embodiment, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosine and further comprise internucleoside linkages, each linkage being a phosphorothioate internucleoside linkage.

[0369] In certain embodiments, the modified oligonucleotide comprises a 5' wing segment described in Sections B1, B2, or B3, a first gap segment consisting of five linked nucleosides having 2'-deoxy sugars, a separator segment at position 10 of SEQ ID NO:2, a second gap segment consisting of four linked nucleosides having 2'-deoxy sugars, and a 3' wing segment described in Sections G1, G2, or G3. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosines. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises an internucleoside linkage, wherein each linkage is a phosphorothioate internucleoside linkage. In an exemplary embodiment, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosine, and further comprises an internucleoside linkage, each linkage being a phosphorothioate internucleoside linkage.

[0370] In certain embodiments, the modified oligonucleotide comprises a sequence that is AT or AU.

[0371] M5. 5-4-1-4-6 modified oligonucleotides In certain embodiments, the modified oligonucleotide comprises a 5' wing segment of five linked nucleosides, a first gap segment of four linked nucleosides having 2'-deoxy sugars, a separator segment at position 10 of SEQ ID NO:2, a second gap segment of four linked nucleosides having 2'-deoxy sugars, and a 3' wing segment of six linked nucleosides. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosines. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises an internucleoside linkage, each of which is a phosphorothioate internucleoside linkage. In an exemplary embodiment, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosine and further comprise internucleoside linkages, each linkage being a phosphorothioate internucleoside linkage.

[0372] In certain embodiments, the modified oligonucleotide comprises a 5' wing segment described in Sections C1, C2, or C3, a first gap segment consisting of four linked nucleosides having 2'-deoxy sugars, a separator segment at position 10 of SEQ ID NO:2, a second gap segment consisting of four linked nucleosides having 2'-deoxy sugars, and a 3' wing segment described in Sections G1, G2, or G3. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosines. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises an internucleoside linkage, wherein each linkage is a phosphorothioate internucleoside linkage. In an exemplary embodiment, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosine, and further comprises an internucleoside linkage, each linkage being a phosphorothioate internucleoside linkage.

[0373] In certain embodiments, the modified oligonucleotide comprises a sequence that is AV or AW.

[0374] M6. 4-5-1-6-4 modified oligonucleotides In certain embodiments, the modified oligonucleotide comprises a 5' wing segment of four linked nucleosides, a first gap segment of five linked nucleosides having 2'-deoxy sugars, a separator segment at position 10 of SEQ ID NO:2, a second gap segment of six linked nucleosides having 2'-deoxy sugars, and a 3' wing segment of four linked nucleosides. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosines. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises an internucleoside linkage, each of which is a phosphorothioate internucleoside linkage. In an exemplary embodiment, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosine and further comprise internucleoside linkages, each linkage being a phosphorothioate internucleoside linkage.

[0375] In certain embodiments, the modified oligonucleotide comprises a 5' wing segment described in Sections B1, B2, or B3, a first gap segment consisting of five linked nucleosides having 2'-deoxy sugars, a separator segment at position 10 of SEQ ID NO:2, a second gap segment consisting of six linked nucleosides having 2'-deoxy sugars, and a 3' wing segment described in Sections E1, E2, or E3. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosines. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises an internucleoside linkage, wherein each linkage is a phosphorothioate internucleoside linkage. In an exemplary embodiment, the modified oligonucleotide of this paragraph further comprises that the cytosines at positions 2, 13, and 20 of SEQ ID NO:2 are each 5-methylcytosine, and further comprises an internucleoside linkage, each linkage being a phosphorothioate internucleoside linkage.

[0376] In certain embodiments, the modified oligonucleotide comprises a sequence that is AX.

[0377] In certain embodiments, one or more modified nucleosides in a wing segment have a modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the modified nucleoside is an LNA nucleoside. In certain embodiments, the modified nucleoside is a 2'-substituted nucleoside. In certain embodiments, the 2'-substituted nucleoside comprises a nucleoside having a bicyclic sugar modification. In certain embodiments, the modified nucleoside is a 2'-MOE nucleoside. In certain embodiments, the modified nucleoside is a constrained ethyl (cEt) nucleoside. In certain embodiments, each modified nucleoside in each wing segment is independently a 2'-MOE nucleoside or a nucleoside having a bicyclic sugar modification, such as a constrained ethyl (cEt) nucleoside or an LNA nucleoside.

[0378] In certain embodiments, the compound or composition comprises a salt of a modified oligonucleotide.

[0379] In certain embodiments, the compound or composition further comprises a pharmaceutically acceptable carrier or diluent.

[0380] In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to an HBV nucleic acid when measured across the entire modified oligonucleotide. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to SEQ ID NO: 1 when measured across the entire modified oligonucleotide.

[0381] In certain embodiments, the compound or modified oligonucleotide is single-stranded.

[0382] In certain embodiments, at least one nucleoside of a modified oligonucleotide comprises a modified sugar. In certain embodiments, at least one modified sugar comprises a 2'-O-methoxyethyl group (2'-O(CH2)2-OCH3). In certain embodiments, the modified sugar comprises a 2'-O-CH3 group.

[0383] In certain embodiments, at least one modified sugar is a bicyclic sugar. In certain embodiments, at least one modified sugar bicyclic sugar comprises a 4'-(CH2)-O-2' bridge, where n is 1 or 2. In certain embodiments, the bicyclic sugar comprises a 4'-CH2-O-2' bridge. In certain embodiments, the bicyclic sugar comprises a 4'-CH(CH3)-O-2' bridge.

[0384] method The present disclosure provides methods of treating a subject having an HBV infection or a disease, disorder, or condition associated with HBV, the methods comprising administering a therapeutically effective amount of a modified oligonucleotide described herein or a pharmaceutical composition comprising the same. In certain embodiments, the modified oligonucleotide comprises any one of SEQ ID NOS: 11-666, or a modified oligonucleotide containing 1, 2, 3, 4, or 5 modifications thereto.

[0385] In certain embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide inhibits expression of HBV mRNA in a subject. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide inhibits HBV DNA levels in a subject. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide inhibits HBV protein and / or antigen levels in a subject. The modified oligonucleotides of the present disclosure, when administered to a subject, can reduce HBV mRNA, DNA, or protein levels, including, for example, but not limited to, HBV antigens such as HBsAg and HBeAb.

[0386] The present disclosure provides a method for treating HBV-associated diseases, disorders, and / or conditions in a subject, the method comprising administering a therapeutically effective amount of any of the pharmaceutical compositions described above to a subject in need thereof to treat the HBV-associated diseases, disorders, and conditions. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human, and the HBV-associated diseases, disorders, and conditions are hepatitis B virus infections caused by human hepatitis B virus. More specifically, the human hepatitis B virus may be of any of the following human geographic genotypes: A (Northwest Europe, North America, Central America), B (Indonesia, China, Vietnam), C (East Asia, Korea, China, Japan, Polynesia, Vietnam), D (Mediterranean region, Middle East, India), E (Africa), F (Native Americans, Polynesia), G (United States, France), or H (Central America).

[0387] In certain embodiments, modified oligonucleotides target a region of HBV nucleic acid. In certain embodiments, modified oligonucleotides targeting a region of HBV nucleic acid have a contiguous nucleobase portion, which is complementary to an equal-length nucleobase portion of the region of the HBV nucleic acid. For example, the portion can be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobase portion complementary to an equal-length portion of the region listed herein.

[0388] Certain embodiments provide methods of treating a disease, disorder, or condition associated with HBV in a subject, the method comprising administering to a subject in need thereof a modified oligonucleotide, e.g., a modified oligonucleotide comprising any one of SEQ ID NOs: 11-666, or a modified oligonucleotide comprising 1, 2, 3, 4, or 5 modifications thereto, or a pharmaceutical composition described herein.

[0389] Certain embodiments provide a method for reducing expression of HBV in a subject, the method comprising administering to the subject a modified oligonucleotide, such as a modified oligonucleotide comprising any one of SEQ ID NOs: 11-666, or a modified oligonucleotide comprising 1, 2, 3, 4, or 5 modifications to those sequences, or a pharmaceutical composition described herein.

[0390] Certain embodiments provide methods for preventing, ameliorating, or treating a disease, disorder, or condition associated with HBV in a subject, the methods comprising administering to the animal a modified oligonucleotide, e.g., a modified oligonucleotide comprising any one of SEQ ID NOs: 11-666, or a modified oligonucleotide comprising 1, 2, 3, 4, or 5 modifications thereto, or a pharmaceutical composition described herein.

[0391] Examples of HBV-related diseases, disorders, or conditions include, but are not limited to, chronic HBV infection, jaundice, liver cancer, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, and HBV viremia. HBV-related conditions or disorders may have symptoms that may include any or all of the following: influenza-like illness, weakness, pain, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain in the liver region of the body, thick or gray stools, generalized itching, and dark urine. These, when combined with a positive test for the presence of hepatitis B virus, hepatitis B virus antigen, or a positive test for the presence of antibodies specific to hepatitis B virus antigen, indicate a HBV-related condition or disorder.

[0392] Certain embodiments provide methods for reducing HBV mRNA expression in a subject, the methods comprising administering to the subject a modified oligonucleotide, e.g., a modified oligonucleotide comprising any one of SEQ ID NOs: 11-666, or a modified oligonucleotide comprising one, two, three, four, or five modifications thereto, or a pharmaceutical composition described herein. In certain embodiments, reducing HBV mRNA expression in a subject prevents, ameliorates, or treats a disease, disorder, or condition associated with HBV. In certain embodiments, reducing HBV mRNA expression in a subject ameliorates or treats HBV infection. In certain embodiments, reducing HBV mRNA expression in a subject prevents, ameliorates, or treats liver disease. In certain embodiments, HBV mRNA expression is reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0393] Certain embodiments provide methods for reducing HBV protein levels in a subject, the methods comprising administering to the subject a modified oligonucleotide, e.g., a modified oligonucleotide comprising any one of SEQ ID NOs: 11-666, or a modified oligonucleotide comprising one, two, three, four, or five modifications thereto, or a pharmaceutical composition described herein. In certain embodiments, reducing the HBV protein level in a subject prevents, ameliorates, or treats an HBV-related disease, disorder, or condition. In certain embodiments, reducing the HBV protein level in a subject ameliorates or treats HBV infection. In certain embodiments, reducing the HBV protein level in a subject prevents, ameliorates, or treats liver disease. In certain embodiments, the HBV protein level is reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0394] Certain embodiments provide methods for reducing HBV DNA levels in a subject, the methods comprising administering to the subject a modified oligonucleotide, such as a modified oligonucleotide comprising any one of SEQ ID NOS: 11-666, or a modified oligonucleotide comprising one, two, three, four, or five modifications to such a sequence, or a pharmaceutical composition described herein. In certain embodiments, reducing HBV DNA levels in the subject prevents, ameliorates, or treats a disease, disorder, or condition associated with HBV. In certain embodiments, the subject may be a mammal, such as a human, and the hepatitis B virus may be human hepatitis B virus. More specifically, the human hepatitis B virus may be one of the following human geographic genotypes: A (Northwest Europe, North America, Central America), B (Indonesia, China, Vietnam), C (East Asia, Korea, China, Japan, Polynesia, Vietnam), D (Mediterranean Region, Middle East, India), E (Africa), F (Native American, Polynesian), G (United States, France), or H (Central America). In certain embodiments, reducing HBV DNA levels in a subject ameliorates or treats HBV infection. In certain embodiments, reducing HBV DNA levels in a subject prevents, ameliorates, or treats liver disease. In certain embodiments, HBV DNA levels are reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0395] Certain embodiments provide methods for reducing HBV antigen levels in a subject, the methods comprising administering to the subject a modified oligonucleotide, e.g., a modified oligonucleotide comprising any one of SEQ ID NOs: 11-666, or a modified oligonucleotide comprising one, two, three, four, or five modifications to such a sequence, or a pharmaceutical composition described herein. In certain embodiments, the antigen is HBsAg or HBeAG. In certain embodiments, reducing the HBV antigen level in a subject prevents, ameliorates, or treats a disease, disorder, or condition associated with HBV. In certain embodiments, reducing the HBV antigen level in a subject prevents, ameliorates, or treats liver disease. In certain embodiments, the HBV antigen level is reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0396] Certain embodiments provide methods for reducing HBV DNA and HBV antigen levels in a subject infected with hepatitis B virus, the methods comprising administering to the subject a modified oligonucleotide, e.g., a modified oligonucleotide comprising any one of SEQ ID NOs: 11-666, or a modified oligonucleotide comprising one, two, three, four, or five modifications to those sequences, or a pharmaceutical composition described herein. In certain embodiments, the antigen is HBsAg or HBeAg. In certain embodiments, the amount of HBV antigen may be sufficiently reduced to result in seroconversion, defined as no serum HBeAg plus serum HBeAb if HBeAg was monitored as the determinant of seroconversion, or no serum HBsAg if HBsAg was monitored as the determinant of seroconversion, as determined by currently available detection limits of commercially available ELISA systems.

