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 and benefits of U.S. Provisional Patent Application No. 63 / 298,092, filed on 10 January 2022, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Hepatitis B is a viral disease transmitted vertically (parenterally) from an infected or carrier mother to her child through contact with contaminated materials such as blood or blood products, contaminated needles, sexual contact, and other means. The World Health Organization estimates that more than 2 billion people worldwide are infected, with approximately 4 million acute cases and 1 million deaths annually, and 350 to 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] HBV, a virus, is a liver-targeting double-stranded virus that infects only humans and non-human primates. Viral replication primarily occurs in the liver, with some replication also occurring 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 a characteristic antigen-antibody pattern progresses over time. The first detectable viral marker is HBsAg, followed by hepatitis B e antigen (HBeAg) and HBV DNA. Titers may be high during the incubation period, but HIV DNA and HBeAg levels begin to decrease with the onset of the 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 and 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 individuals who have been previously infected.

[0004] The current recommended treatments 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 (INFa), pegylated interferon alfa-2a (Peg-IFN2a), entecavir, and tenofovir. While entecavir and tenofovir, which are nucleoside and nucleotide therapies, have been successful in reducing viral load, the HBeAg seroconversion rate and HBsAg clearance rate are much lower than those achieved with IFNa therapy. Other similar therapies, including lamivudine (3TC), telbivudine (LdT), and adefovir, are also used, but resistance to nucleoside / nucleotide therapies generally emerges, and their therapeutic efficacy is limited.

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

[0006] Antisense technology is emerging as an effective means of reducing the expression of specific gene products and may prove to have unique utility in many therapeutic and diagnostic applications. Antisense therapy differs from nucleoside therapy in that it directly targets the transcription of HBV antigens and can lower serum HBeAg and serum HBsAg levels. Because multiple overlapping transcripts are produced during HBV infection, a type of antisense oligomer may also reduce HBV DNA in addition to both HBeAg and HBsAg. Therefore, antisense technology is emerging as an effective means of reducing the expression of specific gene products and may prove to have unique utility in many therapeutic, diagnostic, and research applications for HBV regulation.

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

[0008] This disclosure extends to the principle of HBV antisense oligonucleotides by providing an improved modified oligonucleotide structure, which provides a further segmented gap structure using one or more separators.

[0009] This disclosure is based, at least in part, on the discovery that discontinuous gap segments adjacent to the 5' wing segment and the 3' wing segment in modified oligonucleotides result in improved activity (e.g., a decrease in serum HBsAg or HBeAg levels) compared to conventional antisense oligonucleotides with continuous gaps. One or more separator segments directly positioned between gap segments can improve the activity of antisense oligonucleotides, a fact that clearly contradicts the current doctrine in the art that separator segments can lead to activity degradation. Unexpectedly, the inventors found that positioning separator segments at specific locations in full-length antisense oligonucleotides improved activity, while separator segments at other locations resulted in decreased activity or no change in activity. Furthermore, they found that only specific types of nucleoside modifications in separator segments resulted in improved activity, while other types of nucleoside modifications in separator segments resulted in decreased activity or no change in activity. While not constrained by theory, certain combinations of nucleoside modifications and arrangements of separator segments within full-length antisense oligonucleotides improve the binding and activity of RNAse H endonucleases without compromising complementarity with HBV target sequences.

[0010] This disclosure is based, at least in part, on the finding that certain nucleoside modifications in the 5' and 3' wing segments reduce in vivo toxicity (e.g., a decrease in ALT levels, a decrease in surrogate values ​​for hepatotoxicity, or a decrease in CC30 (the cytotoxic concentration that reduces cell viability by up to 30%)). Unexpectedly, the inventors found that certain types of modified nucleosides in the 5' wing segment increase in vivo toxicity, while certain types of modified nucleosides in the 3' wing segment decrease it. Furthermore, the location of the modified nucleosides within the 5' and 3' wing segments also contributes to the in vivo toxicity. While not bound by theory, certain combinations of nucleoside modifications and their placement within the 5' and 3' wing segments contribute to the complementarity of full-length antisense oligonucleotides to the HBV target sequence. Certain combinations of specific types of modifications at specific locations improve complementarity, while other combinations of specific types of modifications at specific locations reduce complementarity, resulting in increased off-target binding and ultimately toxicity.

[0011] This specification provides methods, compounds, and compositions for regulating the expression of HBV mRNA and protein. In certain embodiments, the compounds useful for regulating the expression of HBV mRNA and protein are antisense compounds. In certain embodiments, the antisense compounds are antisense oligonucleotides.

[0012] In certain embodiments, regulation may occur in cells or tissues. In certain embodiments, cells or tissues are cells or tissues 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 reductions may occur in a time-dependent or dose-dependent manner.

[0013] Also provided are methods, compounds, and compositions useful for preventing, treating, and improving diseases, disorders, and conditions. In certain embodiments, such HBV-related diseases, disorders, and conditions are liver diseases. In certain embodiments, such liver diseases, disorders, and conditions include jaundice, liver cancer, hepatic inflammation, hepatic fibrosis, inflammation, cirrhosis, hepatic failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, and liver disease-related transplantation. In certain embodiments, such 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] Such 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 disorders include aging, tobacco use, exposure to sunlight and ionizing radiation, contact with certain chemicals, infection with certain viruses and bacteria, certain hormone therapies, 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 disorders 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 treatment method includes administering an HBV antisense compound to an individual in need. In certain embodiments, the treatment method includes administering an HBV antisense oligonucleotide to an individual in need. [Brief explanation of the drawing]

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

[0017] [Figure 2-1] Figure 2 shows a table of exemplary modified oligonucleotides of this disclosure. The modifications at each position in the modified oligonucleotide sequence are read using the description in Figure 1. AUS1010 to AUS1714 (SEQ ID NOs. 11 to 666) represent modified oligonucleotide sequences. AUS1233 (SEQ ID NOs. 10), also referred to as AUS1138, is a 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] Figure 3 shows a schematic diagram of the solid-phase synthesis process used to produce modified oligonucleotides.

[0019] [Figure 4] Figure 4 shows a schematic diagram of the modified oligonucleotide administration regimen and HBsAg sampling schedule for HBV Tg mice.

[0020] [Figure 5] Figure 5 shows a schematic diagram of the modified oligonucleotide administration regimen and HBsAg sampling schedule for C57BL / 6 Tg mice.

[0021] [Figure 6] Figure 6 shows a schematic diagram of the modified oligonucleotide administration regimen for C57BL / 6 Tg mice, as well as the sampling schedule for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.

[0022] [Figure 7] Figure 7 shows a schematic diagram of the modified oligonucleotide administration regimen and HBsAg sampling schedule for pAAV-1.2HBV-GTA HDI-HBV mice.

[0023] [Figure 8]Figure 8 shows a schematic diagram of the modified oligonucleotide administration regimen for C57BL / 6 Tg mice, as well as the sampling schedule for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.

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

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

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

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

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

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

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

[0031] [Figure 16] Figure 16 shows Table 15.3, which represents serum HBsAg levels at days 3, 7, 10, 14, and 21 in GT-C 1.0HBV transgenic mice after subcutaneous treatment with 30 mg / kg of the modified oligonucleotide AUS1434, AUS1435, AUS1440, AUS1436, AUS1437, AUS1438, AUS1439, AUS1441, AUS1422, AUS1423, AUS1424, AUS1425, AUS1426, AUS1427, AUS1428, AUS1429, AUS1430, AUS1431, AUS1432, or AUS1433. Mean values ​​are expressed as log10.

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

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

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

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

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

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

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

[0039] [Figure 24] Figure 24 shows GT-D HDI after subcutaneous treatment with 60 mg / kg of the eASO compound 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. Table 16.4 shows serum HBsAg levels in HBV mice at days 3, 7, 10, and 14. Mean values ​​are expressed as log10.

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

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

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

[0043] [Figure 28] Figure 28 shows Table 16.8, which represents serum HBsAg levels at days 3, 7, 10, 14, and 21 in GT-D HDI HBV mice after subcutaneous treatment with 40 mg / kg of the eASO compound AUS1434, AUS1435, AUS1440, AUS1436, AUS1437, AUS1438, AUS1439, AUS1441, AUS1422, AUS1423, AUS1424, AUS1425, AUS1426, AUS1427, AUS1428, AUS1429, AUS1430, AUS1431, AUS1432, or AUS1433. Mean values ​​are expressed as 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, A Table 16.9 shows serum HBsAg levels at days 3, 7, 10, 14, and 21 in GT-D HDI HBV (1.5ug / ml pcDNA3.1 preS2 / S plasmid) mice after subcutaneous treatment with US1474, AUS1475, AUS1476, AUS1477, AUS1478, AUS1479, AUS1480, AUS1481, AUS1482, AUS1483, AUS1488, AUS1489, AUS1490, AUS1443, AUS1444, or AUS1445 at 40 mg / kg. Mean values ​​are expressed as log10.

[0045] [Figure 30] Figure 30 shows Table 16.10, which represents serum HBsAg levels at days 3, 7, 10, 14, 21, and 28 in GT-D HDI HBV (1.5ug / 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. Mean values ​​are expressed as log10.

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

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

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

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

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

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

[0052] [Figure 37] Figure 37 shows Table 16.17, which represents serum HBsAg levels at days 3, 7, 10, 14, and 21 in GT-A HDI HBV (1.5 ug / ml pAAV-1.2 HBV plasmid) mice after subcutaneous treatment with 40 mg / kg of the eASO compound AUS1396, AUS1422, AUS1423, AUS1424, AUS1425, AUS1426, AUS1427, AUS1428, AUS1429, AUS1430, AUS1431, AUS1432, or AUS1433. Mean values ​​are expressed as 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, AUS14 Table 16.18 shows serum HBsAg levels at days 3, 7, 10, 14, and 21 in GT-A HDI HBV (1.5ug / ml pAAV-1.2HBV plasmid) mice after subcutaneous treatment with 73, AUS1474, AUS1475, AUS1476, AUS1477, AUS1478, AUS1479, AUS1480, AUS1481, AUS1482, AUS1483, AUS1488, AUS1489, AUS1490, AUS1443, or AUS1434 at 40 mg / kg. Mean values ​​are expressed as log10.

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

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

[0056] [Figure 41] Figure 41 shows Table 18.2, which illustrates serum ALT levels in WT male C57BL / 6 mice on days 5, 7, 10, 14, 17, and 21, after subcutaneous treatment with 60 mg / kg of the eASO compound AUS1443, AUS1444, AUS1445, AUS1446, AUS1447, AUS1448, AUS1449, AUS1450, AUS1452, AUS1453, AUS1454, AUS1455, AUS1456, or AUS1457 on days 0, 2, and 4. [Modes for carrying out the invention]

[0057] It should be understood that both the above general description and the following detailed description are merely illustrative and explanatory and do not constitute limitations of the claimed invention. In this specification, the use of the singular includes the plural unless otherwise specified. Where used herein, the use of “or” means “and / or” unless otherwise specified. Furthermore, the use of the term “including,” as well as other forms of the term such as “includes” and “included,” is not limiting. Also, unless otherwise specified, terms such as “element” or “component” include both elements and components containing one unit, and elements and components containing multiple subunits.

[0058] Paragraph headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein. However, all documents or parts of documents cited herein, including but not limited to patents, patent applications, articles, books and papers, are expressly incorporated herein by reference, both in whole and in part, with respect to the parts of documents considered herein.

[0059] definition Unless otherwise specified, the technical terms, procedures, and techniques used herein in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry are publicly known and universally used in the art. Standard techniques may be used for chemical synthesis and chemical analysis. Where permitted, all patents, applications, published applications, and other publications, GENBANK accession numbers, and relevant sequence information obtainable through databases such as the National Center for Biotechnology Information (NCBI), as well as other data referred to through the disclosures herein, are incorporated by reference to and throughout the portions of the documents considered herein.

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

[0061] "2'-O-methoxyethyl" (and further, 2'-MOE and 2'-O(CH2)2-OCH3) refers to the O-methoxyethyl modification at the 2' position of the furanose ring. Sugars modified with 2'-O-methoxyethyl are modified sugars.

[0062] "2'-MOE nucleoside" (and further, 2'-O-methoxyethyl nucleoside) refers to a nucleoside containing a 2'-MOE modified sugar moiety.

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

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

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

[0066] "5-methylcytosine" refers to cytosine modified by the addition of a methyl group at the 5th position. 5-methylcytosine is a modified nucleic acid base.

[0067] "Approximately" means within ±7% of the value. For example, if it states, "The compound affected at least approximately 70% of HBV inhibition," it suggests that HBV levels were inhibited within the range of 63% to 77%.

[0068] An "acceptable safety profile" refers to a pattern of side effects that falls within clinically acceptable limits.

[0069] A "pharmaceutical active 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 that targets HBV is a pharmaceutical active ingredient.

[0070] An "active target region" refers to a target region targeted by one or more active antisense compounds. An "active antisense compound" refers to an antisense compound that reduces the level of a target nucleic acid or protein.

[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 be as short as 45 days or as long as 6 months. For most people, the infection causes mild to moderate discomfort, but the body's immune response wins the fight against the virus, and it disappears on its own. However, in people with a dysfunctional immune system, such as those with AIDS, undergoing chemotherapy, taking immunosuppressants, or taking steroids, acute HBV infection can lead to very serious problems, progressing to more severe conditions such as fulminant liver failure.

[0072] "Simultaneous administration" refers to the co-administration of two drugs in any manner that results in both pharmacological effects appearing simultaneously in the patient. Co-administration does not require both drugs to be administered in a single pharmaceutical composition, in the same dosage form, or via the same route of administration. The effects of both drugs themselves do not need to appear simultaneously. The effects only need to overlap for a certain period and do not need to have the same extent.

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

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

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

[0076] "Animals" 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] An "antibody" is a molecule characterized by its ability to react specifically with an antigen in some way, while antibodies and antigens are defined by other characteristics. An antibody is a complete antibody molecule, or, for example, a heavy chain, a light chain, F ab Region, and F c It can also refer to any fragment or region of a domain or area.

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

[0079] An "antisense compound" refers to an oligomeric compound that can hybridize to a target nucleic acid via hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds such as antisense oligonucleotides, siRNA, shRNA, snoRNA, miRNA, and satellite repeats.

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

[0081] "Antisense mechanisms" are all those mechanisms involved in the hybridization of target nucleic acids and compounds, and the result or effect of hybridization is either the destruction or occupation of the target, for example, accompanied by the simultaneous stalling of cellular machinery involved in transcription or splicing.

[0082] An "antisense oligonucleotide" refers to a single-stranded oligonucleotide that has a nucleic acid base sequence that enables hybridization to a corresponding region or segment of a target nucleic acid.

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

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

[0085] A "bicyclic sugar" refers to a furanose ring modified by a bridge between two non-geminal carbon atoms. Bicyclic sugars are modified sugars.

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

[0087] "Cap structure" or "end cap portion" refers to a chemical modification incorporated into one of the ends of an antisense compound.

[0088] "cEt" or "restricted ethyl" refers to a bicyclic sugar moiety containing a bridge linking a 4'-carbon and a 2'-carbon, in which case the bridge has the following formula: 4'-CH(CH3)-O-2'.

[0089] A "restricted ethyl nucleoside" (or cEt nucleoside) refers to a nucleoside containing a bicyclic sugar moiety with a 4'-CH(CH3)-O-2' bridge.

[0090] A "chemically distinct region" refers to a region of an antisense compound that is chemically different in some respect from another region of the same antisense compound. For example, a region containing a 2'-O-methoxyethyl nucleotide is chemically different from a region containing a nucleotide that does not have the 2'-O-methoxyethyl modification.

[0091] A "chimeric antisense compound" refers to an antisense compound having at least two chemically distinct regions, each having multiple subunits.

[0092] Chronic hepatitis B infection occurs when a person initially suffers from an acute infection and is subsequently unable to fight off that infection. Whether the disease becomes chronic or completely resolves depends primarily on the age of the infected person. Approximately 90% of infants infected at birth progress to chronic disease. However, the risk of chronic infection decreases with age, with 20% to 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 target of the embodiments of the present invention, the ASO compositions of the present invention can also treat HBV-related conditions such as inflammation, fibrosis, cirrhosis, liver cancer, and serum hepatitis.

[0093] "Co-administration" means administering two or more pharmaceuticals to an individual. These two or more pharmaceuticals may be present in a single pharmaceutical composition or in separate pharmaceutical compositions. Each of the two or more pharmaceuticals may be administered via the same or different route of administration. Co-administration includes parallel or sequential administration.

[0094] "Complementarity" refers to the ability to form pairs between the nucleic acid bases of a first nucleic acid and a second nucleic acid.

[0095] "To follow" means that an individual adheres to the recommended treatment method.

[0096] Please understand that "comprise," "comrises," and "comprising" imply the inclusion of the described process or element, or group of processes or elements, but do not imply the exclusion of any other process or element, or group of processes or elements.

[0097] "Continuous nucleic acid bases" refers to nucleic acid bases that are immediately adjacent to each other.

[0098] "Healing" refers to a method or process of restoring health, or a prescribed treatment for a disease.

[0099] A "deoxyribonucleotide" refers to a nucleotide that has a hydrogen atom at the 2' position of the sugar portion. Deoxyribonucleotides may be modified using any of the various substituents.

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

[0101] A "diluent" refers to a component in a composition that is pharmacologically necessary or desirable, but does not have pharmacological activity. For example, in the case of drugs for injection, the diluent may be a liquid, such as physiological saline.

[0102] A "dosage unit" refers to the form in which a drug is provided, such as a pill, a tablet, or any other dosage unit known in the art.

[0103] "Dose" means a specific amount of a drug provided in a single dose or over a specific period of time. In certain embodiments, the dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is preferred, the desired dose requires a volume that cannot be readily accommodated in a single injection. In such embodiments, the desired dose may be achieved using two or more injections. In certain embodiments, the dose may be administered in two or more injections to minimize the individual's injection site reaction. In other embodiments, the drug is administered by intravenous infusion over a long period of time or continuously. The dose may be expressed as the amount of the drug per hour, day, week, or month.

