Modulation of hepatitis b virus (HBV) expression

Abstract

The invention relates to modulation of hepatitis B virus (HBV) expression. Chimeric antisense compounds and methods for reducing HBV mRNA, DNA and protein expression are disclosed. Such methods, compounds, and compositions are useful for treating, preventing, or ameliorating HBV-related diseases, disorders, or conditions.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202380024461.X, filed on January 10, 2023, entitled "Regulation of Hepatitis B Virus (HBV) Expression".

[0002] Cross-referencing related applications

[0003] This application claims priority and benefit to U.S. Provisional Application No. 63 / 298,092, filed January 10, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0004] This invention relates to the regulation of hepatitis B virus (HBV) expression. Specifically, this invention discloses chimeric antisense compounds and methods for reducing HBV mRNA, DNA, and protein expression. The methods, compounds, and compositions can be used to treat, prevent, or improve HBV-related diseases, conditions, or symptoms. Background Technology

[0005] Hepatitis B is a viral disease transmitted through contaminated materials (such as blood and blood products), contaminated needles, parenteral transmission, sexual transmission, and vertical transmission from an infected or carrier mother to her offspring. The World Health Organization estimates that more than 2 billion people worldwide are infected, with approximately 4 million acute cases, 1 million deaths, and 350-400 million chronic carriers annually (WHO: Geographic Prevalence of Hepatitis B, 2004. http: / / www.who.int / vaccines-surveillance / graphics / htmls / hepbprev.htm).

[0006] The virus, HBV, is a double-stranded hepatotropic virus that infects only humans and non-human primates. Viral replication occurs primarily in the liver, and to a lesser extent in the kidneys, pancreas, bone marrow, and spleen (Hepatitis B virus biology. Microbiol Mol Biol Rev. 64: 2000; 51-68.). The virus and immune markers are detectable in the blood, and the characteristic antigen-antibody pattern has evolved over time. The first detectable viral marker was HBsAg, followed by hepatitis B e antigen (HBeAg) and HBV DNA. During incubation, titers may be high, but HBV DNA and HBeAg levels begin to decline at the onset of disease and may become undetectable at peak clinical disease (Hepatitis B virus infection—natural history and clinical consequences. *New England Journal of Medicine*, 350: 2004; 1118-1129). HBeAg is a detectable viral marker in the blood and is associated with active viral replication, and therefore with high viral load and infectivity (Hepatitis B e antigen—the dangerous end game of hepatitis B. *New England Journal of Medicine*, 347: 2002; 208-210). The presence of anti-HBsAb and anti-HBcAb (IgG) indicates recovery and immunity in previously infected individuals.

[0007] Currently, the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) recommend treatments for chronic HBV infection including interferon-alpha (INFa), peg-IFN2a, entecavir, and tenofovir. Nucleoside and nucleotide therapies, including entecavir and tenofovir, have been successful in reducing viral load, but the rates of HBeAg seroconversion and HBsAg loss are even lower than those achieved with IFN2a therapy. Other similar therapies have also been used, including lamivudine (3TC), telbivudine (LdT), and adefovir, but resistance to nucleoside / nucleotide therapies typically limits their efficacy.

[0008] Therefore, there is a need in this field to discover and develop new antiviral therapies. Additionally, there is a need for novel anti-HBV therapies capable of increasing the seroconversion rates of HBeAg and HBsAg. Recent clinical studies have found a correlation between seroconversion and decreased HBeAg levels (Fried et al. (2008) *Hepatology* 47:428) and decreased HBsAg levels (Moucari et al. (2009) *Hepatology* 49:1151). Decreased antigen levels may have allowed for immune control of HBV infection, as high antigen levels are thought to induce immune tolerance. Current nucleoside therapies against HBV significantly reduce HBV serum levels but have little effect on HBeAg and HBsAg levels.

[0009] Antisense technology is emerging as an effective means of reducing the expression of specific gene products and is therefore proving uniquely useful in many therapeutic and diagnostic applications. Unlike nucleoside therapy, antisense therapy can directly target transcripts associated with HBV antigens, thereby reducing serum HBeAg and HBsAg levels. Due to the multiple overlapping transcripts produced during HBV infection, a single antisense oligomer also has the potential to reduce HBV DNA beyond HBeAg and HBsAg. Therefore, antisense technology is becoming an effective means of reducing the expression of certain gene products and is thus proving uniquely useful in many therapeutic, diagnostic, and research applications for modulating HBV.

[0010] HBV antisense oligonucleotides with a single gap segment directly side-attached between the 5' and 3' wings have been developed (WO 2012 / 145697). However, many HBV antisense oligonucleotides with this structure exhibit minimal efficacy in reducing serum HBsAg levels and / or causing safety concerns in patients with chronic hepatitis B. Therefore, there is a need in the art for improved HBV antisense oligonucleotides. Summary of the Invention

[0011] This disclosure extends the principle of HBV antisense oligonucleotides by using one or more separators to provide an enhanced modified oligonucleotide structure that provides additional segmentation gap structures.

[0012] This disclosure is based, at least in part, on the discovery that discontinuous gap segments flanked between the 5' and 3' wings of a modified oligonucleotide provide improved activity (e.g., reduced serum HBsAg or HBeAg levels) compared to conventional antisense oligonucleotides with continuous gaps. One or more separator segments placed directly between the gap segments can provide improved activity of the antisense oligonucleotide, contrary to current teachings in the art, suggesting that separator segments may lead to undesirable activity. The inventors have unexpectedly discovered that placing separator segments at specific locations within a full-length antisense oligonucleotide provides improved activity, while separator segments at other locations result in reduced or unchanged activity. Furthermore, only specific types of nucleotide modifications within the separator segments provide improved activity, while other types of nucleotide modifications result in reduced or unchanged activity. Without being bound by theory, specific combinations of nucleotide modifications and the positioning of separator segments within a full-length antisense oligonucleotide improve the binding and activity of RNase H endonuclease without impairing complementarity with the HBV target sequence.

[0013] This disclosure is based, at least in part, on the discovery that specific nucleoside modifications to the 5' and 3' wings reduce in vivo toxicity (e.g., decreased ALT levels, proxy for hepatotoxicity, or CC30 (cytotoxic concentration that reduces cell viability by 30%)). The inventors have unexpectedly discovered that specific types of modified nucleosides in the 5' wings increase in vivo toxicity, while specific types of modified nucleosides in the 3' wings decrease in vivo toxicity. Furthermore, the localization of modified nucleosides within the 5' and 3' wings also contributes to in vivo toxicity. Without being bound by theory, specific combinations of nucleoside modifications and localization within the 5' and 3' wings contribute to the complementarity of the full-length antisense oligonucleotide with the HBV target sequence. While combinations of specific types of modifications at certain locations improve complementarity, other combinations of specific types of modifications and locations decrease complementarity, thereby increasing off-target binding, which ultimately leads to toxicity.

[0014] This document provides methods, compounds, and compositions for regulating the expression of HBV mRNA and protein. In some embodiments, the compounds used to regulate the expression of HBV mRNA and protein are antisense compounds. In some embodiments, the antisense compounds are antisense oligonucleotides.

[0015] In some embodiments, the modulation may occur in cells or tissues. In some embodiments, the cells or tissues are those of an animal. In some embodiments, the animal is a human. In some embodiments, HBV mRNA levels are reduced. In some embodiments, HBV DNA levels are reduced. In some embodiments, HBV protein levels are reduced. In some embodiments, HBV antigen levels are reduced. In some embodiments, HBV s antigen (HBsAg) levels are reduced. In some embodiments, HBV e antigen (HBeAg) levels are reduced. Such reductions may occur in a time-dependent or dose-dependent manner.

[0016] Methods, compounds, and compositions are also provided for the prevention, treatment, and improvement of diseases, conditions, and symptoms. In some embodiments, such HBV-related diseases, conditions, and symptoms are liver diseases. In some embodiments, such liver diseases, conditions, and symptoms include jaundice, hepatocellular carcinoma, liver inflammation, liver fibrosis, inflammation, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic lymphohistiocytosis, serum hepatitis, HBV viremia, and liver disease-related transplantation. In some embodiments, such HBV-related diseases, conditions, and symptoms are hyperproliferative diseases, conditions, and symptoms. In some embodiments, such hyperproliferative diseases, conditions, and symptoms include cancer and associated malignancies and metastases. In some embodiments, such cancers include hepatocellular carcinoma and hepatocellular carcinoma (HCC).

[0017] These diseases, conditions, and symptoms may share one or more common risk factors, causes, or outcomes. Some risk factors and causes for the development of liver disease or proliferative disorders include: increasing age; 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. Some symptoms and outcomes associated with the development of liver disease or proliferative 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 area of ​​the body, clay-colored or gray stools, generalized itching, and dark urine.

[0018] In some embodiments, the method of treatment includes administering an HBV antisense compound to an individual in need. In some embodiments, the method of treatment includes administering an HBV antisense oligonucleotide to an individual in need. Attached Figure Description

[0019] Figure 1 Provided located at Figure 2 The diagram shows an illustration of nucleoside modifications at each position in the sequence. The "Examples" column describes each type of nucleoside modification.

[0020] Figure 2 A table of exemplary modified oligonucleotides of this disclosure is shown. Using Figure 1 The legend in the diagram reads out the modifications at each position of the modified oligonucleotide sequence. AUS1010 to AUS1714 (SEQ ID NO: 11 to SEQ ID NO: 666) represent the modified oligonucleotide sequences. AUS1233 (SEQ ID NO: 10), also known as AUS1138, is the reference modified oligonucleotide sequence.

[0021] Figure 3 A schematic diagram of a solid-phase synthesis method for producing modified oligonucleotides is shown.

[0022] Figure 4 A schematic diagram of the modified oligonucleotide dosing regimen and HBsAg level sampling schedule for HBV Tg mice is shown.

[0023] Figure 5 A schematic diagram of the modified oligonucleotide dosing regimen and HBsAg level sampling schedule for C57BL / 6 Tg mice is shown.

[0024] Figure 6 A schematic diagram of the modified oligonucleotide dosing regimen and the sampling schedule for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in C57BL / 6 Tg mice is shown.

[0025] Figure 7 A schematic diagram of the modified oligonucleotide dosing regimen and HBsAg level sampling schedule for pAAV-1.2HBV-GTA HDI-HBV mice is shown.

[0026] Figure 8 A schematic diagram of the modified oligonucleotide dosing regimen and the sampling schedule for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in C57BL / 6 Tg mice is shown.

[0027] Figure 9A schematic diagram of an exemplary modified oligonucleotide dosing regimen and HBsAg level sampling schedule for pcDNA3.1-preS2-GTD HDI-HBV mice is shown.

[0028] Figure 10 A schematic diagram of an exemplary modified oligonucleotide dosing regimen and HBsAg level sampling schedule for pcDNA3.1-preS2-GTA HDI-HBV mice is shown.

[0029] Figure 11 A schematic diagram of an exemplary modified oligonucleotide dosing regimen and HBsAg level sampling schedule for GTA HBV Tg mice is shown.

[0030] Figure 12 A schematic diagram of an exemplary modified oligonucleotide dosing regimen and HBsAg level sampling schedule for GTD AAV-HBV Tg mice is shown.

[0031] Figure 13 A schematic diagram of an exemplary modified oligonucleotide dosing regimen for C57BL / 6 Tg mice and a timetable for sampling alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels is shown.

[0032] Figure 14 Table 15.1 shows the serum HBsAg levels in GT-C 1.0HBV transgenic mice on days 3, 7, 10, 14, 21, and 28 after subcutaneous treatment with 40 mg / kg of modified oligonucleotides AUS1233, AUS1683, AUS1684, AUS1685, AUS1220, AUS1322, AUS1323, or AUS1324. Mean values ​​are expressed as log... 10 express.

[0033] Figure 15 Table 15.2 shows the serum HBsAg levels in GT-C 1.0HBV transgenic mice on days 3, 7, 10, 14, 21, and 28 after subcutaneous treatment with 40 mg / kg of modified oligonucleotides AUS1169, AUS1171, AUS1170, AUS1168, or AUS1322. Mean values ​​are expressed as log... 10 express.

[0034] Figure 16Table 15.3 shows the serum HBsAg levels in GT-C 1.0HBV transgenic mice on days 3, 7, 10, 14, and 21 after subcutaneous treatment with 30 mg / kg of modified oligonucleotides 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 log... 10 express.

[0035] Figure 17 Table 15.4 shows the serum HBsAg levels in GT-C 1.0HBV transgenic mice on days 3, 7, 10, 14, and 21 after subcutaneous treatment with 30 mg / kg of modified oligonucleotides AUS1463, AUS1466, AUS1476, AUS1472, AUS1489, or AUS1459. Mean values ​​are expressed as log... 10 express.

[0036] Figure 18 Table 15.5 shows the serum HBsAg levels in GT-C 1.0HBV transgenic mice on days 3, 7, 10, 14, 21, and 28 after subcutaneous treatment with 30 mg / kg of modified oligonucleotides AUS1492, AUS1495, AUS1494, AUS1493, AUS1482, or AUS1441. Mean values ​​are expressed as log... 10 express.

[0037] Figure 19 Table 15.6 shows the serum HBsAg levels in GT-C 1.0HBV transgenic mice on days 3, 7, 10, 14, and 21 after subcutaneous treatment with 40 mg / kg of modified oligonucleotides AUS1693, AUS1694, AUS1695, AUS1696, AUS1697, AUS1698, AUS1699, or AUS1700. Mean values ​​are expressed as log... 10 express.

[0038] Figure 20Table 15.7 shows the serum HBsAg levels in GT-C 1.0HBV transgenic mice on days 3, 7, 10, 14, 21, and 28 after treatment with modified oligonucleotides. Rows 1 and 2 show the HBsAg levels after a subcutaneous dose of AUS1493 or AUS1233 at day 0. Rows 3 and 4 show the HBsAg levels after a subcutaneous dose of AUS1493 or AUS1233 at day 0. Mean values ​​are expressed in logarithmic order. 10 express.

[0039] Figure 21 Table 16.1 shows the serum HBsAg levels in GT-D HDI HBV mice on days 3, 7, 10, 14, 21, and 28 after subcutaneous treatment with 60 mg / kg of eASO compounds AUS1683, AUS1684, AUS1685, AUS1220, AUS1322, AUS1323, or AUS1324. Mean values ​​are expressed as log... 10 express.

[0040] Figure 22 Table 16.2 shows the serum HBsAg levels in GT-D HDIHBV mice on days 3, 7, 10, 14, 21, and 28 after subcutaneous treatment with 60 mg / kg of eASO compounds AUS1169, AUS1171, AUS1170, or AUS1168. Mean values ​​are expressed as log... 10 express.

[0041] Figure 23 Table 16.3 shows the serum HBsAg levels in GT-D HDI HBV mice on days 3, 7, 10, 14, and 21 after subcutaneous treatment with 60 mg / kg of eASO compounds AUS1173, AUS1174, AUS1175, or AUS1176. Mean values ​​are expressed as log... 10 express.

[0042] Figure 24Table 16.4 shows the results of GT-D HDI treatment on days 3, 7, 10, and 14 after subcutaneous treatment with 60 mg / kg of eASO compounds AUS1173, AUS1177, AUS1178, AUS1179, AUS1180, AUS1181, AUS1182, AUS1194, AUS1184, AUS1185, AUS1186, AUS1187, AUS1188, AUS1189, AUS1190, AUS1191, AUS1192, AUS1193, AUS1183, AUS1239, AUS1325, AUS1326, AUS1327, AUS1328, AUS1329, AUS1175, AUS1361, or AUS1362. Serum HBsAg levels in HBV mice. Mean values ​​are expressed as logarithms. 10 express.

[0043] Figure 25 Table 16.5 shows the serum HBsAg levels in GT-D HDIHBV mice on days 3, 7, 10, 14, and 22 after subcutaneous treatment with 60 mg / kg of eASO compounds AUS1322, AUS1382, AUS1383, AUS1384, or AUS1385. Mean values ​​are expressed as log... 10 express.

[0044] Figure 26 Table 16.6 shows the serum HBsAg levels in GT-D HDI HBV mice on days 3, 7, 10, 14, and 21 after subcutaneous treatment with 60 mg / kg of eASO compounds AUS1322, AUS1388, AUS1389, AUS1390, AUS1392, AUS1393, AUS1396, AUS1403, AUS1409, AUS1411, AUS1413, AUS1414, or AUS1415. Mean values ​​are expressed as log... 10 express.

[0045] Figure 27 Table 16.7 shows the serum HBsAg levels in GT-D HDI HBV mice on days 3, 7, 10, and 14 after subcutaneous treatment with 60 mg / kg of eASO compounds AUS1387, AUS1326, AUS1328, AUS1388, AUS1390, AUS1403, or AUS1360. Mean values ​​are expressed as log... 10 express.

[0046] Figure 28 Table 16.8 shows the serum HBsAg levels in GT-D HDI HBV mice on days 3, 7, 10, 14, and 21 after subcutaneous treatment with 40 mg / kg of eASO compounds 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 log... 10 express.

[0047] Figure 29 Table 16.9 shows the eASO compounds AUS1444, AUS1458, AUS1459, AUS1460, AUS1461, AUS1462, AUS1463, AUS1464, AUS1465, AUS1466, AUS1467, AUS1468, AUS1469, AUS1470, AUS1471, AUS1472, AUS1473, and AUS1444 at 40 mg / kg. 74. Serum HBsAg levels in GT-D HDI HBV (1.5 ug / ml pcDNA3.1 preS2 / S plasmid) mice on days 3, 7, 10, 14, and 21 after subcutaneous treatment with AUS1475, AUS1476, AUS1477, AUS1478, AUS1479, AUS1480, AUS1481, AUS1482, AUS1483, AUS1488, AUS1489, AUS1490, AUS1443, AUS1444, or AUS1445. Mean values ​​are expressed as log... 10 express.