[0397] Certain embodiments provide methods of treating a subject with an HBV-associated disease, disorder, or condition, the method comprising: a) identifying the subject with the HBV-associated disease, disorder, or condition; and b) administering to the subject a therapeutically effective amount of a modified oligonucleotide or pharmaceutical composition described herein. In certain embodiments, the therapeutically effective amount of the modified oligonucleotide or pharmaceutical composition administered to the subject treats or reduces the HBV-associated disease, disorder, or condition in the subject. In certain embodiments, the HBV-associated disease, disorder, or condition is liver disease. In certain embodiments, the associated disease, disorder, or condition is chronic HBV infection, jaundice, liver cancer, e.g., hepatocellular carcinoma, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, transplant-associated liver disease, or any combination thereof.

[0398] Certain embodiments provide methods of treating a subject having an HBV-associated disease, disorder, or condition, the method comprising: a) identifying the subject having the HBV-associated disease, disorder, or condition; and b) administering to the subject a therapeutically effective amount of a modified oligonucleotide or pharmaceutical composition described herein. In certain embodiments, the therapeutically effective amount of the modified oligonucleotide or pharmaceutical composition administered to the subject treats or reduces the HBV-associated disease, disorder, or condition in the subject. In certain embodiments, the HBV-associated disease, disorder, or condition is liver disease. In certain embodiments, the associated disease, disorder, or condition is chronic HBV infection, jaundice, liver cancer, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, or transplant-associated liver disease.

[0399] In certain embodiments, the HBV has the sequence set forth in GenBank Accession U95551.1 (SEQ ID NO: 3) or any variant or fragment thereof. In certain embodiments, the HBV has the sequence set forth in SEQ ID NOs: 667-674.

[0400] In certain embodiments, the subject is a human.

[0401] In certain embodiments, the subject is a monkey, eg, a cynomolgus monkey.

[0402] In certain embodiments, the subject is a rodent, such as, for example, a mouse or a rat.

[0403] In certain embodiments, the modified oligonucleotide or pharmaceutical composition is designated as the first agent. In certain embodiments, the method includes administering the first agent and one or more second agents. In certain embodiments, the first agent and one or more second agents are co-administered. In certain embodiments, the first agent and one or more second agents are co-administered sequentially or simultaneously. In certain embodiments, the first agent and one or more second agents are not co-administered.

[0404] In certain embodiments, the one or more second agents are also compounds or compositions described herein. In certain embodiments, the one or more second agents are different from the compounds or compositions described herein. Examples of the one or more second agents include, but are not limited to, anti-inflammatory agents, chemotherapeutic agents, or anti-infective agents. In certain embodiments, the disease includes liver cancer, and the one or more second agents include a chemotherapeutic agent such as gemcitabine (Gemzar), oxaliplatin (Eloxatin), cisplatin, doxorubicin, 5-fluorouracil, capecitabine (Xeloda), or mitoxantrone (Novantrone). In certain embodiments, the disease includes liver disease, and the one or more second agents include a corticosteroid, a diuretic, a beta-blocker, or a combination thereof.

[0405] The modified oligonucleotides of the present disclosure and pharmaceutical compositions comprising the same can be administered to a subject by any suitable route of administration.Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), intraperitoneal (into a body cavity), and transmucosal administration.

[0406] In certain embodiments, the administration comprises parenteral administration, hi certain embodiments, the administration comprises subcutaneous administration, hi certain embodiments, the administration comprises intravenous injection or infusion.

[0407] Certain embodiments provide methods for reducing the amount of HBV mRNA, DNA, protein, and / or HBV antigen in a subject infected with hepatitis B virus, the method comprising administering a therapeutically effective amount of the pharmaceutical composition described above to a subject in need thereof, thereby reducing the hepatitis B virus infection and hepatitis B antigen compared to the amount of HBV mRNA, protein, and HBV antigen in the subject before treatment. In some embodiments, the subject may be a human, and the hepatitis B virus may be human hepatitis B virus. More specifically, the human hepatitis B virus may be of any of the following human geographic genotypes: A (Northwest Europe, North America, Central America), B (Indonesia, China, Vietnam), C (East Asia, Korea, China, Japan, Polynesia, Vietnam), D (Mediterranean region, Middle East, India), E (Africa), F (Native Americans, Polynesia), G (United States, France), or H (Central America).

[0408] The present disclosure provides a method for reducing the amount of HBV mRNA, DNA, protein, and / or HBV antigen, or a combination thereof, in a subject infected with hepatitis B virus, the method comprising administering to the subject a therapeutically effective amount of the modified oligonucleotide described above or a pharmaceutical composition comprising the same, thereby reducing hepatitis B virus infection and hepatitis B antigen compared to the amount of HBV mRNA, protein, and / or HBV antigen in the subject before treatment. In certain embodiments, the amount of mRNA is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to the amount before administration of the modified antisense oligonucleotide or pharmaceutical composition. In certain embodiments, the amount of mRNA is reduced by at least 50% compared to the amount before administration of the modified antisense oligonucleotide or pharmaceutical composition. In certain embodiments, the amount of mRNA is reduced by at least 60% compared to the amount before administration of the modified antisense oligonucleotide or pharmaceutical composition. In certain embodiments, the amount of mRNA is reduced by at least 70% compared with the amount before administration of modified antisense oligonucleotide or pharmaceutical composition.In certain embodiments, the amount of mRNA is reduced by at least 80% compared with the amount before administration of modified antisense oligonucleotide or pharmaceutical composition.In certain embodiments, the amount of mRNA is reduced by at least 90% compared with the amount before administration of modified antisense oligonucleotide or pharmaceutical composition.

[0409] The present disclosure provides a method for reducing the amount of HBV mRNA, DNA, protein, and / or HBV antigen in a subject infected with hepatitis B virus, the method comprising administering to the subject a therapeutically effective amount of the modified oligonucleotide or pharmaceutical composition described above, thereby reducing hepatitis B virus infection and hepatitis B antigen compared to the amount of HBV virus, mRNA, DNA, protein, and / or HBV antigen in the subject before treatment, wherein the amount of mRNA is reduced by at least 75% compared to the amount before administration of the modified oligonucleotide or pharmaceutical composition. In a specific embodiment, a method for reducing the amount of HBV virus, mRNA, DNA, protein, and / or HBV antigen in a subject infected with hepatitis B virus, the method comprising administering to the subject a therapeutically effective amount of the modified oligonucleotide or pharmaceutical composition described above, thereby reducing hepatitis B virus infection and hepatitis B antigen compared to the amount of hepatitis B virus and HBV antigen in the subject before treatment, wherein the amount of mRNA is reduced by at least 80% compared to the amount before administration of the modified oligonucleotide or pharmaceutical composition. In certain embodiments, a method is provided for reducing the amount of HBV virus, mRNA, DNA, protein, and / or HBV antigen in a subject infected with hepatitis B virus, the method comprising administering to the subject a therapeutically effective amount of the above-described modified oligonucleotide or pharmaceutical composition, thereby reducing hepatitis B virus infection and hepatitis B antigen compared to the amount of HBV virus, mRNA, DNA, protein, and / or HBV antigen in the subject before treatment, wherein the amount of mRNA is reduced by at least 85% compared to the amount before administration of the modified oligonucleotide or pharmaceutical composition.In certain embodiments, a method is provided for reducing the amount of HBV virus, mRNA, DNA, protein, and / or HBV antigen in a subject infected with hepatitis B virus, the method comprising administering to the subject a therapeutically effective amount of the above-described modified oligonucleotide or pharmaceutical composition, thereby reducing hepatitis B virus infection and hepatitis B antigen compared to the amount of HBV virus, mRNA, DNA, protein, and / or HBV antigen in the subject before treatment, wherein the amount of mRNA is reduced by at least 90% compared to the amount before administration of the modified oligonucleotide or pharmaceutical composition. In certain embodiments, a method is provided for reducing the amount of HBV virus, mRNA, DNA, protein, and / or HBV antigen in a subject infected with hepatitis B virus, the method comprising administering to the subject a therapeutically effective amount of the above-described modified oligonucleotide or pharmaceutical composition, thereby reducing hepatitis B virus infection, mRNA, protein, and / or hepatitis B antigen compared to the amount of HBV mRNA, protein, and / or HBV antigen in the subject before treatment, wherein the amount of mRNA is reduced by at least 95% compared to the amount before administration of the modified oligonucleotide or pharmaceutical composition. In related methods, the HBV antigen may be HBsAg or HBeAg, and more particularly, the amount of HBV antigen may be sufficiently reduced to result in seroconversion, defined as no serum HBeAg plus serum HBeAb if HBeAg was being monitored as a determinant of seroconversion, or no serum HBsAg if HBsAg was being monitored as a determinant of seroconversion, as determined by currently available detection limits of commercially available ELISA systems.

[0410] The present disclosure provides a method for promoting seroconversion of Hepatitis B virus in a mammal infected with HBV, the method comprising administering to a subject infected with Hepatitis B virus a therapeutically effective amount of a modified oligonucleotide, such as a modified oligonucleotide comprising any one of SEQ ID NOs: 11-666, or a modified oligonucleotide comprising 1, 2, 3, 4, or 5 modifications to such a sequence, or a therapeutically effective amount of the pharmaceutical composition described above; monitoring the presence of HBeAg+HBeAb in a serum sample from the subject, or the presence of HBsAg in a serum sample from the subject, as determined by the current detection limit of a commercially available ELISA system; and wherein the absence of HBeAg+HBeAb in the serum sample if HBeAg was monitored as a determinant of seroconversion, or the absence of HBsAg in the serum sample if HBsAg was monitored as a determinant of seroconversion, indicates seroconversion in the subject.

[0411] Certain embodiments provide for the use of a modified oligonucleotide, such as a modified oligonucleotide comprising any one of SEQ ID NOs: 11-666, or a modified oligonucleotide comprising 1, 2, 3, 4, or 5 modifications thereto, or a pharmaceutical composition described herein, to prevent, ameliorate, or treat liver disease or a symptom thereof in a subject.

[0412] In some embodiments, EC of cells treated with modified oligonucleotides (e.g., SEQ ID NOS: 11-666) 50 was measured as a surrogate for the efficacy of the modified oligonucleotide. 50 In some embodiments, the EC 50is less than 250nM, less than 200nM, less than 150nM, less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 65nM, less than 60nM, less than 55nM, less than 50nM, less than 49nM, less than 47nM, less than 46nM, less than 45nM, less than 44nM, less than 43nM, less than 42nM, less than 41nM, less than 40nM, less than 39nM, less than 38nM, less than 37nM, less than 36nM, less than 35nM, less than 34nM, less than 33nM, less than 32nM, less than 31nM, less than 30nM, less than 29nM, less than 28nM, less than 27nM, less than 26nM, less than 25 nM, less than 24 nM, less than 23 nM, less than 22 nM, less than 21 nM, less than 20 nM, less than 19 nM, less than 18 nM, less than 17 nM, less than 16 nM, less than 15 nM, less than 14 nM, less than 13 nM, less than 12 nM, less than 11 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM.