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

[0105] "Duration" refers to the period during which an activity or event persists. In certain embodiments, the duration of treatment is the period during which a dose of the drug is administered.

[0106] In the context of regulating activity or treating or preventing a condition, “effective dose” means administering to a subject requiring such regulation, treatment, or prevention an amount of the active ingredient effective in regulating the effect or treating, preventing, or improving the condition, either as a single dose or as part of a series. The effective dose varies 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, the assessment of the medical condition, and other relevant factors.

[0107] "Effectiveness" refers to the ability to produce a desired effect.

[0108] "Expression" encompasses all functions in which the information encoded by a gene is converted into structures that exist and function within the cell. Such structures are not limited to, but include, the products of transcription and translation.

[0109] The term “fragment” as applied to polynucleotides means a nucleotide sequence that is shorter in length than a reference nucleic acid sequence or reference nucleotide sequence, and will be understood to consist of, essentially consist of, and / or consist of, a sequence of consecutive nucleotides that is identical or nearly identical (e.g., 60%, 70%, 80%, 90%, 92%, 95%, 98%, or 99% identical) to the reference nucleic acid sequence or reference nucleotide sequence. Such nucleic acid fragments according to the present invention may, where appropriate, be contained within a larger polynucleotide of which they are components. In some embodiments, such fragments may contain, essentially consist 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 consecutive nucleotides of the nucleic acid sequence or nucleotide sequence according to the present invention.

[0110] "Completely complementary" or "100% complementary" means that each nucleic acid base of the first nucleic acid has a complementary nucleic acid base in the 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 containing a continuous sequence of nucleosides, in which case each nucleoside is essentially a sugar-modified nucleoside with uniform modification.

[0112] A "gapmer" refers to a chimeric antisense compound in which an internal region containing multiple nucleosides that support RNase H cleavage is located between one or more external regions containing nucleosides, and in this case, the nucleosides containing the internal region may be chemically distinct from the nucleosides or the nucleosides containing the external regions. The internal region may also be called a "gap," and the 5' external region and 3' external region may each be called a "wing."

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

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

[0115] "HBV antigen" means any antigen or protein of the hepatitis B virus, including core proteins such as "hepatitis B core antigen" or "HBcAG" and "hepatitis B E antigen" or "HBeAG," as well as envelope proteins such as "HBV surface antigen" or "HBsAg" or "HBsAG."

[0116] "Hepatitis B E antigen," or "HBeAg," or "HBeAG," is a non-particulate form of the HBV core protein that is secreted. HBV antigens HBeAg and HBcAg share a primary amino acid sequence and therefore exhibit cross-reactivity at the T-cell level. While HBeAg is not required for viral assembly or replication, studies suggest it may be necessary 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). Conversely, infection of young woodchucks with WHeAg-negative mutants resulted in a much lower rate of chronic HBV infection (Cote et al (2000) Hepatology 31:190). HBeAg may function as a tolerance agent by inactivating core-specific T cells via deletion or clonal anergy (Milich et al (1998) J.Immunol.160:8102). There is a positive correlation between the reduction in HBV viral load and antigen during antiviral therapy and seroconversion with HBeAg antibodies and the decrease in T cell expression of 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] "HBV mRNA" refers to any messenger RNA expressed by the hepatitis B virus.

[0118] "HBV nucleic acid" or "HBV DNA" means any nucleic acid that codes for HBV. For example, in certain embodiments, examples include, but are not limited to, the HBV genome or a portion thereof, or any viral DNA sequence that codes for any RNA sequence transcribed from viral DNA containing any mRNA sequence that codes for the HBV protein.

[0119] "HBV protein" refers to any protein secreted by the hepatitis B virus. The term encompasses a variety of HBV antigens, including core proteins such as "hepatitis E antigen," "HBeAg," or "HBeAG," and envelope proteins such as "HBV surface antigen" or "HBsAg."

[0120] "HBV surface antigen," or "HBsAg," or "HBsAG," is the envelope protein of infectious HBV virus particles, but it is also secreted in non-infectious particles, and its serum levels are 1000 times higher than those of HBV virus particles. Serum levels of HBsAg in infected humans or animals can be as high as 1000 gg / 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 response time is 8.3 days (Chulanov et al (2003) J. Med. Virol. 69:313). When HBsAg is internalized by bone marrow dendritic cells, the upregulation of the co-stimulatory molecule (i.e., B7) is inhibited, and T cell stimuli are inhibited (den Brouw et al (2008) Immunology 126:280). In addition, dendritic cells from chronically infected patients show deficiencies in the expression of the co-stimulatory molecule, 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 show altered tetramer binding. These CD8 cells may have a TCR topology that gives partial tolerance or neglect, although they are not anergies (Reignat et al (2002) J. Exp. Med. 195:1089). Furthermore, if serum HBsAg levels decrease by more than 1 log at week 24, a sustained viral response (SVR, defined as HBV DNA being undetectable by PCR one year after treatment) is highly likely to occur during Peg-IFNa2a treatment (92%) (Moucari et al (2009) Hepatology 49:1151).

[0121] "Hepatitis B-related condition" or "HPV-related condition" means any disease, biological condition, medical condition, or event that is worsened, caused, associated with, related to, or traceable to hepatitis B infection, exposure, or disease. The term "hepatitis B-related condition" includes chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocytic inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, liver diseases related to transplantation, and conditions having symptoms that may include any or all of the following: influenza-like illness, malaise, pain, headache, fever, anorexia, diarrhea, nausea and vomiting, pain in the liver region of the body, clay-colored or gray stools, generalized itching, and dark urine when associated with a positive test for the presence of hepatitis B virus, a positive test for hepatitis B virus antigen, or a positive test for antibodies specific to hepatitis B virus antigen.

[0122] "Hybridization" means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, antisense compounds and nucleic acid targets. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, antisense oligonucleotides and nucleic acid targets.

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

[0124] "Identifying animals infected with HBV" means identifying animals diagnosed with HBV, but not limited to identifying animals with any symptoms of HBV infection, including chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, transplant-related liver disease, and any or all of the following: This means: when a positive test result is associated with the presence of hepatitis B virus, hepatitis B virus antigen, or antibodies specific to hepatitis B virus antigen, influenza-like illness, weakness, pain, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain in the liver region of the body, viscous or gray stools, generalized itching, and dark urine.

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

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

[0127] "Individual compliance" refers to adherence to treatments recommended or prescribed by the individual.

[0128] Terms such as "induce," "inhibit," "potentiate," "elevate," "increase," and "decrease" generally indicate a quantitative difference between two states. These terms may refer to a statistically significant difference between two states. For example, "an amount effective in inhibiting HBV activity or expression" means that the level of HBV activity or expression in a treated sample is quantitatively and statistically significant compared to the level of HBV activity or expression in untreated cells. These terms apply, for example, to levels of expression and levels of activity. As used herein, the terms "inhibit" or "decrease" or their grammatical variations refer to a decrease or decline in activity of a particular level or level of 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, the inhibition or reduction results in little to no detectable activity (at most a small amount, e.g., less than about 10% or even less than 5%).

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

[0130] "Inhibiting expression or activity" refers to a reduction or blockage of expression or activity, and does not necessarily mean a complete elimination of expression or activity.

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

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

[0133] "Intraperitoneal administration" refers to administration via injection or infusion into the abdominal cavity.

[0134] "Intravenous administration" means administering the drug intravenously.

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

[0136] "Bound deoxynucleoside" refers to deoxyribonucleic acid bases (A, G, C, T, U) that are linked by phosphate esters to form nucleotides.

[0137] A "bound nucleoside" refers to adjacent nucleosides that are bound together by an internucleoside bond. Examples of bound nucleosides include bound nucleotides, such as phosphodiester bonds, which contain a phosphate atom in the bond.

[0138] "Locked nucleic acid," "LNA," or "LNA nucleoside" refers to a nucleic acid monomer that has a bridge connecting two carbon atoms between the 4' and 2' positions of a nucleoside sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugars are, 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, and (D) aminooxy(4'-CH2-O- Examples include 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 crosslink between the 4' and 2' positions of a sugar, in which case each crosslink 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 )- comprises 1 or 2-4 bonded groups independently selected from, where x is 0, 1 or 2, and n is 1, 2, 3 or 4, R 1 and R 2 Each of these independently consists of H, a protecting group, a hydroxyl group, and C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkinyl, substituted C2-C 12 Alkinyl, 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 , NJ I J 2 SJ 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 2 These are H and C1-C, respectively, independently. 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C12 Alkenyl, C2-C 12 Alkinyl, substituted C2-C 12 Alkinyl, 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 group or a protecting group.

[0140] Examples of 4'-2' bridging groups included in the definition of LNA are not limited to one of the following equations:--[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 Furthermore, other crosslinking groups included in 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', and 4'-CH2-ON(R 1 )-2', and 4'-CH2-N(R 1 )-O-2'-bridged, where R 1 and R 2 Each of these independently represents H, a protecting group, or C1-C 12 It is alkyl.

[0141] Furthermore, the definition of LNA according to the present invention also includes LNA in which the 2'-hydroxyl group of the ribosyl sugar ring is bonded to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy(4'-CH2-O-2') bridge and creating a bicyclic sugar moiety. The bridge may also be a methylene(-CH2-) group bonding the 2' oxygen atom and the 4' carbon atom, in which case the term methyleneoxy(4'-CH2-O-2')LNA is used. Moreover, in the case of a bicyclic sugar moiety having an ethylene bridge group at this position, the term ethyleneoxy(4'-CH2CH2-O-2')LNA is used. Isomers of α-L-methyleneoxy(4'-CH2-O-2') and methyleneoxy(4'-CH2-O-2')LNA are also included within the definition of LNA as used herein.

[0142] "Maximum plasma concentration" or "C max "Maximum concentration" refers to the highest concentration of a drug in the plasma after the drug dose has been administered to the target. Methods for measuring drug concentration are known to those skilled in the art, and include, in particular, liquid chromatography and tandem mass spectrometry.

[0143] A "mismatch" or "non-complementary nucleic acid base" refers to a situation where a nucleic acid base of the first nucleic acid cannot pair with the corresponding nucleic acid base of the second nucleic acid or target nucleic acid.

[0144] "Modified nucleoside bonds" refer to substitutions or changes from the natural nucleoside bond (i.e., phosphodiester nucleoside bonds).

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

[0146] A "modified nucleoside" refers to a nucleoside that independently possesses a modified sugar moiety and / or a modified nucleic acid base.

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

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

[0149] "Modified sugar" means substitution and / or any change from a natural sugar moiety.

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

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

[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. Examples of 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 (miRNA).

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

[0155] "Nucleic acid base complementarity" refers to nucleic acid bases that can form base pairs with other nucleic acid bases. 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 nucleic acid bases refer to nucleic acid bases of an antisense compound that can form base pairs with the nucleic acid bases of the target nucleic acid. For example, if a nucleic acid base at a specific position in an antisense compound can form a hydrogen bond with a nucleic acid base at a specific position in the target nucleic acid, the positions of the hydrogen bonds between the oligonucleotide and the target nucleic acid are considered complementary in terms of the nucleic acid base pair.

[0156] "Nucleic acid base sequence" refers to a continuous sequence of nucleic acid bases, independent of any sugars, bonds, and / or nucleic acid base modifications.

[0157] A "nucleoside" refers to a nucleic acid base linked to a sugar. Nucleosides include deoxynucleosides such as deoxyribonucleosides.

[0158] "Nucleoside mimetic" includes structures used to substitute sugars or sugars and bases, but does not necessarily include structures used to substitute bonds at one or more positions in oligomeric compounds, such as sugar mimetic of morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo, or tricyclo, e.g., nucleoside mimetic having a non-furanose sugar unit. Nucleotide mimetic includes structures used to substitute nucleosides and bonds at one or more positions in oligomeric compounds, such as peptide nucleic acids or morpholino (morpholino linked by -N(H)-C(=O)-O- or other non-phosphodiester bonds). Sugar substitutes overlap with the somewhat broader term nucleoside mimetic, but are intended to indicate substitution of sugar units (furanose rings) only. The tetrahydropyranyl ring provided herein is an example of a sugar substitute, in which case the furanose sugar group is substituted with a tetrahydropyranyl ring system. A "mimetic" refers to a group that is substituted for a sugar, a nucleic acid base, and / or a nucleoside bond. Generally, mimetic groups are used in place of a sugar or a sugar-nucleoside bond combination, while the nucleic acid base is retained for hybridization to a selected target.

[0159] A "nucleotide" refers to a nucleoside in which a phosphate group is covalently bonded to the sugar portion of the nucleoside.

[0160] "Off-target effects" refer to undesirable or harmful biological effects related to the regulation of RNA expression or protein expression of genes other than the intended target nucleic acid.

[0161] An "oligomeric compound" refers to a polymer or linked monomer subunit that can hybridize to at least a nucleic acid molecular region.

[0162] "Oligonucleoside" refers to an oligonucleotide whose nucleoside bonds do not contain a phosphorus atom.

[0163] An "oligonucleotide" refers to a polymer of linked nucleosides, where each nucleoside may or may not be modified independently of the others.

[0164] "Pareral administration" refers to administration by injection (e.g., bolus injection) or intravenous infusion. Parenteral administration includes subcutaneous, intravenous, intramuscular, intra-arterial, intraperitoneal, or intracranial administration, such as subarachnoid or intraventricular administration.

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

[0166] "Pharmacologically acceptable carrier" means a medium or diluent that does not interfere with the structure of the oligonucleotide. With certain such carriers, pharmaceutical compositions can be formulated for oral administration by subjects, for example, as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges.

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

[0168] A "pharmaceutically acceptable salt" means a physiologically and pharmaceutically acceptable salt of an antisense compound, i.e., a salt that retains the desired biological activity of the parent oligonucleotide and does not impart undesirable toxic effects to it.

[0169] A "pharmaceutical" is 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.

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

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

[0172] "Segment" means the nucleobases of a defined number of consecutive (i.e., linked) nucleic acids. In certain embodiments, the segment is the nucleobases of a defined number of consecutive target nucleic acids. In certain embodiments, the segment is the nucleobases of a defined number of consecutive antisense compounds.

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

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

[0175] "Preventive effective amount" refers to the amount of a pharmaceutical product that provides a preventive or prophylactic benefit to an animal.

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

[0177] "Region" is defined as a portion of a target nucleic acid having at least one distinguishable structure, function, or property.

[0178] ? A "ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of the sugar portion. Ribonucleotides can be modified using any of the various substituents.

[0179] "Salt" refers to a physiologically and pharmaceutically acceptable salt of an antisense compound, that is, a salt that retains the desired biological activity of the parent oligonucleotide and does not impart undesirable toxic effects to it.

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

[0181] A “separator” is a segment or region that separates two gap regions in a chimeric antisense compound and is located between the two gap regions. The separator segment may have one or more nucleosides, in which case the nucleosides are chemically distinct from the nucleosides containing the gaps. The separator segment may include nucleosides modified to confer properties such as enhanced inhibitory activity, increased binding affinity to target nucleic acids, reduced in vivo toxicity, or resistance to degradation by nucleases in vivo. The chimeric antisense compound may include one or more separator segments. The separator may be referred to as a “separator,” “separator region,” or “separator segment.” The exemplary chimeric antisense compounds of this disclosure include one, two, three, four, five, or six separator segments.

[0182] "Seroconversion" is defined as the presence of serum HBeAb (no serum HBeAg) when HBeAg was monitored as a determinant of seroconversion, and as the presence of serum HBeAb when HBsAg was monitored as a determinant of seroconversion, based on the currently available detection limits of commercially available ELISA systems.

[0183] The “shortened” or “cleaved” versions of the antisense oligonucleotides taught herein are characterized by the deletion of one, two, or more nucleosides.

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

[0185] As used herein, “significant” means a measurable or observable result, such as a significant outcome. For example, a significant improvement or a significant decrease generally refers to a measurable or observable result, such as a measurable or observable improvement or decrease.

[0186] As used herein, “site” is defined as the specific location of a nucleic acid base within a target nucleic acid.

[0187] "Slowing progression" means a decrease in the development of the disease in question.

[0188] "Specifically hybridizable" refers to an antisense compound that exhibits sufficient complementarity between the antisense oligonucleotide and the target nucleic acid, and induces the desired effect under conditions where specific binding is desirable, i.e., physiological conditions in in vivo assays and therapeutic treatments, while exhibiting minimal or no effect on non-target nucleic acids. "Stringent hybridization conditions" or "stringent conditions" refer to conditions under which the oligomer compound hybridizes to its target sequence but only slightly 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 beneath the skin. "Target" refers to the human or non-human animal selected for the procedure or treatment.

[0191] A "target" refers to a protein whose regulation is desired.

[0192] A "target gene" refers to a gene that codes for a target.

[0193] "Targeting" refers to the process of designing and selecting antisense compounds that specifically hybridize with 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 nucleic acids that can be targeted by antisense compounds.

[0195] The term "target region" refers to the portion of a target nucleic acid that is targeted by one or more antisense compounds.

[0196] The "target segment" refers to the nucleotide sequence of the target nucleic acid targeted by the antisense compound. The "5' target site" refers to the 5' terminal nucleotide of the target segment. The "3' target site" refers to the 3' terminal nucleotide of the target segment.

[0197] "Therapeutic dose" refers to the amount of a drug that provides a therapeutic benefit to an individual.

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

[0199] "Unmodified" nucleic acid bases refer to the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0200] "Unmodified nucleotide" refers to a nucleotide composed of a native nucleic acid base, a sugar moiety, and an internucleoside bond. 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] The "effective target segment" is defined as at least eight nucleic acid base portions (i.e., eight consecutive nucleic acid bases) of the target region targeted by the active oligomer compound.