[0048] Figure 30 Table 16.10 shows the serum HBsAg levels in GT-D HDI HBV (1.5 ug / ml pcDNA3.1 preS2 / S plasmid) mice on days 3, 7, 10, 14, 21, and 28 after subcutaneous treatment with 15 mg / kg or 45 mg / kg of eASO compounds AUS1492, AUS1493, AUS1233, AUS1492, or AUS1493. Mean values ​​are expressed as log... 10 express.

[0049] Figure 31Table 16.11 shows the serum HBsAg levels in GT-A HDI HBV (1.5 ug / ml pcDNA3.1 preS2 / S plasmid) mice on days 3, 7, 10, 14, 21, and 28 after subcutaneous treatment with 40 mg / kg of eASO compounds AUS1492, AUS1493, AUS1233, AUS1492, or AUS1493. Mean values ​​are expressed as log... 10 express.

[0050] Figure 32 Table 16.12 shows the serum HBsAg levels in GT-A HDI HBV (1.5 ug / ml pcDNA3.1 preS2 / S plasmid) mice on days 3, 7, 10, 14, and 21 after subcutaneous treatment with 15 or 45 mg / kg of eASO compounds AUS1441, AUS1466, AUS1472, AUS1488, or AUS1489. Mean values ​​are expressed as log... 10 express.

[0051] Figure 33 Table 16.13 shows the serum HBsAg levels in GT-D HDI HBV (1.5 ug / ml pcDNA3.1 preS2 / S plasmid) mice on days 3, 7, 10, 14, 21, and 28 after subcutaneous treatment with 15 or 45 mg / kg of eASO compounds AUS1495, AUS1494, AUS1233, AUS1495, AUS1494, AUS1492, or AUS1441. Mean values ​​are expressed as log... 10 express.

[0052] Figure 34 Table 16.14 shows the serum HBsAg levels in GT-A HDI HBV (1.5 ug / ml pcDNA3.1 preS2 / S plasmid) mice on days 3, 7, 10, 14, 21, and 28 after subcutaneous treatment with 15 or 45 mg / kg of eASO compounds AUS1495, AUS1494, AUS1233, AUS1495, AUS1494, AUS1492, AUS1482, or AUS1441. Mean values ​​are expressed as log... 10 express.

[0053] Figure 35Table 16.15 shows the serum HBsAg levels in GT-B HDI HBV (0.5 ug / ml pcDNA3.1 preS2 / S plasmid) mice on days 3, 7, 10, 14, and 21 after subcutaneous treatment with the eASO compound AUS1684 at 40 mg / kg. Mean values ​​are expressed as log... 10 express.

[0054] Figure 36 Table 16.16 shows the serum HBsAg levels in GT-A 1.2HBV mice on days 3, 7, 10, 14, 21, and 28 after subcutaneous treatment with 45 mg / kg of eASO compounds AUS1492 or AUS1493. Mean values ​​are expressed as log... 10 express.

[0055] Figure 37 Table 16.17 shows the serum HBsAg levels in mice treated subcutaneously with 40 mg / kg of eASO compounds AUS1396, AUS1422, AUS1423, AUS1424, AUS1425, AUS1426, AUS1427, AUS1428, AUS1429, AUS1430, AUS1431, AUS1432, or AUS1433 on days 3, 7, 10, 14, and 21. Mean values ​​are expressed as log... 10 express.

[0056] Figure 38Table 16.18 shows the results of using eASO compounds AUS1444, AUS1458, AUS1459, AUS1460, AUS1461, AUS1462, AUS1463, AUS1464, AUS1465, AUS1466, AUS1467, AUS1468, AUS1469, AUS1470, AUS1471, AUS1472, AUS1473, and AUS1474 at 40 mg / kg. Serum HBsAg levels in GT-A HDIHBV (1.5 ug / ml pAAV-1.2HBV plasmid) mice on days 3, 7, 10, 14, and 21 after subcutaneous treatment with US1474, AUS1475, AUS1476, AUS1477, AUS1478, AUS1479, AUS1480, AUS1481, AUS1482, AUS1483, AUS1488, AUS1489, AUS1490, AUS1443, or AUS1434. Mean values ​​are expressed as log... 10 express.

[0057] Figure 39 Table 17 shows the serum levels of HBsAg, HBeAg, and HBV DNA in GT-D AAV HBV (rAAV-HBV1.3-mer WT replicon) mice on days 3, 7, 10, 14, 21, and 28 after subcutaneous treatment with 40 mg / kg of the eASO compound AUS1493. Mean values ​​are expressed as log... 10 express.

[0058] Figure 40 Table 18.1 shows the dosage of eASO compounds AUS1434, AUS1435, AUS1440, AUS1436, AUS1437, AUS1438, AUS1439, AUS1441, AUS1178, AUS1190, AUS1188, AUS1192, AUS1422, AUS1423, AUS1424, AUS1425, AUS1426, and AUS1426 at 60 mg / kg on days 0, 2, and 4. Serum ALT levels in male WT C57BL / 6 mice on days 5, 7, 10, 14, 17, and 21 following subcutaneous treatment with S1427, AUS1428, AUS1429, AUS1430, AUS1431, AUS1432, AUS1433, AUS1360, AUS1401, AUS1361, AUS1362, or AUS1411.

[0059] Figure 41Table 18.2 shows the serum ALT levels in male WT C57BL / 6 mice on days 5, 7, 10, 14, 17, and 21 after subcutaneous treatment with 60 mg / kg of eASO compounds AUS1443, AUS1444, AUS1445, AUS1446, AUS1447, AUS1448, AUS1449, AUS1450, AUS1452, AUS1453, AUS1454, AUS1455, AUS1456, or AUS1457 on days 0, 2, and 4. Detailed Implementation

[0060] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and do not limit the claimed invention. In this document, the singular is used to include the plural unless otherwise expressly stated. As used herein, the word “or” means “and / or” unless otherwise stated. Furthermore, the use of the term “including” and other forms such as “includes” and “included” is not limiting. Additionally, unless otherwise expressly stated, terms such as “element” or “component” cover both elements and components comprising one unit and elements and components comprising more than one subunit.

[0061] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, and papers, are hereby expressly incorporated by reference to portions thereof as discussed herein.

[0062] definition

[0063] Unless specifically defined, the nomenclature, procedures, and techniques used in conjunction with the analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are those well-known and commonly used in the art. Standard techniques are applicable to chemical synthesis and chemical analysis. Where permitted, all patents, applications, published applications, and other publications, GENBANK accession numbers and associated sequence information, and other data referenced throughout this disclosure, available from databases such as the National Center for Biotechnology Information (NCBI), are incorporated herein by reference in part, and in their entirety, of the documents discussed herein.

[0064] Unless otherwise stated, the following terms have the following meanings:

[0065] "2'-O-methoxyethyl" (also known as 2'-MOE and 2'-O(CH2)2-OCH3) refers to the O-methoxy-ethyl modification at the 2' position of the furanose ring. Sugars modified with 2'-O-methoxyethyl are modified sugars.

[0066] "2'-MOE nucleoside" (also known as 2'-O-methoxyethyl nucleoside) refers to a nucleoside that includes a sugar moiety modified with 2'-MOE.

[0067] "2'-substituted nucleosides" refers to nucleosides that include a substituent at the 2' position of the furanoyl ring, rather than H or OH. In some embodiments, 2'-substituted nucleosides comprise nucleosides modified with bicyclic sugars.

[0068] "3' target site" refers to the nucleotide that is complementary to the 3' nucleotide of a specific antisense compound in the target nucleic acid.

[0069] "5' target site" refers to the nucleotide that is complementary to the 5' nucleotide of a specific antisense compound in the target nucleic acid.

[0070] "5-Methylcytosine" refers to cytosine modified with a methyl group attached to the 5-position. 5-Methylcytosine is a modified nucleobase.

[0071] "Approximately" means within ± 7% of the value. For example, if the statement is "the compound affects at least approximately 70% of the inhibition of HBV," it implies that HBV levels are inhibited in the range of 63% to 77%.

[0072] "Acceptable safety profile" refers to the pattern of side effects within clinically acceptable limits.

[0073] "Active agent" means one or more substances in a pharmaceutical composition that provide therapeutic benefit when administered to an individual. For example, in some embodiments, an antisense oligonucleotide targeting HBV is an active agent.

[0074] "Active target region" refers to the target region targeted by one or more active antisense compounds. "Active antisense compound" refers to an antisense compound that reduces the level of the target nucleic acid or protein.

[0075] An "acute hepatitis B infection" occurs when someone exposed to the hepatitis B virus begins to show signs and symptoms of viral hepatitis. This period, known as the incubation period, averages 90 days but can be as short as 45 days or as long as 6 months. For most people, this infection causes mild to moderate discomfort that resolves on its own as the body's immune response successfully fights the virus. However, some people, particularly those with compromised immune systems such as those with AIDS, undergoing chemotherapy, taking immunosuppressants, or taking steroids, experience very serious problems due to acute HBV infection and subsequently develop more severe symptoms, such as fulminant hepatic failure.

[0076] "Concomitant administration" refers to the combined administration of two drugs in any manner, where the pharmacological effects of both drugs are simultaneously observed in the patient's body. Concomitant administration does not require the two drugs to be administered as a single drug combination, in the same dosage form, or via the same route of administration. The effects of the two drugs do not need to appear simultaneously. The effects only need to overlap for a certain period of time and do not need to be mutually prolonging.

[0077] "Administration" means the provision of a medicine to an individual, and includes, but is not limited to, administration by a medical professional and self-administration.

[0078] “Pharmaceutical” means an active substance that can provide therapeutic benefit when administered to an animal. “First pharmaceutical agent” means a therapeutic compound described herein. For example, a first pharmaceutical agent may be an antisense oligonucleotide targeting HBV. “Second pharmaceutical agent” means a second therapeutic compound (e.g., a second antisense oligonucleotide targeting HBV) and / or a non-HBV therapeutic compound described herein.

[0079] "Improvement" refers to a reduction in at least one indicator of the severity of a symptom or disease. The severity of an indicator can be determined by subjective or objective measurements known to those skilled in the art.

[0080] "Animal" means human or non-human animal, including but not limited to mice, rats, rabbits, dogs, cats, pigs and non-human primates, including but not limited to monkeys and chimpanzees.

[0081] An "antibody" is a molecule characterized by its specific reaction with an antigen in a certain way, where both the antibody and the antigen are defined in another way. An antibody can refer to the complete antibody molecule or any fragment or region thereof, such as the heavy chain, light chain, or F1 chain. ab District and F c district.

[0082] "Antisense activity" means any detectable or measurable activity attributable to the hybridization of an antisense compound with its target nucleic acid. In some embodiments, antisense activity is a reduction in the amount or expression of the target nucleic acid or protein encoded by such target nucleic acid.

[0083] "Antisense compounds" refer to oligomeric compounds that can undergo hybridization with target nucleic acids via hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds such as antisense oligonucleotides, siRNA, shRNA, snoRNA, miRNA, and satellite repeat sequences.

[0084] "Antisense inhibition" refers to the reduction in the level of the target nucleic acid in the presence of an antisense compound that complements the target nucleic acid, compared to the level in the absence of an antisense compound.

[0085] "Antisense mechanisms" are all those mechanisms that involve the hybridization of a compound with a target nucleic acid, where the result or effect of hybridization is target degradation or target occupation, accompanied by the stagnation of cellular mechanisms such as transcription or splicing.

[0086] "Antisense oligonucleotide" refers to a single-stranded oligonucleotide with a nucleobase sequence that allows hybridization with the corresponding region or segment of a target nucleic acid.

[0087] The area under the curve, or AUC, is the integral of the change in drug concentration in plasma over time. AUC can be determined for the total time for which data is available, such as until the drug is no longer detectable (AUC). 0-t The area under the curve extrapolated from time 0 to infinity (AUC) 0-¥ ), or for a specific truncated time window, such as 24 hours after application (AUC) 0-24 ).

[0088] "Base complementarity" refers to the ability of an antisense oligonucleotide to precisely pair (i.e., hybridize) with the corresponding nucleobases in a target nucleic acid, and is mediated by Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen binding between the corresponding nucleobases.

[0089] "Bicyclic sugar" refers to a furanose ring modified by bridging two non-homogeneous carbon atoms. Bicyclic sugars are modified sugars.

[0090] "Body weight" refers to the total weight of an animal, including all tissues, including adipose tissue.

[0091] "Cap structure" or "terminal cap portion" refers to a chemical modification at any end of an antisense compound.

[0092] “cEt” or “constrained ethyl” means a bicyclic sugar moiety comprising a bridge connecting a 4'-carbon and a 2'-carbon, wherein the bridge has the formula: 4'-CH(CH3)-O-2'.

[0093] "Restricted ethyl nucleoside" (also cEt nucleoside) refers to a nucleoside that includes a bicyclic sugar moiety comprising a 4'-CH(CH3)-O-2' bridge.

[0094] A “chemically distinct region” refers to a region of an antisense compound that is chemically different from another region of the same antisense compound in some respects. For example, a region of a nucleotide with 2'-O-methoxyethyl modification is chemically different from a region of a nucleotide without 2'-O-methoxyethyl modification.

[0095] "Chimeric antisense compounds" refers to antisense compounds that have at least two chemically distinct regions, each with multiple subunits.

[0096] "Chronic hepatitis B infection" occurs when a person initially has an acute infection but subsequently becomes unable to fight it off. Whether the disease is chronic or completely remission depends largely on the age of the infected person. Approximately 90% of infants infected at birth will progress to chronic disease. However, the risk of chronic infection decreases with age, resulting in 20%-50% of children and less than 10% of older children or adults progressing from acute infection to chronic infection. Although the ASO compositions of the present invention can also treat HBV-related conditions such as inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, etc., chronic HBV infection remains the primary treatment target of embodiments of the present invention.

[0097] "Co-administration" means administering two or more drugs to an individual. The two or more drugs may be in a single drug composition or in a separate drug composition. Each of the two or more drugs may be administered via the same or different routes of administration. Co-administration encompasses parallel or sequential administration.

[0098] "Complementarity" refers to the ability of the nucleobases of the first nucleic acid to pair with those of the second nucleic acid.

[0099] "Compliance" refers to an individual's adherence to recommended treatments.

[0100] "comprise / comprises / comprising" is to be understood as implying the inclusion of the stated steps or elements or groups of steps or elements, but does not exclude any other steps or elements or groups of steps or elements.

[0101] "Continuous nucleobases" refers to nucleobases that are adjacent to each other.

[0102] "Cure" refers to the method or process of restoring health or treating a disease with a prescription.

[0103] "Deoxyribonucleotide" refers to a nucleotide that has a hydrogen atom at the 2' position of the sugar moiety. Deoxyribonucleotides can be modified with any substituent from a variety of substituents.

[0104] "Design" or "design to" refers to the process of designing oligomers that specifically hybridize with selected nucleic acid molecules.

[0105] "Diluent" refers to a component in a composition that lacks pharmacological activity but is pharmaceutically necessary or desirable. For example, in injectable drugs, the diluent can be a liquid, such as a saline solution.

[0106] "Dosage unit" means the form in which the medicine is provided, such as pills, tablets, or other dosage units known in the art.

[0107] "Dosage" means a specified quantity of a drug agent delivered in a single administration or over a specified period of time. In some embodiments, the dosage may be administered in two or more boluses, tablets, or injections. For example, in some embodiments where subcutaneous administration is desired, the desired dosage requires a volume that is not easily contained in a single injection. In such embodiments, the desired dosage may be achieved using two or more injections. In some embodiments, the dosage may be administered in two or more injections to minimize injection site reactions in an individual. In other embodiments, the drug agent is administered by infusion over an extended period of time or continuously. A dosage may be stated as the amount of drug agent per hour, per day, per week, or per month.

[0108] A "dosing regimen" is a combination of doses designed to achieve one or more desired effects.

[0109] "Duration" refers to the period of time during which an activity or event continues. In some embodiments, the duration of treatment is the period of time during which a certain dose of the agent is administered.

[0110] In the context of modulating activity or treating or preventing symptoms, "effective amount" means the amount of active ingredient administered, either as a single dose or as part of a series, to a subject requiring such modulation, treatment, or prevention that is effective in modulating the effect or in treating, preventing, or improving the symptoms. Effective amount will vary depending on the health and physical condition of the subject being treated, the subject's classification group, the formulation of the composition, assessment of the medical condition, and other relevant factors.

[0111] "Efficacy" refers to the ability to produce the desired effect.

[0112] "Expression" encompasses all the functions of translating the coding information of a gene into structures that exist and operate within the cell. These structures include, but are not limited to, the products of transcription and translation.

[0113] The term "fragment" applied to polynucleotides will be understood to mean a nucleotide sequence that is shorter than a reference nucleic acid or nucleotide sequence and comprises, substantially constitutes, and / or is composed of, a nucleotide sequence that is identical or substantially identical (e.g., 60%, 70%, 80%, 90%, 92%, 95%, 98%, or 99% identical) to, a reference nucleic acid or nucleotide sequence. Where appropriate, such nucleic acid fragments according to the invention may be included in larger polynucleotides in which said nucleic acid fragments are components. In some embodiments, such fragments may comprise, substantially constitutes, and / or is composed of, oligonucleotides 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 or nucleotide sequence according to the invention.

[0114] "Completely complementary" or "100% complementary" means that each nucleobase of the first nucleic acid has a complementary nucleobase in the second nucleic acid. In some embodiments, the first nucleic acid is an antisense compound, and the target nucleic acid is the second nucleic acid.

[0115] "Fully modified motif" refers to an antisense compound consisting of a continuous sequence of nucleosides, wherein essentially each nucleoside is a sugar-modified nucleoside with uniform modification.