[0413] In some embodiments, EC of cells treated with modified oligonucleotides (e.g., SEQ ID NOS: 11-666) 50 The ratio of EC50 of cells treated with a reference oligonucleotide (e.g., SEQ ID NO: 10) to EC50 of cells treated with a reference oligonucleotide (e.g., SEQ ID NO: 10) is calculated as a measure of efficacy in reducing HBsAg levels. In some embodiments, the ratio is from about 0.05 to about 250. In some embodiments, the EC 50The ratios are less than 250, less than 150nM, less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 65nM, less than 60nM, less than 55nM, less than 50nM, less than 49nM, less than 47nM, less than 46nM, less than 45nM, less than 44nM, less than 43nM, less than 42nM, less than 41nM, less than 40nM, less than 39nM, less than 38nM, less than 37nM, less than 36nM, less than 35nM, less than 34nM, less than 33nM, less than 32nM, less than 31nM, less than 30nM, less than 29nM, less than 28nM, less than 27nM, less than 26nM, less than 2 less than 5 nM, less than 24 nM, less than 23 nM, less than 22 nM, less than 21 nM, less than 20 nM, less than 19 nM, less than 18 nM, less than 17 nM, less than 16 nM, less than 15 nM, less than 14 nM, less than 13 nM, less than 12 nM, less than 11 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2, or less than 0.1.

[0414] In certain embodiments, a compound or composition described herein, when delivered to HepG2.2.1 cells, has an in vitro IC of less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 65 nM, less than 60 nM, less than 55 nM, less than 50 nM, less than 49 nM, less than 47 nM, or less than 46 nM. 50 It is effective to have at least one of the following:

[0415] In certain embodiments, a compound or composition described herein, when delivered to HepG2.2.1 cells, has an in vitro IC of less than 250 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 35 nM, less than 34 nM, less than 33 nM, less than 32 nM, or less than 31 nM. 50 It is effective to have at least one of the following:

[0416] In certain embodiments, a compound or composition described herein, when delivered to HepG2.2.1 cells, has an in vitro IC of less than 20 μM, less than 10 μM, less than 9.5 μM, less than 9.0 μM, less than 8.5 μM, less than 8.0 μM, less than 7.5 μM, less than 7.0 μM, less than 6.5 μM, less than 6.0 μM, less than 5.5 μM, less than 5.0 μM, less than 4.5 μM, less than 4.0 μM, less than 3.5 μM, less than 3.0 μM, or less than 2.5 μM. 50 It is effective to have at least one of the following:

[0417] In some embodiments, the MTT CC25 value (nM) is used as a measure of cytotoxicity. In some embodiments, the MTT CC25 of cells treated with modified oligonucleotides described herein is about 10 nM to about 250 nM. In some embodiments, the MTT CC25 is greater than 10 nM, greater than 15 nM, greater than 20 nM, greater than 25 nM, greater than 30 nM, greater than 35 nM, greater than 40 nM, greater than 45 nM, greater than 50 nM, greater than 55 nM, greater than 60 nM, greater than 65 nM, greater than 70 nM, greater than 75 nM, greater than 80 nM, greater than 85 nM, greater than 90 nM, greater than 95 nM, or greater than 95 nM. higher than 100 nM, higher than 110 nM, higher than 120 nM, higher than 130 nM, higher than 140 nM, higher than 150 nM, higher than 160 nM, higher than 170 nM, higher than 180 nM, higher than 190 nM, higher than 200 nM, higher than 210 nM, higher than 220 nM, higher than 230 nM, higher than 240 nM, or higher than 250 nM.

[0418] In some embodiments, CCK8 CC30 (nM) is used as a measure of cytotoxicity. In some embodiments, the CCK8 CC30 of cells treated with modified oligonucleotides described herein is between about 10 nM and about 250 nM. In some embodiments, the CCK8 CC30 is greater than 10 nM, greater than 15 nM, greater than 20 nM, greater than 25 nM, greater than 30 nM, greater than 35 nM, greater than 40 nM, greater than 45 nM, greater than 50 nM, greater than 55 nM, greater than 60 nM, greater than 65 nM, greater than 70 nM, greater than 75 nM, greater than 80 nM, greater than 85 nM, greater than 90 nM, greater than 95 nM, or greater than 95 nM. higher than 100 nM, higher than 110 nM, higher than 120 nM, higher than 130 nM, higher than 140 nM, higher than 150 nM, higher than 160 nM, higher than 170 nM, higher than 180 nM, higher than 190 nM, higher than 200 nM, higher than 210 nM, higher than 220 nM, higher than 230 nM, higher than 240 nM, or higher than 250 nM.

[0419] In some embodiments, the "C / E ratio" is used as a measure of efficacy in reducing HBsAg levels compared to cytotoxicity. In some embodiments, the C / E ratio is calculated by taking the ratio of the MTT CC25 (nM) of the modified oligonucleotide to the MTT CC25 (nM) of the reference oligonucleotide (e.g., SEQ ID NO: 10), and dividing this by the HBsAg EC (nM) of the modified oligonucleotide (e.g., SEQ ID NOs: 11-666). 50 In some embodiments, the C / E ratio is calculated by taking the ratio of CCK8 CC25 (nM) of the modified oligonucleotide / CCK8 CC25 (nM) of the reference oligonucleotide (e.g., SEQ ID NO: 10) and dividing this by the HBsAg EC50 (nM) of the modified oligonucleotide (e.g., SEQ ID NOs: 11-666). 50In some embodiments, the C / E ratio is calculated by dividing the C / E ratio by the HBsAg EC50 of the reference oligonucleotide (SEQ ID NO: 10). In some embodiments, the C / E ratio is greater than 1, greater than 1.1, greater than 1.2, greater than 1.3, greater than 1.4, greater than 1.5, greater than 1.6, greater than 1.7, greater than 1.8, greater than 1.9, greater than 2.0, greater than 2.1, greater than 2.2, greater than 2.3, greater than 2.4, greater than 2.5, greater than 2.6, greater than 2.7, greater than 2.8, greater than 2.9, greater than 3.0, greater than 10, greater than 11, greater than 12, greater than 13, greater than 14, greater than 15, greater than 16, greater than 17, greater than 18, greater than 19, greater than 20, greater than 21, greater than 22, greater than 23, greater than 24, greater than 25, greater than 30, greater than 35, greater than 40, greater than 45, or greater than 50.

[0420] In certain embodiments, the compounds or compositions described herein are highly tolerable, as indicated by at least one of a 4-fold or less, 3-fold or less, or 2-fold or less increase in ALT or AST levels, or a 30% or less, 20% or less, 15% or less, 12% or less, 10% or less, 5% or less, or 2% or less increase in liver, spleen, or kidney weight compared to animals treated with saline. In certain embodiments, the compounds or compositions described herein are highly tolerable, as indicated by no increase in ALT or AST compared to animals treated with saline. In certain embodiments, the compounds or compositions described herein are highly tolerable, as indicated by no increase in liver, spleen, or kidney weight compared to animals treated with saline.

[0421] In some embodiments, animals treated with a compound or composition comprising a modified oligonucleotide (e.g., SEQ ID NOs: 11-666) have ALT levels that are reduced by about 1.1-fold to about 10-fold compared to animals treated with a reference compound (e.g., SEQ ID NO: 10). In some embodiments, animals treated with a compound or composition comprising a modified oligonucleotide (e.g., SEQ ID NOs: 11-666) have ALT levels that are reduced by at least 1.25-fold compared to animals treated with a reference compound (e.g., SEQ ID NO: 10). In some embodiments, animals treated with a compound or composition comprising a modified oligonucleotide (SEQ ID NOs: 11-666) have ALT levels that are reduced by 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, or 2.3-fold compared to animals treated with a reference compound (e.g., SEQ ID NO: 10). , 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4.0x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, 4.9x, or 5.0x decreased ALT values.

[0422] In some embodiments, animals treated with a compound or composition comprising a modified oligonucleotide (e.g., SEQ ID NO: 11-666) have AST values ​​that are reduced by about 1.1-fold to about 10-fold compared to animals treated with a reference compound (e.g., SEQ ID NO: 10). In some embodiments, animals treated with a compound or composition comprising a modified oligonucleotide (e.g., SEQ ID NO: 11-666) have AST values ​​that are reduced by at least 1.25-fold compared to animals treated with a reference compound (e.g., SEQ ID NO: 10). In some embodiments, animals treated with a compound or composition comprising a modified oligonucleotide (SEQ ID NO: 11-666) have AST values ​​that are reduced by 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4.0-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold, or 5.0-fold compared to animals treated with a reference compound (e.g., SEQ ID NO: 10).

[0423] Certain embodiments provide for the use of a modified oligonucleotide, e.g., a modified oligonucleotide comprising any one of SEQ ID NOs: 11-666, or a pharmaceutical composition described herein, for the manufacture of a medicament for treating, ameliorating, delaying, or preventing a disease, disorder, or condition associated with HBV in an animal.

[0424] Certain embodiments provide for the use of a modified oligonucleotide, e.g., a modified oligonucleotide comprising any one of SEQ ID NOs: 11-666, or a pharmaceutical composition described herein, for the manufacture of a medicament for treating, ameliorating, delaying, or preventing liver disease in an animal.

[0425] Kits and Manufactured Products The present disclosure provides kits containing the modified oligonucleotides described herein, and kits containing pharmaceutical compositions containing the same. In some embodiments, the modified oligonucleotides include those containing one of SEQ ID NOs: 11-666, or those containing one, two, three, four, or five modifications to those sequences.

[0426] Certain embodiments provide kits for treating, preventing, or ameliorating a disease, disorder, or condition associated with HBV, as described herein, wherein the kit includes a) a compound or composition described herein, e.g., a composition comprising a modified oligonucleotide comprising any one of SEQ ID NOS: 11-666, or a modified oligonucleotide comprising 1, 2, 3, 4, or 5 modifications thereto, and optionally b) an additional agent or treatment described herein. The kit further includes instructions or a label for using the kit to treat, prevent, or ameliorate a disease, disorder, or condition associated with HBV.

[0427] The compositions comprising modified oligonucleotides may be lyophilized and then packaged in a kit, or may be provided in a solution together with a pharmaceutically acceptable carrier, excipient diluent.

[0428] In certain embodiments, the kit further comprises at least one additional agent for treating a disease, disorder, or condition associated with HBV.

[0429] Medication and Administration The present disclosure provides methods comprising administering to a subject in need thereof a therapeutically effective amount of a modified oligonucleotide, for example, a modified oligonucleotide comprising any one of SEQ ID NOs: 11-666, or a modified oligonucleotide comprising 1, 2, 3, 4, or 5 modifications to such a sequence, or a pharmaceutical composition comprising the same.

[0430] In certain embodiments, for example, a therapeutically effective amount of a modified oligonucleotide or pharmaceutical composition comprising the same comprises a dose at which a maximum log 10 serum HBsAg reduction of at least -0.2, at least -0.3, at least -0.4, at least -0.5, at least -0.7, at least -1.0, at least -1.5, at least -2.0, at least -2.5, at least -3.0, at least -3.5, at least -4.0, or at least -4.5 is observed. In some embodiments, the maximum log 10 serum HBsAg reduction is observed 1, 2, 3, 4, 5, 6, 7, 8, 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, or 35 days after administration of the therapeutically effective amount.

[0431] In certain embodiments, for example, a therapeutically effective amount of a modified oligonucleotide or pharmaceutical composition comprising the same comprises a dose that reduces HBsAg levels in the serum of a subject by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. In some embodiments, the reduction in serum HBsAg is observed 1, 2, 3, 4, 5, 6, 7, 8, 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, or 35 days after administration of the therapeutically effective amount.

[0432] In certain embodiments, for example, a therapeutically effective amount of a modified oligonucleotide or pharmaceutical composition comprising the same comprises a dose at which a maximum log 10 serum HBeAg reduction of at least -0.2, at least -0.3, at least -0.4, at least -0.5, at least -0.7, at least -1.0, at least -1.5, at least -2.0, at least -2.5, at least -3.0, at least -3.5, at least -4.0, or at least -4.5 is observed. In some embodiments, the maximum log 10 serum HBeAg reduction is observed 1, 2, 3, 4, 5, 6, 7, 8, 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, or 35 days after administration of the therapeutically effective amount.

[0433] In certain embodiments, for example, a therapeutically effective amount of a modified oligonucleotide or pharmaceutical composition comprising the same comprises a dose that reduces serum HBeAg levels in a subject by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. In some embodiments, the reduction in serum HBeAg is observed 1, 2, 3, 4, 5, 6, 7, 8, 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, or 35 days after administration of the therapeutically effective amount.