[0202] A “wing” is a terminal segment of a chimeric antisense oligonucleotide modified to confer properties to the oligonucleotide, such as enhanced inhibitory activity, increased binding affinity to target nucleic acids, reduced in vivo toxicity, or resistance to in vivo degradation by nucleases. A wing may also be referred to as a “wing,” “wing region,” or “wing segment.” As used herein, a wing comprises at least two linked nucleosides, a subset of which may contain one or more deoxynucleosides, but the entire wing does not consist solely of deoxynucleosides.

[0203] The chimeric antisense compounds of this disclosure include a 5' wing segment (W1) located at the 5' end of a chimeric antisense oligonucleotide, wherein the 3' terminal residue of W1 is not a deoxynucleoside. The chimeric antisense compounds of this disclosure also include a 3' wing segment (W2) located at the 3' end of a chimeric antisense oligonucleotide, wherein the 5' terminal residue of W2 is not a deoxynucleoside.

[0204] The 5' wing (W1) begins at the 5' end of the chimeric antisense oligonucleotide, extends from 5' in the 3' direction, and ends at the first nucleoside, which is directly ligated to the deoxynucleoside of the first gap, rather than to 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 from 3' to 5', and ends at the first nucleoside, which is directly ligated to the deoxynucleoside of the gap rather than the deoxynucleoside, thus indicating the 3' end of the final gap and the 5' end of W2.

[0206] Exemplary chimeric antisense oligonucleotides This disclosure provides at least the following exemplary chimeric antisense oligonucleotides.

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

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

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

[0210] In some embodiments, the modified oligonucleotide is 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 is in the 5' to 3' direction.

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

[0213] In some embodiments, the modified oligonucleotide is 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, one or more of G1, G2, G3, G4, G5, G6 and / or G7 contain 1 to 10 linked deoxynucleosides.

[0217] In some embodiments, 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 nucleic acid bases long. In some embodiments, the modified oligonucleotide is at least 20 nucleic acid bases long. In some embodiments, the modified oligonucleotide is 20 nucleic acid bases long.

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

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

[0221] In some embodiments, the modified oligonucleotide is a modified oligonucleotide of formula I, where W1 contains four linked nucleosides, G1 contains five linked deoxynucleosides, S1 contains one linked nucleoside, G2 contains five linked deoxynucleosides, and W2 contains five linked nucleosides.

[0222] In some embodiments, the modified oligonucleotide is of formula I, and the length of G1-S1-G2 is the length of 8 to 12 nucleic acid bases.

[0223] The following embodiments relate to any of formulas I to VI: In some embodiments, G1, G2, G3, G4, G5, G6, and G7 may contain nucleosides that have been modified to a 2'-deoxynucleoside. In some embodiments, one or more of G1, G2, G3, G4, G5, G6, and G7 contain nucleosides that have been modified with 2'-deoxy-5-methylcytidine sugar. In some embodiments, S1, S2, S3, S4, S5, and / or S6 contain nucleosides that have been modified with 2'-O-methoxyethyl sugar. In some embodiments, S1, S2, S3, S4, S5, and / or S6 contain nucleosides that have been modified with 5-methylcytidine. In some embodiments, S1, S2, S3, S4, S5, and / or S6 contain nucleosides that have been modified with 2'-O-methyl sugar. In some embodiments, S1, S2, S3, S4, S5, and / or S6 contain nucleosides with 2'-OH sugar modifications. In some embodiments, S1, S2, S3, S4, S5, and / or S6 contain nucleosides with 2'-fluoro sugar modifications. In some embodiments, S1, S2, S3, S4, S5, and / or S6 contain nucleosides with 2'-fluoro-arabino nucleic acid (2'-fluoro-ANA) sugar modifications. In some embodiments, S1, S2, S3, S4, S5, and / or S6 contain glycol nucleic acid (GNA). In some embodiments, S1, S2, S3, S4, S5, and / or S6 contain LNA. In some embodiments, W1 contains a nucleoside with 2'-deoxy sugar modifications (for example, the 2'-deoxy sugar modifications are located at positions 2 and / or 5 of the sequence corresponding to SEQ ID NO: 2). In some embodiments, W1 contains a nucleoside containing a 2'-O-methoxyethyl sugar modification (for example, the 2'-O-methoxyethyl sugar modification is located at positions 1, 2, 3, 4, and / or 5 of the sequence corresponding to SEQ ID NO: 2). In some embodiments, W1 contains 2'-O-methoxyethyl 5-methylcytidine at position 2 of the sequence corresponding to SEQ ID NO: 2. In some embodiments, W1 contains a nucleoside containing a 2'-O-methyl sugar modification (for example, the 2'-O-methyl sugar modification is located at positions 1, 2, 3, 4, and / or 5 of the sequence corresponding to SEQ ID NO: 2).In some embodiments, W1 contains 2'-O-methyl5-methylcytidine at position 2 of the sequence corresponding to SEQ ID NO: 2. In some embodiments, W1 contains a nucleoside containing a 2'-fluorosaccharide modification. In some embodiments, W1 contains a nucleoside containing a 2'-fluoroarabinonucleotide (2'-fluoro-ANA) modification. In some embodiments, W1 contains glycol nucleic acid (GNA) (e.g., GNA is at position 2 of the sequence corresponding to SEQ ID NO: 2). In some embodiments, W1 contains a modified nucleoside, in which case the modified nucleoside is locked nucleic acid (LNA) (e.g., LNA is 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'-deoxysaccharide modification (e.g., the 2'-deoxysaccharide modification is 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 (for example, the 2'-O-methoxyethyl sugar modification is located at positions 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 (for example, the 2'-O-methyl sugar modification is located at positions 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-arabino nucleic acid (2'-fluoro-ANA) modification. In some embodiments, W2 contains a modified nucleoside, in which case the modified nucleoside is glycol nucleic acid (GNA). In some embodiments, W2 contains a modified nucleoside, in which case the modified nucleoside is locked nucleic acid (LNA) (for example, the LNA is located at positions 16, 17, 18, 19, and / or 20 of the sequence corresponding to SEQ ID NO: 2).

[0224] Exemplary target sequences and exemplary modified oligonucleotides Specific embodiments provide methods, compounds, and compositions for inhibiting HBV mRNA expression.

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

[0226] In certain embodiments, the HBV nucleic acid is the sequence described in GENBANK accession number 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 at positions 1583-1602 of SEQ ID NO: 3.

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

[0228] In some embodiments, the HBV target includes sequences 1, 4, and 675-838. In some embodiments, the HBV target includes the sequence CTTGG TCATG GGCCA TCAG (sequence number 1). In some embodiments, the HBV target includes the sequence GCACT TCGCT TCACC TCTGC (sequence number 4).

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

[0230] In certain embodiments, the HBV target comprises a sequence, a portion thereof, or a variant thereof, as listed in Sequence ID No. 3. 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 the HBV nucleic acid.

[0231] In certain embodiments, the HBV target comprises the sequence listed 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 listed 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 length 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 that bind to the HBV target sequence. In certain embodiments, the compound or composition comprises a modified oligonucleotide of length 20 linked nucleosides that bind to the HBV target sequence.

[0234] In a particular embodiment, the modified oligonucleotide of 20 linked nucleosides has the nucleic acid base sequence GCAGA GGTGA AGCGA AGTGC (SEQ ID NO: 2). A chart showing the positions of the nucleic acid bases in SEQ ID NO: 2 is shown below. [Table 3]

[0235] In a particular embodiment, the modified oligonucleotide of 20 linked nucleosides has the nucleic acid base sequence GTGAA GCGAA GTGCA CACGG (SEQ ID NO: 5). A chart showing the positions of the nucleic acid bases in SEQ ID NO: 5 is shown below. [Table 4]

[0236] In certain embodiments, the compound comprises a modified oligonucleotide as described herein. Examples of modified oligonucleotides, but not limited to those shown in Figures 1-2, include those shown herein. In some embodiments, the compound comprises a modified oligonucleotide containing one of the sequences AUS1010-AUS1714. In some embodiments, the compound comprises a modified oligonucleotide containing one of the sequences SEQ ID NOs 11-666, or a modified oligonucleotide containing one, two, three, four, or five modifications to those sequences.

[0237] In certain embodiments, the compound includes a modified oligonucleotide containing one of the following 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 alphabetical identifier, AUS identifier, and sequence number corresponding to the sequences of the modified oligonucleotides. [Table 5-1] [Table 5-2]

[0238] Characteristics of modified oligonucleotides In a particular embodiment, the compound comprises a modified oligonucleotide of 20 linked nucleosides, consisting of: a first gap segment of linked nucleosides, a second gap segment of linked nucleosides, a separator segment of linked nucleosides to the first and second gap segments, a 5' wing segment of linked nucleosides, and a 3' wing segment of linked nucleosides. In this case, the first gap segment, the separator segment, and the second gap segment are positioned 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, the separator segment, and the second gap segment together consist of 8, 9, 10, 11, or 12 linked nucleosides.

[0239] In a particular embodiment, the compound comprises a modified oligonucleotide consisting of 20 linked nucleosides: a first gap segment consisting of linked nucleosides, a second gap segment consisting of linked nucleosides, a separator segment consisting of nucleosides linked between the first and second gap segments (the first gap segment, the separator segment, and the second gap segment together consist of 8, 9, 10, 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, the second gap segment, and the separator segment are positioned 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, the first gap segment consists of a linked nucleoside containing a 2'-deoxy sugar, the second gap segment consists of a linked nucleoside containing a 2'-deoxy sugar, the separator segment consists of a single nucleoside containing a 2'-O(CH2)2-OCH3 sugar or a 2'-OCH3 sugar, the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 1, and the modified oligonucleotide has the nucleic acid base sequence of SEQ ID NO: 2 (GCAGA GGTGA AGCGA AGTGC).

[0240] In a particular embodiment, the compound comprises a modified oligonucleotide consisting of 20 linked nucleosides: a first gap segment consisting of linked nucleosides, a second gap segment consisting of linked nucleosides, a separator segment consisting of nucleosides linked between the first and second gap segments (the first gap segment, the separator segment, and the second gap segment together consist of 8, 9, 10, 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, the second gap segment, and the separator segment are positioned 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, the first gap segment consists of linked nucleosides containing a 2'-deoxy sugar, the second gap segment consists of linked nucleosides containing a 2'-deoxy sugar, the separator segment consists of one nucleoside containing a 2'-O(CH2)2-OCH3 sugar or a 2'-OCH3 sugar, at least one of the nucleosides in the 5' wing segment contains a 2'-O(CH2)2-OCH3 sugar, at least one of the nucleosides in the 3' wing segment contains a 2'-O(CH2)2-OCH3 sugar, the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 1, and the modified oligonucleotide is 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 nucleosides in the 5' wing segment contain 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 contains two nucleosides, each containing a 2'-deoxy sugar. In certain embodiments, the nucleosides at positions 2 and 3 of SEQ ID NO: 2 contain a 2'-deoxy sugar. In certain embodiments, the nucleosides at positions 2 and 4 of SEQ ID NO: 2 contain a 2'-deoxy sugar. In certain embodiments, the nucleosides at positions 3 and 4 of SEQ ID NO: 2 contain a 2'-deoxy sugar. In certain embodiments, the nucleosides at positions 2 and 5 of SEQ ID NO: 2 contain a 2'-deoxy sugar.

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

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

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

[0245] B2. 5' wing segment w / 4 linked nucleosides, 2'-deoxy combination In certain embodiments, the 5' wing segment consists of four linked nucleosides, in which case one of the linked nucleosides contains a nucleoside having a 2'-deoxy sugar. In certain embodiments, the 5' wing segment consists of four linked nucleosides, in which case two of the linked nucleosides contain a nucleoside having a 2'-deoxy sugar. In certain embodiments, the 5' wing segment consists of four linked nucleosides, in which case 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 a particular embodiment, the 5' wing segment consists of four linked nucleosides, in which case one of the four linked nucleosides contains a nucleoside having a 2'-deoxy sugar, one of the four linked nucleosides contains locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

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

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

[0248] B3. 5' wing segment with 4 linked nucleosides, combined with 2'-LNA In certain embodiments, the 5' wing segment consists of four linked nucleosides, in which case one of the four linked nucleosides contains locked nucleic acid. In certain embodiments, the 5' wing segment consists of four linked nucleosides, in which case two of the four linked nucleosides contain locked nucleic acid. In certain embodiments, the 5' wing segment consists of four linked nucleosides, in which case one of the four linked nucleosides contains a 4'-CH2-O-2' sugar. In certain embodiments, the 5' wing segment consists of four linked nucleosides, in which case two of the four linked nucleosides contain a 4'-CH2-O-2' sugar.

[0249] In certain embodiments, the 5' wing segment consists of four linked nucleosides, in which case one of the four linked nucleosides contains 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 four linked nucleosides, in which case 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.

[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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

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

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

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

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

[0256] C3. 5' wing segment with 5 linked nucleosides, combined with 2'-LNA In certain embodiments, the 5' wing segment consists of five linked nucleosides, in which case one of the five linked nucleosides contains locked nucleic acid. In certain embodiments, the 5' wing segment consists of five linked nucleosides, in which case two of the five linked nucleosides contain locked nucleic acid. In certain embodiments, the 5' wing segment consists of five linked nucleosides, in which case one of the five linked nucleosides contains a 4'-CH2-O-2' sugar. In certain embodiments, the 5' wing segment consists of five linked nucleosides, in which case two of the five linked nucleosides contain a 4'-CH2-O-2' sugar.

[0257] In a particular embodiment, the 5' wing segment consists of five linked nucleosides, in which case one of the five linked nucleosides contains locked nucleic acid, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In a particular embodiment, the 5' wing segment consists of five linked nucleosides, in which case 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.

[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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0259] In a particular embodiment, the 5' wing segment consists of five linked nucleosides, where the nucleosides at positions 1 and 3 of SEQ ID NO: 2 contain locked nucleic acid, 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 nucleosides in the 3' wing segment contain a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the 3' wing segment contains two nucleosides, each of which has a 2'-deoxy sugar.

[0261] In certain embodiments, one, two, three, or four of the nucleosides in the 3'-wing segment include the sugar modifications described herein. In certain embodiments, one, two, three, or four of the nucleosides in the 3'-wing segment include a bicyclic sugar. In certain embodiments, one, two, three, or four of the nucleosides in the 3'-wing segment include a restricted ethyl sugar.

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

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

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

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

[0266] E2. 3' wing segment w / 4 linked nucleosides, 2'-deoxy combination In certain embodiments, the 3' wing segment consists of four linked nucleosides, in which case one of the linked nucleosides contains a nucleoside having a 2'-deoxy sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, in which case two of the linked nucleosides contain a nucleoside having a 2'-deoxy sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, in which case 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 a particular embodiment, the 3' wing segment consists of four linked nucleosides, in which case one of the four linked nucleosides contains a nucleoside having a 2'-deoxy sugar, one of the four linked nucleosides contains locked nucleic acid, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0267] E3. 3' wing segment with 4 linked nucleosides, combined with 2'-LNA In certain embodiments, the 3' wing segment consists of four linked nucleosides, in which case one of the four linked nucleosides contains locked nucleic acid. In certain embodiments, the 3' wing segment consists of four linked nucleosides, in which case two of the four linked nucleosides contain locked nucleic acid. In certain embodiments, the 3' wing segment consists of four linked nucleosides, in which case one of the four linked nucleosides contains a 4'-CH2-O-2' sugar. In certain embodiments, the 3' wing segment consists of four linked nucleosides, in which case two of the four linked nucleosides contain a 4'-CH2-O-2' sugar.

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

[0270] In a particular embodiment, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 16 and 18 of SEQ ID NO: 2 contain locked nucleic acids, and the other two linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0272] In a particular embodiment, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 16 and 19 of SEQ ID NO: 2 contain locked nucleic acids, and the other two linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0274] In a particular embodiment, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 16 and 20 of SEQ ID NO: 2 contain locked nucleic acids, and the other two linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0276] In a particular embodiment, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 17 and 18 of SEQ ID NO: 2 contain locked nucleic acid, 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0278] In a particular embodiment, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 17 and 19 of SEQ ID NO: 2 contain locked nucleic acids, and the other two linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0280] In a particular embodiment, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 17 and 20 of SEQ ID NO: 2 contain locked nucleic acids, and the other two linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0282] In a particular embodiment, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 18 and 19 of SEQ ID NO: 2 contain locked nucleic acid, 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0284] In a particular embodiment, the 3' wing segment consists of four linked nucleosides, where the nucleosides at positions 18 and 20 of SEQ ID NO: 2 contain locked nucleic acids, and the other two linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

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

[0289] In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 contain locked nucleic acids. In certain embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 contain 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 having a 2'-O(CH2)2-OCH3 sugar.

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

[0292] F2. 3' wing segment w / 5 linked nucleosides, 2'-deoxy combination In certain embodiments, the 3' wing segment consists of five linked nucleosides, in which case one of the linked nucleosides contains a nucleoside having a 2'-deoxy sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, in which case two of the linked nucleosides contain a nucleoside having a 2'-deoxy sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, in which case 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 a particular embodiment, the 3' wing segment consists of five linked nucleosides, in which case one of the five linked nucleosides contains a nucleoside having a 2'-deoxy sugar, one of the five linked nucleosides contains locked nucleic acid, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0293] F3. 3' wing segment with 5 linked nucleosides, combined with 2'-LNA In certain embodiments, the 3' wing segment consists of five linked nucleosides, in which case one of the five linked nucleosides contains locked nucleic acid. In certain embodiments, the 3' wing segment consists of five linked nucleosides, in which case two of the five linked nucleosides contain locked nucleic acid. In certain embodiments, the 3' wing segment consists of five linked nucleosides, in which case one of the five linked nucleosides contains 4'-CH2-O-2' sugar. In certain embodiments, the 3' wing segment consists of five linked nucleosides, in which case two of the five linked nucleosides contain 4'-CH2-O-2' sugar.