[0116] A "gapmer" refers to a chimeric antisense compound in which an inner region containing multiple nucleotides supporting RNase H cleavage is located between an outer region containing one or more nucleotides, wherein the nucleotides constituting the inner region may be chemically different from the one or more nucleotides constituting the outer region. The inner region may be referred to as a "gap," and each of the 5' and 3' outer regions may be referred to as a "wing."

[0117] An "interstitial space" is an internal segment of a chimeric antisense compound that includes one or more linked deoxyribonucleotides and is located between the 5' wing (W1) and the 3' wing (W2). An interstitial space may be referred to as an "interstitial space," "interstitial region," or "interstitial segment."

[0118] "HBV" refers to mammalian hepatitis B virus, including human hepatitis B virus. The term encompasses the geographic genotype of hepatitis B virus, particularly human hepatitis B virus, as well as variant strains of the geographic genotype of hepatitis B virus.

[0119] "HBV antigen" refers to any hepatitis B virus antigen or protein, 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".

[0120] Hepatitis B e antigen (HBeAg) or HBeAG is the secreted, non-particulate form of the HBV core protein. The HBV antigens HBeAg and HBcAg share a primary amino acid sequence and therefore exhibit cross-reactivity at the T cell level. Viral assembly or replication does not require HBeAg, but studies suggest that establishing chronic infection may require it. Neonatal infection with HBeAg-negative mutants typically results in fulminant acute rather than chronic HBV infection (Terezawa et al. (1991), Pediatr. Res. 29:5), while infection of young marmots with WHeAg-negative mutants results in a much lower rate of chronic WHV infection (Cote et al. (2000), Hepatology 31:190). HBeAg may act as a tolerogen by inactivating core-specific T cells through deletion or clonal non-reactivity (Milich et al. (1998), Journal of Immunology 160:8102). Following antiviral therapy and HBeAg seroconversion, a positive correlation exists between the reduction in HBV viral load and antigen of inhibitory receptor programmed death-1 (PD-1; also known as PDCD1) (i.e., the negative regulator of T cell activation) and the reduction in T cell expression (Evans et al. (2008) Hepatology 48:759).

[0121] "HBV mRNA" refers to any messenger RNA expressed by the hepatitis B virus.

[0122] "HBV nucleic acid" or "HBV DNA" means any nucleic acid that encodes HBV. For example, in some embodiments, HBV nucleic acid includes, but is not limited to, any viral DNA sequence encoding the HBV genome or a portion thereof, or any RNA sequence transcribed from viral DNA containing any mRNA sequence encoding HBV proteins.

[0123] "HBV protein" refers to any protein secreted by the hepatitis B virus. The term covers various HBV antigens, including core proteins such as "hepatitis E antigen," "HBeAg," or "HBeAG," and envelope proteins such as "HBV surface antigen" or "HBsAg."

[0124] HBV surface antigen, or HBsAg, or HBsAG, is the envelope protein of infectious HBV viral particles, but it is also secreted as a non-infectious particle at serum levels that are 1000 times higher than those of HBV viral particles. Serum levels of HBsAg in infected humans or animals can reach up to 1000 gg / mL (Kann and Gehrlich (1998), *Topley & Wilson's Microbiology and Microbial Infections*, 9th ed. 745). In acute HBV infection, serum levels or serum t... % The half-life of HBsAg is 8.3 days (Chulanov et al. (2003) *Journal of Medical Virology* 69: 313). Internalized HBsAg in myeloid dendritic cells inhibits the upregulation of costimulatory molecules (i.e., B7) and suppresses T cell stimulation capacity (den Brouw et al. (2008) *Immunology* 126:280), and dendritic cells from patients with chronic infection also show expression of costimulatory molecules, secretion of IL-12, and defects in T cell stimulation in the presence of HBsAg (Zheng et al. (2004) *Journal of Viral Hepatitis* 11:217). HBsAg-specific CD8 cells from CHB patients show altered tetramer binding. These CD8 cells are not unresponsive, but may possess a TCR topology that confers partial tolerance or ignorance (Reignate et al. (2002) Journal of Experimental Medicine 195:1089). Furthermore, a decrease in serum HBsAg > 1 log at week 24 has a high predictive value (92%) for sustained virological response (SVR – defined as undetectable HBV DNA by PCR 1 year after treatment) during Peg-IFNa2a therapy (Moucari et al. (2009) Hepatology 49:1151).

[0125] "Hepatitis B-related symptoms" or "HBV-related symptoms" refers to any disease, biological condition, medical condition, or event caused, related to, or traceable to hepatitis B infection, exposure, or exacerbation of the disease. The term "hepatitis B-related symptoms" includes chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serological hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serological hepatitis, HBV viremia, transplant-related liver disease, and symptoms that may include any or all of the following: flu-like illness, weakness, pain, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain in the liver area of ​​the body, clay-colored or gray stools, generalized itching, and dark urine, accompanied by a positive test for the presence of hepatitis B virus, a positive test for hepatitis B virus antigen, or a positive test for the presence of antibodies specific to hepatitis B virus antigen.

[0126] "Hybridization" refers to the annealing of complementary nucleic acid molecules. In some embodiments, the complementary nucleic acid molecules include, but are not limited to, antisense compounds and nucleic acid targets. In some embodiments, the complementary nucleic acid molecules include, but are not limited to, antisense oligonucleotides and nucleic acid targets.

[0127] As used herein, the terms "IC50" or "IC" are used in this context. 50 "IC50" refers to the concentration of a drug at which cell viability is reduced by half. Therefore, IC50... 50 It is a measure of the effectiveness of a drug in inhibiting biological processes.

[0128] “Identifying animals with HBV infection” means identifying animals that have been diagnosed with HBV; or identifying animals exhibiting any symptoms of HBV infection, including but not limited to chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serological hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serological hepatitis, HBV viremia, transplant-related liver disease, and symptoms that may include any or all of the following: flu-like illness, weakness, pain, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain in the liver area of ​​the body, clay-colored or gray feces, generalized itching and dark urine, accompanied by a positive test for the presence of hepatitis B virus, hepatitis B virus antigen, or a positive test for the presence of antibodies specific to hepatitis B virus antigen.

[0129] "Closest" means that there are no intermediate elements between the adjacent elements.

[0130] "Individual" refers to a human or non-human animal selected for treatment or therapy.

[0131] "Individual compliance behavior" refers to an individual's adherence to recommended or prescribed therapies.

[0132] Terms such as “inducing,” “inhibiting,” “enhancing,” “increasing,” “adding,” and “reducing” generally indicate a quantitative difference between two states. These terms can refer to a statistically significant difference between two states. For example, “the amount of HBV activity or expression effectively inhibited” means that the level of HBV activity or expression in the treated sample will be quantitatively different from the level of HBV activity or expression in untreated cells, and may be statistically significant. These terms apply to, for example, expression levels and activity levels. As used herein, the terms “inhibit” or “reduce”, or their grammatical variations, mean a reduction or decrease in a specified level or activity 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, inhibition or reduction results in the presence of very little perceptible activity, or essentially no perceptible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0133] "Inhibition of HBV" means reducing the level or expression of HBV mRNA, DNA, and / or protein. In some embodiments, HBV is inhibited in the presence of an antisense compound containing an antisense oligonucleotide targeting HBV, compared to the expression levels of HBV mRNA, DNA, and / or protein in the absence of an HBV antisense compound such as an antisense oligonucleotide.

[0134] "Inhibition of expression or activity" refers to the reduction or blockage of expression or activity, and does not necessarily indicate the complete elimination of expression or activity.

[0135] "Injection site reaction" refers to inflammation or abnormal redness of the skin at the injection site in an individual.

[0136] "Nucleoside bond" refers to the chemical bond between nucleosides.

[0137] "Intraperitoneal administration" means administration via infusion or injection into the peritoneum.

[0138] "Intravenous administration" means administration via a vein.

[0139] "Extended" antisense oligonucleotides are antisense oligonucleotides that have one or more additional nucleosides relative to the antisense oligonucleotides disclosed herein.

[0140] "Linked deoxynucleosides" refers to deoxyribonucleic acid bases (A, G, C, T, U) linked by phosphate esters to form nucleotides.

[0141] "Linked nucleosides" refers to adjacent nucleosides linked together by an internucleotide bond. Examples of linked nucleosides are nucleotides in which the bond involves a phosphate ester group atom, such as a phosphodiester bond.

[0142] "Locked nucleic acid" or "LNA" or "LNA nucleotide" refers to a nucleic acid monomer with a bridge connecting two carbon atoms between the 4' and 2' positions of the nucleotide sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugars include, but are not limited to: (A) α-L-methyleneoxy(4'-CH2-O-2')LNA; (B) β-D-methyleneoxy(4'-CH2-O-2')-LNA; (C) ethyleneoxy(4'-(CH2)2-O-2')LNA; (D) aminooxy(4'-CH2-ON(R)-2')LNA; and (E) oxyamino(4'-CH2-N(R)-O-2')LNA; as described below.

[0143]

[0144] As used herein, LNA compounds include, but are not limited to, compounds having at least one bridge between the 4' and 2' positions of the sugar, wherein each bridge independently comprises one or two to four independently linked groups selected from: -[C(R 1 (R) 2 )]n-、-C(R 1 )=C(R 2 )-、-C(R 1 )=N-、-C(=NR 1 )-, -C(=O)-, -C(=S)-, -O-, -Si(R 1 )2-、-S(=O) x -and-N(R) 1 )-; where: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R 1 and R 2 Independently, it is H, protecting group, hydroxyl group, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 alkenyl, C2-C 12 Alkyne group, substituted C2-C 12 alkynyl group, C5-C 20 Aryl, substituted C5-C 20 Aryl, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, C5-C7 alicyclic, substituted C5-C7 alicyclic, halogen, OJ 1NJ I J 2 SJ 1 N3, COOJ 1 Acyl group (C(=O)-H), substituted acyl group, CN, sulfonyl group (S(=O)2-J) 1 ), or sulfoxide (S(=O)-J 1 ); and each J 1 and J 2 Independently, H, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 alkenyl, C2-C 12 Alkyne group, substituted C2-C 12 alkynyl group, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic, substituted heterocyclic, C1-C 12 Aminoalkyl, substituted C1-C 12 Aminoalkyl groups or protecting groups.

[0145] Examples of 4'-2' bridging groups covered within the definition of LNA include, but are not limited to, one of the following formulas: --[C(R 1 (R) 2 )] n -、-[C(R 1 (R) 2 )] n -O-、-C(R 1 (R) 2 )-N(R 1 )-O- or —C(R 1 (R) 2 )-ON(R 1 In addition, the definition of LNA also includes other bridging groups such as 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'-bridge, where each R 1 and R 2 Independently, it is H, a protecting group, or C1-C. 12 alkyl.

[0146] The definition of LNA according to the present invention also includes LNA in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH2-O-2') bridge to form a bicyclic sugar moiety. The bridge can also be a methylene (-CH2-) group connecting the 2' oxygen atom and the 4' carbon atom; for said group, the term methyleneoxy (4'-CH2-O-2')LNA is used. Furthermore, in the case of a bicyclic sugar moiety having an ethylene bridging group in this position, the term vinyloxy (4'-CH2CH2-O-2')LNA is used. As used herein, isomers of α-L-methyleneoxy (4'-CH2-O-2'), i.e., methyleneoxy (4'-CH2-O-2')LNA, are also covered within the definition of LNA.

[0147] "Maximum plasma concentration" or "C" max "Drug concentration" refers to the highest concentration of the drug in the plasma after administration of a drug dose to a subject. Methods for measuring drug concentration are known to those skilled in the art, and in particular include liquid chromatography and tandem mass spectrometry.

[0148] "Mismatch" or "non-complementary nucleobases" refers to a situation where the nucleobases of the first nucleic acid cannot pair with the corresponding nucleobases of the second or target nucleic acid.

[0149] "Modified nucleoside interbonds" refers to the substitution or any alteration of naturally occurring nucleoside interbonds (i.e., phosphodiester nucleoside interbonds).

[0150] "Modified nucleobases" refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. "Unmodified nucleobases" refers to purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0151] "Modified nucleosides" refers to nucleosides that independently possess modified sugar moieties and / or modified nucleobases.

[0152] "Modified nucleotides" refers to nucleotides that independently possess modified sugar moieties, modified nucleoside bonds, or modified nucleobases.

[0153] "Modified oligonucleotide" means an oligonucleotide that includes at least one modified nucleoside bond, a modified sugar and / or a modified nucleobase.

[0154] "Modified sugar" means the substitution and / or any alteration of the natural sugar portion.

[0155] "Monomer" refers to a single unit of an oligomer. Monomers include, but are not limited to, naturally occurring or modified nucleosides and nucleotides.

[0156] "Modal" refers to the pattern of unmodified and modified nucleosides in an antisense compound. "Natural sugar moiety" refers to the sugar moiety present in DNA (2'-H) or RNA (2'-OH). "Naturally present internucleotide bond" refers to the 3' to 5' phosphodiester bond.

[0157] "Non-complementary nucleobases" refer to a pair of nucleobases that do not form hydrogen bonds with each other or support hybridization in other ways.

[0158] "Nucleic acid" refers to molecules that contain monomeric nucleotides. Nucleic acids include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering RNA (siRNA), and microRNA (miRNA).

[0159] "Nucleobase" refers to the heterocyclic portion that can pair with a base of another nucleic acid.

[0160] "Nucleobase complementarity" refers to nucleobases that can pair with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In some embodiments, complementary nucleobases refer to the nucleobases of an antisense compound that can pair with the nucleobases of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound can hydrogen bond with a nucleobase at a certain position of the target nucleic acid, the position of the hydrogen bond between the oligonucleotide and the target nucleic acid is considered complementary at said nucleobase pair.

[0161] "Nucleobase sequence" refers to the sequence of consecutive nucleobases that is independent of any sugar, bond, and / or nucleobase modifications.

[0162] "Nucleoside" refers to a nucleobase linked to a sugar. Nucleosides include deoxynucleosides, such as deoxyribonucleoside.

[0163] "Nucleoside mimics" encompass those structures that replace sugars or sugar and bases and are not necessarily linked at one or more positions in an oligomer, such as nucleoside mimics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic, or tricyclic sugar mimics, for example, non-furanose sugar units. Nucleotide mimics encompass those structures that replace bonds at one or more positions in a nucleoside and oligomer, such as peptide nucleic acids or morpholino (morpholino linked by -N(H)-C(=O)-O- or other non-phosphodiester bonds). Sugar substitutions overlap with the slightly broader term nucleoside mimics but are intended only to indicate the substitution of sugar units (furanose rings). The tetrahydropyranyl rings provided herein illustrate examples of sugar substitutions in which the furanose sugar group has been replaced by a tetrahydropyranyl ring system. "Mimetic" refers to a group substituted by a sugar, nucleobase, and / or nucleoside inter-unit bond. Typically, analogs are used instead of sugars or sugar-nucleoside bonds, and nucleobases are maintained to hybridize with the selected target.

[0164] "Nucleotide" refers to a nucleoside that has a phosphate ester group covalently linked to the sugar portion of the nucleoside.

[0165] "Off-target effects" refer to unwanted or harmful biological effects associated with the regulation of RNA or the protein expression of genes other than their intended target nucleic acids.

[0166] "Oligomers" refers to polymers of linked monomeric subunits that can hybridize with at least one region of a nucleic acid molecule.

[0167] "Oligonucleotide" refers to an oligonucleotide in which the nucleoside internucleotide bond does not contain a phosphorus atom.

[0168] "Oligonucleotide" refers to a polymer of linked nucleosides, each of which may be modified or unmodified independently of the others.

[0169] "Parenteral administration" means administration by injection (e.g., bolus injection) or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intraperitoneal administration, or intracranial administration, such as intrathecal or intraventricular administration.

[0170] The term "peptide" refers to a molecule formed by linking at least two amino acids together via an amide bond. As used herein, "peptide" can refer to both polypeptides and proteins.

[0171] "Pharmaceutically acceptable carriers" refers to culture media or diluents that do not interfere with the structure of oligonucleotides. Some such carriers enable pharmaceutical compositions to be formulated into, for example, tablets, pills, sugar-coated pills, capsules, liquids, gels, syrups, pastes, suspensions, and lozenges for oral administration to subjects.

[0172] "Pharmaceutically acceptable derivatives" encompass pharmaceutically acceptable salts, conjugates, prodrugs, or isomers of the compounds described herein.

[0173] "Pharmaceutical acceptable salt" means a physiologically and pharmaceutically acceptable salt of an antisense compound, that is, a salt that retains the desired biological activity of the parent oligonucleotide without conferring undesirable toxicological effects.

[0174] "Pharmaceutical" means a substance that provides therapeutic benefit when administered to an individual. For example, in some embodiments, an antisense oligonucleotide targeting HBV is a pharmaceutical.

[0175] "Pharmaceutical composition" means a mixture of substances suitable for administration to a subject. For example, a pharmaceutical composition may include an antisense oligonucleotide and a sterile aqueous solution. In some embodiments, the pharmaceutical composition exhibits activity in certain cell lines in a free uptake assay.

[0176] A "thiophosphate bond" refers to a bond between nucleosides, where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A thiophosphate bond is a modified nucleoside bond.

[0177] "Partial" means a defined number of consecutive (i.e., linked) nucleobases of a nucleic acid. In some embodiments, a partial portion is a defined number of consecutive nucleobases of the target nucleic acid. In some embodiments, a partial portion is a defined number of consecutive nucleobases of the antisense compound.

[0178] "Prevention" refers to delaying or preventing the onset or development of symptoms or diseases for a period of time ranging from hours to days, preferably weeks to months.

[0179] "Prodrug" refers to a therapeutic agent prepared in an inactive form, which is converted into its active form (i.e., a drug) in the body or its cells by the action of endogenous enzymes or other chemicals and / or conditions.

[0180] "Effective dose" refers to the amount of a drug that provides preventive or therapeutic benefits to animals.