[0434] In certain embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the same may be administered in an amount of 10 mg to 1500 mg, 10 mg to 1000 mg, 10 mg to 900 mg, 10 mg to 800 mg, 10 mg to 700 mg, 10 mg to 600 mg, 10 mg to 500 mg, 10 mg to 400 mg, 10 mg to 300 mg, 10 mg to 200 mg, 10 mg to 100 mg, 5 mg to 600 mg, 10 mg to 700 mg, 10 mg to 800 mg, 10 mg to 900 mg, 10 mg to 100 mg, 10 mg to 1500 mg, 10 mg to 10 ... 0mg~1500mg, 50mg~1000mg, 50mg~900mg, 50mg~800mg, 50mg~700mg, 50mg~600mg, 50mg~500mg, 50 mg~400mg, 50mg~300mg, 50mg~200mg, 50mg~100mg, 100mg~1500mg, 100mg~1000mg, 100mg~900mg, 1 00mg~800mg, 100mg~700mg, 100mg~600mg, 100mg~500mg, 100mg~400mg, 100mg~300mg, 100mg~200 mg, 200mg~1500mg, 200mg~1000mg, 200mg~900mg, 200mg~800mg, 200mg~700mg, 200mg~600mg, 200m The modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 50 mg to 1500 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 50 mg to 1000 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 50 mg to 700 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 50 mg to 700 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 50 mg to 500 mg of the modified oligonucleotide.In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 50 mg to 450 mg of modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 50 mg to 300 mg of modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 50 mg to 200 mg of modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 100 mg to 1500 mg of modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 100 mg to 1000 mg of modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 100 mg to 700 mg of modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 100 mg to 500 mg of modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 100 mg to 450 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 100 mg to 300 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 100 mg to 200 mg of the modified oligonucleotide.

[0435] In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered at a concentration of 0.01 mg / kg to 30.0 mg / kg, 0.01 mg / kg to 27.0 mg / kg, 0.01 mg / kg to 25.0 mg / kg, 0.01 mg / kg to 22.0 mg / kg, 0.01 mg / kg to 20.0 mg / kg, 0.01 0.01mg / kg~15.0mg / kg, 0.01mg / kg~10.0mg / kg, 0.01mg / kg~8.0mg / kg, 0.01mg / kg~5.0mg / kg, 0.01mg / kg~4.0mg / kg, 0.01mg / kg~ 3.0mg / kg, 0.01mg / kg~2.0mg / kg, 0.01mg / kg~1.0mg / kg, 0.01mg / kg~0.5mg / kg, 0.01mg / kg~0.1mg / kg, 0.1mg / kg~30.0mg / kg, 0.1 mg / kg~27.0mg / kg, 0.1mg / kg~25.0mg / kg, 0.1mg / kg~22.0mg / kg, 0.1mg / kg~20.0mg / kg, 0.1mg / kg~18.0mg / kg, 0.1mg / kg~15.0mg / kg, 0.1mg / kg~12.0mg / kg, 0.1mg / kg~10.0mg / kg, 0.1mg / kg~8.0mg / kg, 0.1mg / kg~5.0mg / kg, 0.1mg / kg~4.0mg / kg, 0.1mg / kg~3. 0mg / kg, 0.1mg / kg~2.0mg / kg, 0.1mg / kg~1.0mg / kg, 0.1mg / kg~0.5mg / kg, 1.0mg / kg~30.0mg / kg, 1.0mg / kg~27.0mg / kg, 1.0mg / kg ~25.0mg / kg, 1.0mg / kg~22.0mg / kg, 1.0mg / kg~20.0mg / kg, 1.0mg / kg~18.0mg / kg, 1.0mg / kg~15.0mg / kg, 1.0mg / kg~12.0mg / kg, 1 .0mg / kg~10.0mg / kg, 1.0mg / kg~8.0mg / kg, 1.0mg / kg~5.0mg / kg, 1.0mg / kg~4.0mg / kg, 1.0mg / kg~3.0mg / kg, 1.0mg / kg~2.0mg / kg , 5.0mg / kg~30.0mg / kg, 5.0mg / kg~27.0mg / kg, 5.0mg / kg~25.0mg / kg, 5.0mg / kg~22.0mg / kg, 5.0mg / kg~20.0mg / kg, 5.0mg / kg~18.The modified oligonucleotide may be administered at a dose ranging from 0 mg / kg, 5.0 mg / kg to 15.0 mg / kg, 5.0 mg / kg to 10.0 mg / kg, 5.0 mg / kg to 7.0 mg / kg, 10.0 mg / kg to 30.0 mg / kg, 10.0 mg / kg to 27.0 mg / kg, 10.0 mg / kg to 25.0 mg / kg, 10.0 mg / kg to 22.0 mg / kg, 10.0 mg / kg to 20.0 mg / kg, 10.0 mg / kg to 18.0 mg / kg, 10.0 mg / kg to 15.0 mg / kg, 15.0 mg / kg to 30.0 mg / kg, 15.0 mg / kg to 25.0 mg / kg, 15.0 mg / kg to 20.0 mg / kg, 20 mg / kg to 30 mg / kg, or 25 mg / kg to 30 mg / kg. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered at a dose ranging from 0.01 mg / kg to 30 mg / kg of modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered at a dose ranging from 0.1 mg / kg to 25 mg / kg of modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered at a dose ranging from 1.0 mg / kg to 20 mg / kg of modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered at a dose ranging from 5.0 mg / kg to 20 mg / kg of modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered at a dose ranging from 1.0 mg / kg to 15 mg / kg of modified oligonucleotide. In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered at a dose ranging from 1.0 mg / kg to 5.0 mg / kg of modified oligonucleotide.

[0436] In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every 11 days, every 12 days, every 13 days, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 2 months, every 3 months, or every 4 months.

[0437] In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every two weeks, every three weeks, or monthly.

[0438] In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising same is administered to a subject for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

[0439] In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to the subject until a particular outcome is achieved, for example, until HBsAg and / or HBeAg in the subject's serum is reduced. In certain embodiments, administering the modified oligonucleotide or pharmaceutical composition comprising the same to the subject continues until HBsAg and / or HBeAg in the subject's serum is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to before administration of the modified oligonucleotide or pharmaceutical composition comprising the same.

[0440] In certain embodiments, the modified oligonucleotide or pharmaceutical composition comprising same is administered to a subject once daily, twice daily, three times daily, or four times daily.

[0441] In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every day. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every two days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every three days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every four days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every five days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every six days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every seventh day. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every eighth day. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every ninth day. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every ten days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every eleven days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every 12 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every 13 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every two weeks. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every three weeks. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject monthly. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every two months. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every three months. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every four months.In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every five months. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject every six months. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject more than once per year. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject more than once every two years. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to the subject more than once every two years or more.

[0442] In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to a subject once every 1 to 30 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to a subject once every 1 to 22 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to a subject once every 1 to 15 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to a subject once every 1 to 8 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to a subject once every 1 to 5 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to a subject once every 1 to 4 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to a subject once every 1 to 3 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to a subject once every 10 to 20 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to a subject once every 5 to 15 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered to a subject once every 15 to 30 days.

[0443] In certain embodiments, the modified oligonucleotide, or pharmaceutical composition comprising the same, is administered at least once every 36 hours. In certain embodiments, the modified oligonucleotide, or pharmaceutical composition comprising the same, is administered at least once every 48 hours. In certain embodiments, the modified oligonucleotide, or pharmaceutical composition comprising the same, is administered at least once every 60 hours. In certain embodiments, the modified oligonucleotide, or pharmaceutical composition comprising the same, is administered at least once every 72 hours. In certain embodiments, the modified oligonucleotide, or pharmaceutical composition comprising the same, is administered at least once every 84 hours. In certain embodiments, the modified oligonucleotide, or pharmaceutical composition comprising the same, is administered at least once every 96 hours. In certain embodiments, the modified oligonucleotide, or pharmaceutical composition comprising the same, is administered at least once every 5 days. In certain embodiments, the modified oligonucleotide, or pharmaceutical composition comprising the same, is administered at least once every 6 days. In certain embodiments, the modified oligonucleotide, or pharmaceutical composition comprising the same, is administered at least once every 7 days. In certain embodiments, the modified oligonucleotide, or pharmaceutical composition comprising the same, is administered at least once every 8-10 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered at least once every 10 to 12 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered at least once every 12 to 15 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered at least once every 15 to 25 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition comprising the same, is administered at least once every 20 to 30 days.

[0444] In some embodiments, the administration of the modified oligonucleotide or pharmaceutical composition comprising the same includes a drug holiday. For example, the modified oligonucleotide or pharmaceutical composition comprising the same is administered to a subject every three days, followed by a one-week, two-week, three-week, or one-month break, after which administration resumes. Those skilled in the art will understand that this drug holiday is exemplary. Other lengths and frequencies of drug holidays are also contemplated within the scope of the present disclosure. As a further example, the modified oligonucleotide or pharmaceutical composition comprising the same may be administered to a subject until the HBsAg or HbeAg level falls below a certain threshold or until HBV infection is no longer detectable in the subject, followed by a drug holiday, and then administration resumes when HBsAg or HbeAg is again detected in the subject's serum.

[0445] In certain embodiments, a single dose of the modified oligonucleotide or a pharmaceutical composition comprising the same is administered to the subject, hi other embodiments, multiple doses of the modified oligonucleotide or a pharmaceutical composition comprising the same are administered to the subject.

[0446] In certain embodiments, the administration of a modified oligonucleotide or a pharmaceutical composition comprising the same may include a dosing schedule in which the modified oligonucleotide or a pharmaceutical composition comprising the same is administered more frequently initially, followed by less frequent administration. Such a dosing schedule has the advantage that it can be used to maintain steady-state liver concentrations of the modified oligonucleotides described herein. For example, if the hepatic half-life of a modified oligonucleotide is 3-4 weeks, an initial dosing schedule including loading doses on days 1 and 4, followed by weekly maintenance doses starting on day 8 (e.g., days 8, 15, and 22, etc.) can be used to achieve steady-state liver concentrations of the modified oligonucleotides of the present disclosure.

[0447] In certain embodiments, administration of the modified oligonucleotide, or pharmaceutical composition comprising same, comprises a loading dose followed by a loading maintenance dose.

[0448] The term "loading dose" refers to one or more doses of a modified oligonucleotide or pharmaceutical composition that are administered in addition to a dosing regimen or that are higher than other doses in a dosing regimen. As used herein, a "loading dose" can refer to one or more doses of a modified oligonucleotide or pharmaceutical composition that are at the same or lower concentration (if administered in addition to a regular dosing regimen), or that are at a higher concentration than the doses of modified oligonucleotide or pharmaceutical composition administered as part of the dosing regimen (if administered in place of a regular dosing regimen). A loading dose may be administered more frequently than a maintenance dose in a dosing regimen.

[0449] As used herein, the term "maintenance dose" refers to repeated, regular administration of a therapeutic agent. As used herein, a maintenance dose does not include a loading dose, and in some embodiments, a loading dose may be administered in addition to, for example, before, the maintenance dose.

[0450] In the exemplary dosing regimen comprising loading dose and maintenance dose, modified oligonucleotide or pharmaceutical composition is administered at a higher loading dose, followed by a lower maintenance dose.For example, the loading dose can be about 1.1X, 1.2X, 1.3X, 1.4X, 1.5X, 1.6X, 1.7X, 1.8X, 1.9X, 2.0X, 2.1X, 2.2X, 2.3X, 2.4X, 2.5X, 3.0X, 3.5X, 4.0X, 4.5X or 5X higher than the maintenance dose.Alternatively, or additionally, the loading dose can be administered more frequently than the maintenance dose.For example, the loading dose can be administered every day, every 2 days, every 3 days, every 4 days or every 5 days for at least 1, 2, 3, 4 or 5 times, followed by the maintenance dose every week, every 10 days, every 14 days, etc. Those skilled in the art will understand that if necessary, a loading dose can be administered again to a subject who has been administered according to a maintenance dosing schedule.As a further exemplary dosing schedule, a subject can be administered a loading dose on days 1 and 4, followed by a weekly maintenance dose on day 8.