[0294] In a particular embodiment, the 3' wing segment consists of five linked nucleosides, in which case one of the five linked nucleosides contains locked nucleic acid, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In a particular embodiment, the 3' wing segment consists of five linked nucleosides, in which case three of the five linked nucleosides contain locked nucleic acids, and the other two linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In a particular embodiment, the 3' wing segment consists of five linked nucleosides, in which case 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 a particular embodiment, the 3' wing segment consists of five linked nucleosides, in which case 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.

[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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0296] In a particular embodiment, the 3' wing segment consists of five linked nucleosides, where the nucleosides at positions 16 and 18 of SEQ ID NO: 2 contain locked nucleic acids, and the other three linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0298] In a particular embodiment, the 3' wing segment consists of five linked nucleosides, where the nucleosides at positions 16 and 19 of SEQ ID NO: 2 contain locked nucleic acids, and the other three linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0300] In a particular embodiment, the 3' wing segment consists of five linked nucleosides, where the nucleosides at positions 16 and 20 of SEQ ID NO: 2 contain locked nucleic acids, and the other three linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0302] In a particular embodiment, the 3' wing segment consists of five linked nucleosides, where the nucleosides at positions 17 and 18 of SEQ ID NO: 2 contain locked nucleic acid, 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0304] In a particular embodiment, the 3' wing segment consists of five linked nucleosides, where the nucleosides at positions 17 and 19 of SEQ ID NO: 2 contain locked nucleic acids, and the other three linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0306] In a particular embodiment, the 3' wing segment consists of five linked nucleosides, where the nucleosides at positions 17 and 20 of SEQ ID NO: 2 contain locked nucleic acids, and the other three linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0308] In a particular embodiment, the 3' wing segment consists of five linked nucleosides, where the nucleosides at positions 18 and 19 of SEQ ID NO: 2 contain locked nucleic acid, 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0310] In a particular embodiment, the 3' wing segment consists of five linked nucleosides, where the nucleosides at positions 18 and 20 of SEQ ID NO: 2 contain locked nucleic acids, and the other three linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0312] In a particular embodiment, the 3' wing segment consists of five linked nucleosides, where the nucleosides at positions 19 and 20 of SEQ ID NO: 2 contain locked nucleic acids, and the other three linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0314] In a particular embodiment, the 3' wing segment consists of five linked nucleosides, where the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 contain locked nucleic acids, and the other two linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

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

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

[0318] G2. 3' wing segment w / 6 linked nucleosides, 2'-deoxy combination In certain embodiments, the 3' wing segment consists of six linked nucleosides, in which case one of the linked nucleosides contains a nucleoside having a 2'-deoxy sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, in which case two of the linked nucleosides contain a nucleoside having a 2'-deoxy sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, in which case 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 a particular embodiment, the 3' wing segment consists of six linked nucleosides, in which case one of the six linked nucleosides contains a nucleoside having a 2'-deoxy sugar, one of the six linked nucleosides contains locked nucleic acid, and the other four linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In a particular embodiment, the 3' wing segment consists of six linked nucleosides, in which case one of the six linked nucleosides contains a nucleoside having a 2'-deoxy sugar, two of the five linked nucleosides contain 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 6 linked nucleosides, combined with 2'-LNA In certain embodiments, the 3' wing segment consists of six linked nucleosides, in which case one of the six linked nucleosides contains locked nucleic acid. In certain embodiments, the 3' wing segment consists of six linked nucleosides, in which case two of the six linked nucleosides contain locked nucleic acid. In certain embodiments, the 3' wing segment consists of six linked nucleosides, in which case one of the six linked nucleosides contains 4'-CH2-O-2' sugar. In certain embodiments, the 3' wing segment consists of six linked nucleosides, in which case two of the six linked nucleosides contain 4'-CH2-O-2' sugar.

[0320] In a particular embodiment, the 3' wing segment consists of six linked nucleosides, in which case one of the six linked nucleosides contains locked nucleic acid, and the other five linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In a particular embodiment, the 3' wing segment consists of six linked nucleosides, in which case three of the six linked nucleosides contain locked nucleic acids, and the other three linked nucleosides each contain a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In a particular embodiment, the 3' wing segment consists of six linked nucleosides, in which case 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 a particular embodiment, the 3' wing segment consists of six linked nucleosides, in which case 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.

[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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0322] In a particular embodiment, the 3' wing segment consists of six linked nucleosides, where the nucleosides at positions 16 and 18 of SEQ ID NO: 2 contain locked nucleic acids, and the other four linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0324] In a particular embodiment, the 3' wing segment consists of six linked nucleosides, where the nucleosides at positions 16 and 19 of SEQ ID NO: 2 contain locked nucleic acids, and the other four linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0326] In a particular embodiment, the 3' wing segment consists of six linked nucleosides, where the nucleosides at positions 16 and 20 of SEQ ID NO: 2 contain locked nucleic acids, and the other four linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0328] In a particular embodiment, the 3' wing segment consists of six linked nucleosides, where the nucleosides at positions 17 and 18 of SEQ ID NO: 2 contain locked nucleic acids, and the other four linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0330] In a particular embodiment, the 3' wing segment consists of six linked nucleosides, where the nucleosides at positions 17 and 19 of SEQ ID NO: 2 contain locked nucleic acids, and the other four linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0332] In a particular embodiment, the 3' wing segment consists of six linked nucleosides, where the nucleosides at positions 17 and 20 of SEQ ID NO: 2 contain locked nucleic acids, and the other four linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0334] In a particular embodiment, the 3' wing segment consists of six linked nucleosides, where the nucleosides at positions 18 and 19 of SEQ ID NO: 2 contain locked nucleic acids, and the other four linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0336] In a particular embodiment, the 3' wing segment consists of six linked nucleosides, where the nucleosides at positions 18 and 20 of SEQ ID NO: 2 contain locked nucleic acids, and the other four linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0338] In a particular embodiment, the 3' wing segment consists of six linked nucleosides, where the nucleosides at positions 19 and 20 of SEQ ID NO: 2 contain locked nucleic acids, and the other four linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

[0340] In a particular embodiment, the 3' wing segment consists of six linked nucleosides, where the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 contain locked nucleic acids, and the other three linked nucleosides each contain nucleosides 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 contain locked nucleic acids. 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 contain a 4'-CH2-O-2' sugar.

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

[0343] H-0. Gap Segment In some embodiments, the modified oligonucleotide contains or comprises at least one gap segment. In some embodiments, the modified oligonucleotide contains or comprises at least two gap segments. In some embodiments, the modified oligonucleotide contains 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 segment, 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. In some embodiments, the modified oligonucleotide contains or comprises two gap segments. In some embodiments, the modified oligonucleotide contains or comprises three gap segments. In some embodiments, the modified oligonucleotide contains or comprises four gap segments. In some embodiments, the modified oligonucleotide contains or comprises five gap segments. In some embodiments, the modified oligonucleotide contains or comprises six gap segments. In some embodiments, the modified oligonucleotide contains or consists of seven gap segments.

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

[0345] H. First gap segment In certain embodiments, the first gap segment contains or comprises 1, 2, 3, 4, 5, 6, 7, or 8 linked nucleosides. In certain embodiments, the first gap segment contains or comprises 4 or 5 linked nucleosides. In certain embodiments, the first gap segment contains or comprises 4 linked nucleosides. In certain embodiments, the first gap segment contains or comprises 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 contains or comprises 1, 2, 3, 4, 5, 6, 7, or 8 linked nucleosides. In certain embodiments, the second gap segment contains or comprises 4 or 5 linked nucleosides. In certain embodiments, the first gap segment contains or comprises 4 linked nucleosides. In certain embodiments, the first gap segment contains or comprises 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 modifications. Exemplary modifications of the nucleoside include, but are not limited to, 2'-methoxyethyl nucleoside, 2'-O-methyl nucleoside, 2'OH nucleoside, 2'-fluoro 2'-deoxynucleoside, 2'-F-arabino nucleic 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 containing a 2'-OCH3 sugar. In certain embodiments, the separator segment consists of a single nucleoside containing a 2'-O(CH2)2-OCH3 sugar.

[0353] In certain embodiments, the separator segment is located at position 7, 8, 9, 10, 11, 12, 13, or 14 of sequence number 2. In certain embodiments, the separator segment is located at position 9, 10, or 11 of sequence number 2. In certain embodiments, the separator segment is located at position 10 of sequence number 2.

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

[0355] In certain embodiments, the separator segment consists of a single nucleoside containing a 2'-O(CH2)2-OCH3 sugar. In certain embodiments, the separator segment consists of a single nucleoside containing a 2'-OCH3 sugar. In certain embodiments, the separator segment is located at position 7, 8, 9, 10, 11, 12, 13, or 14 of SEQ ID NO: 2. In certain embodiments, the separator segment is located at position 9, 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 skeleton In certain embodiments, the modified oligonucleotide is a single-strand modified oligonucleotide. In certain embodiments, at least one nucleoside bond is a modified nucleoside bond. In certain embodiments, each nucleoside bond is a phosphorothioate nucleoside bond.

[0357] L. Modified nucleic acid bases In certain embodiments, the modified oligonucleotide contains at least one modified nucleic acid base. In certain embodiments, the modified oligonucleotide contains one, two, or three modified nucleic acid bases. In certain embodiments, the modified nucleic acid base 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 consisting of five linked nucleosides, a first gap segment consisting of four linked nucleosides having a 2'-deoxy sugar, a separator segment at position 10 of SEQ ID NO: 2, a second gap segment consisting of five linked nucleosides having a 2'-deoxy sugar, and a 3'-wing segment consisting of five linked nucleosides. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, respectively. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises internucleoside bonds, each bond being a phosphorothioate internucleoside bond. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, and further comprises internucleoside bonds, each of which is a phosphorothioate internucleoside bond.

[0359] In certain embodiments, the modified oligonucleotide comprises a 5'-wing segment as described in Section C1, C2, or C3, a first gap segment consisting of four linked nucleosides having a 2'-deoxy sugar, a separator segment at position 10 of SEQ ID NO: 2, a second gap segment consisting of five linked nucleosides having a 2'-deoxy sugar, and a 3'-wing segment as described in Section F1, F2, or F3. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, respectively. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises internucleoside bonds, each bond being a phosphorothioate internucleoside bond. In exemplary embodiments, the modified oligonucleotides of this paragraph further include 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, and further include internucleoside bonds, each of which is a phosphorothioate internucleoside bond.

[0360] In certain embodiments, the 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 consisting of four linked nucleosides, a first gap segment consisting of five linked nucleosides having a 2'-deoxy sugar, a separator segment at position 10 of SEQ ID NO: 2, a second gap segment consisting of five linked nucleosides having a 2'-deoxy sugar, and a 3'-wing segment consisting of five linked nucleosides. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, respectively. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises internucleoside bonds, each bond being a phosphorothioate internucleoside bond. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, and further comprises internucleoside bonds, each of which is a phosphorothioate internucleoside bond.

[0362] In certain embodiments, the modified oligonucleotide comprises a 5'-wing segment as described in Section B1, B2, or B3, a first gap segment consisting of five linked nucleosides having a 2'-deoxy sugar, a separator segment at position 10 of SEQ ID NO: 2, a second gap segment consisting of five linked nucleosides having a 2'-deoxy sugar, and a 3'-wing segment as described in Section F1, F2, or F3. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, respectively. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises internucleoside bonds, each bond being a phosphorothioate internucleoside bond. In exemplary embodiments, the modified oligonucleotides of this paragraph further include 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, and further include internucleoside bonds, each of which is a phosphorothioate internucleoside bond.

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

[0364] The following is an example annotation of the location and segment of AO (AUS1493 or Sequence ID 456). [Table 6]

[0365] M3. 5-4-1-6-4 modified oligonucleotides In certain embodiments, the modified oligonucleotide comprises a 5'-wing segment consisting of five linked nucleosides, a first gap segment consisting of four linked nucleosides having a 2'-deoxy sugar, a separator segment at position 10 of SEQ ID NO: 2, a second gap segment consisting of six linked nucleosides having a 2'-deoxy sugar, and a 3'-wing segment consisting of four linked nucleosides. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, respectively. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises internucleoside bonds, each bond being a phosphorothioate internucleoside bond. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, and further comprises internucleoside bonds, each of which is a phosphorothioate internucleoside bond.

[0366] In certain embodiments, the modified oligonucleotide comprises a 5'-wing segment as described in Section C1, C2, or C3, a first gap segment consisting of four linked nucleosides having a 2'-deoxy sugar, a separator segment at position 10 of SEQ ID NO: 2, a second gap segment consisting of six linked nucleosides having a 2'-deoxy sugar, and a 3'-wing segment as described in Section E1, E2, or E3. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, respectively. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises internucleoside bonds, each bond being a phosphorothioate internucleoside bond. In exemplary embodiments, the modified oligonucleotides of this paragraph further include 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, and further include internucleoside bonds, each of which is a phosphorothioate internucleoside bond.

[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 consisting of four linked nucleosides, a first gap segment consisting of five linked nucleosides having a 2'-deoxy sugar, a separator segment at position 10 of SEQ ID NO: 2, a second gap segment consisting of four linked nucleosides having a 2'-deoxy sugar, and a 3'-wing segment consisting of six linked nucleosides. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, respectively. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises internucleoside bonds, each bond being a phosphorothioate internucleoside bond. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, and further comprises internucleoside bonds, each of which is a phosphorothioate internucleoside bond.

[0369] In certain embodiments, the modified oligonucleotide comprises a 5'-wing segment as described in Section B1, B2, or B3, a first gap segment consisting of five linked nucleosides having a 2'-deoxy sugar, a separator segment at position 10 of SEQ ID NO: 2, a second gap segment consisting of four linked nucleosides having a 2'-deoxy sugar, and a 3'-wing segment as described in Section G1, G2, or G3. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, respectively. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises internucleoside bonds, each bond being a phosphorothioate internucleoside bond. In exemplary embodiments, the modified oligonucleotides of this paragraph further include 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, and further include internucleoside bonds, each of which is a phosphorothioate internucleoside bond.

[0370] In certain embodiments, the modified oligonucleotide includes 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 consisting of five linked nucleosides, a first gap segment consisting of four linked nucleosides having a 2'-deoxy sugar, a separator segment at position 10 of SEQ ID NO: 2, a second gap segment consisting of four linked nucleosides having a 2'-deoxy sugar, and a 3'-wing segment consisting of six linked nucleosides. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, respectively. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises internucleoside bonds, each bond being a phosphorothioate internucleoside bond. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, and further comprises internucleoside bonds, each of which is a phosphorothioate internucleoside bond.

[0372] In certain embodiments, the modified oligonucleotide comprises a 5'-wing segment as described in sections C1, C2, or C3, a first gap segment consisting of four linked nucleosides having a 2'-deoxy sugar, a separator segment at position 10 of SEQ ID NO: 2, a second gap segment consisting of four linked nucleosides having a 2'-deoxy sugar, and a 3'-wing segment as described in sections G1, G2, or G3. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, respectively. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises internucleoside bonds, each bond being a phosphorothioate internucleoside bond. In exemplary embodiments, the modified oligonucleotides of this paragraph further include 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, and further include internucleoside bonds, each of which is a phosphorothioate internucleoside bond.

[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 consisting of four linked nucleosides, a first gap segment consisting of five linked nucleosides having a 2'-deoxy sugar, a separator segment at position 10 of SEQ ID NO: 2, a second gap segment consisting of six linked nucleosides having a 2'-deoxy sugar, and a 3'-wing segment consisting of four linked nucleosides. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, respectively. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises internucleoside bonds, each bond being a phosphorothioate internucleoside bond. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, and further comprises internucleoside bonds, each of which is a phosphorothioate internucleoside bond.

[0375] In certain embodiments, the modified oligonucleotide comprises a 5'-wing segment as described in Section B1, B2, or B3, a first gap segment consisting of five linked nucleosides having a 2'-deoxy sugar, a separator segment at position 10 of SEQ ID NO: 2, a second gap segment consisting of six linked nucleosides having a 2'-deoxy sugar, and a 3'-wing segment as described in Section E1, E2, or E3. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, respectively. In exemplary embodiments, the modified oligonucleotide of this paragraph further comprises internucleoside bonds, each bond being a phosphorothioate internucleoside bond. In exemplary embodiments, the modified oligonucleotides of this paragraph further include 5-methylcytosine at positions 2, 13, and 20 of SEQ ID NO: 2, and further include internucleoside bonds, each of which is a phosphorothioate internucleoside bond.

[0376] In certain embodiments, the modified oligonucleotide includes the sequence AX.

[0377] In certain embodiments, one or more modified nucleosides in the 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 includes 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 restricted 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 restricted 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 nucleic acid sequence of the modified oligonucleotide is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to HBV nucleic acid 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 the modified oligonucleotide contains a modified sugar. In certain embodiments, at least one modified sugar contains a 2'-O-methoxyethyl group (2'-O(CH2)2-OCH3). In certain embodiments, the modified sugar contains a 2'-O-CH3 group.

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

[0384] method This disclosure provides a method for treating subjects having HBV infection or a disease, disorder, or condition related to HBV, the method comprising administering a therapeutically effective amount of a modified oligonucleotide described herein or a pharmaceutical composition containing such modified oligonucleotide. In certain embodiments, the modified oligonucleotide comprises any one sequence from SEQ ID NOs: 11 to 666, or comprises one, two, three, four, or five modifications to those sequences.

[0385] In certain embodiments, a modified oligonucleotide, or a pharmaceutical composition containing the same modified oligonucleotide, inhibits the expression of HBV mRNA in a subject. In certain embodiments, a modified oligonucleotide, or a pharmaceutical composition containing the same modified oligonucleotide, inhibits the level of HBV DNA in a subject. In certain embodiments, a modified oligonucleotide, or a pharmaceutical composition containing the same modified oligonucleotide, inhibits the protein and / or antigen levels of HBV in a subject. When administered to a subject, the modified oligonucleotides of this disclosure can reduce the levels of HBV mRNA, DNA, or protein, including, for example, HBV antigens such as HBsAg and HBeAb.