[0181] "Recommended therapy" refers to a treatment plan recommended by medical professionals for the treatment, improvement, or prevention of disease.

[0182] A “region” is defined as a portion of a target nucleic acid that has at least one identifiable structure, function, or characteristic.

[0183] "Ribosonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of the sugar moiety. Ribonucleotides can be modified with any substituent from a variety of substituents.

[0184] “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 without conferring undesirable toxicological effects.

[0185] A “segment” is defined as a smaller or sub-region within a target nucleic acid.

[0186] A "separator" is a segment or region that separates two gap regions and is located between the two gap regions in a chimeric antisense compound. The separator segment may have one or more nucleosides, wherein the nucleosides are chemically different from the nucleosides comprising the gap. The separator segment includes nucleosides modified to impart properties such as enhanced inhibitory activity, increased binding affinity to target nucleic acids, reduced in vivo toxicity, or resistance to degradation by in vivo nucleases. A chimeric antisense compound may include one or more separator segments. A separator may be referred to as a "separator," a "separator region," or a "separator segment." Exemplary chimeric antisense compounds of this disclosure include one, two, three, four, five, or six separator segments.

[0187] If HBeAg is monitored as a determinant of seroconversion, then "seroconversion" is defined as the absence of serum HBeAg plus the presence of serum HBeAb; or if HBsAg is monitored as a determinant of seroconversion, then said seroconversion is defined as the absence of serum HBsAg, as determined by the currently available detection limits of commercial ELISA systems.

[0188] The “shortened” or “truncated” versions of the antisense oligonucleotides taught in this article have one, two or more missing nucleosides.

[0189] "Side effects" refers to a physiological response to treatment that is attributable to an effect other than the desired outcome. In some embodiments, side effects 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 can indicate hepatotoxicity or abnormal liver function. For example, increased bilirubin can indicate hepatotoxicity or abnormal liver function.

[0190] As used in this article, “significant” means a measurable or observable result, such as a significant improvement or reduction.

[0191] As used in this article, "site" is defined as a unique nucleobase position within the target nucleic acid.

[0192] "Slowing the progression" means reducing the development of the disease.

[0193] "Specifically hybridizable" refers to an antisense compound that has sufficient complementarity between the antisense oligonucleotide and the target nucleic acid to induce the desired effect, while exhibiting minimal or no effect on non-target nucleic acids under the conditions where specific binding is expected, i.e., physiological conditions under which it is measured in vivo and used in therapeutic applications. "Strong hybridization conditions" or "strict conditions" refer to conditions under which the oligomer will hybridize with its target sequence, but with a minimum number of other sequences.

[0194] As used in this article, "statistical significance" refers to a measurable or observable parameter that is unlikely to occur by chance.

[0195] "Subcutaneous application" means application just beneath the skin. "Subject" refers to a human or non-human animal selected for treatment or therapy.

[0196] "Target" refers to the protein that is expected to be regulated.

[0197] "Target gene" refers to the gene that encodes the target.

[0198] “Targeting” refers to the process of designing and selecting antisense compounds that will specifically hybridize with the target nucleic acid and induce the desired effect.

[0199] "Target nucleic acid", "target RNA", "target RNA transcript" and "nucleic acid target" all refer to nucleic acids that can be targeted by antisense compounds.

[0200] "Target area" refers to a portion of the target nucleic acid that targets one or more antisense compounds.

[0201] "Target region" refers to the nucleotide sequence of the target nucleic acid that targets the antisense compound. "5' target site" refers to the 5th nucleotide of the target region. "3' target site" refers to the 3rd nucleotide of the target region.

[0202] "Therapeutic effective dose" refers to the amount of medication that provides therapeutic benefit to an individual.

[0203] "Treatment" refers to the application of a composition to achieve a change or improvement in a disease or symptom.

[0204] "Unmodified" nucleobases refer to purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0205] "Unmodified nucleotide" means a nucleotide that contains a naturally occurring nucleobase, a sugar moiety, and a nucleoside bond. In some embodiments, the unmodified nucleotide is an RNA nucleotide (i.e., 3-D-ribonucleoside) or a DNA nucleotide (i.e., 3-D-deoxyribonucleoside).

[0206] "Validated target segment" is defined as at least 8 nucleobase segments (i.e., 8 consecutive nucleobases) of the target region of an active oligomer compound.

[0207] A "wing" is the terminal segment of a chimeric antisense oligonucleotide, which is modified to endow the oligonucleotide with 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. A wing may 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 the linked nucleosides may include one or more deoxynucleosides, but the entire wing may not consist solely of deoxynucleosides.

[0208] The chimeric antisense compounds disclosed herein include a 5'-winged region (W1) located at the 5' end of the chimeric antisense oligonucleotide, and the residue at the 3' end of W1 cannot be a deoxynucleoside. The chimeric antisense compounds disclosed herein also include a 3'-winged region (W2) located at the 3' end of the chimeric antisense oligonucleotide, and the 5'-terminal residue of W2 cannot be a deoxynucleoside.

[0209] The 5' wing (W1) begins at the 5' end of the chimeric antisense oligonucleotide extending in the 5' to 3' direction and terminates at the first nucleoside of the deoxynucleoside that is not directly linked to the deoxynucleoside of the first gap, thus indicating the 3' end of W1 and the 5' end of the first gap (G1).

[0210] The 3' wing (W2) begins at the 3' end of the chimeric antisense oligonucleotide extending in the 3' to 5' direction and terminates at the first nucleotide of the deoxynucleotide that is not directly linked to the interstitial deoxynucleotide, thus indicating the 3' and 5' ends of the last interstitial space of W2.

[0211] Exemplary chimeric antisense oligonucleotides

[0212] This disclosure provides at least the following exemplary chimeric antisense oligonucleotides.

[0213] In one aspect, a modified oligonucleotide is provided, said modified oligonucleotide comprising, from 5' to 3':

[0214] 5' W1 - G1 - S1 - G2 - W2 3' (Formula I)

[0215] in:

[0216] W1 is the 5' wing section;

[0217] W2 is the 3' wing section;

[0218] G1 is the first gap section;

[0219] S1 is the first dividing sub-segment;

[0220] G2 is the second gap section;

[0221] - is a nucleoside internucleotide bond; and

[0222] At least one nucleoside of the oligonucleotide is modified.

[0223] In some embodiments, the modified oligonucleotide from 5' to 3' comprises:

[0224] 5' W1 - G1 - S1 - G2 - S2 - G3 - W2 3' (Formula II)

[0225] in:

[0226] S2 is the second dividing sub-segment; and

[0227] G3 is the third gap section.

[0228] In some embodiments, the modified oligonucleotide from 5' to 3' comprises:

[0229] 5' W1 - G1 - S1 - G2 - S2 - G3 - S3 - G4 - W2 3' (Formula III)

[0230] in:

[0231] S3 is the third dividing sub-segment; and

[0232] G4 is the fourth gap section.

[0233] In some embodiments, the modified oligonucleotide from 5' to 3' comprises:

[0234] 5' W1 – G1 – S1 – G2 – S2 – G3 – S3 – G4 – S4 – G5 – W2 3’ (Formula IV)

[0235] in:

[0236] S4 is the fourth dividing sub-segment; and

[0237] G5 is the fifth gap section.

[0238] In some embodiments, the modified oligonucleotide from 5' to 3' comprises:

[0239] 5' W1 – G1 – S1 – G2 – S2 – G3 – S3 – G4 – S4 – G5 – S5 – G6 – W2 3’ (Formula V)

[0240] in:

[0241] S5 is the fifth dividing sub-segment; and

[0242] G6 is the sixth gap section.

[0243] In some embodiments, the modified oligonucleotide from 5' to 3' comprises:

[0244] 5' W1 – G1 – S1 – G2 – S2 – G3 – S3 – G4 – S4 – G5 – S5 – G6 – S6 –G7 – W2 3’ (Formula VI)

[0245] in:

[0246] S6 is the sixth dividing sub-segment; and

[0247] G7 is the seventh gap section.

[0248] In some embodiments, W1 comprises 2 to 25 linked nucleosides. In some embodiments, W1 comprises one or more linked deoxynucleosides.

[0249] In some embodiments, W2 comprises 2 to 35 linked nucleosides. In some embodiments, W2 comprises one or more linked deoxynucleosides.

[0250] In some embodiments, any one or more of G1, G2, G3, G4, G5, G6 and / or G7 comprises 1 to 10 linked deoxynucleosides.

[0251] In some embodiments, any one or more of S1, S2, S3, S4, S5 and / or S6 include one, two, three, four or five linked deoxynucleosides.

[0252] In some embodiments, the modified oligonucleotide is 18-50 nucleotides in length. In some embodiments, the modified oligonucleotide is at least 20 nucleotides in length. In some embodiments, the modified oligonucleotide is 20 nucleotides in length.

[0253] In some embodiments, the modified oligonucleotide has Formula I, and W1 comprises 4-6 linked nucleosides, G1 comprises 1-6 linked deoxynucleosides, S1 comprises 1 linked nucleoside, G2 comprises 1-6 linked deoxynucleosides, and W2 comprises 4-6 linked nucleosides.

[0254] In some embodiments, the modified oligonucleotide has Formula I, and W1 comprises 4-6 linked nucleosides, G1 comprises 5 linked deoxynucleosides, S1 comprises 1 linked nucleoside, G2 comprises 5 linked deoxynucleosides, and W2 comprises 4-6 linked nucleosides.

[0255] In some embodiments, the modified oligonucleotide has Formula I, and W1 comprises 4 linked nucleosides, G1 comprises 5 linked deoxynucleosides, S1 comprises 1 linked nucleoside, G2 comprises 5 linked deoxynucleosides, and W2 comprises 5 linked nucleosides.

[0256] In some embodiments, the modified oligonucleotide has Formula I, and the length of G1-S1-G2 is between 8 and 12 nucleotides.

[0257] The following embodiments relate to any one of formulas I-VI: In some embodiments, G1, G2, G3, G4, G5, G6, and G7 may include nucleosides containing modifications of 2'-deoxynucleosides. In some embodiments, any one or more of G1, G2, G3, G4, G5, G6, and G7 include nucleosides containing modifications of 2'-deoxy-5-methylcytidine sugar. In some embodiments, S1, S2, S3, S4, S5, and / or S6 include nucleosides containing modifications of 2'-O-methoxyethyl sugar. In some embodiments, S1, S2, S3, S4, S5, and / or S6 include nucleosides containing 5-methylcytidine. In some embodiments, S1, S2, S3, S4, S5, and / or S6 include nucleosides containing modifications of 2'-O-methyl sugar. In some embodiments, S1, S2, S3, S4, S5, and / or S6 include nucleosides containing modifications of 2'-OH sugar. In some embodiments, S1, S2, S3, S4, S5, and / or S6 comprise nucleosides modified with 2'-fluorosugars. In some embodiments, S1, S2, S3, S4, S5, and / or S6 comprise nucleosides modified with 2'-fluoro-arabinonucleotide (2'-fluoro-ANA) sugars. In some embodiments, S1, S2, S3, S4, S5, and / or S6 comprise glycolic acid (GNA). In some embodiments, S1, S2, S3, S4, S5, and / or S6 comprise LNA. In some embodiments, W1 comprises nucleosides modified with 2'-deoxysugars (e.g., wherein the 2'-deoxysugar modification is located at positions 2 and / or 5 of the sequence corresponding to SEQ ID NO: 2). In some embodiments, W1 comprises nucleosides modified with 2'-O-methoxyethylsugars (e.g., wherein the 2'-O-methoxyethylsugar 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 includes 2'-O-methoxyethyl 5-methylcytidine at position 2 of the sequence corresponding to SEQ ID NO: 2. In some embodiments, W1 includes a nucleoside containing a 2'-O-methyl sugar modification (e.g., wherein 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 includes 2'-O-methyl 5-methylcytidine at position 2 of the sequence corresponding to SEQ ID NO: 2. In some embodiments, W1 includes a nucleoside containing a 2'-fluorosugar modification. In some embodiments, W1 includes a nucleoside containing a 2'-fluoro-arabinonucleotide (2'-fluoro-ANA) modification. In some embodiments, W1 includes ethylene glycol nucleic acid (GNA) (e.g., wherein the GNA is located at position 2 of the sequence corresponding to SEQ ID NO: 2).In some embodiments, W1 comprises a modified nucleoside, wherein the modified nucleoside is a locked nucleic acid (LNA) (e.g., wherein the LNA is located at position 1 and / or 3 of the sequence corresponding to SEQ ID NO: 2). In some embodiments, W2 comprises a nucleoside containing a 2'-deoxysugar modification (e.g., wherein the 2'-deoxysugar modification is located at position 15 and / or 16 of the sequence corresponding to SEQ ID NO: 2). In some embodiments, W2 comprises a nucleoside containing a 2'-O-methoxyethylsugar modification (e.g., wherein the 2'-O-methoxyethylsugar 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 comprises a nucleoside containing a 2'-O-methylsugar modification (e.g., wherein the 2'-O-methylsugar 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 comprises a nucleoside containing a 2'-fluorosugar modification. In some embodiments, W2 comprises a nucleoside modified with 2'-fluoro-arabinonucleotide (2'-fluoro-ANA). In some embodiments, W2 comprises a modified nucleoside, wherein the modified nucleoside is ethylene glycol nucleic acid (GNA). In some embodiments, W2 comprises a modified nucleoside, wherein the modified nucleoside is locked nucleic acid (LNA) (e.g., wherein the LNA is located at positions 16, 17, 18, 19 and / or 20 of the sequence corresponding to SEQ ID NO: 2).

[0258] Exemplary target sequences and exemplary modified oligonucleotides

[0259] Some embodiments provide methods, compounds, and compositions for inhibiting HBV mRNA expression.

[0260] Some embodiments provide antisense compounds that target HBV nucleic acid sequences. Exemplary HBV nucleic acid sequences include, but are not limited to, the sequences shown in Table 1.

[0261] Table 1. Exemplary HBV sequences

[0262]

[0263] In some embodiments, the HBV nucleic acid is the sequence shown in GENBANK accession number U95551.1 (incorporated herein as SEQ ID NO: 3). In some embodiments, the antisense compound targets the sequence described 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.

[0264] Exemplary HBV nucleic acid target sequences include, but are not limited to, the sequences shown in Table 2.

[0265] Table 2. Exemplary HBV target sequences

[0266]

[0267]

[0268]

[0269]

[0270]

[0271] In some embodiments, the HBV target includes the sequences of SEQ ID NO: 1, 4, 675-838. In some embodiments, the HBV target includes the sequence of CTTGG TCATG GGCCA TCAG (SEQ ID NO: 1). In some embodiments, the HBV target includes the sequence of GCACT TCGCT TCACC TCTGC (SEQ ID NO: 4).

[0272] In some embodiments, the compounds provided herein comprise modified oligonucleotides. In some embodiments, the compounds comprise modified oligonucleotides and conjugates as described herein. In some embodiments, the modified oligonucleotides are pharmaceutically acceptable derivatives.

[0273] In some embodiments, the HBV target includes the sequence described in SEQ ID NO:3 or a portion thereof or a variant thereof. In some 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.

[0274] In some embodiments, the HBV target includes the sequence described in SEQ ID NO: 1. In some 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.

[0275] In some embodiments, the HBV target includes the sequence described in SEQ ID NO: 4. In some 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.

[0276] In some embodiments, the compound or composition comprises a modified oligonucleotide of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 linked nucleosides that bind to the HBV target sequence. In some embodiments, the compound or composition comprises a modified oligonucleotide of 20 linked nucleosides that bind to the HBV target sequence.

[0277] In some embodiments, the modified oligonucleotides of the 20 linked nucleosides have the nucleobase sequence GCAGA GGTGAAGCGA AGTGC (SEQ ID NO: 2). A diagram illustrating the nucleobase positions of SEQ ID NO: 2 is shown below:

[0278]

[0279] In some embodiments, the modified oligonucleotides of the 20 linked nucleosides have the nucleotide sequence GTGAA GCGAA GTGCACACGG (SEQ ID NO: 5). A diagram showing the nucleotide positions of SEQ ID NO: 5 is shown below:

[0280]

[0281] In some embodiments, the compounds include the modified oligonucleotides described herein. Exemplary modified oligonucleotides include, but are not limited to, those described herein. Figures 1-2 Those shown. In some embodiments, the compound comprises a modified oligonucleotide comprising a sequence of any one of AUS1010 to AUS1714. In some embodiments, the compound comprises a modified oligonucleotide comprising a sequence of any one of SEQ ID NO: 11 to 666, or a modified oligonucleotide comprising one, two, three, four, or five modifications to said modified oligonucleotide.

[0282] In some embodiments, the compound comprises a modified oligonucleotide containing any 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 letter identifier, AUS identifier, and SEQ ID NO corresponding to the sequence of the modified oligonucleotide.

[0283] Table 3. Nomenclature of Modified Oligonucleotides

[0284]

[0285]

[0286] Characteristics of modified oligonucleotides

[0287] In some embodiments, the compound comprises a modified oligonucleotide consisting of 20 linked nucleosides: a first gap segment composed of linked nucleosides; a second gap segment composed of linked nucleosides; a separator segment composed of nucleosides linked to the first and second gap segments; a 5' wing segment composed of linked nucleosides; and a 3' wing segment composed of linked nucleosides; wherein the first gap segment, the separator segment, and the second gap segment are positioned between the 5' wing segment and the 3' wing segment; wherein the first gap segment is linked to the 5' wing segment, and the second gap segment is linked to the 3' wing segment; wherein the first gap segment, the separator segment, and the second gap segment together consist of 8, 9, 10, 11, or 12 linked nucleosides.