[0451] Antisense Compounds Oligomeric compounds include but are not limited to oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimics, antisense compounds, antisense oligonucleotides and siRNA.Oligomeric compounds can be " antisense " to target nucleic acid, which means that they can hybridize to target nucleic acid through hydrogen bonds.

[0452] In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of a target segment of a targeted target nucleic acid. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of a target segment of a targeted target nucleic acid.

[0453] In certain embodiments, antisense oligonucleotides targeting HBV nucleic acids may be shortened or truncated. For example, one subunit may be deleted from the 5' end (5' truncation) or from the 3' end (3' truncation). Shortened or truncated antisense compounds targeting HBV nucleic acids may have two subunits deleted from the 5' end of the antisense compound or two subunits deleted from the 3' end of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, one nucleoside deleted from the 5' end and one nucleoside deleted from the 3' end of the antisense compound.

[0454] In an extended antisense compound, when one additional subunit is present, the additional subunit may be located at the 5'-end or 3'-end of the antisense compound. When two or more additional subunits are present, the additional subunits may be adjacent to each other, for example, two subunits may be added to the 5'-end of the antisense compound (5'-addition) or two subunits may be added to the 3'-end of the antisense compound (3'-addition). Alternatively, the additional subunits may be dispersed throughout the antisense compound, for example, one subunit may be added to the 5'-end and one subunit may be added to the 3'-end of the antisense compound.

[0455] It is also possible to increase or decrease the length of antisense compounds, such as antisense oligonucleotides, and / or introduce mismatch bases without losing activity. See, for example, Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992). In this paper, a series of antisense oligonucleotides ranging from 13 to 25 nucleobases in length were tested for their ability to induce target RNA cleavage in an oocyte injection model. Antisense oligonucleotides 25 nucleobases long with 8 or 11 mismatch bases near the end of the antisense oligonucleotide were able to induce specific cleavage of target mRNA, albeit to a lesser extent than antisense oligonucleotides without mismatches. Similarly, target-specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with one or three mismatches.

[0456] Gautschi et al. (Natl. Cancer Inst. 93:463-471, March 2001) demonstrated that an oligonucleotide 100% complementary to bcl-2 mRNA and containing three mismatches to bcl-xL mRNA reduced the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide showed potent antitumor activity in vivo.

[0457] Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a tandem 14-nucleobase antisense oligonucleotide series, as well as 28-nucleobase and 42-nucleobase antisense oligonucleotides composed of two or three tandem antisense oligonucleotide sequences, for their ability to terminate human DHFR translation in a rabbit reticulocyte assay. Each of the three 14-nucleobase antisense oligonucleotides alone was able to inhibit translation, although to a moderate degree compared with the 28-nucleobase or 42-nucleobase antisense oligonucleotides.

[0458] Antisense compound motif In certain embodiments, antisense compounds targeted to HBV nucleic acids have chemically modified subunits arranged in a pattern or motif that confers properties to the antisense compound, such as, for example, enhanced inhibitory activity, increased binding affinity for the target nucleic acid, or resistance to degradation by nucleases in vivo.

[0459] Typically, chimeric antisense compounds contain at least one region modified to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and / or increased inhibitory activity. A second region of the chimeric antisense compound may optionally function as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

[0460] Antisense compounds with a gapmer motif are considered chimeric antisense compounds. In a gapmer, an internal region containing nucleotides that support RNase H cleavage is positioned between external regions containing nucleotides that are chemically distinct from the nucleosides of the internal region. In antisense oligonucleotides with a gapmer motif, the gap segment generally serves as a substrate for endonuclease cleavage, while the wing segments contain modified nucleosides. In certain embodiments, gapmer regions are distinguished by the type of sugar moiety, each of which comprises a distinct region. The types of sugar moieties used to distinguish gapmer regions include, in some embodiments, β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides may include, inter alia, 2'-MOE and 2'-O-CH3), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides may include those with a constrained ethyl). In certain embodiments, the nucleosides in the wings may contain several modified sugar moieties, including, for example, 2'-MOE and bicyclic sugar moieties such as, for example, constrained ethyl or LNA. In certain embodiments, the wings may contain several modified and unmodified sugar moieties. In certain embodiments, the wings may contain various combinations of 2'-MOE nucleosides, bicyclic sugar moieties such as, for example, constrained ethyl nucleosides or LNS nucleosides, and 2'-deoxynucleosides.

[0461] Each distinct region may comprise uniform, variant, or alternative sugar moieties. A wing-gap-wing motif is often described as "XYZ," where "X" represents the length of the 5' wing, "Y" represents the length of the gap, and "Z" represents the length of the 3' wing. "X" and "Z" may comprise uniform, variant, or alternative sugar moieties. In certain embodiments, "X" and "Y" may comprise one or more 2'-deoxynucleosides. "Y" may comprise a 2'-deoxynucleoside. As used herein, a gapmer described as XYZ has a configuration in which a gap is positioned immediately adjacent to each of the 5'-wing and 3'-wing. Thus, there are no intervening nucleotides between the 5'-wing and the gap or between the gap and the 3'-wing. Any of the antisense compounds described herein may have a gapmer motif. In certain embodiments, "X" and "Z" are the same, and in other embodiments, they are different. In certain embodiments, "Y" is 8 to 15 nucleosides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 nucleosides, or more.

[0462] Target Nucleic Acids, Target Regions, and Nucleotide Sequences In certain embodiments, nucleotide sequences encoding HBV include, but are not limited to, GENBANK Accession U95551.1 (incorporated herein as SEQ ID NO:1).

[0463] It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, internucleoside linkage, or nucleobase, and thus, an antisense compound defined by a SEQ ID NO may contain, independently, one or more modifications to a sugar moiety, internucleoside linkage, or nucleobase.

[0464] In certain embodiments, the target region is a structurally defined region of the target nucleic acid. For example, the target region may include a 3'UTR, a 5'UTR, an exon, an intron, an exon / intron junction, a coding region, a translation initiation region, a translation termination region, or other defined nucleic acid region. Structurally defined regions of HBV can be obtained by accession number from a sequence database, such as NCBI, and such information is incorporated herein by reference. In certain embodiments, the target region may include the sequence from the 5' target site of one target segment within the target region to the 3' target site of another target segment within the same target region.

[0465] Targeting involves determining at least one target segment to which the antisense compound hybridizes to produce a desired effect. In certain embodiments, the desired effect is a reduction in the level of mRNA of the target nucleic acid. In certain embodiments, the desired effect is a reduction in the level of a protein encoded by the target nucleic acid or a change in a phenotype associated with the target nucleic acid.

[0466] A target region may contain one or more target segments. Multiple target segments within a target region may overlap, or may not overlap. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain embodiments, target segments within a target region are separated by a number of nucleotides on the target nucleic acid, i.e., 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides, about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides, or no more than about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides, or a range defined by any two of the foregoing values. In certain embodiments, target segments within a target region are separated by 5 nucleotides or less on the target nucleic acid. In certain embodiments, the target segments are contiguous. Target regions defined by a range having a starting nucleic acid that is either a 5' target site or a 3' target site listed herein are contemplated.

[0467] Suitable target segments may be present in 5'UTR, coding region, 3'UTR, intron, exon, or exon / intron junction. Suitable target segments may also include target segments containing start codons or stop codons. Suitable target segments may specifically exclude certain structurally defined regions, such as start codons or stop codons.

[0468] Determining suitable target segments can include comparing the sequence of target nucleic acid with other sequences throughout the genome.For example, the BLAST algorithm can be used to identify the similarity region between different nucleic acids.This comparison can prevent the selection of antisense compound sequences that may nonspecifically hybridize with sequences other than the selected target nucleic acid (i.e., non-target sequences or off-target sequences).

[0469] The activity of antisense compounds within the active target region (e.g., activity as defined by the percent reduction in target nucleic acid levels) can vary. In certain embodiments, a reduction in HBV mRNA levels indicates inhibition of HBV expression. A reduction in HBV protein levels also indicates inhibition of target mRNA expression. Additionally, phenotypic changes indicate inhibition of HBV expression. In certain embodiments, reduced fatigue, reduced flu-like symptoms, increased appetite, reduced nausea, reduced joint pain, reduced jaundice, reduced abdominal pain, reduced weakness, reduced weight loss, reduced breast enlargement in men, reduced palmar rash, reduced difficulty clotting blood, reduced liver cirrhosis, reduced spider veins on the skin, increased absorption of vitamins A and D, reduced tumor growth, reduced tumor volume, reduced headache, reduced fever, reduced diarrhea, reduced pain in the liver region of the body, clay-colored or gray stools, reduced itching, reduced dark urine, and reduced nausea and vomiting can indicate inhibition of HBV expression. In certain embodiments, an improvement in symptoms associated with HBV-related conditions, diseases, and disorders can indicate inhibition of HBV expression. In certain embodiments, a decrease in cirrhosis is indicative of inhibition of HBV expression. In certain embodiments, a decrease in liver cancer markers may be indicative of inhibition of HBV expression.

[0470] Hybridization In some embodiments, hybridization occurs between the antisense compounds disclosed herein and HBV nucleic acids. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of nucleic acid molecules.

[0471] Hybridization can occur under a variety of conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

[0472] Methods for determining whether a sequence is capable of specifically hybridizing to a target nucleic acid are known in the art. In certain embodiments, the antisense compounds provided herein are capable of specifically hybridizing to HBV nucleic acids.

[0473] Complementarity An antisense compound and a target nucleic acid are complementary to one another when a sufficient number of the nucleobases of the antisense compound are capable of hydrogen bonding with corresponding nucleobases of the target nucleic acid, thereby producing the desired effect (e.g., antisense inhibition of a target nucleic acid, such as an HBV nucleic acid).

[0474] Non-complementary nucleobases between an antisense compound and an HBV nucleic acid are acceptable, provided that the antisense compound is capable of specifically hybridizing to the target nucleic acid. Furthermore, an antisense compound may hybridize across one or more segments of an HBV nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch, or hairpin structure).

[0475] In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to an HBV nucleic acid, target region, target segment, or specified portion thereof, or are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target nucleic acid, target region, target segment, or specified portion thereof. The percent complementarity between a target nucleic acid and an antisense compound can be determined using routine methods.

[0476] For example, 18 of the 20 nucleobases of an antisense compound are complementary to target region, thereby specifically hybridizing, and the antisense compound has 90% complementarity.In this embodiment, the remaining non-complementary nucleobases can be clustered with complementary nucleobases, or can be interspersed with complementary nucleobases, and do not need to be contiguous with each other or contiguous with complementary nucleobases.Therefore, an 18-nucleobase long antisense compound has four non-complementary nucleobases flanked by two regions that are completely complementary to target nucleic acid, and has 77.8% complementarity with target nucleic acid as a whole, and therefore falls within the scope of the present invention. The percentage complementarity of target nucleic acid region and antisense compound can be routinely determined using BLAST program (basic local alignment search tool) and PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) known in the art.The percentage homology, sequence identity or complementarity can be determined by default setting of Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), for example, using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0477] In certain embodiments, the antisense compounds provided herein, or specific portions thereof, are fully complementary (i.e., 100% complementary) to a target nucleic acid, or a specific portion thereof. For example, an antisense compound may be fully complementary to an HBV nucleic acid, or a target region, target segment, or target sequence thereof. As used herein, "fully complementary" means that each nucleobase of an antisense compound can precisely base pair with the corresponding nucleobase of a target nucleic acid. For example, a 20-nucleobase antisense compound is fully complementary to a 400-nucleobase long target sequence, provided that there is a corresponding 20-nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. The term "fully complementary" can also be used to refer to a specific portion of a first nucleic acid and / or a second nucleic acid. For example, a 20-nucleobase portion of a 30-nucleobase antisense compound can be "fully complementary" to a 400-nucleobase long target sequence. A 20 nucleobase portion of a 30 nucleobase oligonucleotide is fully complementary to a target sequence if the target sequence has a corresponding 20 nucleobase portion, where each nucleobase is complementary to a 20 nucleobase portion of an antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

[0478] The non-complementary nucleobase may be located at the 5'-end or 3'-end of the antisense compound. Alternatively, the non-complementary nucleobase may be located at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e., linked) or non-contiguous. In one embodiment, the non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.