[0386] This disclosure provides a method for treating HBV-related diseases, disorders, and / or conditions in a subject, the method comprising administering a therapeutically effective amount of any of the above-described pharmaceutical compositions to a subject in need to treat HBV-related diseases, disorders, and conditions. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human, and the HBV-related diseases, disorders, and conditions are hepatitis B virus infections derived from human hepatitis B virus. More specifically, the human hepatitis B virus may be any of the following human geographical genotypes: A (Northwestern Europe, North America, Central America), B (Indonesia, China, Vietnam), C (East Asia, South 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, the modified oligonucleotide targets a region of HBV nucleic acid. In certain embodiments, the modified oligonucleotide targeting a region of HBV nucleic acid has a continuous nucleic acid base moiety, the nucleic acid base moiety being complementary to a nucleic acid base moiety of equal length to the region of HBV nucleic acid. For example, the moiety may consist of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 continuous nucleic acid base moieties complementary to the regions of equal length listed herein.

[0388] Certain embodiments provide a method for treating an HBV-related disease, disorder, or condition in a subject, the method comprising administering to the subject in need a modified oligonucleotide, such as a modified oligonucleotide containing one of SEQ ID NOs: 11-666, or a modified oligonucleotide containing one, two, three, four, or five modifications to their sequence, or a pharmaceutical composition described herein.

[0389] A particular embodiment provides a method for reducing HBV expression in a subject, the method comprising administering to the subject a modified oligonucleotide, such as a modified oligonucleotide containing one of sequence numbers 11 to 666, or a modified oligonucleotide containing one, two, three, four, or five modifications to the sequence thereof, or a pharmaceutical composition described herein.

[0390] Certain embodiments provide a method for preventing, improving or treating an HBV-related disease, disorder, or condition in a subject, the method comprising administering to an animal a modified oligonucleotide, such as a modified oligonucleotide containing one of SEQ ID NOs. 11 to 666, or a modified oligonucleotide containing one, two, three, four, or five modifications to their sequence, 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, hepatitis, hepatic fibrosis, cirrhosis, hepatic failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, and HBV viremia. HBV-related conditions or disorders may have symptoms that include one 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, viscous or gray stools, generalized itching, and dark urine. These indicate an HBV-related condition or disorder when combined with a positive test for the presence of hepatitis B virus, hepatitis B virus antigen, or antibodies specific to hepatitis B virus antigen.

[0392] Certain embodiments provide a method for reducing HBV mRNA expression in a subject, the method comprising administering to the subject a modified oligonucleotide, such as a modified oligonucleotide containing one of sequence numbers 11-666, or a modified oligonucleotide containing one, two, three, four, or five modifications to their sequences, or a pharmaceutical composition described herein. In certain embodiments, the reduction of HBV mRNA expression in a subject prevents, improves, or treats HBV-related diseases, disorders, or conditions. In certain embodiments, the reduction of HBV mRNA expression in a subject improves or treats HBV infections. In certain embodiments, the reduction of HBV mRNA expression in a subject prevents, improves, or treats liver diseases. 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 a method for reducing the level of HBV protein in a subject, the method comprising administering to the subject a modified oligonucleotide, such as a modified oligonucleotide containing one of sequence numbers 11-666, or a modified oligonucleotide containing one, two, three, four, or five modifications to their sequence, or a pharmaceutical composition described herein. In certain embodiments, the reduction of the level of HBV protein in a subject prevents, improves, or treats HBV-related diseases, disorders, or conditions. In certain embodiments, the reduction of the level of HBV protein in a subject improves or treats HBV infection. In certain embodiments, the reduction of the level of HBV protein in a subject prevents, improves, or treats liver disease. In certain embodiments, the level of HBV protein 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 a method for reducing the level of HBV DNA in a subject, the method comprising administering to the subject a modified oligonucleotide, such as a modified oligonucleotide containing one of sequence numbers 11-666, or a modified oligonucleotide containing one, two, three, four, or five modifications to their sequences, or a pharmaceutical composition described herein. In certain embodiments, the reduction in the level of HBV DNA in the subject prevents, improves, or treats HBV-related diseases, disorders, or conditions. 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 any of the following human geographical genotypes: A (Northwestern Europe, North America, Central America), B (Indonesia, China, Vietnam), C (East Asia, South 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). In certain embodiments, a reduction in the level of HBV DNA in a subject improves or treats HBV infection. In certain embodiments, a reduction in the level of HBV DNA in a subject prevents, improves, or treats liver disease. In certain embodiments, the level of HBV DNA is 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 a method for reducing the level of HBV antigen in a subject, the method comprising administering to the subject a modified oligonucleotide, such as a modified oligonucleotide containing one of sequence numbers 11-666, or a modified oligonucleotide containing one, two, three, four, or five modifications to their sequence, or a pharmaceutical composition described herein. In certain embodiments, the antigen is HBsAg or HBeAG. In certain embodiments, the reduction in the level of HBV antigen in a subject prevents, improves, or treats HBV-related diseases, disorders, or conditions. In certain embodiments, the reduction in the level of HBV antigen in a subject prevents, improves, or treats liver disease. In certain embodiments, the level of HBV antigen 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 a method for reducing the levels of HBV DNA and HBV antigen in subjects infected with hepatitis B virus, the method comprising administering to the subjects a modified oligonucleotide, such as a modified oligonucleotide containing one of sequence numbers 11-666, or a modified oligonucleotide containing one, two, three, four, or five modifications to their 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 reduced sufficiently to produce seroconversion, which is defined as serum HBeAg-free + serum HBeAg-present when HBeAg was monitored as a determinant of seroconversion, and serum HBsAg-free when HBsAg was monitored as a determinant of seroconversion, as determined by the currently available detection limits of a commercially available ELISA system.

[0397] Certain embodiments provide a method for treating a subject having an HBV-related disease, disorder, or condition, the method comprising: a) identifying the subject having an HBV-related disease, disorder, or condition; and b) administering to the subject a therapeutically effective amount of the 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-related disease, disorder, or condition in the subject. In certain embodiments, the HBV-related disease, disorder, or condition is liver disease. In certain embodiments, the related disease, disorder, or condition is chronic HBV infection, jaundice, liver cancer such as hepatocellular carcinoma, liver inflammation, hepatic fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, transplant-related liver disease, or any combination thereof.

[0398] Certain embodiments provide a method for treating a subject having an HBV-related disease, disorder, or condition, the method comprising: a) identifying the subject having an HBV-related disease, disorder, or condition; and b) administering to the subject a therapeutically effective amount of the 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-related disease, disorder, or condition in the subject. In certain embodiments, the HBV-related disease, disorder, or condition is liver disease. In certain embodiments, the related 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-related liver disease.

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

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

[0401] In certain embodiments, the subject is a monkey, such as a crab-eating macaque.

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

[0403] In certain embodiments, a modified oligonucleotide or pharmaceutical composition is designated as the first agent. In certain embodiments, the method comprises 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, one or more second agents are compounds or compositions described herein. In certain embodiments, one or more second agents are different from compounds or compositions described herein. Examples of 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 one or more second agents include, for example, chemotherapeutic agents such as gemcitabine (Gemzar), oxaliplatin (Eloxatin), cisplatin, doxorubicin, 5-fluorouracil, capecitabine (Xeloda), or mitoxantrone (Novantrone). In certain embodiments, the disease includes liver disease, and one or more second agents include corticosteroids, diuretics, beta-blockers, or combinations thereof.

[0405] The modified oligonucleotides of this disclosure and pharmaceutical compositions comprising them can be administered to a subject by any preferred route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), intraperitoneal (intracavitary), and transmucosal administration.

[0406] In certain embodiments, administration includes parenteral administration. In certain embodiments, administration includes subcutaneous administration. In certain embodiments, administration includes intravenous injection or infusion.

[0407] Certain embodiments provide 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 a therapeutically effective dose of the above-described pharmaceutical composition to a subject in need, thereby reducing 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 human, and the hepatitis B virus may be human hepatitis B virus. More specifically, the human hepatitis B virus may be any of the following human geographical genotypes: A (Northwestern Europe, North America, Central America), B (Indonesia, China, Vietnam), C (East Asia, South 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] This 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 a therapeutically effective amount of the modified oligonucleotide or a pharmaceutical composition containing the same to the subject to reduce 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 to the amount of modified antisense oligonucleotide or pharmaceutical composition before administration. In certain embodiments, the amount of mRNA is reduced by at least 80% compared to the amount of modified antisense oligonucleotide or pharmaceutical composition before administration. In certain embodiments, the amount of mRNA is reduced by at least 90% compared to the amount of modified antisense oligonucleotide or pharmaceutical composition before administration.

[0409] This disclosure provides a method for reducing the amounts of HBV mRNA, DNA, protein, and / or HBV antigen in a subject infected with hepatitis B virus, the method comprising administering a therapeutically effective dose of the modified oligonucleotide or pharmaceutical composition described above to the subject to reduce hepatitis B virus infection and hepatitis B antigen compared to the amounts of HBV virus, mRNA, DNA, protein, and / or HBV antigen in the subject before treatment, in which case 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 particular embodiment, the method reduces the amounts of HBV virus, mRNA, DNA, protein, and / or HBV antigen in a subject infected with hepatitis B virus, the method comprising administering a therapeutically effective dose of the modified oligonucleotide or pharmaceutical composition described above to the subject to reduce hepatitis B virus infection and hepatitis B antigen compared to the amounts of hepatitis B virus and HBV antigen in the subject before treatment, in which case 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, the method reduces the amount of HBV virus, 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 modified oligonucleotide or pharmaceutical composition described above to the subject to reduce 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, in which case 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, the method reduces the amount of HBV virus, 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 modified oligonucleotide or pharmaceutical composition described above to the subject to reduce 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, in which case 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, the method reduces the amount of HBV virus, 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 modified oligonucleotide or pharmaceutical composition described above to the subject to reduce 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, in which case the amount of mRNA is reduced by at least 95% compared to the amount before administration of the modified oligonucleotide or pharmaceutical composition. In the relevant methods, the HBV antigen may be HBsAg or HBeAg, and more specifically, the amount of HBV antigen may be reduced sufficiently to produce seroconversion, and seroconversion is defined as serum HBeAg absent + serum HBeAb present when HBeAg is monitored as a determinant of seroconversion, when determined by the currently available detection limits of a commercially available ELISA system, and as serum HBsAg absent when HBsAg is monitored as a determinant of seroconversion.

[0410] This disclosure provides a method for promoting seroconversion of hepatitis B virus antibodies in mammals infected with HBV, the method comprising administering a therapeutically effective amount of a modified oligonucleotide, such as a modified oligonucleotide containing one of sequence numbers 11 to 666, or a modified oligonucleotide containing one, two, three, four, or five modifications to their sequences, or a therapeutically effective amount of the above-mentioned pharmaceutical composition, to a subject infected with hepatitis B virus, and monitoring the presence of HBeAg + HBeAb in the subject's serum sample, or monitoring the presence of HBsAg in the subject's serum sample, as determined by the current detection limits of a commercially available ELISA system, and if HBeAg was being monitored as a determinant of seroconversion, the absence of HBeAg + the presence of HBeAb in the serum sample, or if HBsAg was being monitored as a determinant of seroconversion, the absence of HBsAg in the serum sample, indicates seroconversion in the subject.

[0411] Certain embodiments provide the use of modified oligonucleotides, such as modified oligonucleotides containing any one of SEQ ID NOs: 11-666, or modified oligonucleotides containing one, two, three, four, or five modifications to their sequences, or pharmaceutical compositions described herein, for preventing, improving, or treating liver disease or its symptoms in a subject.

[0412] In some embodiments, the EC of cells treated with modified oligonucleotides (e.g., SEQ ID NOs: 11-666) 50 This was measured as a surrogate for the effectiveness of the modified oligonucleotide. In some embodiments, EC 50 The EC range is approximately 0.1 nM to approximately 250 nM. In some embodiments, 50This includes 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, The levels are less than 25nM, less than 24nM, less than 23nM, less than 22nM, less than 21nM, less than 20nM, less than 19nM, less than 18nM, less than 17nM, less than 16nM, less than 15nM, less than 14nM, less than 13nM, less than 12nM, less than 11nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, less than 1nM, less than 0.9nM, less than 0.8nM, less than 0.7nM, less than 0.6nM, less than 0.5nM, less than 0.4nM, less than 0.3nM, less than 0.2nM, or less than 0.1nM.

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

[0414] In certain embodiments, the compounds or compositions described herein, when delivered to HepG2.2.1 cells, produce in vitro ICs 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, and less than 46 nM. 50 It is valid by having at least one of the following.

[0415] In certain embodiments, the compounds or compositions described herein, when delivered to HepG2.2.1 cells, produce in vitro ICs 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, and less than 31 nM. 50 It is valid by having at least one of the following.

[0416] In certain embodiments, when the compounds or compositions described herein are delivered to HepG2.2.1 cells, they produce 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, and less than 2.5 μM. 50 It is valid by having 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 the modified oligonucleotides described herein is about 10 nM to about 250 nM. In some embodiments, the MTT CC25 is higher than 10 nM, higher than 15 nM, higher than 20 nM, higher than 25 nM, higher than 30 nM, higher than 35 nM, higher than 40 nM, higher than 45 nM, higher than 50 nM, higher than 55 nM, higher than 60 nM, higher than 65 nM, higher than 70 nM, higher than 75 nM, higher than 80 nM, higher than 85 nM, higher than 90 nM, higher than 95 nM It is also high, higher than 100nM, higher than 110nM, higher than 120nM, higher than 130nM, higher than 140nM, higher than 150nM, higher than 160nM, higher than 170nM, higher than 180nM, higher than 190nM, higher than 200nM, higher than 210nM, higher than 220nM, higher than 230nM, higher than 240nM, or higher than 250nM.

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

[0419] In some embodiments, the "C / E ratio" is used as a measure of effectiveness in reducing HBsAg levels compared to cytotoxicity. In some embodiments, the C / E ratio is taken as the ratio of MTT CC25(nM) of the modified oligonucleotide to the MTT CC25(nM) of the reference oligonucleotide (e.g., SEQ ID NO: 10), and this is used to measure the HBsAg EC of the modified oligonucleotide (e.g., SEQ ID NOs: 11-666) (nM). 50 The C / E ratio is calculated by dividing by the ratio of HBsAg EC50 of the reference oligonucleotide (SEQ ID NO: 10). In some embodiments, the C / E ratio is taken as the ratio of CCK8 CC25(nM) of the modified oligonucleotide / CCK8 CC25(nM) of the reference oligonucleotide (e.g., SEQ ID NO: 10), and this is divided by the ratio of (nM) HBsAg EC50 of the modified oligonucleotide (e.g., SEQ ID NOs: 11-666). 50It is calculated by dividing by the ratio of HBsAg EC50 of the reference oligonucleotide (SEQ ID NO: 10). In some embodiments, the C / E ratio is approximately 0.01 to approximately 50. 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 having at least one of the following: an increase in ALT or AST values ​​of 4 times or less, 3 times or less, or 2 times or less compared to animals treated with physiological saline, or an increase in liver, spleen, or kidney weight of 30% or less, 20% or less, 15% or less, 12% or less, 10% or less, 5% or less, or 2% or less. In certain embodiments, the compounds or compositions described herein are highly tolerable, as indicated by the absence of an increase in ALT or AST compared to animals treated with physiological saline. In certain embodiments, the compounds or compositions described herein are highly tolerable, as indicated by the absence of an increase in liver, spleen, or kidney weight compared to animals treated with physiological saline.

[0421] In some embodiments, animals treated with a compound or composition containing modified oligonucleotides (e.g., SEQ ID NOs: 11-666) have ALT values ​​that are approximately 1.1 to 10 times lower compared to animals treated with a reference compound (e.g., SEQ ID NOs: 10). In some embodiments, animals treated with a compound or composition containing modified oligonucleotides (e.g., SEQ ID NOs: 11-666) have ALT values ​​that are at least 1.25 times lower compared to animals treated with a reference compound (e.g., SEQ ID NOs: 10). In some embodiments, animals treated with a compound or composition containing modified oligonucleotides (e.g., SEQ ID NOs: 11-666) have ALT values ​​that are 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, and 2.3 times lower compared to animals treated with a reference compound (e.g., SEQ ID NOs: 10). It has an ALT value that is reduced by 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, or 5.0 times.

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

[0423] Certain embodiments provide the use of modified oligonucleotides, such as any one of SEQ ID NOs: 11-666, or the pharmaceutical compositions described herein, for the manufacture of pharmaceuticals for treating, improving, delaying, or preventing HBV-related diseases, disorders, or conditions in animals.

[0424] Certain embodiments provide the use of modified oligonucleotides, such as any one of SEQ ID NOs: 11-666, or the pharmaceutical compositions described herein, for the manufacture of pharmaceuticals for treating, improving, delaying, or preventing liver disease in animals.

[0425] Kits and manufactured products This disclosure provides kits comprising modified oligonucleotides described herein, and kits comprising pharmaceutical compositions comprising such modified oligonucleotides. In some embodiments, the modified oligonucleotides comprise any one sequence from SEQ ID NOs: 11 to 666, or modified oligonucleotides comprising one, two, three, four, or five modifications to those sequences.

[0426] Certain embodiments provide a kit for treating, preventing or improving HBV-related diseases, disorders or conditions, as described herein, the kit comprising a) a modified oligonucleotide, for example, containing one of the sequences of SEQ ID NOs: 11-666, or a composition containing a modified oligonucleotide with one, two, three, four, or five modifications to those sequences, and optionally, b) additional agents or treatments as described herein. The kit further comprises instructions or labels for using the kit to treat, prevent or improve HBV-related diseases, disorders or conditions.

[0427] Compositions containing modified oligonucleotides may be lyophilized and then packaged in kits, or they may be provided in solution with pharmaceutically acceptable carriers and excipient diluents.