[0288] In some embodiments, the compound comprises a modified oligonucleotide consisting of 20 linked nucleosides: a first gap segment composed of linked nucleosides; a second gap segment composed of linked nucleosides; a separator segment composed of nucleosides linked between the first gap segment and the second gap segment; wherein 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 composed of linked nucleosides; and a 3' wing segment. The segment is composed of linked nucleosides; wherein the first gap segment, the second gap segment, and the separator segment are located between the 5' wing segment and the 3' wing segment; wherein the first gap segment is connected to the 5' wing segment, and the second gap segment is connected to the 3' wing segment; wherein the first gap segment is composed of a linked nucleoside having a 2'-deoxy sugar; wherein the second gap segment is composed of a linked nucleoside having a 2'-deoxy sugar; wherein the separator segment is composed of a nucleoside comprising a 2'-O(CH2)2-OCH3 sugar or a 2'-OCH3 sugar, wherein the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 1; and wherein the modified oligonucleotide has the nucleobase sequence of SEQ ID NO: 2 (GCAGA GGTGA AGCGA AGTGC).

[0289] In some embodiments, the compound comprises a modified oligonucleotide consisting of 20 linked nucleosides: a first gap segment composed of linked nucleosides; a second gap segment composed of linked nucleosides; a separator segment composed of nucleosides linked between the first gap segment and the second gap segment; wherein 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 composed of linked nucleosides; and a 3' wing segment composed of linked nucleosides; wherein the first gap segment, the second gap segment, and the separator segment are positioned within the... Between the 5' wing segment and the 3' wing segment; wherein the first gap segment is connected to the 5' wing segment, and the second gap segment is connected to the 3' wing segment; wherein the first gap segment is composed of a nucleoside linked by a 2'-deoxy sugar; wherein the second gap segment is composed of a nucleoside linked by a 2'-deoxy sugar; wherein the separator segment is composed of a nucleoside comprising a 2'-O(CH2)2-OCH3 sugar or a 2'-OCH3 sugar, wherein at least one nucleoside in the 5' wing segment comprises a 2'-O(CH2)2-OCH3 sugar; and wherein at least one nucleoside in the 3' wing segment comprises a 2'-O(CH2)2-OCH3 sugar; wherein the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 1; and wherein the modified oligonucleotide has the nucleobase sequence of SEQ ID NO: 2 (GCAGA GGTGA AGCGA AGTGC).

[0290] A. General 5' wing section

[0291] In some embodiments, at least one nucleoside in the 5' wing segment comprises a 2'-O(CH2)2-OCH3 sugar. In some embodiments, each nucleoside in the 5' wing segment comprises a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment comprises a nucleoside having a 2'-deoxy sugar. In some embodiments, the 5' wing segment comprises a nucleoside having a 2'-deoxy sugar, and other nucleosides in the 5' wing segment comprise a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the nucleoside at position 2 of SEQ ID NO: 2 comprises a 2'-deoxy sugar. In some embodiments, the nucleoside at position 3 of SEQ ID NO: 2 comprises a 2'-deoxy sugar. In some embodiments, the nucleoside at position 4 of SEQ ID NO: 2 comprises a 2'-deoxy sugar. In some embodiments, the 5' wing segment further comprises two nucleosides, each having a 2'-deoxy sugar. In some embodiments, the nucleosides located at positions 2 and 3 of SEQ ID NO: 2 comprise 2'-deoxy sugars. In some embodiments, the nucleosides located at positions 2 and 4 of SEQ ID NO: 2 comprise 2'-deoxy sugars. In some embodiments, the nucleosides located at positions 3 and 4 of SEQ ID NO: 2 comprise 2'-deoxy sugars. In some embodiments, the nucleosides located at positions 2 and 5 of SEQ ID NO: 2 comprise 2'-deoxy sugars.

[0292] In some embodiments, one, two, three, or four nucleotides in the 5' wing region comprise the sugar modifications described herein. In some embodiments, one, two, three, or four nucleotides in the 5' wing region comprise bicyclic sugars. In some embodiments, one, two, three, or four nucleotides in the 5' wing region comprise restricted ethyl sugars.

[0293] In some embodiments, one, two, three, or four nucleotides in the 5' wing segment comprise locked nucleic acids. In some embodiments, one, two, three, or four nucleotides in the 5' wing segment comprise a 4'-CH2-O-2' sugar. In some embodiments, two nucleotides in the 5' wing segment comprise locked nucleic acids. In some embodiments, two nucleotides in the 5' wing segment comprise a 4'-CH2-O-2' sugar. In some embodiments, the nucleotides located at positions 1 and 3 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the nucleotides located at positions 1 and 3 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar.

[0294] B1. 5' Wing with 4 linked nucleosides

[0295] In some embodiments, the 5' wing segment is composed of four linked nucleosides. In some embodiments, the 5' wing segment is composed of four linked nucleosides, wherein each of the four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment is composed of four linked nucleosides, wherein three of the four linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment is composed of four linked nucleosides, wherein two of the four linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment is composed of four linked nucleosides, wherein one of the four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0296] B2. A 5'-winged region with four linked nucleosides, 2'-deoxy combination

[0297] In some embodiments, the 5' wing segment is composed of four linked nucleosides, wherein one of the linked nucleosides comprises a nucleoside having a 2'-deoxy sugar. In some embodiments, the 5' wing segment is composed of four linked nucleosides, wherein two of the linked nucleosides comprise nucleosides having a 2'-deoxy sugar. In some embodiments, the 5' wing segment is composed of four linked nucleosides, wherein one of the four linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment is composed of four linked nucleosides, wherein one of the four linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, one of the four linked nucleosides comprises a locked nucleic acid, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein one of the four linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, two of the four linked nucleosides comprise locked nucleic acids, and the other linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0298] In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein the nucleoside at position 2 of SEQ ID NO: 2 comprises a 2'-deoxy sugar. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein the nucleoside at position 2 of SEQ ID NO: 2 comprises a 2'-deoxy sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein the nucleoside at position 3 of SEQ ID NO: 2 comprises a 2'-deoxy sugar. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein the nucleoside at position 3 of SEQ ID NO: 2 comprises a 2'-deoxy sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein the nucleoside at position 4 of SEQ ID NO: 2 comprises a 2'-deoxy sugar. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein the nucleoside at position 4 of SEQ ID NO: 2 comprises a 2'-deoxy sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0299] In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 2 and 3 of SEQ ID NO: 2 comprise 2'-deoxy sugars. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 2 and 3 of SEQ ID NO: 2 comprise 2'-deoxy sugars, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 2 and 4 of SEQ ID NO: 2 comprise 2'-deoxy sugars. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 2 and 4 of SEQ ID NO: 2 comprise 2'-deoxy sugars, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 3 and 4 of SEQ ID NO: 2 comprise 2'-deoxy sugars. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein the nucleosides at positions 3 and 4 of SEQ ID NO: 2 comprise 2'-deoxy sugars, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0300] B3. A 5'-winged region with four linked nucleosides, 2'-LNA combination

[0301] In some embodiments, the 5' wing region consists of four linked nucleosides, wherein one of the four linked nucleosides comprises a locked nucleic acid. In some embodiments, the 5' wing region consists of four linked nucleosides, wherein two of the four linked nucleosides comprise a locked nucleic acid. In some embodiments, the 5' wing region consists of four linked nucleosides, wherein one of the four linked nucleosides comprises a 4'-CH2-O-2' sugar. In some embodiments, the 5' wing region consists of four linked nucleosides, wherein two of the four linked nucleosides comprise a 4'-CH2-O-2' sugar.

[0302] In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein one of the four linked nucleosides comprises a locked nucleic acid, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein two of the four linked nucleosides comprise a locked nucleic acid, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein one of the four linked nucleosides comprises a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of four linked nucleosides, wherein two of the four linked nucleosides comprise 4'-CH2-O-2' sugars, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0303] In some embodiments, the 5' wing region consists of four linked nucleosides, and the nucleosides at positions 1 and 3 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 5' wing region consists of four linked nucleosides, and the nucleosides at positions 1 and 3 of SEQ ID NO: 2 include 4'-CH2-O-2' sugars.

[0304] In some embodiments, the 5' wing segment consists of four linked nucleosides, and the nucleosides at positions 1 and 3 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of four linked nucleosides, and the nucleosides at positions 1 and 3 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0305] C1. 5' Wing with 5 linked nucleosides

[0306] In some embodiments, the 5' wing segment is composed of five linked nucleosides. In some embodiments, the 5' wing segment is composed of five linked nucleosides, wherein each of the five linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment is composed of five linked nucleosides, wherein four of the five linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment is composed of five linked nucleosides, wherein three of the five linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment is composed of five linked nucleosides, wherein two of the five linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein one of the five linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0307] C2. A 5'-winged region with 5 linked nucleosides, 2'-deoxy combination

[0308] In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein one of the linked nucleosides comprises a nucleoside having a 2'-deoxy sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein two of the linked nucleosides comprise a nucleoside having a 2'-deoxy sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein one of the five linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein one of the five linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, one of the five linked nucleosides comprises a locked nucleic acid, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein one of the five linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, two of the five linked nucleosides comprise locked nucleic acids, and the other two linked nucleosides comprise nucleosides having a 2'-O(CH2)2-OCH3 sugar.

[0309] In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleoside at position 2 of SEQ ID NO: 2 comprises a 2'-deoxy sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleoside at position 2 of SEQ ID NO: 2 comprises a 2'-deoxy sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleoside at position 3 of SEQ ID NO: 2 comprises a 2'-deoxy sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleoside at position 3 of SEQ ID NO: 2 comprises a 2'-deoxy sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleoside at position 4 of SEQ ID NO: 2 comprises a 2'-deoxy sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleoside at position 4 of SEQ ID NO: 2 comprises a 2'-deoxy sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0310] In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 2 and 3 of SEQ ID NO: 2 comprise 2'-deoxy sugars. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 2 and 3 of SEQ ID NO: 2 comprise 2'-deoxy sugars, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 2 and 4 of SEQ ID NO: 2 comprise 2'-deoxy sugars. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 2 and 4 of SEQ ID NO: 2 comprise 2'-deoxy sugars, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 3 and 4 of SEQ ID NO: 2 comprise 2'-deoxy sugars. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein the nucleosides at positions 3 and 4 of SEQ ID NO: 2 comprise 2'-deoxy sugars, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0311] C3. A 5'-winged region with 5 linked nucleosides, 2'-LNA combination

[0312] In some embodiments, the 5' wing segment is composed of five linked nucleosides, wherein one of the five linked nucleosides comprises a locked nucleic acid. In some embodiments, the 5' wing segment is composed of five linked nucleosides, wherein two of the five linked nucleosides comprise a locked nucleic acid. In some embodiments, the 5' wing segment is composed of five linked nucleosides, wherein one of the five linked nucleosides comprises a 4'-CH2-O-2' sugar. In some embodiments, the 5' wing segment is composed of five linked nucleosides, wherein two of the five linked nucleosides comprise a 4'-CH2-O-2' sugar.

[0313] In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein one of the linked nucleosides comprises a locked nucleic acid, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein two of the linked nucleosides comprise a locked nucleic acid, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein one of the linked nucleosides comprises a 4'-CH2-O-2' sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, wherein two of the five linked nucleosides comprise 4'-CH2-O-2' sugars, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0314] In some embodiments, the 5' wing region consists of five linked nucleosides, and the nucleosides at positions 1 and 3 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 5' wing region consists of five linked nucleosides, and the nucleosides at positions 1 and 3 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0315] In some embodiments, the 5' wing segment consists of five linked nucleosides, and the nucleosides at positions 1 and 3 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 5' wing segment consists of five linked nucleosides, and the nucleosides at positions 1 and 3 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0316] D. Generally, the 3' wing section

[0317] In some embodiments, at least one nucleotide in the 3' wing segment comprises a 2'-O(CH2)2-OCH3 sugar. In some embodiments, each nucleotide in the 3' wing segment comprises a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment comprises a nucleotide having a 2'-deoxy sugar. In some embodiments, the 3' wing segment comprises a nucleotide having a 2'-deoxy sugar, and other nucleotides in the 3' wing segment comprise a 2''-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment further comprises two nucleotides, each having a 2'-deoxy sugar.

[0318] In some embodiments, one, two, three, or four nucleotides in the 3' wing region comprise the sugar modifications described herein. In some embodiments, one, two, three, or four nucleotides in the 3' wing region comprise bicyclic sugars. In some embodiments, one, two, three, or four nucleotides in the 3' wing region comprise restricted ethyl sugars.

[0319] In some embodiments, one, two, three, or four nucleotides in the 3' wing segment comprise locked nucleic acids. In some embodiments, one, two, three, or four nucleotides in the 3' wing segment comprise a 4'-CH2-O-2' sugar. In some embodiments, two nucleotides in the 3' wing segment comprise locked nucleic acids. In some embodiments, two nucleotides in the 3' wing segment comprise a 4'-CH2-O-2' sugar. In some embodiments, three nucleotides in the 3' wing segment comprise locked nucleic acids. In some embodiments, three nucleotides in the 3' wing segment comprise a 4'-CH2-O-2' sugar.

[0320] In some embodiments, the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars. In some embodiments, the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars. In some embodiments, the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars. In some embodiments, the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars. In some embodiments, the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars. In some embodiments, the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars. In some embodiments, the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars. In some embodiments, the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars. In some embodiments, the nucleosides located at positions 19 and 20 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the nucleosides located at positions 19 and 20 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars.

[0321] In some embodiments, the nucleosides located at positions 17, 18, and 19 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the nucleosides located at positions 17, 18, and 19 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars. In some embodiments, the nucleosides located at positions 17, 19, and 20 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the nucleosides located at positions 17, 19, and 20 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars.

[0322] E1. 3' Wing with 4 linked nucleosides

[0323] In some embodiments, the 3' wing segment is composed of four linked nucleosides. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein each of the four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein three of the four linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein two of the four linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein one of the four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0324] E2. A 3'-winged region with four linked nucleosides, 2'-deoxy combination

[0325] In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein one of the linked nucleosides comprises a nucleoside having a 2'-deoxy sugar. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein two of the linked nucleosides comprise nucleosides having a 2'-deoxy sugar. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein one of the four linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein one of the four linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, one of the four linked nucleosides comprises a locked nucleic acid, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein one of the four linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, two of the four linked nucleosides comprise locked nucleic acids, and the other linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0326] E3. A 3'-winged region with four linked nucleosides, 2'-LNA combination

[0327] In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein one of the four linked nucleosides comprises a locked nucleic acid. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein two of the four linked nucleosides comprise a locked nucleic acid. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein one of the four linked nucleosides comprises a 4'-CH2-O-2' sugar. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein two of the four linked nucleosides comprise a 4'-CH2-O-2' sugar.

[0328] In some embodiments, the 3' wing segment consists of four linked nucleosides, wherein one of the four linked nucleosides comprises a locked nucleic acid, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of four linked nucleosides, wherein two of the four linked nucleosides comprise a locked nucleic acid, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of four linked nucleosides, wherein three of the four linked nucleosides comprise a locked nucleic acid, and the other linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein one of the four linked nucleosides comprises a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein two of the four linked nucleosides comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of four linked nucleosides, wherein three of the four linked nucleosides comprise a 4'-CH2-O-2' sugar, and the other linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0329] In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars.

[0330] In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0331] In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 include 4'-CH2-O-2' sugars.

[0332] In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0333] In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 include 4'-CH2-O-2' sugars.

[0334] In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0335] In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars.

[0336] In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0337] In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 include 4'-CH2-O-2' sugars.

[0338] In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0339] In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 include 4'-CH2-O-2' sugars.

[0340] In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0341] In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 include 4'-CH2-O-2' sugars.

[0342] In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0343] In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 include 4'-CH2-O-2' sugars.

[0344] In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0345] In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 include 4'-CH2-O-2' sugars.

[0346] In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0347] In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise locked nucleic acids. In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise 4'-CH2-O-2' sugars.

[0348] In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 include locked nucleic acids, and the other linked nucleosides include nucleosides having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 include a 4'-CH2-O-2' sugar, and the other linked nucleosides include nucleosides having a 2'-O(CH2)2-OCH3 sugar.

[0349] In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of four linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 include 4'-CH2-O-2' sugars.

[0350] In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 include locked nucleic acids, while the other linked nucleosides include nucleosides having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of four linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 include a 4'-CH2-O-2' sugar, while the other linked nucleosides include nucleosides having a 2'-O(CH2)2-OCH3 sugar.

[0351] F1. 3' Wing with 5 linked nucleosides

[0352] In some embodiments, the 3' wing segment is composed of five linked nucleosides. In some embodiments, the 3' wing segment is composed of five linked nucleosides, wherein each of the five linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of five linked nucleosides, wherein four of the five linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of five linked nucleosides, wherein three of the five linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of five linked nucleosides, wherein two of the five linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, wherein one of the five linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0353] F2. A 3'-winged region with 5 linked nucleosides, 2'-deoxy combination

[0354] In some embodiments, the 3' wing segment consists of five linked nucleosides, wherein one of the linked nucleosides comprises a nucleoside having a 2'-deoxy sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, wherein two of the linked nucleosides comprise nucleosides having a 2'-deoxy sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, wherein one of the five linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, wherein one of the five linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, one of the five linked nucleosides comprises a locked nucleic acid, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, wherein one of the five linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, two of the five linked nucleosides comprise locked nucleic acids, and the other two linked nucleosides comprise nucleosides having a 2'-O(CH2)2-OCH3 sugar.

[0355] F3. A 3' wing region with 5 linked nucleosides, 2'-LNA combination

[0356] In some embodiments, the 3' wing segment is composed of five linked nucleosides, wherein one of the five linked nucleosides comprises a locked nucleic acid. In some embodiments, the 3' wing segment is composed of five linked nucleosides, wherein two of the five linked nucleosides comprise a locked nucleic acid. In some embodiments, the 3' wing segment is composed of five linked nucleosides, wherein one of the five linked nucleosides comprises a 4'-CH2-O-2' sugar. In some embodiments, the 3' wing segment is composed of five linked nucleosides, wherein two of the five linked nucleosides comprise a 4'-CH2-O-2' sugar.