[0479] In certain embodiments, antisense compounds that are 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length, or that are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length, contain no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase to a target nucleic acid, e.g., an HBV nucleic acid, or a specified portion thereof. In certain embodiments, antisense compounds that are 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length, or that are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length, contain no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase to a target nucleic acid, e.g., an HBV nucleic acid, or a specified portion thereof.

[0480] The provided antisense compounds also include antisense compounds that are complementary to a portion of a target nucleic acid. As used herein, a "portion" refers to a specified number of contiguous (i.e., linked) nucleobases within a region or segment of a target nucleic acid. A "portion" can also refer to a specified number of contiguous nucleobases of an antisense compound. In certain embodiments, an antisense compound is complementary to at least an 8-nucleobase portion of a target segment. In certain embodiments, an antisense compound is complementary to at least a 9-nucleobase portion of a target segment. In certain embodiments, an antisense compound is complementary to at least a 10-nucleobase portion of a target segment. In certain embodiments, an antisense compound is complementary to at least an 11-nucleobase portion of a target segment. In certain embodiments, an antisense compound is complementary to at least a 12-nucleobase portion of a target segment. In certain embodiments, an antisense compound is complementary to at least a 13-nucleobase portion of a target segment. In certain embodiments, an antisense compound is complementary to at least a 14-nucleobase portion of a target segment. In certain embodiments, an antisense compound is complementary to at least a 15-nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range of nucleobases defined by any two of these values.

[0481] identity The antisense compounds provided herein may have a specified percent identity with a specific nucleotide sequence, SEQ ID NO:, or compound or part thereof.As used herein, an antisense compound is identical to a sequence disclosed herein if it has the same nucleic acid base pairing ability.For example, RNA containing uracil instead of thymidine in the disclosed DNA sequence is considered identical to the DNA, since both uracil and thymidine pair with adenine.Shortened and extended versions of the antisense compounds described herein and compounds with non-identical bases compared to the antisense compounds provided herein are also anticipated.The non-identical bases may be adjacent to each other or distributed throughout the antisense compound.The percent identity of an antisense compound is calculated according to the number of identical bases that form base pairs compared to the sequence being compared.

[0482] As used herein, a "complementary" polynucleotide is one that can base pair according to the standard Watson-Crick complementarity rules. Specifically, purines pair with pyrimidines, forming pairs such as cytosine and guanine (G:C), thymine and adenine (A:T) in DNA, and uracil and adenine (A:U) in RNA. For example, the sequence "AGT" binds to the complementary sequence "TCA." It should be understood that two polynucleotides can hybridize to each other even if they are not completely complementary to each other, as long as each has at least one region that is substantially complementary to the other.

[0483] As used herein, the terms "substantially complementary" or "partially complementary" mean that two nucleic acid sequences are complementary over at least about 50%, 60%, 70%, 80%, or 90% of their nucleotides.

[0484] In some embodiments, two nucleic acid sequences may be complementary over at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of their nucleotides. In some embodiments, two nucleic acid sequences may be 60%-100% complementary, 70%-100% complementary, 80%-100% complementary, 90%-100% complementary, 60%-90% complementary, 60%-80% complementary, 60%-70% complementary, 70%-90% complementary, 70%-80% complementary, 80%-100% complementary, or 80%-90% complementary.

[0485] The terms "substantially complementary" and "partially complementary" can also mean that two nucleic acid sequences are capable of hybridizing under high stringency conditions, such conditions being known in the art.

[0486] As used herein, the term "identity" means that sequences are compared to each other as follows: To determine the percent identity of two nucleic acid sequences, the sequences are first aligned to each other, and then these sequences can be compared. For this purpose, for example, gaps can be inserted into the sequence of the first nucleic acid sequence, and the nucleotide can be compared with the corresponding position in the second nucleic acid sequence. If a position in the first nucleic acid sequence is occupied by the same nucleotide as a position in the second sequence, the two sequences are identical at this position. The percent identity between two sequences is a function of the number of identical positions divided by the total number of positions compared in the examined sequences.

[0487] The "percent identity" of an aligned segment of a test sequence and a reference sequence is the percentage of identical elements shared by the two aligned sequences divided by the total number of elements in the reference sequence segment, i.e., the total number of elements in the entire reference sequence or a specified portion of the reference sequence that is smaller.

[0488] The percent identity of two sequences can be determined using a mathematical algorithm. A preferred example of a mathematical algorithm that can be used to compare two sequences is, but is not limited to, the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877. Such an algorithm is incorporated into the NBLAST program, which can be used to identify sequences with a desired identity to the sequences of the present invention. To obtain gapped alignment, as described herein, the "Gapped BLAST" program can be used as described in Altschul et al. (1997), Nucleic Acids Res, 25:3389-3402. When using BLAST and Gapped BLAST programs, the preset parameters of the particular program (e.g., NBLAST) can be used. Sequences may be further aligned using the Genetic Computing Group's GAP (global alignment program) version 9, using a preset (BLOSUM62) matrix (values ​​-4 to +11) with a gap open penalty of -12 (for the first zero in the gap) and a gap extension penalty of -4 (for each additional consecutive zero in the gap). After alignment, percent identity is calculated by expressing the number of matches as a percent content of nucleic acids in the claimed sequence. The methods described for determining percent identity of two nucleic acid sequences can be correspondingly used for encoded amino acid sequences, if desired.

[0489] Methods useful for determining sequence identity are also disclosed in Guide to Huge Computers (Martin J. Bishop, ed., Academic Press, San Diego (1994)), and Carillo, H., and Lipton, D., (Applied Math 48:1073 (1988)). More specifically, preferred computer programs for determining sequence identity include, but are not limited to, the Basic Local Alignment Search Tool (BLAST) program, publicly available from the National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institutes of Health, Bethesda, Md. 20894 (BLAST Manual, Altschul et al., NCBI, NLM, NIH; (Altschul et al. al., J. Mol. Biol. 215:403-410 (1990)). Versions 2.0 and higher of the BLAST program allow for the introduction of gaps (deletions and insertions) in the alignment, and for peptide sequences, BLASTX can be used to determine sequence identity, and for polynucleotide sequences, BLASTN can be used to determine sequence identity. The percent identity can be 70% or greater, e.g., at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, or 100% identity.

[0490] qualification A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of a nucleoside is typically a heterocyclic base moiety. A nucleotide is a nucleoside that further includes a phosphate group covalently linked to the sugar portion of the nucleoside. For nucleosides containing a pentofuranosyl sugar, the phosphate group may be attached to the 2', 3', or 5' hydroxyl moiety of the sugar. Oligonucleotides are formed by covalently linking adjacent nucleosides to one another to form linear polymeric oligonucleotides. Within the oligonucleotide structure, the phosphate groups are generally considered to form the internucleoside linkages of the oligonucleotide.

[0491] Modifications to antisense compounds include substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because they possess desirable properties, such as enhanced cellular uptake, increased affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0492] Chemically modified nucleosides can also be employed to increase the binding affinity of shortened or truncated antisense oligonucleotides to their target nucleic acids. As a result, shorter antisense compounds containing such chemically modified nucleosides often achieve comparable results.

[0493] Modified internucleoside linkages The natural internucleoside linkage in RNA and DNA is a 3'-5' phosphodiester linkage. Antisense compounds having one or more modified, i.e., non-natural, internucleoside linkages are often chosen over antisense compounds having natural internucleoside linkages because of desirable properties such as increased cellular uptake, increased affinity for the target nucleic acid, and increased stability in the presence of nucleases.

[0494] The oligonucleotide with modified internucleoside bond includes the internucleoside bond that holds phosphorus atom and the internucleoside bond that does not have phosphorus atom.Representative internucleoside bond that contains phosphorus includes but is not limited to phosphodiester, phosphotriester, methylphosphonate, phosphoramidate and phosphorothioate.The method of making phosphorus-containing bond and non-phosphorus-containing bond is known.

[0495] In certain embodiments, antisense compounds targeted to HBV nucleic acids contain one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of the antisense compounds is a phosphorothioate internucleoside linkage.

[0496] modified sugar moiety The antisense compounds provided herein may optionally contain one or more nucleosides, in which the sugar group is modified. Such sugar-modified nucleosides may confer enhanced nuclease stability, enhanced binding affinity, or some other beneficial biological property to the antisense compound. In certain embodiments, the nucleoside comprises a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and 2' substituents), bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNAs), and the modification of ribosyl ring oxygen atoms with S, N(R), or C(R 1 )(R 2 )(R=H, C1-C 12Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleosides (see PCT International Patent Application Publication No. WO2008 / 101157, published 8 / 21 / 08, for other disclosed 5',2'-bissubstituted nucleosides), further substitution at the 2-position, substitution of the ribosyl ring oxygen atom with S (see published U.S. Patent Application No. US2005 / 0130923, published June 16, 2005), and 5'-substitution of BNA (see PCT International Patent Application Publication No. WO2007 / 134181, published 11 / 22 / 07, for LNA substituted with, for example, a 5'-methyl or 5'-vinyl group).

[0497] Examples of nucleosides having modified sugar moieties include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH, and 2'-O(CH)OCH substituents. The 2'-position substituent can be allyl, amino, azido, thio, O-allyl, O-C-C 10 Alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n ), wherein R m and R n are each independently H or substituted or unsubstituted C-C 10 It is alkyl.

[0498] As used herein, "bicyclic nucleoside" refers to a modified nucleoside containing a bicyclic sugar moiety. Examples of bicyclic nucleosides include, but are not limited to, nucleosides containing a bridge between the 4'-ribosyl ring atom and the 2'-ribosyl ring atom. In certain embodiments, the antisense compounds provided herein contain one or more bicyclic nucleosides, wherein the bridge comprises a 4'-2' bicyclic nucleoside. Examples of such 4'-2' bicyclic nucleosides include, but are not limited to, one of the following formulas: 4'-(CH2)-O-2' (LNA), 4'-(CH2)-S-2, 4'-(CH2)2-O-2' (ENA), 4'-CH(CH3)-O-2' (cEt), and 4'-CH(CHOCH3)-O-2', and analogs thereof (see U.S. Pat. No. 7,399,845, issued July 15, 2008), 4'-C(CH3)(CH3)-O-2', and analogs thereof. Log (see published PCT International Patent Application Publication No. WO2009 / 006478, published January 8, 2009), 4'-CH2-N(OCH3)-2' and analogs thereof (see published PCT International Patent Application Publication No. WO2008 / 150729, published December 11, 2008), 4'-CH2-ON(CH3)-2' (see published U.S. Patent Application No. US2004 / 0171570, published September 2, 2004), 4'-CH2-N(R)-O-2', where R is HCl-C 12alkyl, or those which are protecting groups (see U.S. Pat. No. 7,427,672, issued Sep. 23, 2008), 4'-CH2-C(H)(CH3)-2' (see Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134), and 4'-CH2-C(=CH2)-2' and analogs thereof (see published PCT International Patent Application Publication No. WO2008 / 154401, published Dec. 8, 2008).Also, for example, Singh et al.,Chem.Commun.,1998,4,455-456;Koshkin et al.,Tetrahedron,1998,54,3607-3630;Wahlestedt et al.,Proc.Natl.Acad.Sci.USA,2000,97,5633-5638;Kumar et al. al.,Bioorg.Med.Chem.Lett.,1998,8,2219-2222;Singh et al.,J.Org.Chem.,1998,63,10035-10039;Srivastava et al.,J.Am.Chem.Soc.,129(26)8362-8379(Jul.4,2007);Elayadi et al.,Curr.Opinion Invens.Drugs,2001,2,558-561;Braasch et al.,Chem.Biol.,2001,8,1-7;Orum et al.,Curr.Opinion Mol. Ther., 2001, 3,239-243; U.S. Patent Nos. 6,670,461, 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 7,399,845; published PCT International Patent Application Publications WO2004 / 106356, WO94 / 14226, WO2005 / 021570, and WO2007 / 134181; U.S. Patent Publication US2004 / 0171 570, US2007 / 0287831, and US2008 / 0039618; and U.S. patent applications 12 / 129,154, 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787, and 61 / 099,844; and PCT international patent applications PCT / US2008 / 064591, PCT / US2008 / 066154, and PCT / US2008 / 068922. Each of the foregoing bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see PCT International Patent Application PCT / DK98 / 00393, published March 25, 1999 as WO 99 / 14226).