[0428] In certain embodiments, the kit further includes at least one additional agent for treating HBV-related diseases, disorders, or conditions.

[0429] Medication and administration This disclosure provides a method comprising administering a therapeutically effective amount of a modified oligonucleotide, for example, a modified oligonucleotide containing one of sequence numbers 11 to 666, or a modified oligonucleotide containing one, two, three, four, or five modifications to their sequence, or a pharmaceutical composition containing the same, to a subject in need.

[0430] In certain embodiments, for example, a therapeutically effective dose of a modified oligonucleotide or a pharmaceutical composition containing the same includes a dose that results in an observed maximum log10 serum HBsAg decrease 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. In some embodiments, the maximum log10 serum HBsAg decrease 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 dose.

[0431] In certain embodiments, for example, a therapeutically effective dose of a modified oligonucleotide or a pharmaceutical composition containing the same includes a dose that reduces the HBsAg level in the target serum 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 dose.

[0432] In certain embodiments, for example, a therapeutically effective dose of a modified oligonucleotide or a pharmaceutical composition containing the same includes a dose that results in an observed maximum log10 serum HBeAg decrease 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. In some embodiments, the maximum log10 serum HBeAg decrease 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 dose.

[0433] In certain embodiments, for example, a therapeutically effective dose of a modified oligonucleotide or a pharmaceutical composition containing the same includes a dose that reduces the HBeAg level in the target serum 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 dose.

[0434] In certain embodiments, modified oligonucleotides or pharmaceutical compositions containing them are available in doses 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, and 5 mg. 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 is administered to the subject in doses ranging from 50 mg to 500 mg, 200 mg to 400 mg, 200 mg to 300 mg, 300 mg to 1500 mg, 300 mg to 1000 mg, 300 mg to 900 mg, 300 mg to 800 mg, 300 mg to 700 mg, 300 mg to 600 mg, 300 mg to 500 mg, or 300 mg to 400 mg. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in doses ranging from 50 mg to 1500 mg of modified oligonucleotides. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in doses ranging from 50 mg to 1000 mg of modified oligonucleotides. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in doses ranging from 50 mg to 700 mg of modified oligonucleotides. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in doses ranging from 50 mg to 500 mg of the modified oligonucleotide.In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in a dose ranging from 50 mg to 450 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in a dose ranging from 50 mg to 300 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in a dose ranging from 50 mg to 200 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in a dose ranging from 100 mg to 1500 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in a dose ranging from 100 mg to 1000 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in a dose ranging from 100 mg to 700 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in a dose ranging from 100 mg to 500 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in a dose ranging from 100 mg to 450 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in a dose ranging from 100 mg to 300 mg of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject in a dose ranging from 100 mg to 200 mg of the modified oligonucleotide.

[0435] In certain embodiments, modified oligonucleotides or pharmaceutical compositions containing the same are available in concentrations 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, and 0.01 mg / kg. 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.It is administered in doses within the range of modified oligonucleotides: 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 a pharmaceutical composition containing the same is administered in doses ranging from 0.01 mg / kg to 30 mg / kg. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered in doses ranging from 0.1 mg / kg to 25 mg / kg. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered in doses ranging from 1.0 mg / kg to 20 mg / kg. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered in doses ranging from 5.0 mg / kg to 20 mg / kg. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered in doses ranging from 1.0 mg / kg to 15 mg / kg. In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered in doses ranging from 1.0 mg / kg to 5.0 mg / kg.

[0436] In certain embodiments, a modified oligonucleotide or a pharmaceutical composition containing the same is administered to a subject daily, every two days, every three days, every four days, every five days, every six days, every seven days, every eight days, every nine days, every ten days, every eleven days, every twelve days, every thirteen days, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every eleven weeks, every twelve weeks, every thirteen weeks, every two months, every three months, or every four months.

[0437] In certain embodiments, the modified oligonucleotide or a pharmaceutical composition containing the same is administered to the subject daily, every two days, every three days, every four days, every five days, every six days, every seven days, every eight days, every nine days, every ten days, every two weeks, every three weeks, or monthly.

[0438] In certain embodiments, a modified oligonucleotide or a pharmaceutical composition containing the same is administered to a subject for at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least one year, at least 1.5 years, at least two years, at least three years, at least four years, or at least five years.

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

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

[0441] In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject daily. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every two days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every three days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every four days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every five days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every six days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every seven days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every eight days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every nine days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every ten days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every eleven days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every 12 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every 13 days. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every two weeks. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every three weeks. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every month. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every two months. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every three months. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every four months.In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every five months. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject every six months. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject at least twice a year. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject at least twice every two years. In certain embodiments, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to the subject at least twice every two years or more.

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

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

[0444] In some embodiments, the administration of a modified oligonucleotide, or a pharmaceutical composition containing the same, includes a drug-free period. For example, the modified oligonucleotide, or a pharmaceutical composition containing the same, is administered to a subject every three days, followed by a period of one, two, three weeks, or one month without administration, after which administration is resumed. Those skilled in the art will understand that this drug-free period is illustrative. Other durations and frequencies of drug-free periods are also expected within the scope of this disclosure. As a further example, the modified oligonucleotide, or a pharmaceutical composition containing the same, may be administered to a subject until the level of HBsAg or HbeAg falls below a certain threshold, or until HBV infection is no longer detected in the subject, followed by a drug-free period, after which administration is resumed if HBsAg or HbeAg is detected again in the subject's serum.

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

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

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

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

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

[0450] In an exemplary dosing regimen including a loading dose and a maintenance dose, the modified oligonucleotide or pharmaceutical composition is administered at a higher loading dose, followed by a lower maintenance dose. For example, the loading dose may 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 may be administered more frequently than the maintenance dose. For example, the loading dose may be administered daily, every two days, every three days, every four days, or every five days for at least one, two, three, four, or five doses, followed by a maintenance dose weekly, every ten days, every fourteen days, etc. Those skilled in the art will understand that, if necessary, a loading dose may be administered again to a subject who has been administered according to a maintenance dosing schedule. In a further exemplary dosing schedule, a subject may be administered a loading dose on days 1 and 4, followed by a weekly maintenance dose on day 8.

[0451] Antisense compounds Oligomer compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetic compounds, antisense compounds, antisense oligonucleotides, and siRNAs. The oligomeric compound may be "antisense" with respect to the target nucleic acid, meaning that it can hybridize to the target nucleic acid via hydrogen bonding.

[0452] In certain embodiments, the antisense compound, when described in the 5'-3' direction, has a nucleic acid base sequence that includes the reverse complement of the target segment of the target nucleic acid. In certain such embodiments, the antisense oligonucleotide, when described in the 5'-3' direction, has a nucleic acid base sequence that includes the reverse complement of the target segment of the target nucleic acid.

[0453] In certain embodiments, the antisense oligonucleotide targeting HBV nucleic acid may be truncated or cleaved. For example, one subunit may be deleted from the 5' end (5' cleavage) or from the 3' end (3' cleavage). The truncated or cleaved antisense compound targeting HBV nucleic acid 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, if one additional subunit is present, the additional subunit may be located at the 5' or 3' end of the antisense compound. If two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound, two subunits may be added to the 5' end (5' addition), or two subunits may be added to the 3' end (3' addition). Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound, one subunit may be added to the 5' end and one subunit to the 3' end.

[0455] It is also possible to increase or decrease the length of antisense compounds, such as antisense oligonucleotides, and / or introduce mismatched bases without losing their 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 in length from 13 to 25 nucleic acid bases were validated for their ability to induce target RNA cleavage in an oocyte injection model. Antisense oligonucleotides with 25 nucleic acid bases in length, containing 8 or 11 mismatched bases near the end, were able to induce specific cleavage of target mRNA, albeit to a lower degree than antisense oligonucleotides without mismatches. Similarly, target-specific cleavage was achieved using 13 nucleic acid base antisense oligonucleotides, some containing 1 or 3 mismatched bases.

[0456] Gautschi et al. (Natl. Cancer Inst. 93:463-471, March 2001) demonstrated that an oligonucleotide that was 100% complementary to bcl-2 mRNA and had three mismatches with bc1-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) investigated the ability of tandem 14-nucleotide antisense oligonucleotides, and 28-nucleotide and 42-nucleotide antisense oligonucleotides composed of two or three sequences of each tandem antisense oligonucleotide, to halt human DHFR translation in a rabbit reticulocyte assay. Each of the three 14-nucleotide antisense oligonucleotides individually was able to inhibit translation, albeit to a moderate level compared to the 28-nucleotide or 42-nucleotide antisense oligonucleotides.

[0458] Antisense compound motif In certain embodiments, the antisense compound targeting HBV nucleic acid has chemically modified subunits arranged in patterns or motifs that confer properties to the antisense compound, such as enhanced inhibitory activity, increased binding affinity to the target nucleic acid, or resistance to degradation by nucleases in vivo.

[0459] Typically, a chimeric antisense compound contains at least one modified region that confers increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity to target nucleic acids, and / or increased inhibitory activity. The second region of the chimeric antisense compound may optionally function as a substrate for RNase H, a cellular endonuclease that cleaves the RNA strand of an RNA:DNA double-stranded molecule.

[0460] Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer, an internal region having multiple nucleotides that support RNaseH cleavage is located between an external region having multiple nucleotides that are chemically distinct from the nucleoside of the internal region. In antisense oligonucleotides having a gapmer motif, the gap segment generally functions as a substrate for endonuclease cleavage, while the wing segment contains a modified nucleoside. In certain embodiments, the gapmer region is identified by the type of sugar moiety, each comprising distinctly different regions. 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, in particular, 2'-MOE and 2'-O-CH3), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides may include those having a restricted ethyl group). In certain embodiments, the nucleoside in the wing may include several modified sugar moieties, such as 2'-MOE and bicyclic sugar moieties such as restricted ethyl or LNA. In certain embodiments, the wing may include several modified and unmodified sugar moieties. In certain embodiments, the wing may include various combinations of 2'-MOE nucleosides, bicyclic sugar moieties such as restricted ethyl nucleoside or LNS nucleoside, and 2'-deoxynucleosides.

[0461] Each separate region may contain a uniform sugar moiety, a variant sugar moiety, or an alternative sugar moiety. The wing-gap-wing motif is often written 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 contain uniform, variant, or alternative sugar moieties. In certain embodiments, "X" and "Y" may contain one or more 2'-deoxynucleosides. "Y" may contain a 2'-deoxynucleoside. As used herein, a gapmer described as XYZ has a configuration such that the gap is located immediately adjacent to the 5'-wing and the 3'-wing, respectively. Therefore, there are no nucleotides interposing 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 identical, while in other embodiments they are different. In certain embodiments, "Y" is 8 to 15 nucleosides. X, Y, or Z may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 nucleosides, or any of these.

[0462] Target nucleic acid, target region, and nucleotide sequence In certain embodiments, the nucleotide sequence encoding HBV may include, but is not limited to, GENBANK accession U95551.1 (incorporated herein as Sequence ID No. 1).

[0463] It should be understood that the sequences described in each sequence number of the examples contained herein are independent of any modifications to the sugar moiety, nucleoside bond, or nucleic acid base. However, the antisense compounds defined by the sequence numbers may independently contain one or more modifications to the sugar moiety, nucleoside bond, or nucleic acid base.

[0464] In certain embodiments, the target region is a structurally defined region of the target nucleic acid. For example, the target region may encompass the 3'UTR, 5'UTR, exons, introns, exon / intron junctions, coding regions, translation start regions, translation termination regions, or other defined nucleic acid regions. 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 encompass 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 in which the antisense compound hybridizes to produce the desired effect. In certain embodiments, the desired effect is a decrease in the mRNA level of the target nucleic acid. In certain embodiments, the desired effect is a decrease in the level of the protein encoded by the target nucleic acid, or a phenotypic change related to the target nucleic acid.

[0466] The target region may contain one or more target segments. Multiple target segments within the target region may overlap, or they may not overlap. In certain embodiments, the target segments within the target region are separated by approximately 300 nucleotides or less. In certain embodiments, the target segment within the target region is separated by a number of nucleotides on the target nucleic acid, namely 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides, approximately 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides, or 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 or fewer nucleotides, or nucleotides within a range defined by any two of the aforementioned values. In certain embodiments, target segments within a target region are separated by 5 nucleotides, or approximately 5 nucleotides or less, on the target nucleic acid. In certain embodiments, the target segments are contiguous. A target region is expected, defined by a range having an initiating nucleic acid, which is either a 5' target site or a 3' target site as enumerated herein.

[0467] A suitable target segment may be located within the 5'UTR, coding region, 3'UTR, intron, exon, or exon / intron junction. A target segment containing a start codon or stop codon is also a suitable target segment. A suitable target segment may specifically exclude certain structurally defined regions, such as start codons or stop codons.

[0468] Determining an appropriate target segment may involve comparing the target nucleic acid sequence with other sequences across the entire genome. For example, the BLAST algorithm may be used to identify regions of similarity between different nucleic acids. Performing this comparison can prevent the selection of antisense compound sequences that may nonspecifically hybridize to sequences other than the selected target nucleic acid (i.e., non-target or off-target sequences).

[0469] The activity of antisense compounds within the active target region (e.g., activity defined by the percentage reduction in the target nucleic acid level) may vary. In certain embodiments, a decrease in HBV mRNA levels indicates inhibition of HBV expression. A decrease in HBV protein levels also indicates inhibition of target mRNA expression. Furthermore, phenotypic changes indicate inhibition of HBV expression. In certain embodiments, reduced fatigue, decreased flu-like symptoms, increased appetite, decreased nausea, decreased arthralgia, decreased jaundice, decreased abdominal pain, decreased weakness, decreased weight loss, decreased mammary gland enlargement in males, decreased palmar rash, decreased blood clotting difficulties, decreased cirrhosis, decreased spider veins on the skin, increased absorption of vitamins A and D, decreased tumor growth, decreased tumor volume, decreased headache, decreased fever, decreased diarrhea, decreased pain in the liver region of the body, clay-colored or gray stools, decreased itching, decreased dark urine, and decreased nausea and vomiting may indicate inhibition of HBV expression. In certain embodiments, improvement of symptoms associated with HBV-related conditions, diseases, and disorders may indicate inhibition of HBV expression. In certain embodiments, a reduction in cirrhosis indicates inhibition of HBV expression. In certain embodiments, a decrease in liver cancer markers may indicate 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 between complementary nucleic acid bases of the nucleic acid molecule (e.g., Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds).

[0471] Hybridization can occur under a variety of conditions. The stringent conditions are sequence-dependent and determined by the properties and composition of the nucleic acid molecules being hybridized.

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

[0473] Complementarity Antisense compounds and target nucleic acids are complementary when a sufficient number of nucleic acid bases in the antisense compound can form hydrogen bonds with the corresponding nucleic acid bases of the target nucleic acid, thereby producing the desired effect (e.g., antisense inhibition of the target nucleic acid, such as HBV nucleic acid).

[0474] Non-complementary nucleic acid bases between the antisense compound and the HBV nucleic acid may be acceptable, provided that the antisense compound can specifically hybridize to the target nucleic acid. Furthermore, the antisense compound may hybridize across one or more segments of the HBV nucleic acid such that intervening or adjacent segments do not participate in the hybridization event (e.g., loop structure, mismatch, or hairpin structure).

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

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

[0477] In certain embodiments, the antisense compounds, or specific portions thereof, provided herein are fully complementary (i.e., 100% complementary) to the target nucleic acid, or specific portions thereof. For example, an antisense compound may be fully complementary to the HBV nucleic acid, or its target region, target segment, or target sequence. As used herein, “fully complementary” means that each nucleic acid base of the antisense compound can accurately form base pairs with the corresponding nucleic acid base of the target nucleic acid. For example, a 20-nucleotide antisense compound is fully complementary to a 400-nucleotide target sequence, insofar as there is a corresponding 20-nucleotide portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used with respect to specific portions of a first nucleic acid and / or a second nucleic acid. For example, a 20-nucleotide portion of a 30-nucleotide antisense compound may be “fully complementary” to a 400-nucleotide target sequence. The 20-nucleotide portion of a 30-nucleotide oligonucleotide is perfectly complementary to the target sequence if the target sequence has a corresponding 20-nucleotide portion, in which case each nucleic acid base is complementary to the 20-nucleotide portion of the antisense compound. At the same time, the entire 30-nucleotide antisense compound may or may not be perfectly complementary to the target sequence, depending on whether the remaining 10 nucleic acid bases of the antisense compound are also complementary to the target sequence.

[0478] The non-complementary nucleic acid base may be located at the 5' or 3' end of the antisense compound. Alternatively, the non-complementary nucleic acid base may be located within the antisense compound. If two or more non-complementary nucleic acid bases are present, they may be contiguous (i.e., linked) or discontinuous. In one embodiment, the non-complementary nucleic acid base is located within the wing segment of the gapmer antisense oligonucleotide.

[0479] In certain embodiments, an antisense compound having a length of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleic acid bases, or an antisense compound having a length of up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleic acid bases, contains 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer non-complementary nucleic acid bases with respect to a target nucleic acid, such as HBV nucleic acid or a particular portion thereof. In certain embodiments, an antisense compound having a length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleic acid bases, or an antisense compound having a length of up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleic acid bases, contains 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer non-complementary nucleic acid bases with respect to a target nucleic acid, such as HBV nucleic acid or a particular portion thereof.