[0357] In some embodiments, the 3' wing segment consists of five linked nucleosides, wherein one of the five linked nucleosides comprises a locked nucleic acid, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, wherein two of the five linked nucleosides comprise a locked nucleic acid, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, wherein three of the five linked nucleosides comprise a locked nucleic acid, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of five linked nucleosides, wherein one of the five linked nucleosides comprises a 4'-CH2-O-2' sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of five linked nucleosides, wherein two of the five linked nucleosides comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of five linked nucleosides, wherein three of the five linked nucleosides comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0358] In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0359] In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0360] In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0361] In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0362] In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0363] In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0364] In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0365] In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0366] In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0367] In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0368] In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0369] In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0370] In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0371] In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0372] In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0373] In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0374] In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0375] In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0376] In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 include 4'-CH2-O-2' sugars.

[0377] In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0378] In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of five linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 include 4'-CH2-O-2' sugars.

[0379] In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of five linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other two linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0380] G1. 3' Wing with 6 linked nucleosides

[0381] In some embodiments, the 3' wing segment is composed of six linked nucleosides. In some embodiments, the 3' wing segment is composed of six linked nucleosides, wherein each of the six linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of six linked nucleosides, wherein five of the six linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of six linked nucleosides, wherein four of the six linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of six linked nucleosides, wherein three of the six linked nucleosides comprise a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of six linked nucleosides, wherein two of the six linked nucleosides comprise nucleosides having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of six linked nucleosides, wherein one of the six linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0382] G2. A 3'-winged region with 6 linked nucleosides, 2'-deoxy combination

[0383] In some embodiments, the 3' wing segment consists of six linked nucleosides, wherein one of the linked nucleosides comprises a nucleoside having a 2'-deoxy sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, wherein two of the linked nucleosides comprise nucleosides having a 2'-deoxy sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, wherein one of the six linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, and each of the other five linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, wherein one of the six linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, one of the six linked nucleosides comprises a locked nucleic acid, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, wherein one of the five linked nucleosides comprises a nucleoside having a 2'-deoxy sugar, two of the six linked nucleosides comprise locked nucleic acids, and the other three linked nucleosides comprise nucleosides having a 2'-O(CH2)2-OCH3 sugar.

[0384] G3. A 3' wing region with 6 linked nucleosides, 2'-LNA combination

[0385] In some embodiments, the 3' wing segment is composed of six linked nucleosides, wherein one of the six linked nucleosides comprises a locked nucleic acid. In some embodiments, the 3' wing segment is composed of six linked nucleosides, wherein two of the six linked nucleosides comprise a locked nucleic acid. In some embodiments, the 3' wing segment is composed of six linked nucleosides, wherein one of the six linked nucleosides comprises a 4'-CH2-O-2' sugar. In some embodiments, the 3' wing segment is composed of six linked nucleosides, wherein two of the six linked nucleosides comprise a 4'-CH2-O-2' sugar.

[0386] In some embodiments, the 3' wing segment comprises six linked nucleosides, wherein one of the six linked nucleosides comprises a locked nucleic acid, and each of the other five linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment comprises six linked nucleosides, wherein two of the six linked nucleosides comprise a locked nucleic acid, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment comprises six linked nucleosides, wherein three of the six linked nucleosides comprise a locked nucleic acid, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of six linked nucleosides, wherein one of the six linked nucleosides comprises a 4'-CH2-O-2' sugar, and each of the other five linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of six linked nucleosides, wherein two of the six linked nucleosides comprise a 4'-CH2-O-2' sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment is composed of six linked nucleosides, wherein three of the six linked nucleosides comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0387] In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0388] In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 16 and 18 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0389] In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0390] In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 16 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0391] In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0392] In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 16 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0393] In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0394] In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17 and 18 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0395] In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0396] In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0397] In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0398] In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0399] In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0400] In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 18 and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0401] In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0402] In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 18 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0403] In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 include 4'CH2-O-2' sugars.

[0404] In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 19 and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other four linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0405] In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 include 4'-CH2-O-2' sugars.

[0406] In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17, 18, and 19 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0407] In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 include locked nucleic acids. In some embodiments, the 3' wing region consists of six linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 include 4'-CH2-O-2' sugars.

[0408] In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 comprise locked nucleic acids, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the 3' wing segment consists of six linked nucleosides, and the nucleosides at positions 17, 19, and 20 of SEQ ID NO: 2 comprise a 4'-CH2-O-2' sugar, and each of the other three linked nucleosides comprises a nucleoside having a 2'-O(CH2)2-OCH3 sugar.

[0409] H-0. Gap section

[0410] In some embodiments, the modified oligonucleotide includes or consists of at least one gap segment. In some embodiments, the modified oligonucleotide includes or consists of at least two gap segments. In some embodiments, the modified oligonucleotide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 gap segments. The gap segments are consecutively referred to as a first gap segment, a second gap segment, a third gap segment, a fourth gap segment, a fifth gap segment, a sixth gap segment, a seventh gap segment, an eighth gap segment, a ninth gap segment, or a tenth gap segment, wherein the first gap segment is closest to the 5' end of the modified oligonucleotide, and the last gap segment is closest to the 3' end of the modified oligonucleotide. In some embodiments, the modified oligonucleotide includes or consists of two gap segments. In some embodiments, the modified oligonucleotide includes or consists of three gap segments. In some embodiments, the modified oligonucleotide includes or consists of four gap segments. In some embodiments, the modified oligonucleotide includes or consists of five gap segments. In some embodiments, the modified oligonucleotide comprises or consists of six interstitial segments. In some embodiments, the modified oligonucleotide comprises or consists of seven interstitial segments.

[0411] In some embodiments, the gap segment comprises 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.

[0412] H. First gap section

[0413] In some embodiments, the first gap segment includes or is composed of one, two, three, four, five, six, seven, or eight linked nucleosides. In some embodiments, the first gap segment includes or is composed of four or five linked nucleosides. In some embodiments, the first gap segment includes or is composed of four linked nucleosides. In some embodiments, the first gap segment includes or is composed of five linked nucleosides.

[0414] In some embodiments, the first gap segment comprises or consists of one, two, three, four, five, six, seven, or eight linked nucleosides, each linked nucleoside having a 2'-deoxy sugar. In some embodiments, the first gap segment comprises or consists of four or five linked nucleosides, each linked nucleoside having a 2'-deoxy sugar. In some embodiments, the first gap segment comprises or consists of four linked nucleosides, each linked nucleoside having a 2'-deoxy sugar. In some embodiments, the first gap segment comprises or consists of five linked nucleosides, each linked nucleoside having a 2'-deoxy sugar.

[0415] In some embodiments, the first gap segments each having a 2'-deoxyglucose are positions 6, 7, 8, and 9 of SEQ ID NO: 2. In some embodiments, the first gap segments each having a 2'-deoxyglucose are positions 5, 6, 7, 8, and 9 of SEQ ID NO: 2. In some embodiments, the first gap segments each having a 2'-deoxyglucose are positions 6, 7, 8, 9, and 10 of SEQ ID NO: 2.

[0416] I. Second gap section

[0417] In some embodiments, the second gap segment comprises or consists of one, two, three, four, five, six, seven, or eight linked nucleosides. In some embodiments, the second gap segment comprises or consists of four or five linked nucleosides. In some embodiments, the first gap segment comprises or consists of four linked nucleosides. In some embodiments, the first gap segment comprises or consists of five linked nucleosides.

[0418] In some embodiments, the second gap segment comprises or consists of one, two, three, four, five, six, seven, or eight linked nucleosides, each linked nucleoside having a 2'-deoxy sugar. In some embodiments, the second gap segment comprises or consists of four linked nucleosides, each linked nucleoside having a 2'-deoxy sugar. In some embodiments, the second gap segment comprises or consists of five linked nucleosides, each linked nucleoside having a 2'-deoxy sugar. In some embodiments, the second gap segment comprises or consists of six linked nucleosides, each linked nucleoside having a 2'-deoxy sugar.

[0419] In some embodiments, the second gap segments each having a 2'-deoxyglucose are positions 11, 12, 13, and 14 of SEQ ID NO: 2. In some embodiments, the second gap segments each having a 2'-deoxyglucose are positions 11, 12, 13, 14, and 15 of SEQ ID NO: 2. In some embodiments, the second gap segments each having a 2'-deoxyglucose are positions 11, 12, 13, 14, 15, and 16 of SEQ ID NO: 2.

[0420] J. Separating sub-segments

[0421] In some embodiments, the separator segment includes 0, 1, 2, 3, 4, or 5 linked nucleosides. In some embodiments, the separator segment includes 1 nucleoside. In some embodiments, the nucleoside includes modifications. Exemplary modifications of the nucleoside include, but are not limited to, 2'-methoxyethyl nucleoside, 2'-O-methyl nucleoside, 2'OH nucleoside, 2'-fluoro2'-deoxy nucleoside, 2'-F-arabinonucleotide (2'-F-ANA), ethylene glycol nucleic acid (GNA), or locked nucleic acid (LNA).

[0422] In some embodiments, the separator segment consists of a single nucleoside comprising a 2'-OCH3 sugar. In some embodiments, the separator segment consists of a single nucleoside comprising a 2'-O(CH2)2-OCH3 sugar.

[0423] In some embodiments, the separator segment is located at position 7, 8, 9, 10, 11, 12, 13, or 14 of SEQ ID NO: 2. In some embodiments, the separator segment is located at position 9, 10, or 11 of SEQ ID NO: 2. In some embodiments, the separator segment is located at position 10 of SEQ ID NO: 2.

[0424] In some embodiments, the separator segment is located at position 10 of SEQ ID NO: 2 and consists of a single nucleoside comprising a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the separator segment consists of a single nucleoside comprising a 2'-OCH3 sugar. In some embodiments, the separator segment is located at position 7, 8, 9, 10, 11, 12, 13, or 14 of SEQ ID NO: 2. In some embodiments, the separator segment is located at position 9, 10, or 11 of SEQ ID NO: 2. In some embodiments, the separator segment is located at position 10 of SEQ ID NO: 2.

[0425] In some embodiments, the separator segment comprises a single nucleoside, said nucleoside comprising a 2'-O(CH2)2-OCH3 sugar. In some embodiments, the separator segment comprises a single nucleoside, said nucleoside comprising a 2'-OCH3 sugar. In some embodiments, the separator segment is located at position 7, 8, 9, 10, 11, 12, 13, or 14 of SEQ ID NO: 2. In some embodiments, the separator segment is located at position 9, 10, or 11 of SEQ ID NO: 2. In some embodiments, the separator segment is located at position 10 of SEQ ID NO: 2.

[0426] K. Oligonucleotide backbone

[0427] In some embodiments, the modified oligonucleotide is a single-stranded modified oligonucleotide. In some embodiments, at least one internucleotide bond is a modified internucleotide bond. In some embodiments, each internucleotide bond is a phosphate thioester internucleotide bond.

[0428] L. Modified nucleobases

[0429] In some embodiments, the modified oligonucleotide comprises at least one modified nucleobase. In some embodiments, the modified oligonucleotide comprises one, two, or three modified nucleobases. In some embodiments, the modified nucleobase is 5-methylcytosine. In some embodiments, the cytosine at positions 2, 13, and 20 of SEQ ID NO: 2 is each 5-methylcytosine.

[0430] M1. 5-4-1-5-5 modified oligonucleotides

[0431] In some embodiments, the modified oligonucleotide comprises: a 5'-wing segment consisting of five linked nucleosides; a first spacer segment consisting of four linked nucleosides having a 2'-deoxy sugar; a spacer segment located at position 10 of SEQ ID NO: 2; a second spacer segment consisting of five linked nucleosides having a 2'-deoxy sugar; and a 3'-wing segment consisting of five linked nucleosides. In an exemplary embodiment, the modified oligonucleotide of this segment further comprises cytosines located at positions 2, 13, and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine. In an exemplary embodiment, the modified oligonucleotide of this segment further comprises internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond. In an exemplary embodiment, the modified oligonucleotide of this paragraph further includes cytosine at positions 2, 13 and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine, and further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond.

[0432] In some embodiments, the modified oligonucleotide includes: a 5'-wing segment as described in sections C1, C2, or C3; a first spacer segment consisting of four linked nucleosides having a 2'-deoxy sugar; a spacer segment at position 10 of SEQ ID NO: 2; a second spacer segment consisting of five linked nucleosides having a 2'-deoxy sugar; and a 3'-wing segment as described in sections F1, F2, or F3. In an exemplary embodiment, the modified oligonucleotide of this segment further includes cytosine at positions 2, 13, and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine. In an exemplary embodiment, the modified oligonucleotide of this segment further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond. In an exemplary embodiment, the modified oligonucleotide of this paragraph further includes cytosine at positions 2, 13 and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine, and further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond.

[0433] In some embodiments, the modified oligonucleotide includes 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.

[0434] M2. 4-5-1-5-5 modified oligonucleotides

[0435] In some embodiments, the modified oligonucleotide comprises: a 5'-wing segment consisting of four linked nucleosides; a first spacer segment consisting of five linked nucleosides having a 2'-deoxy sugar; a spacer segment located at position 10 of SEQ ID NO: 2; a second spacer segment consisting of five linked nucleosides having a 2'-deoxy sugar; and a 3'-wing segment consisting of five linked nucleosides. In an exemplary embodiment, the modified oligonucleotide of this segment further comprises cytosines located at positions 2, 13, and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine. In an exemplary embodiment, the modified oligonucleotide of this segment further comprises internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond. In an exemplary embodiment, the modified oligonucleotide of this paragraph further includes cytosine at positions 2, 13 and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine, and further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond.

[0436] In some embodiments, the modified oligonucleotide includes: a 5'-wing segment as described in sections B1, B2, or B3; a first spacer segment consisting of five linked nucleosides having a 2'-deoxy sugar; a spacer segment at position 10 of SEQ ID NO: 2; a second spacer segment consisting of five linked nucleosides having a 2'-deoxy sugar; and a 3'-wing segment as described in sections F1, F2, or F3. In an exemplary embodiment, the modified oligonucleotide of this segment further includes cytosine at positions 2, 13, and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine. In an exemplary embodiment, the modified oligonucleotide of this segment further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond. In an exemplary embodiment, the modified oligonucleotide of this paragraph further includes cytosine at positions 2, 13 and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine, and further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond.

[0437] In some 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.

[0438] The following shows exemplary annotations of the location and segment of AO (AUS 1493 or SEQ ID NO:456).

[0439]

[0440] M3. 5-4-1-6-4 modified oligonucleotides

[0441] In some embodiments, the modified oligonucleotide comprises: a 5'-wing segment consisting of five linked nucleosides; a first spacer segment consisting of four linked nucleosides having a 2'-deoxy sugar; a spacer segment located at position 10 of SEQ ID NO: 2; a second spacer segment consisting of six linked nucleosides having a 2'-deoxy sugar; and a 3'-wing segment consisting of four linked nucleosides. In an exemplary embodiment, the modified oligonucleotide of this segment further comprises cytosines located at positions 2, 13, and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine. In an exemplary embodiment, the modified oligonucleotide of this segment further comprises internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond. In an exemplary embodiment, the modified oligonucleotide of this paragraph further includes cytosine at positions 2, 13 and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine, and further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond.

[0442] In some embodiments, the modified oligonucleotide includes: a 5'-wing segment as described in sections C1, C2, or C3; a first spacer segment consisting of four linked nucleosides having a 2'-deoxy sugar; a spacer segment at position 10 of SEQ ID NO: 2; a second spacer segment consisting of six linked nucleosides having a 2'-deoxy sugar; and a 3'-wing segment as described in sections E1, E2, or E3. In an exemplary embodiment, the modified oligonucleotide of this segment further includes cytosine at positions 2, 13, and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine. In an exemplary embodiment, the modified oligonucleotide of this segment further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond. In an exemplary embodiment, the modified oligonucleotide of this paragraph further includes cytosine at positions 2, 13 and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine, and further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond.

[0443] In some embodiments, the modified oligonucleotide includes a sequence that is AP, AQ, AR, or AS.

[0444] M4. 4-5-1-4-6 modified oligonucleotides

[0445] In some embodiments, the modified oligonucleotide comprises: a 5'-wing segment consisting of four linked nucleosides; a first spacer segment consisting of five linked nucleosides having a 2'-deoxy sugar; a spacer segment located at position 10 of SEQ ID NO: 2; a second spacer segment consisting of four linked nucleosides having a 2'-deoxy sugar; and a 3'-wing segment consisting of six linked nucleosides. In an exemplary embodiment, the modified oligonucleotide of this segment further comprises cytosines located at positions 2, 13, and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine. In an exemplary embodiment, the modified oligonucleotide of this segment further comprises internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond. In an exemplary embodiment, the modified oligonucleotide of this paragraph further includes cytosine at positions 2, 13 and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine, and further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond.

[0446] In some embodiments, the modified oligonucleotide includes: a 5'-wing segment as described in sections B1, B2, or B3; a first spacer segment consisting of five linked nucleosides having a 2'-deoxy sugar; a spacer segment at position 10 of SEQ ID NO: 2; a second spacer 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 an exemplary embodiment, the modified oligonucleotide of this segment further includes cytosine at positions 2, 13, and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine. In an exemplary embodiment, the modified oligonucleotide of this segment further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond. In an exemplary embodiment, the modified oligonucleotide of this paragraph further includes cytosine at positions 2, 13 and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine, and further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond.

[0447] In some embodiments, the modified oligonucleotide includes a sequence, which is AT or AU.