[0499] In certain embodiments, the bicyclic sugar moiety of a BNA nucleoside includes, but is not limited to, compounds having at least one bridge between the 4' and 2' positions of the pentofuranosyl sugar moiety, wherein the bridge is independently selected from the group consisting of -[C(R a )(R b )] n -, -C(R a )=C(R b )-, -C(R a )=N-, -C(=NR a )-, -C(=O)-, -C(=S)-, -O-, -Si(R a )2-, -S(=O) x - and -N(R a )-, or 2 to 4 linked groups independently selected from During the ceremony, x is 0, 1, or 2; n is 1, 2, 3, or 4; R a and R b are each independently H, a protecting group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ 1 , N.J. 1 J 2 , S.J. 1 , N3, COOJ 1 , acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J 1 ), or sulfoxyl (S(=O)-J 1 ), and J 1 and J. 2are independently H, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C 12 Aminoalkyl, substituted C1-C 12 aminoalkyl, or a protecting group.

[0500] In certain embodiments, the bridge of the bicyclic sugar moiety is —[C(R a )(R b )] n -, -[C(R a )(R b )] n -O-, -C(R a )(R b )-N(R)-O-, or -C(R a )(R b In certain embodiments, the bridges are 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-ON(R)-2', and 4'-CH2-N(R)-O-2'-, where each R is independently H, a protecting group, or C1-C 12 It is alkyl.

[0501] In certain embodiments, bicyclic nucleosides are further defined by their isomeric configuration. For example, nucleosides comprising 4'-2' methylene-oxy bridges can be in α-L configuration or β-D configuration. In the past, α-L-methyleneoxy (4'-CH2-O-2') BNAs have been incorporated into antisense oligonucleotides, and the antisense oligonucleotides have shown antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 63 65-63 72).

[0502] In certain embodiments, bicyclic nucleosides include, but are not limited to: As shown below, (A) α-L-methyleneoxy (4'-CH2-O-2') BNA, (B) β-D-methyleneoxy (4'-CH2-O-2') BNA, (C) ethyleneoxy (4'-(CH2)2-O-2') BNA, (D) aminooxy (4'-CH2-ON(R)-2') BNA, (E) oxyamino (4'-CH2-N(R)-O-2') BNA, (F) methyl(methyleneoxy) (4'-CH(CH)-O-2') BNA, (G) methylene-thio (4'-CH-S-2') BNA, (H) methylene-amino (4'-CH-N(R)-2') BNA, (I) methyl carbocyclic (4'-CH-CH(CH)-2') BNA, and (J) propylene carbocyclic (4'-(CH)-2') BNA. [ka] wherein Bx is a base moiety and R is independently H, a protecting group, or C1-C 12 It is alkyl.

[0503] In certain embodiments, the bicyclic nucleoside conforms to Formula I: [ka] During the ceremony, Bx is a heterocyclic base moiety; -Q a -Q b -Q c- is -CH2-N(R c )-CH2-, -C(=O)-N(R c )-CH2-, -CH2-ON(R c )-, -CH2-N(R c )-O-, or -N(R c )-O-CH2, R c is C1-C 12 an alkyl, or amino protecting group, and T a and T b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a support medium.

[0504] In certain embodiments, the bicyclic nucleoside has Formula II: [ka] During the ceremony, Bx is a heterocyclic base moiety; T a and T b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a support medium; Z a is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, substituted amido, thiol, or substituted thio.

[0505] In one embodiment, the substituents are each independently selected from halogen, oxo, hydroxyl, OJ c , N.J. c J d , S.J. c , N3, OC(=X)J c , and N.J. e C(=X)NJ c J d and wherein J is a substituted or unsubstituted group, c , J d , and J.e are each independently H, C1-C6 alkyl, or substituted C1-C6 alkyl; X is 0 or NJ c is.

[0506] In certain embodiments, the bicyclic nucleoside has Formula III: [ka] During the ceremony, Bx is a heterocyclic base moiety; T a and T b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a support medium; Z b is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, or substituted acyl (C(=O)-).

[0507] In certain embodiments, the bicyclic nucleoside has formula IV: [ka] During the ceremony, Bx is a heterocyclic base moiety; T a and T b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a support medium; R d is C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; q a , q b , q c and q dare each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl, C1-C6 alkoxyl, substituted C1-C6 alkoxyl, acyl, substituted acyl, C1-C6 aminoalkyl, or substituted C1-C6 aminoalkyl.

[0508] In certain embodiments, the bicyclic nucleoside has Formula V: [ka] During the ceremony, Bx is a heterocyclic base moiety; T a and T b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a support medium; q a , q b , q e , and q f are each independently hydrogen, halogen, or C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C1-C 12 Alkoxy, substituted C1-C 12 Alkoxy, OJ j , S.J. j , SOJ j , SO2J j , N.J. j J k , N3, CN, C(=O)OJ j , C(=O)NJ j J k , C(=O)J j , OC(=O)NJ j J k , N(H)C(=NH)NJ j J k , N(H)C(=O)NJ j Jk , or N(H)C(=S)NJ j J k and or q e and q f are both =C(q g )(q h ) and q g and q h are each independently H, halogen, or C1-C 12 Alkyl or substituted C1-C 12 It is alkyl.

[0509] The synthesis and preparation of methyleneoxy (4'-CH2-O-2') BNA monomers of adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil have been reported, along with their oligomerization and nucleic acid recognition capabilities (see, for example, Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and their preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0510] Methyleneoxy(4'-CH2-O-2')BNA, methyleneoxy(4'-CH2-O-2')BNA, and 2'-thio-BNA have also been prepared (see, for example, Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been reported (see, for example, Wengel et al., International Publication No. 99 / 14226). In addition, the synthesis of 2'-amino-BNA, a novel conformationally restricted high-affinity oligonucleotide analog, has also been reported in the art (see, for example, Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Additionally, 2'-amino-BNAs and 2'-methylamino-BNAs have also been generated, and the thermal stability of duplexes with complementary RNA and DNA strands has also been previously reported.

[0511] In certain embodiments, the bicyclic nucleoside has formula VI: [ka] During the ceremony, Bx is a heterocyclic base moiety; T a and T b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a support medium; q i , q j , q k and q l are each independently H, halogen, or C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C1-C 12 Alkoxyl, substituted C1-C 12 Alkoxyl, OJ j , S.J. j , SOJ j , SO2J j , N.J. j J k , N3, CN, C(=O)OJ j , C(=O)NJ j J k , C(=O)J j , OC(=O)NJ j J k , N(H)C(=NH)NJ j J k , N(H)C(=O)NJ j J k , or N(H)C(=S)NJ j J k and q i and q j , or q 1 and q k are both =C(q g )(q h) where q g and q h are each independently H, halogen, or C1-C 12 Alkyl or substituted C1-C 12 It is alkyl.

[0512] One carbocyclic bicyclic nucleoside with a 4'-(CH2)3-2' bridge and an alkenyl analog, the bridge 4'-CH=CH-CH2-2', has also been reported (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides, along with their oligomerization and biochemical studies, have also been reported (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

[0513] As used herein, "bicyclic nucleoside" refers to a nucleoside that includes a bridge linking two carbon atoms of the sugar ring, thereby forming a bicyclic sugar moiety. In certain embodiments, the bridge links the 2' carbon to another carbon of the sugar ring.

[0514] As used herein, "4'-2' bicyclic nucleoside" or "4' to 2' bicyclic nucleoside" refers to a bicyclic nucleoside containing a furanose ring that contains a bridge connecting the 2' and 4' carbon atoms.

[0515] As used herein, "monocyclic nucleoside" refers to a nucleoside that includes a modified sugar moiety that is not a bicyclic sugar moiety. In certain embodiments, the sugar moiety or sugar moiety analog of the nucleoside may be modified or substituted at any position.

[0516] As used herein, "2'-modified sugar" refers to a furanosyl sugar modified at the 2' position. In certain embodiments, such modifications include a substituent selected from the following: halides, including, but not limited to, substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted aminoalkyl, substituted and unsubstituted alkyl, substituted and unsubstituted aryl, and substituted and unsubstituted alkynyl. In certain embodiments, the 2' modification is selected from a substituent, including, but not limited to, O[(CH2) n O] m CH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, OCH2C(=O)N(H)CH3, and O(CH2) n ON[(CH2) n CH3]2, where n and m are 1 to 10. Other 2'-substituents may be selected from: C1-C 12Alkyl, substituted alkyl, alkenyl, alkynyl, 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 cleaving group, reporter group, intercalator, group for improving the pharmacokinetic properties, group for improving the pharmacological properties of antisense compounds, and other substituents with similar properties. In certain embodiments, the modified nucleoside comprises a 2'-MOE side chain (see, e.g., Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitutions have been reported to have improved binding affinity compared to unmodified nucleosides and other modified nucleosides, such as 2'-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides with 2'-MOE substituents have also been shown to be antisense inhibitors of gene expression, with promising properties for in vivo use (see, e.g., Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

[0517] As used herein, "modified tetrahydropyran nucleosides" or "modified THP nucleosides" refers to nucleosides with a six-membered tetrahydropyran "sugar" substituted in place of the pentofuranosyl residue in a normal nucleoside (sugar substitute). Modified THP nucleosides include, but are not limited to, those referred to in the art as hexitol nucleic acids (HNA), anitol nucleic acids (ANA), mannitol nucleic acids (MNA) (see Leumann, CJ. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro-HNA (F-HNA), or those compounds having the following formula X: [ka] wherein, independently for each of said at least one tetrahydropyran nucleoside analog of Formula X: Bx is a heterocyclic base moiety; T 3 and T 4 are each independently an internucleoside linking group that attaches a tetrahydropyran nucleoside analog to an antisense compound, or T 3 and T 4 One of the internucleoside linking groups that connects the tetrahydropyran nucleoside analog to the antisense compound is T 3 and T 4 the other of which is H, a hydroxyl protecting group, an attached conjugate group, or a group at the 5' or 3' terminus, q 1 , q 2 , q 3 , q 4 , q 5 , q 6 , and q 7 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl, and R 1 and R 2one of which is hydrogen and the other is halogen, substituted or unsubstituted alkoxy, NJ 1 J 2 , S.J. 1 , N3, OC(=X)J 1 , OC(=X)NJ 1 J 2 , N.J. 3 C(=X)NJ 1 J 2 and CN, and X is O, S or NJ 1 and J 1 , J 2 , and J. 3 are each independently H or C1-C6 alkyl.

[0518] In certain embodiments, modified THP nucleosides of formula X are provided, wherein q m , q n , q p , q r , q s , q t , and q u Each of q is H. In certain embodiments, q m , q n , q p , q r , q s , q t , and q u is other than H. In certain embodiments, q m , q n , q p , q r , q s , q t , and q u In certain embodiments, THP nucleosides of formula X are provided, wherein at least one of R 1 and R 2 One of R is F. In certain embodiments, R 1 is fluoro, and R 2 is H and R 1 is methoxy, and R 2 is H, and R 1 is methoxyethoxy, and R 2is H.

[0519] As used herein, "2'-modified nucleoside" or "2'-substituted nucleoside" refers to a nucleoside containing a sugar containing a substituent other than H or OH at the 2' position of the furanose ring. 2'-modified nucleosides include, but are not limited to, bicyclic nucleosides in which the bridge connecting two carbon atoms of the sugar ring connects the 2' carbon of the sugar ring to another carbon. Also included are, for example, allyl, amino, azido, thio, O-allyl, O-C1-C 10 Alkyl, -OCF3, O-(CH2)2-OCH3, 2'-O(CH2)2SCH3, O-(CH2)2-ON(R m )(R n ), or O-CH2-C(=O)-N(R m )(R n ) in which R m and R n each independently represents H or a substituted or unsubstituted C-C 10 2'-Modified nucleosides may further contain other modifications, for example, at other positions on the sugar and / or at the nucleobase.

[0520] As used herein, "2'-F" refers to a sugar containing a fluoro group at the 2' position.

[0521] As used herein, "2'-OMe" or "2'-OCH3" or "2'-O-methyl" each refer to a nucleoside containing a sugar that contains an -OCH3 group at the 2' position of the sugar ring.

[0522] As used herein, "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the nucleosides are modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and / or deoxyribonucleosides (DNA).