[0480] The antisense compounds provided also include antisense compounds that are complementary to a portion of the target nucleic acid. As used herein, “portion” refers to a specified number of consecutive (i.e., linked) nucleic acid bases within a region or segment of the target nucleic acid. “Portion” may also refer to a specified number of consecutive nucleic acid bases of the antisense compound. In certain embodiments, the antisense compound is complementary to a portion of at least 8 nucleic acid bases of the target segment. In certain embodiments, the antisense compound is complementary to a portion of at least 9 nucleic acid bases of the target segment. In certain embodiments, the antisense compound is complementary to a portion of at least 10 nucleic acid bases of the target segment. In certain embodiments, the antisense compound is complementary to a portion of at least 11 nucleic acid bases of the target segment. In certain embodiments, the antisense compound is complementary to a portion of at least 12 nucleic acid bases of the target segment. In certain embodiments, the antisense compound is complementary to a portion of at least 13 nucleic acid bases of the target segment. In certain embodiments, the antisense compound is complementary to a portion of at least 14 nucleic acid bases of the target segment. In certain embodiments, the antisense compound is complementary to a portion of at least 15 nucleic acid bases of the target segment. Furthermore, antisense compounds that are complementary to at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid base segments of the target segment, or to nucleic acid base segments within a range defined by any two of these values, are also anticipated.

[0481] identity The antisense compounds provided herein may have a specified percentage of identity with respect to a particular nucleotide sequence, sequence number, or compound or part thereof. When used herein, an antisense compound is identical to a sequence disclosed herein if it has the ability to pair the same nucleic acid bases. For example, RNA containing uracil instead of thymidine in a disclosed DNA sequence is considered identical to the DNA because both uracil and thymidine pair with adenine. Shortened and extended forms of antisense compounds described herein, as well as compounds having bases that are not identical to the antisense compounds provided herein, are also anticipated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. The percentage of identity of an antisense compound is calculated according to the number of bases that form identical base pairs with the sequence being compared.

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

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

[0484] In some embodiments, two nucleic acid sequences may be complementary to 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” may also mean that two nucleic acid sequences can hybridize 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 percentage of identity between two nucleic acid sequences, the sequences are first aligned relative to each other, and then these sequences can be compared. For this purpose, for example, gaps may be inserted into the sequence of the first nucleic acid sequence, and nucleotides can be compared to the corresponding positions in the second nucleic acid sequence. The two sequences are identical at a position if the position in the first nucleic acid sequence is occupied by the same nucleotide as the position in the second sequence. The percentage of identity between two sequences is a function of the number of identical positions divided by the total number of positions compared in the sequences examined.

[0487] The "percentage of identity" of the aligned segments of the test sequence and the reference sequence is the percentage obtained by dividing the number of identical elements shared by the two aligned sequences 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 smaller than the reference sequence.

[0488] The percentage of identity between 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 described in 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 having the desired identity with the sequence of the present invention. To obtain gap alignment, the “Gapped BLAST” program, as described herein, can be used as described in Altschul et al. (1997), Nucleic Acids Res, 25:3389-3402. When using the BLAST program and the Gapped BLAST program, preset parameters of a specific program (e.g., NBLAST) may be used. The sequences may be further aligned using version 9 of the Genetic Computing Group's GAP (global alignment program) with a preset (BLOSUM62) matrix (values ​​-4 to +11) with a gap-opening penalty of -12 (for the first zero in the gap) and a gap-extending penalty of -4 (for each additional consecutive zero in the gap). After alignment, the identity percentage is calculated by expressing the number of matches as the percentage of nucleic acid content in the claimed sequences. The method described for determining the identity percentage of two nucleic acid sequences may be used in correspondence to the encoded amino acid sequences, as necessary.

[0489] Useful methods 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, although not limited to preferred computer programs for determining sequence identity, the Basic Local Alignment Search Tool (BLAST) program (BLAST Manual, Altschul et al., NCBI, NLM, NIH; (Altschul et al.) is publicly available from the National Center Biotechnology Information (NCBI) of the National Library of Medicine, National Institute of Health, Bethesda, Md. 20894. Examples include al., J.Mol.Biol.215:403-410 (1990). Versions 2.0 and above of the BLAST program allow for the introduction of gaps (deletions and insertions) into alignment, and sequence identity can be determined using BLASTX for peptide sequences and BLASTN for polynucleotide sequences. The percentage of identity can be 70% or more, 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 Nucleosides are base-sugar combinations. The nucleic acid base (also known as the base) portion of a nucleoside is usually a heterocyclic base portion. A nucleotide is a nucleoside that further contains a phosphate group covalently bonded to the sugar portion of the nucleoside. For nucleosides containing pentofuranosyl sugars, the phosphate group may be bonded to the 2', 3', or 5' hydroxyl portion of the sugar. Oligonucleotides are formed by covalently bonding adjacent nucleosides to each other to form linear, high-molecular-weight oligonucleotides. Within the oligonucleotide structure, phosphate groups are generally considered to form internucleoside bonds of the oligonucleotide.

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

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

[0493] Inter-modified nucleoside bonding The natural nucleoside bond between RNA and DNA is a 3'-5' phosphodiester bond. Antisense compounds with one or more modifications, i.e., non-natural nucleoside bonds, are often preferred over antisense compounds with natural nucleoside bonds because they possess desirable properties such as increased cellular uptake, enhanced affinity to target nucleic acids, and improved stability in the presence of nucleases.

[0494] Oligonucleotides containing modified nucleoside bonds include nucleoside bonds that retain a phosphorus atom and nucleoside bonds that do not contain a phosphorus atom. Representative phosphorus-containing nucleoside bonds, though not limited to these, include phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates. Methods for producing phosphorus-containing and non-phosphorus-containing bonds are well known.

[0495] In certain embodiments, the antisense compound targeting HBV nucleic acid includes one or more modified nucleoside bonds. In certain embodiments, the modified nucleoside bonds are phosphorothioate bonds. In certain embodiments, each nucleoside bond in the antisense compound is a phosphorothioate nucleoside bond.

[0496] modified sugar moiety The antisense compounds provided herein may optionally comprise one or more nucleosides, in which case the sugar group is modified. Such sugar-modified nucleosides may confer to the antisense compound enhanced nuclease stability, enhanced binding affinity, or any other beneficial biological properties. 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 ribosyl ring oxygen atoms with S, N(R), or C(R) 1 )(R 2 )(R=H, C1-C 12Examples of chemically modified sugars include substitutions of alkyl groups or protecting groups, and combinations thereof. Examples of chemically modified sugars include 2'-F-5'-methyl-substituted nucleosides (see PCT International Patent Application Publication WO2008 / 101157, published 8 / 21 / 08, relating to other disclosed 5',2'-bis-substituted nucleosides), substitution of a ribosyl ring oxygen atom with S, further substituted at the 2 position (see published US Patent Application US2005 / 0130923, published 16 / June 2005), and 5' substitution of BNA (see PCT International Patent Application Publication WO2007 / 134181, published 11 / 22 / 07, where LNA is substituted, for example, with a 5'-methyl or 5'-vinyl group).

[0497] Examples of nucleosides containing a modified sugar moiety include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl(R or S), 4'-S, 2'-F, 2'-OCH3, and 2'-O(CH2)2OCH3 substituents. Substituents at the 2' position include allyl, amino, azide, thio, O-allyl, and O-C1-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 ) may be selected from, and in the formula, R m and R n Each is independently H, or substituted or unsubstituted C1-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, but not limited to, include nucleosides containing a bridge between a 4'-ribosyl ring atom and a 2'-ribosyl ring atom. In certain embodiments, the antisense compounds provided herein comprise one or more bicyclic nucleosides, the bridge comprising a 4'-2' bicyclic nucleoside. Examples of such 4'-2' bicyclic nucleosides include, but are not limited to, one of the following: 4'-(CH2)-O-2'(LNA), 4'-(CH2)-S-2, 4'-(CH2)2-O-2'(ENA), 4'-CH(CH3)-O-2'(cEt), and 4'-CH(CH2OCH3)-O-2', as well as their analogs (see U.S. Patent No. 7,399,845, granted July 15, 2008), 4'-C(CH3)(CH3)-O-2', and its analogs. Log (see published PCT international patent application WO2009 / 006478, published January 8, 2009), 4'-CH2-N(OCH3)-2' and its analogs (see published PCT international patent application WO2008 / 150729, published December 11, 2008), 4'-CH2-ON(CH3)-2' (see published US patent application US2004 / 0171570, published September 2, 2004), 4'-CH2-N(R)-O-2', where R is HC1-C 12Alkyl or protecting groups (see U.S. Patent No. 7,427,672, granted September 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 its analogs (see published PCT international patent application publication WO2008 / 154401, published December 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, and 7,399,845; Published PCT International Patent Applications Publications WO2004 / 106356, WO94 / 14226, WO2005 / 021570, and WO2007 / 134181; U.S. Patent Publication US2004 / 0171 See also 570, US2007 / 0287831, and US2008 / 0039618; and US 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. For example, the aforementioned bicyclic nucleosides having one or more stereochemical sugar conformations, including α-L-ribofuranose and β-D-ribofuranose, can be prepared (see PCT international patent application PCT / DK98 / 00393, published as WO99 / 14226 on March 25, 1999).

[0499] In certain embodiments, the bicyclic sugar moiety of the 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, where in this case the bridge is independently, -[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 )- and includes one or two to four linked groups independently selected from, wherein, 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, a heterocyclic radical, a substituted heterocyclic radical, heteroaryl, a substituted heteroaryl, a C5-C7 alicyclic radical, a substituted C5-C7 alicyclic radical, halogen, OJ 1 , NJ 1 J 2 , SJ 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 2These are H and C1-C, respectively, independently. 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkinyl substitution C2-C 12 Alkinyl, C5-C 20 Aryl, substitution 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 group or a protecting group.

[0500] In certain embodiments, the crosslinking 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 )-ON(R)-. In certain embodiments, the crosslinks 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 isomer configuration. For example, nucleosides containing a 4'-2'-methylene-oxybridge may have either an α-L configuration or a β-D configuration. In the past, α-L-methyleneoxy(4'-CH2-O-2')BNA has been incorporated into antisense oligonucleotides, which have exhibited antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 63 65-63 72).

[0502] In certain embodiments, the bicyclic nucleoside is not limited, 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, Examples include (F) methyl(methyleneoxy)(4'-CH(CH3)-O-2')BNA, (G) methylene-thio(4'-CH2-S-2')BNA, (H) methylene-amino(4'-CH2-N(R)-2')BNA, (I) methyl carbon ring(4'-CH2-CH(CH3)-2')BNA, and (J) propylene carbon ring(4'-(CH2)3-2')BNA: [ka] In the formula, Bx is the base portion, and R is independently H, a protecting group, or C1-C 12 It is alkyl.

[0503] In certain embodiments, the bicyclic nucleoside conforms to the following formula I: [ka] During the ceremony, Bx is the heterocyclic base portion, -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 C1-C 12 It is an alkyl or amino protecting group, and T a and T b Each of these is independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a supporting medium.

[0504] In certain embodiments, the bicyclic nucleoside has formula II: [ka] During the ceremony, Bx is the heterocyclic base portion, T a and T b Each of these is independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a supporting medium. Z a These are 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 amide, thiol, or substituted thio.

[0505] In one embodiment, the substituents are independently halogen, oxo, hydroxyl, and OJ. c , NJ c J d SJ c N3, OC(=X)J c , and NJ e C(=X)NJ c J d It is monosubstituted or polysubstituted with a substituent independently selected from, where J c , J d , and Je Each of these is independently H, C1-C6 alkyl, or substituted C1-C6 alkyl, and X is 0 or NJ. c That is the case.

[0506] In certain embodiments, the bicyclic nucleoside has formula III: [ka] During the ceremony, Bx is the heterocyclic base portion, T a and T b Each of these is independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a supporting medium. Z b These are 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 the heterocyclic base portion, T a and T b Each of these is independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a supporting medium. R d These are 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 dEach of these is 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 the heterocyclic base portion, T a and T b Each of these is independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a supporting medium. q a , q b , q e , and q f These are, independently, hydrogen, halogen, and C1-C. 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkinyl substitution C2-C 12 Alkinyl, C1-C 12 Alkyl, substituted C1-C 12 Alkoxy, OJ j SJ j SOJ j SO2J j , NJ 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 Both are =C(q g )(q h ) and q g and q h These are H, halogen, and C1-C, respectively, and are independent of each other. 12 Alkyl or substituted C1-C 12 It is alkyl.

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

[0510] Methyleneoxy(4'-CH2-O-2')BNA, methyleneoxy(4'-CH2-O-2')BNA, and 2'-thio-BNA have also been synthesized (see, e.g., Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The synthesis of locked nucleoside analogs containing oligodeoxyribonucleotide doubles as substrates for nucleic acid polymerases has also been reported (see, e.g., Wengel et al., International Publication No. 99 / 14226). Furthermore, the synthesis of 2'-amino-BNA, a novel structurally restricted high-affinity oligonucleotide analog, has also been reported in this field (see, e.g., Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino-BNA and 2'-methylamino-BNA have also been produced, and their double-stranded thermal stability with complementary RNA and DNA strands has been reported in the past.

[0511] In certain embodiments, the bicyclic nucleoside has formula VI: [ka] During the ceremony, Bx is the heterocyclic base portion, T a and T b Each of these is independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a supporting medium. q i , q j , q k and q l These are H, halogen, and C1-C, respectively, and are independent of each other. 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkinyl substitution C2-C 12 Alkinyl, C1-C 12 Alkoxyl, substituted C1-C 12 Alkoxyl, OJ j SJ j SOJ j SO2J j , NJ 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 Both are =C(q g )(q h) and in the formula, q g and q h These are H, halogen, and C1-C, respectively, and are independent of each other. 12 Alkyl or substituted C1-C 12 It is alkyl.

[0512] A single carbocyclic nucleoside with 4'-(CH2)3-2' bridges and alkenyl analogs, as well as a bridged 4'-CH=CH-CH2-2' structure, 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 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 that connects two carbon atoms of a sugar ring, thereby forming a bicyclic sugar moiety. In certain embodiments, the bridge connects the 2' carbon of the sugar ring to another carbon.

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

[0515] As used herein, “monocyclic nucleoside” refers to a nucleoside containing 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” means a furanosyl sugar modified at the 2' position. In certain embodiments, such modifications include substituents selected from: substituted and unsubstituted alkoxys, substituted and unsubstituted thioalkyls, substituted and unsubstituted aminoalkyls, substituted and unsubstituted alkyls, substituted and unsubstituted allyls, and substituted and unsubstituted alkynyl halides, but are not limited to those. In certain embodiments, the 2' modification is selected from substituents including, but are 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, alkalyl, aralkyl, O-alkalyl, or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkalyl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving pharmacokinetic properties, and group for improving the pharmacological properties of antisense compounds, as well as other substituents having similar properties. In certain embodiments, the modified nucleoside includes a 2'-MOE side chain (see, for example, Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitutions have been reported to improve binding affinity compared to unmodified nucleosides, as well as to 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 and possess properties that make them promising for in vivo applications (see, for example, 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 nucleoside” or “modified THP nucleoside” means a nucleoside in which a six-membered tetrahydropyran “sugar” is substituted in place of a 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 acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, CJ. Bioorg. & Med. Chem. (2002) 10:841-854), fluoroHNA (F-HNA), or compounds thereof having the following formula X: [ka] In the formula, independently for each of the at least one tetrahydropyrannucleoside analogs of formula X: Bx is the heterocyclic base portion, T 3 and T 4 Each of these is independently a nucleoside-internucleoside bond group that binds the tetrahydropyran nucleoside analog to the antisense compound, or T 3 and T 4 One of them is an internucleoside bond group that binds a tetrahydropyrannucleoside analog to an antisense compound, T 3 and T 4 The other of these is H, a hydroxyl protecting group, a bonded conjugate group, or a 5' or 3' terminal group. q 1 , q 2 , q 3 , q 4 , q 5 , q 6 , and q 7 Each of these is 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 them is hydrogen, and the other is halogen, substituted or unsubstituted alkoxy, NJ 1 J 2 SJ 1 N3, OC(=X)J 1 , OC(=X)NJ 1 J 2 , NJ 3 C(=X)NJ 1 J 2 Selected from , and CN, X is O, S or NJ 1 And J 1 , J 2 , and J 3 Each of these is independently either H or a C1-C6 alkyl group.

[0518] In certain embodiments, a modified THP nucleoside of formula X is provided, where q m , q n , q p , q r , q s , q t , and q u Each of these is H. In a particular embodiment, q m , q n , q p , q r , q s , q t , and q u At least one of them is other than H. In a particular embodiment, q m , q n , q p , q r , q s , q t , and q u At least one of them is methyl. In certain embodiments, a THP nucleoside of formula X is provided, where R 1 and R 2 One of them is F. In a particular embodiment, R 1 is fluoro, and R 2 H is R 1 is methoxy, and R 2 H is H, and R 1 is methoxyethoxy, and R 2H is H.

[0519] As used herein, "2'-modified nucleoside" or "2'-substituted nucleoside" refers to a nucleoside containing a sugar with a substituent other than H or OH at the 2' position of the furanose ring. Examples of 2'-modified nucleosides include, but are not limited to, bicyclic nucleosides, in which the bridge connecting the two carbon atoms of the sugar ring connects the 2' carbon of the sugar ring to another carbon. Examples also include allyl, amino, azide, thio, O-allyl, and 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 Examples include nucleosides containing non-crosslinked 2'-substituted groups such as ), in which case R m and R n Each of these independently represents H, or a substituted or unsubstituted C1-C. 10 It is alkyl. The 2'-modified nucleoside may further contain other modifications, for example, at other positions of the sugar and / or at the nucleic acid base.

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

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

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

[0523] Many other bicyclo and tricyclo sugar substitution ring systems are also known in this field and can be used to modify nucleosides for the purpose of incorporation into antisense compounds (see, e.g., review article: Leumann, JC, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance their activity.

[0524] The method for producing modified sugars is known to those skilled in the art.

[0525] In nucleotides with modified sugar moieties, the nucleic acid base moieties (natural, modified, or a combination thereof) are maintained for the purpose of hybridization with appropriate nucleic acid targets.

[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 nucleotide is located within a gapmer motif. In certain embodiments, the modified sugar moiety is cEt. In certain embodiments, the cEt modified nucleotide is located across 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 formulations. The compositions and methods for formulating pharmaceutical compositions are not limited but depend on many factors, including the route of administration, the severity of the disease, or the dose administered.