[0448] M5. 5-4-1-4-6 modified oligonucleotides

[0449] In some embodiments, the modified oligonucleotide comprises: a 5'-wing segment consisting of five linked nucleosides; a first spacer segment consisting of four linked nucleosides having a 2'-deoxy sugar; a spacer segment located at position 10 of SEQ ID NO: 2; a second spacer segment consisting of four linked nucleosides having a 2'-deoxy sugar; and a 3'-wing segment consisting of six linked nucleosides. In an exemplary embodiment, the modified oligonucleotide of this segment further comprises cytosines located at positions 2, 13, and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine. In an exemplary embodiment, the modified oligonucleotide of this segment further comprises internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond. In an exemplary embodiment, the modified oligonucleotide of this paragraph further includes cytosine at positions 2, 13 and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine, and further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond.

[0450] In some embodiments, the modified oligonucleotide comprises: a 5'-wing segment as described in sections C1, C2, or C3; a first spacer segment consisting of four linked nucleosides having a 2'-deoxy sugar; a spacer segment at position 10 of SEQ ID NO: 2; a second spacer 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 an exemplary embodiment, the modified oligonucleotide of this segment further comprises cytosine at positions 2, 13, and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine. In an exemplary embodiment, the modified oligonucleotide of this segment further comprises internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond. In an exemplary embodiment, the modified oligonucleotide of this paragraph further includes cytosine at positions 2, 13 and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine, and further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond.

[0451] In some embodiments, the modified oligonucleotide includes a sequence, said sequence being AV or AW.

[0452] M6. 4-5-1-6-4 modified oligonucleotides

[0453] In some embodiments, the modified oligonucleotide comprises: a 5'-wing segment consisting of four linked nucleosides; a first spacer segment consisting of five linked nucleosides having a 2'-deoxy sugar; a spacer segment located at position 10 of SEQ ID NO: 2; a second spacer segment consisting of six linked nucleosides having a 2'-deoxy sugar; and a 3'-wing segment consisting of four linked nucleosides. In an exemplary embodiment, the modified oligonucleotide of this segment further comprises cytosines located at positions 2, 13, and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine. In an exemplary embodiment, the modified oligonucleotide of this segment further comprises internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond. In an exemplary embodiment, the modified oligonucleotide of this paragraph further includes cytosine at positions 2, 13 and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine, and further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond.

[0454] In some embodiments, the modified oligonucleotide includes: a 5'-wing segment as described in sections B1, B2, or B3; a first spacer segment consisting of five linked nucleosides having 2'-deoxy sugars; a spacer segment at position 10 of SEQ ID NO: 2; a second spacer segment consisting of six linked nucleosides having 2'-deoxy sugars; and a 3'-wing segment as described in sections E1, E2, or E3. In an exemplary embodiment, the modified oligonucleotide of this segment further includes cytosines at positions 2, 13, and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine. In an exemplary embodiment, the modified oligonucleotide of this segment further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond. In an exemplary embodiment, the modified oligonucleotide of this paragraph further includes cytosine at positions 2, 13 and 20 of SEQ ID NO: 2, each of which is 5-methylcytosine, and further includes internucleotide bonds, wherein each internucleotide bond is a phosphate thioester internucleotide bond.

[0455] In some embodiments, the modified oligonucleotide includes the sequence AX.

[0456] In some embodiments, one or more modified nucleosides in the wing segments have modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified nucleoside is an LNA nucleoside. In some embodiments, the modified nucleoside is a 2'-substituted nucleoside. In some embodiments, the 2'-substituted nucleoside comprises a nucleoside modified with a bicyclic sugar. In some embodiments, the modified nucleoside is a 2'-MOE nucleoside. In some embodiments, the modified nucleoside is a restricted ethyl (cEt) nucleoside. In some embodiments, each modified nucleoside in each wing segment is independently a 2'-MOE nucleoside or nucleoside modified with a bicyclic sugar, such as a restricted ethyl (cEt) nucleoside or an LNA nucleoside.

[0457] In some embodiments, the compound or composition comprises a salt of a modified oligonucleotide.

[0458] In some embodiments, the compound or composition further includes a pharmaceutically acceptable carrier or diluent.

[0459] In some embodiments, the nucleotide sequence of the modified oligonucleotide is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the HBV nucleic acid, as measured throughout the modified oligonucleotide. In some embodiments, the nucleotide sequence of the modified oligonucleotide is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to SEQ ID NO: 1, as measured throughout the modified oligonucleotide.

[0460] In some embodiments, the compound or modified oligonucleotide is single-stranded.

[0461] In some embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified sugar. In some embodiments, at least one modified sugar comprises 2'-O-methoxyethyl (2'-O(CH2)2-OCH3). In some embodiments, the modified sugar comprises a 2'-O-CH3 group.

[0462] In some embodiments, at least one modified sugar is a bicyclic sugar. In some embodiments, at least one modified sugar, i.e., a bicyclic sugar, includes a 4'-(CH2)-O-2' bridge, where n is 1 or 2. In some embodiments, the bicyclic sugar includes a 4'-CH2-O-2' bridge. In some embodiments, the bicyclic sugar includes a 4'-CH(CH3)-O-2' bridge.

[0463] method

[0464] This disclosure provides a method of treating a subject suffering from HBV infection or HBV-related disease, condition, or symptom, the method comprising administering a therapeutically effective amount of a modified oligonucleotide as described herein or a pharmaceutical composition comprising said modified oligonucleotide. In some embodiments, the modified oligonucleotide comprises a sequence of any one of SEQ ID NO: 11-666, or comprises a modified oligonucleotide comprising one, two, three, four, or five modifications to said sequence.

[0465] In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide inhibits HBV mRNA expression in a subject. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide inhibits HBV DNA levels in a subject. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide inhibits HBV protein levels and / or antigen levels in a subject. When administered to a subject, the modified oligonucleotides of this disclosure can reduce the levels of HBV mRNA, DNA, or protein containing HBV antigens such as, but not limited to, HBsAg and HBeAg.

[0466] This disclosure provides a method for treating HBV-related diseases, symptoms, and / or conditions in a subject, the method comprising administering to a subject in need a therapeutically effective amount of any pharmaceutical composition as described above to treat the HBV-related disease, symptoms, and conditions. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, and the HBV-related disease, symptoms, and conditions are hepatitis B virus infection of human hepatitis B virus. More specifically, the human hepatitis B virus can be any of the following human geographic genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean region, Middle East, India); E (Africa); F (Native Americans, Polynesia); G (United States, France); or H (Central America).

[0467] In some embodiments, the modified oligonucleotide targets a region of HBV nucleic acid. In some embodiments, such modified oligonucleotides targeting the region of HBV nucleic acid have a continuous nucleotide motif that is complementary to a nucleotide motif of equal length to the region of HBV nucleic acid. For example, the motif may be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotide motifs complementary to a nucleotide motif of equal length to the region described herein.

[0468] Some embodiments provide methods for treating a subject with HBV-related disease, condition, or symptom, the methods comprising administering to a subject in need a modified oligonucleotide as described herein, such as a modified oligonucleotide comprising any one of SEQ ID NO: 11-666, or a modified oligonucleotide comprising one, two, three, four, or five modifications to said modified oligonucleotide, or a pharmaceutical composition.

[0469] Some embodiments provide a method for reducing HBV expression in a subject, the method comprising administering to the subject a modified oligonucleotide as described herein, such as a modified oligonucleotide comprising any one of SEQ ID NO: 11-666 or a modified oligonucleotide comprising one, two, three, four or five modifications to said modified oligonucleotide, or a pharmaceutical composition.

[0470] Some embodiments provide a method for preventing, improving, or treating HBV-related disease, condition, or symptom in a subject, the method comprising administering to an animal a modified oligonucleotide as described herein, such as a modified oligonucleotide comprising any one of SEQ ID NO: 11-666 or a modified oligonucleotide comprising one, two, three, four, or five modifications to said modified oligonucleotide, or a pharmaceutical composition.

[0471] Examples of HBV-related diseases, symptoms, or conditions include, but are not limited to, chronic HBV infection, jaundice, liver cancer, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serological hepatitis, and HBV viremia. HBV-related symptoms or conditions may include any or all of the following: flu-like illness, weakness, pain, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain in the liver area of ​​the body, clay-colored or gray stools, generalized itching, and dark urine, accompanied by a positive test for the presence of hepatitis B virus, a positive test for hepatitis B virus antigen, or a positive test for the presence of antibodies specific to hepatitis B virus antigen, indicating the presence of HBV-related symptoms or conditions.

[0472] Some embodiments provide a method for reducing HBV mRNA expression in a subject, the method comprising administering to the subject a modified oligonucleotide as described herein, such as a modified oligonucleotide comprising any one of SEQ ID NO: 11-666 or a modified oligonucleotide comprising one, two, three, four, or five modifications to said modified oligonucleotide, or a pharmaceutical composition. In some embodiments, the reduction in HBV mRNA expression in the subject prevents, improves, or treats HBV-related diseases, conditions, or symptoms. In some embodiments, the reduction in HBV mRNA expression in the subject improves or treats HBV infection. In some embodiments, the reduction in HBV mRNA expression in the subject prevents, improves, or treats liver disease. In some 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%.

[0473] Some embodiments provide a method for reducing HBV protein levels in a subject, the method comprising administering to the subject a modified oligonucleotide as described herein, such as a modified oligonucleotide comprising any one of SEQ ID NO: 11-666 or a modified oligonucleotide comprising one, two, three, four, or five modifications to said modified oligonucleotide, or a pharmaceutical composition. In some embodiments, the reduction in HBV protein levels in the subject prevents, improves, or treats HBV-related diseases, conditions, or symptoms. In some embodiments, the reduction in HBV protein levels in the subject improves or treats HBV infection. In some embodiments, the reduction in HBV protein levels in the subject prevents, improves, or treats liver disease. In some embodiments, HBV protein levels are reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0474] Some embodiments provide a method for reducing HBV DNA levels in a subject, the method comprising administering to the subject a modified oligonucleotide as described herein, such as a modified oligonucleotide comprising any one of SEQ ID NO: 11-666 or a modified oligonucleotide comprising one, two, three, four, or five modifications to said modified oligonucleotide, or a pharmaceutical composition. In some embodiments, the reduction in HBV DNA levels in the subject prevents, improves, or treats HBV-related diseases, conditions, or symptoms. In some 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 geographic genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean region, Middle East, India); E (Africa); F (Native Americans, Polynesia); G (United States, France); or H (Central America). In some embodiments, the reduction in HBV DNA levels in the subject improved or treated HBV infection. In some embodiments, the reduction in HBV DNA levels in the subject prevented, improved, or treated liver disease. In some embodiments, HBV DNA levels were reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0475] Some embodiments provide a method for reducing HBV antigen levels in a subject, the method comprising administering to the subject a modified oligonucleotide as described herein, such as a modified oligonucleotide comprising any one of SEQ ID NO: 11-666 or a modified oligonucleotide comprising one, two, three, four, or five modifications to said modified oligonucleotide, or a pharmaceutical composition. In some embodiments, the antigen is HBsAg or HBeAG. In some embodiments, the reduction in HBV antigen levels in the subject prevents, improves, or treats HBV-related diseases, conditions, or symptoms. In some embodiments, the reduction in HBV antigen levels in the subject prevents, improves, or treats liver disease. In some embodiments, HBV antigen levels are reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0476] Some embodiments provide a method for reducing HBV DNA and HBV antigen in a subject infected with hepatitis B virus, the method comprising administering to the subject a modified oligonucleotide as described herein, such as a modified oligonucleotide comprising any one of SEQ ID NO: 11-666 or a modified oligonucleotide comprising one, two, three, four, or five modifications to said modified oligonucleotide, or a pharmaceutical composition. In some embodiments, the antigen is HBsAg or HBeAg. In some embodiments, if HBeAg is monitored as a determinant of seroconversion, the amount of HBV antigen can be sufficiently reduced to produce seroconversion, which is defined as the absence of serum HBeAg plus the presence of serum HBeAb, or if HBsAg is monitored as a determinant of seroconversion, which is defined as the absence of serum HBsAg, as determined by the currently available detection limits of commercial ELISA systems.

[0477] Some embodiments provide a method for treating a subject with an HBV-related disease, condition, or symptom, the method comprising: a) identifying the subject with the HBV-related disease, condition, or symptom, and b) administering to the subject a therapeutically effective amount of the modified oligonucleotide or pharmaceutical composition described herein. In some embodiments, the therapeutically effective amount of the modified oligonucleotide or pharmaceutical composition administered to the subject treats or reduces the subject's HBV-related disease, condition, or symptom, or its symptoms. In some embodiments, the HBV-related disease, condition, or symptom is a liver disease. In some embodiments, the related disease, condition, or symptom is chronic HBV infection, jaundice, liver cancer such as hepatocellular carcinoma, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, transplant-related liver disease, or any combination thereof.

[0478] Some embodiments provide a method for treating a subject with an HBV-related disease, condition, or symptom, the method comprising: a) identifying the subject with the HBV-related disease, condition, or symptom, and b) administering to the subject a therapeutically effective amount of the modified oligonucleotide or pharmaceutical composition described herein. In some embodiments, the therapeutically effective amount of the modified oligonucleotide or pharmaceutical composition administered to the subject treats or reduces the subject's HBV-related disease, condition, or symptom, or its symptoms. In some embodiments, the HBV-related disease, condition, or symptom is a liver disease. In some embodiments, the related disease, condition, or symptom 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.

[0479] In some embodiments, HBV has a sequence as shown in GenBank accession number U95551.1 (SEQ ID NO: 3) or any variant or fragment thereof. In some embodiments, HBV has a sequence as shown in SEQ ID NO: 667-674.

[0480] In some embodiments, the subject is a human being.

[0481] In some embodiments, the subjects are monkeys, such as cynomolgus monkeys.

[0482] In some embodiments, the subjects are rodents, such as mice or rats.

[0483] In some embodiments, a modified oligonucleotide or pharmaceutical composition is designated as a first agent. In some embodiments, the method includes administering the first agent and one or more second agents. In some embodiments, the first agent and one or more second agents are administered concurrently. In some embodiments, the first agent and one or more second agents are administered sequentially or concurrently. In some embodiments, the first agent and one or more second agents are not administered concurrently.

[0484] In some embodiments, one or more second agents are also compounds or compositions described herein. In some 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 a particular embodiment, the disease includes liver cancer, and one or more second agents include chemotherapeutic agents such as gemcitabine (Gemzar), oxaliplatin (Eloxatin), cisplatin, doxorubicin, 5-fluorouracil, capecitabine (Xeloda), or mitoxantrone (Novantrone). In a specific embodiment, the disease includes liver disease, and one or more second agents include corticosteroids, diuretics, beta-blockers, or combinations thereof.

[0485] The modified oligonucleotides of this disclosure and pharmaceutical compositions comprising said modified oligonucleotides may be administered to a subject via any suitable route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), intraperitoneal (into body cavities), and transmucosal administration.

[0486] In some embodiments, administration includes parenteral administration. In some embodiments, administration includes subcutaneous administration. In some embodiments, administration includes intravenous injection or infusion.

[0487] Some embodiments provide a method for reducing the levels of HBV mRNA, DNA, protein, and / or HBV antigen in a subject infected with hepatitis B virus, the method comprising administering to a subject in need a therapeutically effective amount of the pharmaceutical composition described above to reduce hepatitis B virus infection and hepatitis B antigen levels compared to pre-treatment levels of HBV mRNA, protein, and HBV antigen in the subject. In some embodiments, the subject may be a human, and the hepatitis B virus may be human hepatitis B virus. More specifically, the human hepatitis B virus may be any of the following human geographic genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean region, Middle East, India); E (Africa); F (Native Americans, Polynesia); G (United States, France); or H (Central America).

[0488] This disclosure provides a method for reducing the amount of HBV mRNA, DNA, protein, and / or HBV antigen, or combinations thereof, in a subject infected with hepatitis B virus. The method includes administering to the subject a therapeutically effective amount of a modified oligonucleotide as described above, or a pharmaceutical composition comprising said modified oligonucleotide, to reduce hepatitis B virus infection and hepatitis B antigen levels compared to pre-treatment levels of HBV mRNA, protein, and / or HBV antigen. In some 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 prior to administration of the modified antisense oligonucleotide or pharmaceutical composition. In some embodiments, the amount of mRNA is reduced by at least 50% compared to the amount prior to administration of the modified antisense oligonucleotide or pharmaceutical composition. In some embodiments, the amount of mRNA is reduced by at least 60% compared to the amount prior to administration of the modified antisense oligonucleotide or pharmaceutical composition. In some embodiments, the amount of mRNA is reduced by at least 70% compared to the amount prior to application of the modified antisense oligonucleotide or pharmaceutical composition. In some embodiments, the amount of mRNA is reduced by at least 80% compared to the amount prior to application of the modified antisense oligonucleotide or pharmaceutical composition. In some embodiments, the amount of mRNA is reduced by at least 90% compared to the amount prior to application of the modified antisense oligonucleotide or pharmaceutical composition.