[0523] Many other bicyclic and tricyclic sugar substitute ring systems are known in the art and can be used to modify nucleosides for incorporation into antisense compounds (see, for example, review article: Leumann, JC, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.

[0524] Methods for making modified sugars are known to those of skill in the art.

[0525] In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified, or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.

[0526] In certain embodiments, the antisense compound comprises one or more nucleotides having a modified sugar moiety. In certain embodiments, the modified sugar moiety is 2'-MOE. In certain embodiments, the 2'-MOE modified nucleotides are positioned within a gapmer motif. In certain embodiments, the modified sugar moiety is cEt. In certain embodiments, the cEt modified nucleotides are positioned throughout the wings of the gapmer motif.

[0527] Compositions and methods for formulating pharmaceutical compositions Antisense oligonucleotides may be mixed with pharmaceutically acceptable active or inactive substances to prepare pharmaceutical compositions or pharmaceutical preparations.The composition and method for formulating pharmaceutical compositions depend on many criteria, including but not limited to, the route of administration, the extent of disease, or the dosage to be administered.

[0528] Antisense compounds targeting HBV nucleic acids may be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is a suitable diluent for use in parenterally delivered compositions. Thus, in one embodiment, a pharmaceutical composition comprising an antisense compound targeting HBV nucleic acids and a pharmaceutically acceptable diluent is employed in the methods described herein. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.

[0529] Pharmaceutical compositions containing antisense compounds include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotides that can provide (directly or indirectly) biologically active metabolites or residues thereof when administered to animals, including humans.Thus, for example, the present disclosure also covers the pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents.Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts.

[0530] Prodrugs may incorporate additional nucleosides at one or both termini of the antisense compound, and are cleaved by endogenous nucleases within the body to form the active antisense compound.

[0531] The present disclosure provides pharmaceutical compositions comprising a modified oligonucleotide of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the modified oligonucleotide comprises any one of SEQ ID NOs: 11-666. In some embodiments, the modified oligonucleotide comprises any one of SEQ ID NOs: 11-666 and one, two, three, four, or five modifications thereto, as described herein.

[0532] The modified oligonucleotide may be mixed with a pharmaceutically acceptable active or inactive substance to prepare a pharmaceutical composition or pharmaceutical preparation. The composition and method for formulating a pharmaceutical composition depend on many criteria, including but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0533] Pharmaceutical compositions of the present disclosure may optionally include therapeutic agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.

[0534] Antisense compounds targeting HBV nucleic acids may be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is a suitable diluent for use in parenterally delivered compositions. Thus, in one embodiment, a pharmaceutical composition comprising an antisense compound targeting HBV nucleic acids and a pharmaceutically acceptable diluent is employed in the methods described herein. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.

[0535] The pharmaceutical composition comprising modified oligonucleotide can include any pharmaceutically acceptable salt, ester, or salt of such ester, or any other oligonucleotide, which can provide (directly or indirectly) biologically active metabolite or its residue when administered to animals, including humans.Therefore, for example, the present disclosure also covers the pharmaceutically acceptable salts of antisense compounds, prodrugs, the pharmaceutically acceptable salts of such prodrugs, and other biological equivalents.Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salt and potassium salt.

[0536] Modified oligonucleotides formulated as prodrugs are contemplated within the scope of this disclosure. Prodrugs may incorporate additional nucleosides at one or both ends of the antisense compound, and are cleaved by endogenous nucleases in the body to form active antisense compounds.

[0537] A pharmaceutical composition may include any of the reagents described above and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0538] A "pharmaceutical composition" is a formulation containing a modified oligonucleotide described herein in a form suitable for administration to a subject. In certain embodiments, the pharmaceutical composition is in bulk or unit dosage form. The unit dosage form may be in any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single-use syringe, a single pump of an aerosol inhaler, or a vial. The amount of active ingredient (e.g., a formulation of a modified oligonucleotide) in a unit dose of the composition is an effective amount and will vary according to the specific treatment involved. Those skilled in the art will recognize that routine variations in dosage may be necessary depending on the age and condition of the patient. Dosage will also depend on the route of administration. Various routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intrathecal, intranasal, etc. Dosage forms for topical or transdermal administration of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. In certain embodiments, the modified oligonucleotide is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

[0539] "Pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and not biologically or otherwise harmful, useful in preparing pharmaceutical compositions, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use. As used herein, "pharmaceutically acceptable excipient" includes both one and more of such excipients.

[0540] Pharmaceutical compositions are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), intraperitoneal (intrabody cavity), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, intraperitoneal, or subcutaneous administration may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate, and an agent for adjusting tonicity such as sodium chloride or dextrose. pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral or subcutaneous preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials. These preparations can contain antioxidants, buffers, bacteriostats, and solutes to render them isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions can contain suspending agents and thickening agents. The preparations can be in unit / dose or multi-dose containers, for example, sealed ampoules, syringes, and vials, and can be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as saline or water for injection, immediately before use.

[0541] The pharmaceutical compositions described herein may be prepared in a generally known manner, for example, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers containing excipients and / or adjuvants that facilitate processing of the active agent into a pharmaceutically usable preparation. Of course, the appropriate formulation depends on the selected route of administration.

[0542] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0543] Oral compositions generally contain an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of therapeutic oral administration, the active agent may be incorporated into an excipient and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, in which case the agent in the fluid carrier is orally applied, rinsed in the mouth, and expectorated or swallowed. Pharmaceutically compatible binders and / or auxiliary substances may also be included as part of the composition. The tablets, pills, capsules, troches, etc. may contain any of the following ingredients, or agents of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavor.

[0544] For administration by inhalation, the agents are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0545] Pharmaceutical compositions may be prepared using pharmaceutically acceptable carriers that protect modified oligonucleotides from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. For example, biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations are clear to those skilled in the art, and the materials can be obtained commercially. Liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0546] The formulation of oral or parenteral compositions into the form of unit dosage form is particularly advantageous for the ease of administration and uniformity of dosage.As used herein, unit dosage form refers to a physically separate unit that is adapted to be a single dose for the subject to be treated, and each unit contains a predetermined amount of modified oligonucleotide that is calculated to be associated with required pharmaceutical carrier and to produce desired therapeutic effect.The specification of unit dosage form of the present disclosure is determined by and directly depends on the specific characteristics of active agent and the specific therapeutic effect that is achieved.

[0547] The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration.

[0548] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; etc. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.

[0549] Techniques for formulating and administering the disclosed compositions of the present invention can be found in Remingt...

Claims

1. A modified oligonucleotide complementary to a portion of the hepatitis B virus (HBV) genome, comprising sequence number 456.

2. The modified oligonucleotide of claim 1 , wherein at least one internucleoside linkage is a phosphodiester bond.

3. 2. The modified oligonucleotide of claim 1, wherein at least one internucleoside linkage is a modified internucleoside linkage.

4. 4. The modified oligonucleotide of claim 3, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

5. A modified oligonucleotide as described in claim 1, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

6. The modified oligonucleotide described in claim 1, wherein the modified oligonucleotide is linked to a conjugate group.

7. The modified oligonucleotide described in claim 1, wherein the modified oligonucleotide is linked to a stabilizing group.

8. A pharmaceutical composition comprising the modified oligonucleotide according to any one of claims 1 to 7, or a salt thereof, and at least one pharmaceutically acceptable carrier or diluent.

9. A formulation comprising approximately 100 mg / mL, 150 mg / mL, or 200 mg / mL of a modified oligonucleotide described in any one of claims 1 to 7.

10. A pharmaceutical composition for reducing HBV mRNA, HBV DNA, HBV protein, or HBV antigen levels in a subject, comprising the modified oligonucleotide of claim 1.

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

12. A pharmaceutical composition for preventing, treating, ameliorating or delaying the progression of an HBV-related disease, disorder or condition in a subject, comprising a modified oligonucleotide described in claim 1.

13. A pharmaceutical composition for reducing liver toxicity and / or persistence in a subject having an HBV-related disease, disorder or condition, comprising a modified oligonucleotide described in claim 1.

14. 14. The pharmaceutical composition of any one of claims 10 to 13, wherein the subject is administered at least one, at least two, at least three, at least four, or at least five doses of the pharmaceutical composition.

15. The pharmaceutical composition of any one of claims 10 to 13, wherein the subject is administered the pharmaceutical composition on days 1, 4, and 8.

16. 16. The pharmaceutical composition of claim 15, wherein the subject is administered the pharmaceutical composition once a week after day 8.

17. 16. The pharmaceutical composition of claim 15, wherein the amount of each administration is substantially the same.

18. 17. The pharmaceutical composition of claim 16, wherein the amount of each administration is substantially the same.

19. The pharmaceutical composition according to any one of claims 10 to 13, wherein the dosage of the pharmaceutical composition is from 30 mg to 600 mg.

20. 20. The pharmaceutical composition of claim 19, wherein the dose is from 100 mg to 500 mg.

21. 21. The pharmaceutical composition of claim 20, wherein the dose is 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 450 mg, or 500 mg.

22. The pharmaceutical composition of any one of claims 10 to 13, for parenteral, subcutaneous, transdermal, intraocular, intramuscular, or intravenous administration.

23. A pharmaceutical composition described in any one of claims 10 to 13, for administration in combination with a second agent.

24. 14. The pharmaceutical composition of claim 12 or 13, wherein the disease, disorder or condition is a liver disease, disorder or condition.

25. The method of claim 25, wherein the disease, disorder, or condition is jaundice, liver inflammation, liver fibrosis, inflammation, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV infection, HBV viremia, or liver disease-related transplant; or b) the disease or condition is a hyperproliferative condition; 25. The pharmaceutical composition of claim 24.

26. 26. The pharmaceutical composition of claim 25, wherein the hyperproliferative condition is liver cancer.

27. The pharmaceutical composition of any one of claims 10 to 13, wherein the subject is co-infected with hepatitis delta virus (HDV) and HBV.

28. The pharmaceutical composition according to any one of claims 10 to 13, wherein the subject is a human.

29. Use of the modified oligonucleotide described in claim 1 for producing a pharmaceutical composition for reducing the level of HBV mRNA, HBV DNA, HBV protein, or HBV antigen in a subject.

30. The use of claim 29, wherein the HBV antigen is HBsAg or HBeAg.

31. Use of a modified oligonucleotide described in claim 1 for manufacturing a pharmaceutical composition for preventing, treating, ameliorating or delaying the progression of an HBV-related disease, disorder or condition in a subject.

32. Use of a modified oligonucleotide described in claim 1 for producing a pharmaceutical composition for reducing liver toxicity and / or persistence in a subject having an HBV-related disease, disorder or condition.

33. The use of any one of claims 29 to 32, wherein the subject is administered at least one, at least two, at least three, at least four, or at least five doses of the pharmaceutical composition.

34. The use described in any one of claims 29 to 32, wherein the subject is administered the pharmaceutical composition on days 1, 4, and 8.

35. The use of claim 34, wherein the subject is administered the pharmaceutical composition once a week after day 8.

36. The use of claim 34, wherein the amount of each administration is substantially the same.

37. The use of claim 35, wherein the amount of each administration is substantially the same.

38. The use described in any one of claims 29 to 32, wherein the dose of the pharmaceutical composition is 30 mg to 600 mg.

39. The use of claim 38, wherein the dose of the pharmaceutical composition is 100 mg to 500 mg.

40. The use of claim 39, wherein the dose of the pharmaceutical composition is 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 450 mg, or 500 mg.

41. The use of any one of claims 29 to 32, wherein the pharmaceutical composition is for parenteral, subcutaneous, transdermal, intraocular, intramuscular, or intravenous administration.

42. The use of any one of claims 29 to 32, wherein the pharmaceutical composition is for administration in combination with a second agent.

43. The use of claim 31 or 32, wherein the disease, disorder or condition is a liver disease, disorder or condition.

44. A method for treating liver disease, disorder, or condition comprising administering to a patient a therapeutically effective amount of ... b) the disease or condition is a hyperproliferative condition; 44. The use according to claim 43.

45. The use of claim 44, wherein the hyperproliferative condition is liver cancer.

46. ​​The use of any one of claims 29 to 32, wherein the subject is co-infected with hepatitis delta virus infection (HDV) and HBV.

47. The use described in any one of claims 29 to 32, wherein the subject is a human.

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