[0528] Antisense compounds targeting HBV nucleic acid may be used in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. Examples of pharmaceutically acceptable diluents include phosphate-buffered saline (PBS), which is a suitable diluent for use in compositions delivered parenterally. Therefore, in one embodiment, a pharmaceutical composition comprising an antisense compound targeting HBV nucleic acid and a pharmaceutically acceptable diluent is employed in the method described herein. In a particular embodiment, the pharmaceutically acceptable diluent is PBS. In a particular embodiment, the antisense compound is an antisense oligonucleotide.

[0529] Pharmaceutical compositions comprising antisense compounds include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotides, which can (directly or indirectly) provide a biologically active metabolite or residue thereof when administered to animals, including humans. Therefore, for example, this disclosure also covers antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and pharmaceutically acceptable salts of other biological equivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0530] The prodrug may incorporate an additional nucleoside at one or both ends of the antisense compound, which is cleaved by an endogenous nuclease in the body to form an active antisense compound.

[0531] This disclosure provides pharmaceutical compositions comprising the modified oligonucleotides of this disclosure and pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the modified oligonucleotides comprise any one sequence from SEQ ID NOs. 11 to 666. In some embodiments, the modified oligonucleotides comprise any one sequence from SEQ ID NOs. 11 to 666 as described herein, and one, two, three, four, or five modifications to those sequences.

[0532] Modified oligonucleotides may be mixed with pharmaceutically acceptable active or inactive substances to prepare pharmaceutical compositions or pharmaceutical formulations. The compositions and methods for formulating pharmaceutical compositions are not limited but depend on many factors, including the route of administration, the severity of the disease, or the dose administered.

[0533] The pharmaceutical compositions of this disclosure may optionally include therapeutic agents, pharmaceuticals, carriers, adjuvants, dispersants, diluents, and the like.

[0534] Antisense compounds targeting HBV nucleic acid may be used in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. Examples of pharmaceutically acceptable diluents include phosphate-buffered saline (PBS), which is a suitable diluent for use in compositions delivered parenterally. Therefore, in one embodiment, a pharmaceutical composition comprising an antisense compound targeting HBV nucleic acid and a pharmaceutically acceptable diluent is employed in the method described herein. In a particular embodiment, the pharmaceutically acceptable diluent is PBS. In a particular embodiment, the antisense compound is an antisense oligonucleotide.

[0535] Pharmaceutical compositions containing modified oligonucleotides may include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotides that can (directly or indirectly) provide a biologically active metabolite or residue thereof when administered to animals, including humans. Therefore, for example, this disclosure also covers antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and pharmaceutically acceptable salts of other biological equivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0536] Modified oligonucleotides formulated as prodrugs are expected to be within the scope of this disclosure. The prodrug may incorporate additional nucleosides at one or both ends of an antisense compound, which are cleaved by endogenous nucleases in the body to form an active antisense compound.

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

[0538] A "pharmaceutical composition" is a formulation containing the modified oligonucleotides described herein in a form suitable for administration to a subject. In certain embodiments, the pharmaceutical composition is in bulk or unit dosage form. A unit dosage form is any of a variety of forms, including, for example, capsules, IV bags, tablets, single-use syringes, single pumps of aerosol inhalers, or vials. The amount of the active ingredient (e.g., the formulation of the modified oligonucleotides) in a unit dose of the composition is an effective amount and varies according to the specific treatment involved. Those skilled in the art will recognize that it may be necessary to routinely change the dosage depending on the patient's age and condition. The dosage will also depend on the route of administration. Various routes are expected, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, oral cavity, sublingual, intrapleural, intrathecal, and intranasal. 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 any necessary preservatives, buffers, or sprays.

[0539] "Pharmacologically acceptable excipients" means excipients that are generally safe, non-toxic, and not biologically or otherwise harmful, and that are useful in the preparation of pharmaceutical compositions, and that are acceptable not only for human pharmaceutical use but also for veterinary use. As used herein, "pharmaceutically acceptable excipients" includes one or more such excipients.

[0540] Pharmaceutical compositions are formulated to suit 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 (into body cavities), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, intraperitoneal, or subcutaneous applications may contain the following components: sterile diluents such as, for example, water for injection, physiological saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as, for example, benzyl alcohol or methylparaben; antioxidants such as, for example, ascorbic acid or sodium bisulfite; chelating agents such as, for example, ethylenediaminetetraacetic acid; buffering agents such as, for example, acetates, citrates, or phosphates; and agents for adjusting tonicity such as, for example, sodium chloride or dextrose. pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral or subcutaneous preparations may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials. These preparations may contain antioxidants, buffers, bacteriostatic agents, and solutes to be isotonic with the intended recipient's blood. Aqueous and non-aqueous sterile suspensions may contain suspending agents and thickeners. Preparations may be in unit / dose or multi-dose containers, for example, in sealed ampoules, syringes, and vials, and may be stored in a lyophilized state requiring only the addition of a sterile liquid carrier, such as physiological saline or water for injection, immediately before use.

[0541] The pharmaceutical compositions described herein may be manufactured by commonly known methods, for example, by conventional mixing, dissolution, granulation, dragee-making, levigating, emulsification, capsule encapsulation, encapsulation, or lyophilization processes. The pharmaceutical compositions may also be formulated conventionally using one or more pharmaceutically acceptable carriers containing excipients and / or adjuvants that facilitate the processing of the active ingredient into a pharmaceutically usable preparation. Of course, the appropriate formulation depends on the chosen route of administration.

[0542] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, New Jersey, Parsippany), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to pass easily through an injection needle. The composition must be stable under manufacturing and storage conditions and protected from microbial contamination, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity may be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using a surfactant. The action of microorganisms can be inhibited by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Often, the composition preferably contains isotonic agents, such as sugars, polyhydric alcohols such as mannitol and sorbitol, and sodium chloride. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0543] Oral compositions generally contain an inert diluent or a pharmaceutically acceptable food-grade carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For therapeutic oral administration, the activator may be incorporated into the 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 applied orally, rinsed in the mouth and spat out, or swallowed. Pharmaceutically compatible binders and / or auxiliary substances may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or agents of similar properties: binders, e.g., microcrystalline cellulose, tragacanth gum, or gelatin; excipients, e.g., starch, or lactose; disintegrants, e.g., alginic acid, Primogel, or corn starch; lubricants, e.g., magnesium stearate or Sterotes; flow enhancers, e.g., colloidal silicon dioxide; sweeteners, e.g., sucrose, or saccharin; or flavoring agents, e.g., peppermint, methyl salicylate, or orange flavoring.

[0544] For administration by inhalation, the agent is delivered in the form of a suitable spray, such as an aerosol spray from a pressurized container or dispenser containing a gas such as carbon dioxide, or from 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. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may be used. Methods for preparing such formulations are apparent to those skilled in the art, and the materials are commercially available. Liposome suspensions (containing 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. Patent No. 4,522,811.

[0546] Formulating oral or parenteral compositions in the form of unit dosage forms is particularly beneficial for ease of administration and dose uniformity. As used herein, a unit dosage form refers to a physically distinct unit adapted as a single dose to the target to be treated, each unit containing a predetermined amount of modified oligonucleotide calculated to produce the desired therapeutic effect when associated with the required pharmaceutical carrier. The specifications of the unit dosage forms in this disclosure are determined by and directly depend on the inherent characteristics of the activator and the specific therapeutic effect to be achieved.

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

[0548] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Examples of pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of parent compounds formed from non-toxic inorganic or organic acids.

[0549] Techniques for formulating and administering the compositions disclosed in the present invention can be found in Remington: The Science and Practice of Pharmacy, 19th edition, Mack Publishing Co., Easton, PA (1995).

[0550] All percentages and ratios used herein are by weight unless otherwise specified. Other properties and advantages of the present invention are evident from various examples. The provided examples illustrate various components and techniques useful for carrying out the present invention. The examples do not limit the claimed invention. Based on this disclosure, those skilled in the art can identify and employ other components and techniques useful for carrying out the present invention. The modified oligonucleotides of this disclosure may be covalently bonded to one or more moieties or conjugates that enhance the activity, cell distribution, or cell uptake of the resulting antisense oligonucleotide. Typical conjugate groups include cholesterol and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0551] Modified oligonucleotides may also be modified to have one or more stabilizing groups, which are generally attached to one or both ends of the antisense compound to enhance properties such as nuclease stability. Stabilizing groups include cap structures. These terminal modifications can protect antisense compounds with terminal nucleic acids from exonuclease degradation and aid in intracellular delivery and / or localization. The caps may be present at the 5'-end (5'-cap) or the 3'-end (3'-cap), or at both ends. Cap structures are known in the art, for example, inverted deoxy abasic caps. Furthermore, 3' and 5' stabilizing groups that can be used to cap one or both ends of an antisense compound to confer nuclease stability are disclosed in WO03 / 004602, published on January 16, 2003.

[0552] The modified oligonucleotides of this disclosure may be encapsulated within particles or incorporated on the surface of particles. In certain embodiments, the particles are nanoparticles. Exemplary nanoparticles include liposomes, micelles, polymer nanoparticles, lipid-polymer nanoparticles, and polymer micelles.

[0553] In certain embodiments, the nanoparticles include liposomes. Liposomes are spherical vesicles having at least one lipid bilayer and, in some embodiments, an aqueous core. In some embodiments, the lipid bilayer of the liposome may contain phospholipids. A non-limiting example of a phospholipid is phosphatidylcholine, but the lipid bilayer may contain additional lipids, such as phosphatidylethanolamine. Liposomes may be multilayer liposomes, i.e., consisting of several layered lipid bilayers, or single-layer liposomes consisting of a single lipid bilayer. Liposomes may be fabricated in specific size ranges that are viable phagocytic targets. Liposomes may be in size ranges of 20 nm to 100 nm, 100 nm to 400 nm, 1 μM or larger, or 200 nm to 3 μM. Examples of lipidoid and lipid-based formulations are provided in U.S. Published Patent Application 20090023673. In other embodiments, one or more lipids are one or more cationic lipids. Those skilled in the art will recognize which liposomes are suitable for encapsulating the modified oligonucleotides described herein.

[0554] In certain embodiments, the nanoparticles include micelles, which are aggregates of surfactant molecules. Exemplary micelles include aggregates of amphiphilic polymers, polymers, or copolymers in aqueous solution, in which the hydrophilic head portion is in contact with the surrounding solvent, while the hydrophobic tail portion is isolated in the center of the micelle.

[0555] In certain embodiments, the nanoparticles include polymer nanoparticles. Polymer nanoparticles include one or more polymers, such as polyester, poly(orthoester), poly(ethyleneimine), poly(caprolactone), polyanhydride, poly(acrylic acid), polyglycolide, or poly(urethane). In yet another embodiment, one or more polymers include poly(lactic acid) (PLA) or poly(lactic acid-coglycolic acid) (PLGA). In exemplary embodiments, one or more polymers include, for example, polyalkylene glycols such as polyethylene glycol (PEG), or polyalkylene oxides such as polyethylene oxide (PEO).

[0556] In some embodiments, the nanoparticles or a portion thereof are degradable. In other embodiments, the lipids and / or polymers of the nanoparticles are degradable.

[0557] Conjugated antisense compounds Antisense compounds may be covalently bonded to one or more moieties or conjugates, which enhance the activity, cell distribution, or cell uptake of the resulting antisense oligonucleotide. Typical conjugate groups include cholesterol and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0558] Antisense compounds may also be modified to have one or more stabilizing groups, which are generally attached to one or both ends of the antisense compound to enhance properties such as nuclease stability. Stabilizing groups include cap structures. These terminal modifications can protect antisense compounds with terminal nucleic acids from exonuclease degradation and aid in intracellular delivery and / or localization. The caps may be present at the 5'-end (5'-cap) or the 3'-end (3'-cap), or at both ends. Cap structures are known in the art, for example, inverted deoxy abasic caps. Furthermore, 3' and 5' stabilizing groups that can be used to cap one or both ends of an antisense compound to confer nuclease stability are disclosed in WO03 / 004602, published on January 16, 2003.

[0559] Cell culture and antisense compound treatment The effects of antisense compounds on HIV nucleic acid levels, activity, or expression can be tested in vitro in various cell types. Cell types used for such analyses are available from suppliers (e.g., American Type Culture Collection, Manassas, Virginia; Zen-Bio, Inc., Research Triangle Park, North Carolina; Clonetics Corporation, Walkersville, Maryland) and cultured using commercially available reagents (e.g., Invitrogen Life Technologies, Carlsbad, California) according to the supplier's instructions. Exemplary cell types, but not limited to, include HuVEC cells, b.END cells, HepG2 cells, Hep3B cells, and primary hepatocytes.

[0560] In vitro testing of antisense oligonucleotides This specification describes a method for treating cells using antisense oligonucleotides, which can be appropriately modified for treatments using other antisense compounds.

[0561] When the cells reach approximately 60-80% confluence in culture, they may be treated with antisense oligonucleotides.

[0562] One commonly used reagent for introducing antisense oligonucleotides into cultured cells is LIPOFECTIN (Invitrogen, Carlsbad, California), a cationic lipid transfection reagent. Antisense oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM1 (Invitrogen, Carlsbad, California) to obtain the desired final concentration of antisense oligonucleotides and a LIPOFECTIN concentration that may range from 2 to 12 μg / mL per 100 nM antisense oligonucleotide.

[0563] Another reagent used to introduce antisense oligonucleotides into cultured cells is LIPOFECTAMINE (Invitrogen, Carlsbad, California). Antisense oligonucleotides are mixed with LIPOFECTAMINE in OPTI-MEM1 low-serum medium (Invitrogen, Carlsbad, California) to obtain the desired final concentration of antisense oligonucleotides and a LIPOFECTAMINE concentration that can range from 2 to 12 μg / mL per 100 nM antisense oligonucleotide.

[0564] Another technique used to introduce antisense oligonucleotides into cultured cells is electroporation.

[0565] Cells are treated with antisense oligonucleotides by routine methods. Cells may also be harvested 16–24 hours after antisense oligonucleotide treatment, at which point the RNA or protein levels of the target nucleic acid are measured by methods known in the art and described herein. Generally, when treatment is performed on multiple replicas, the data are expressed as the average of the treatments of the replicas.

[0566] The concentration of antisense oligonucleotides used varies depending on the cell line. Methods for determining the optimal antisense oligonucleotide concentration for a particular cell line are known in the art. When transfected with LIPOFECTAMINE, antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM. When transfected using electroporation, antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM.

[0567] RNA isolation RNA analysis may be performed on total cellular RNA or poly(A)+ mRNA. Methods for RNA isolation are known in the art. RNA is prepared using methods known in the art, for example, using TRIZOL reagent (Invitrogen, Carlsbad, California) according to the manufacturer's recommended protocol.

[0568] Analysis of inhibition at target levels or expression Inhibition of HBV nucleic acid levels or expression can be analyzed by various methods known in the art. For example, target nucleic acid levels can be quantified by, for instance, Northern blotting, competitive polymerase chain reaction (PCR), or quantitative real-time PCR. RNA analysis may be performed on total cellular RNA or poly(A)+ mRNA. Methods for RNA isolation are known in the art. Northern blotting is also routine in the art. Quantitative real-time PCR can be easily performed using the commercially available ABI PRISM 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, California, and should be used according to the manufacturer's instructions.

[0569] Quantitative real-time PCR analysis of target RNA levels The target RNA level may be quantified by quantitative real-time PCR using an ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, California) according to the manufacturer's instructions. The method for quantitative real-time PCR is well known in this field.

[0570] Before performing real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction to produce complementary DNA (cDNA), which is then used as a substrate for real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. The reagents for RT and real-time PCR may be obtained from Invitrogen, Inc. (Carlsbad, California). The RT real-time PCR reaction is performed by methods known to those skilled in the art.

[0571] The target amount of gene (or RNA) obtained by real-time PCR is normalized by using the expression level of a gene whose expression is constant, such as cyclophyllin A, or by quantifying total RNA using RIBOGREEN (Invitrogen, ...

Claims

1. A modified oligonucleotide complementary to a portion of the hepatitis B virus (HBV) genome, comprising SEQ ID NO:

456.

2. The modified oligonucleotide according to claim 1, wherein at least one nucleoside bond is a phosphodiester bond.

3. The modified oligonucleotide according to claim 1, wherein at least one nucleoside bond is a modified nucleoside bond.

4. The modified oligonucleotide according to claim 3, wherein the modified nucleoside bond is a phosphorothioate nucleoside bond.

5. The modified oligonucleotide according to claim 1, wherein each nucleoside bond is a phosphorothioate nucleoside bond.

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

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

8. A pharmaceutical composition comprising a 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 the modified oligonucleotide according to any one of claims 1 to 7.

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

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

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

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

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

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

17. The pharmaceutical composition according to claim 15, wherein the amount of each dose is substantially the same.

18. The pharmaceutical composition according to claim 16, wherein the amount of each dose is substantially the same.

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

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

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

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

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

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

26. The pharmaceutical composition according to claim 25, wherein the hyperproliferative state is liver cancer.

27. ​​The pharmaceutical composition according to any one of claims 10 to 13, wherein the subject is co-infected with hepatitis delta virus infection (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 according to claim 1 for producing a pharmaceutical composition for reducing the levels of HBV mRNA, HBV DNA, HBV protein, or HBV antigen in a subject.

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

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

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

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

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

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

36. The use according to claim 34, wherein the amount of each dose is substantially the same.

37. The use according to claim 35, wherein the amount of each dose is substantially the same.

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

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

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

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

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

44. a) The disease, disorder or condition is jaundice, inflammation of the liver, hepatic fibrosis, inflammation, cirrhosis, hepatic failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV infection, HBV viremia, or liver disease-related transplantation; or b) The disease or condition is a hyperproliferative state, The use described in claim 43.

45. The use according to claim 44, wherein the hyperproliferative state is liver cancer.

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

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

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