[0489] This disclosure provides a method for reducing the levels of HBV mRNA, DNA, protein, and / or HBV antigen in a subject infected with hepatitis B virus. The method includes administering to the subject a therapeutically effective amount of a modified oligonucleotide or pharmaceutical composition as described above to reduce hepatitis B virus infection and hepatitis B antigen levels compared to pre-treatment levels of HBV virus, mRNA, DNA, protein, and / or HBV antigen, wherein the amount of mRNA is reduced by at least 75% compared to the amount prior to administration of the modified oligonucleotide or pharmaceutical composition. In some embodiments, the method reduces the levels of HBV virus, mRNA, DNA, protein, and / or HBV antigen in a subject infected with hepatitis B virus, the method including administering to the subject a therapeutically effective amount of a modified oligonucleotide or pharmaceutical composition as described above to reduce hepatitis B virus infection and hepatitis B antigen levels compared to pre-treatment levels of hepatitis B virus and HBV antigen, wherein the amount of mRNA is reduced by at least 80% compared to the amount prior to administration of the modified oligonucleotide or pharmaceutical composition. In some 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 includes administering to the subject a therapeutically effective amount of a modified oligonucleotide or pharmaceutical composition as described above 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, wherein the amount of mRNA is reduced by at least 85% compared to the amount before administration of the modified oligonucleotide or pharmaceutical composition. In some 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 includes administering to the subject a therapeutically effective amount of a modified oligonucleotide or pharmaceutical composition as described above to reduce hepatitis B virus infection and hepatitis B antigen compared to the pre-treatment amounts of HBV virus, mRNA, DNA, protein, and / or HBV antigen, wherein the amount of mRNA is reduced by at least 90% compared to the amount prior to administration of the modified oligonucleotide or pharmaceutical composition.In some 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 includes administering to the subject a therapeutically effective amount of a modified oligonucleotide or pharmaceutical composition as described above to reduce HBV infection, mRNA, protein, and / or HBV antigen compared to the pre-treatment levels in the subject, wherein the amount of mRNA is reduced by at least 95% compared to the level prior to administration of the modified oligonucleotide or pharmaceutical composition. In related methods, the HBV antigen may be HBsAg or HBeAg, and more specifically, if HBeAg is monitored as a determinant of seroconversion, the amount of HBV antigen can be sufficiently reduced to produce seroconversion, defined as the absence of serum HBeAg plus the presence of serum HBeAb, or if HBsAg is monitored as a determinant of seroconversion, the seroconversion is defined as the absence of serum HBsAg, as determined by the currently available detection limits of commercial ELISA systems.

[0490] This disclosure provides a method for promoting seroconversion of hepatitis B virus in mammals infected with HBV, the method comprising administering to a subject infected with hepatitis B a therapeutically effective amount of a modified oligonucleotide as described above, such as a modified oligonucleotide comprising any one of SEQ ID NO: 11-666 or a modified oligonucleotide comprising one, two, three, four or five modifications to said modified oligonucleotide, or a pharmaceutical composition; monitoring the presence of HBeAg plus HBeAb in a serum sample of the subject if HBeAg is monitored as a determinant of seroconversion, or monitoring the presence of HBsAg in a serum sample of the subject such that HBeAg plus HBeAb is absent in the serum sample, or if HBsAg is monitored as a determinant of seroconversion, HBsAg is absent in the serum sample, as determined by the current detection limit of a commercial ELISA system, which is an indicator of seroconversion in the subject.

[0491] Some embodiments provide the use of modified oligonucleotides or pharmaceutical compositions as described herein for the prevention, improvement or treatment of liver disease or its symptoms in a subject, said modified oligonucleotides including, for example, any one of SEQ ID NO: 11-666 or including one, two, three, four or five modifications to said modified oligonucleotides.

[0492] In some embodiments, ECs of cells treated with modified oligonucleotides (e.g., SEQ ID NO: 11-666) 50The EC is measured as a representative of the effectiveness of the modified oligonucleotide. In some embodiments, EC... 50 From approximately 0.1 nM to approximately 250 nM. In some embodiments, EC 50 <250 nM, <200 nM, <150 nM, <100 nM, <90 nM, <80 nM, <70 nM, <65 nM, <60 nM, <55 nM, <50 nM, <49 nM, <47 nM, <46 nM, <45 nM, <44 nM, <43 nM, <42 nM, <41 nM, <40 nM, <39 nM, <38 nM, <37 nM, <36 nM, <35 nM, <34 nM, <33 nM, <32 nM, <31 nM, <30 nM, <29 nM, <28 nM, <27 nM, <26 nM, <25 nM, <24 nM, <23 nM, <22 nM, <21 nM, <20 nM, less than 19 nM, less than 18 nM, less than 17 nM, less than 16 nM, less than 15 nM, less than 14 nM, less than 13 nM, less than 12 nM, less than 11 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM.

[0493] In some embodiments, the EC50 of cells treated with modified oligonucleotides (e.g., SEQ ID NO: 11-666) is calculated. 50 The ratio of EC50 in cells treated with a reference oligonucleotide (e.g., SEQ ID NO: 10) is used as a measure of efficacy in reducing HBsAg levels. In some embodiments, the ratio is from about 0.05 to about 250. In some embodiments, EC50... 50Ratios less than 250, less than 150, less than 100, less than 90, less than 80, less than 70, less than 65, less than 60, less than 55, less than 50, less than 49, less than 47, less than 46, less than 45, less than 44, less than 43, less than 42, less than 41, less than 40, less than 39, less than 38, less than 37, less than 36, less than 35, less than 34, less than 33, less than 32, less than 31, less than 30, less than 29, less than 28, less than 27, less than 26. Less than 25, less than 24, less than 23, less than 22, less than 21, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, less than 1, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1.

[0494] In some embodiments, compounds or compositions as described herein are effective when delivered to HepG2.2.1 cells because of their in vitro IC50 efficacy. 50 At least one of them is less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 65 nM, less than 60 nM, less than 55 nM, less than 50 nM, less than 49 nM, less than 47 nM, or less than 46 nM.

[0495] In some embodiments, compounds or compositions as described herein are effective when delivered to HepG2.2.1 cells because of their in vitro IC50 efficacy. 50 At least one of them is less than 250 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 35 nM, less than 34 nM, less than 33 nM, less than 32 nM, or less than 31 nM.

[0496] In some embodiments, compounds or compositions described herein are effective when delivered to HepG2.2.1 cells as described herein because of their in vitro IC50 efficacy. 50At least one of the following is less than 20 µM, less than 10 µM, less than 9.5 µM, less than 9.0 µM, less than 8.5 µM, less than 8.0 µM, less than 7.5 µM, less than 7.0 µM, less than 6.5 µM, less than 6.0 µM, less than 5.5 µM, less than 5.0 µM, less than 4.5 µM, less than 4.0 µM, less than 3.5 µM, less than 3.0 µM, or less than 2.5 µM.

[0497] 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 from about 10 nM to about 250 nM. In some embodiments, the MTT CC25 is greater than 10 nM, greater than 15 nM, greater than 20 nM, greater than 25 nM, greater than 30 nM, greater than 35 nM, greater than 40 nM, greater than 45 nM, greater than 50 nM, greater than 55 nM, greater than 60 nM, greater than 65 nM, greater than 70 nM, greater than 75 nM, greater than 80 nM, greater than 85 nM, greater than 90 nM, greater than 95 nM, greater than 100 nM, greater than 110 nM, greater than 120 nM, greater than 130 nM, greater than 140 nM, greater than 150 nM, greater than 160 nM, greater than 170 nM, greater than 180 nM, greater than 190 nM, greater than 200 nM, greater than 210 nM, greater than 220 nM, greater than 230 nM, greater than 240 nM, or greater than 250 nM.

[0498] 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 from about 10 nM to about 250 nM. In some embodiments, CCK8 CC30 is greater than 10 nM, greater than 15 nM, greater than 20 nM, greater than 25 nM, greater than 30 nM, greater than 35 nM, greater than 40 nM, greater than 45 nM, greater than 50 nM, greater than 55 nM, greater than 60 nM, greater than 65 nM, greater than 70 nM, greater than 75 nM, greater than 80 nM, greater than 85 nM, greater than 90 nM, greater than 95 nM, greater than 100 nM, greater than 110 nM, greater than 120 nM, greater than 130 nM, greater than 140 nM, greater than 150 nM, greater than 160 nM, greater than 170 nM, greater than 180 nM, greater than 190 nM, greater than 200 nM, greater than 210 nM, greater than 220 nM, greater than 230 nM, greater than 240 nM, or greater than 250 nM.

[0499] In some embodiments, the "C / E ratio" is used as a measure of efficacy in reducing HBsAg levels, relative to cytotoxicity. In some embodiments, this is achieved by taking the ratio of the modified oligonucleotide's MTT CC25 (nM) to the reference oligonucleotide's MTT CC25 (nM) and dividing it by the modified oligonucleotide's HBsAg EC50 (e.g., SEQ ID NO: 10). 50 The C / E ratio is calculated by the ratio of the HBsAg EC50 of the modified oligonucleotide to that of the reference oligonucleotide (e.g., SEQ ID NO: 10). In some embodiments, the C / E ratio is calculated by taking the ratio of the CCK8 CC25 (nM) of the modified oligonucleotide to that of the reference oligonucleotide (e.g., SEQ ID NO: 10) and dividing it by the HBsAg EC50 of the modified oligonucleotide (e.g., SEQ ID NO: 11-666). 50 The C / E ratio is calculated using the ratio of HBsAg EC50 of a reference oligonucleotide (e.g., SEQ ID NO: 10). In some embodiments, the C / E ratio is from about 0.01 to about 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.

[0500] In some embodiments, the compounds or compositions described herein are highly tolerable, as demonstrated by an increase in ALT or AST values ​​of no more than 4, 3, or 2 times, or an increase in liver, spleen, or kidney weight of no more than at least one of 30%, 20%, 15%, 12%, 10%, 5%, or 2%, compared to saline-treated animals. In some embodiments, the compounds or compositions described herein are highly tolerable, as demonstrated by no increase in ALT or AST, compared to saline-treated animals. In some embodiments, the compounds or compositions described herein are highly tolerable, as demonstrated by no increase in liver, spleen, or kidney weight, compared to saline-treated animals.

[0501] In some embodiments, the ALT levels of animals treated with a compound or composition comprising a modified oligonucleotide (e.g., SEQ ID NO: 11-666) are reduced to about 1 / 10 to about 1 / 1.1 of the ALT levels of animals treated with a reference compound (e.g., SEQ ID NO: 10). In some embodiments, the ALT levels of animals treated with a compound or composition comprising a modified oligonucleotide (e.g., SEQ ID NO: 11-666) are reduced to at least 1 / 1.25 of the ALT levels of animals treated with a reference compound (e.g., SEQ ID NO: 10). In some embodiments, the ALT levels of animals treated with a compound or composition comprising a modified oligonucleotide (e.g., SEQ ID NO: 11-666) are reduced to about 1 / 10 to about 1 / 1.1 of the ALT levels of animals treated with a reference compound (e.g., SEQ ID NO: 10). 10) The ALT levels of the treated animals were 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2.0, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, and 1 / 2. 9, 1 / 3.0, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4.0, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, or 1 / 5.0.

[0502] In some embodiments, the AST levels of animals treated with a compound or composition comprising a modified oligonucleotide (e.g., SEQ ID NO: 11-666) are reduced to about 1 / 10 to about 1 / 1.1 of the AST levels of animals treated with a reference compound (e.g., SEQ ID NO: 10). In some embodiments, the AST levels of animals treated with a compound or composition comprising a modified oligonucleotide (e.g., SEQ ID NO: 11-666) are reduced to at least 1 / 1.25 of the AST levels of animals treated with a reference compound (e.g., SEQ ID NO: 10). In some embodiments, the AST levels of animals treated with a compound or composition comprising a modified oligonucleotide (e.g., SEQ ID NO: 11-666) are reduced to about 1 / 10 to about 1 / 1.1 of the AST levels of animals treated with a reference compound (e.g., SEQ ID NO: 10). 10) AST levels in treated animals at 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2.0, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, and 1 / 2. 9, 1 / 3.0, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4.0, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, or 1 / 5.0.

[0503] Some embodiments provide for the use of modified oligonucleotides or pharmaceutical compositions as described herein for the preparation of medicaments for the treatment, improvement, delay, or prevention of HBV-related diseases, symptoms, or conditions in animals, said modified oligonucleotides including, for example, any of the modified oligonucleotides in SEQ ID NO: 11-666.

[0504] Some embodiments provide the use of modified oligonucleotides or pharmaceutical compositions as described herein for the preparation of medicaments for treating, improving, delaying or preventing liver diseases in animals, said modified oligonucleotides including, for example, any of the modified oligonucleotides in SEQ ID NO: 11-666.

[0505] reagent kits and products

[0506] This disclosure provides a kit comprising the modified oligonucleotides described herein and a pharmaceutical composition comprising said modified oligonucleotides. In some embodiments, the modified oligonucleotides comprise sequences of any one of SEQ ID NO: 11-666, or comprise modified oligonucleotides comprising one, two, three, four, or five modifications to said sequences.

[0507] Some embodiments provide a kit for treating, preventing, or improving HBV-related diseases, conditions, or symptoms as described herein, wherein the kit comprises: a) a compound or composition as described herein, said composition comprising, for example, a modified oligonucleotide comprising a sequence comprising any one of SEQ ID NO: 11-666 or a composition comprising one, two, three, four, or five modifications of said modified oligonucleotide; and optionally b) another agent or therapy as described herein. The kit may further comprise instructions or labeling for using the kit to treat, prevent, or improve HBV-related diseases, conditions, or symptoms.

[0508] Compositions containing modified oligonucleotides may be lyophilized prior to packaging in a kit, or may be provided in a solution containing a pharmaceutically acceptable carrier, excipient, or diluent.

[0509] In some embodiments, the kit further includes at least one additional agent for treating HBV-related diseases, conditions, or symptoms.

[0510] Administration and application

[0511] This disclosure provides a method comprising administering to a subject in need a therapeutically effective dose of a modified oligonucleotide, such as a modified oligonucleotide comprising any one of SEQ ID NO: 11-666 or a modified oligonucleotide comprising one, two, three, four or five modifications to said modified oligonucleotide, or a pharmaceutical composition comprising said modified oligonucleotide.

[0512] In some embodiments, a therapeutically effective dose of, for example, a modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide, includes a dose in which a maximum log10 reduction in serum HBsAg is observed to be 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, a decrease in maximum log10 serum HBsAg was observed at 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.

[0513] In some embodiments, a therapeutically effective dose of, for example, a modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide, includes a dose that reduces serum HBsAg levels in a subject by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. In some embodiments, a reduction in serum HBsAg is observed on days 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 after administration of the therapeutically effective dose.

[0514] In some embodiments, a therapeutically effective dose of, for example, a modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide, includes a dose in which a maximum log10 reduction in serum HBeAg is observed to be 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, a decrease in maximum log10 serum HBeAg was observed at 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.

[0515] In some embodiments, a therapeutically effective dose of, for example, a modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide, includes a dose that reduces the HBeAg level in the subject's 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, a reduction in serum HBeAg is observed on days 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 after administration of the therapeutically effective dose.

[0516] In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered to the subject in doses within the following ranges: 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, 50 mg to 1500 mg, 50 mg to 1000 mg, 50 mg to 900 mg, 50 mg to 800 mg, 50 mg to 700 mg, 50 mg to 600 mg, 50 mg to 500 mg, 50 mg to 400 mg, 50 mg to 300 mg, 50 mg to 200 mg, 50 mg to 100 mg, 100 mg to 2 ... mg to 1500 mg, 100 mg to 1000 mg, 100 mg to 900 mg, 100 mg to 800 mg, 100 mg to 700 mg, 100 mg to 600 mg, 100 mg to 500 mg, 100 mg to 400 mg, 100 mg to 300 mg, 100 mg to 200 mg, 200 mg to 1500 mg, 200 mg to 1000 mg, 200 mg to 900 mg, 200 mg to 800 mg, 200 mg to 700 mg, 200 mg to 600 mg, 200 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 700 mg, 300 mg to 1500 mg, 300 mg to 1000 mg, 300 mg to 900 mg, 300 mg to 8 ... 50 mg to 600 mg, 300 mg to 500 mg, or 300 mg to 400 mg. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject in a dose ranging from 50 mg to 1500 mg. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject in a dose ranging from 50 mg to 1000 mg. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject in a dose ranging from 50 mg to 700 mg.In some embodiments, a subject is administered a dose of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide in the range of 50 mg to 500 mg. In some embodiments, a subject is administered a dose of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide in the range of 50 mg to 450 mg. In some embodiments, a subject is administered a dose of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide in the range of 50 mg to 300 mg. In some embodiments, a subject is administered a dose of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide in the range of 50 mg to 200 mg. In some embodiments, a subject is administered a dose of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide in the range of 100 mg to 1500 mg. In some embodiments, a subject is administered a dose of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide in the range of 100 mg to 1000 mg. In some embodiments, a subject is administered a dose of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide in the range of 100 mg to 700 mg. In some embodiments, a subject is administered a dose of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide in the range of 100 mg to 500 mg. In some embodiments, a subject is administered a dose of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide in the range of 100 mg to 450 mg. In some embodiments, a subject is administered a dose of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide in the range of 100 mg to 300 mg. In some embodiments, a dose of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered to a subject in the range of 100 mg to 200 mg.

[0517] In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered in doses of the modified oligonucleotide within the following ranges: 0.01 mg / kg to 30.0 mg / kg, 0.01 mg / kg to 27.0 mg / kg, 0.01 mg / kg to 25.0 mg / kg, 0.01 mg / kg to 22.0 mg / kg, 0.01 mg / kg to 20.0 mg / kg, 0.01 mg / kg to 15.0 mg / kg, 0.01 mg / kg to 10.0 mg / kg, 0.01 mg / kg to 8.0 mg / kg, 0.01 mg / kg to 5.0 mg / kg, 0.01 mg / kg to 4.0 mg / kg, 0.01 mg / kg to 3.0 mg / kg, 0.01 mg / kg to 2.0 mg / kg, 0.01 mg / kg to 1 ...3.0 mg / kg, 0.01 mg / kg to 3.0 mg / kg, 0.01 mg / kg to 3. mg / kg to 0.5 mg / kg, 0.01 mg / kg to 0.1 mg / kg, 0.1 mg / kg to 30.0 mg / kg, 0.1 mg / kg to 27.0 mg / kg, 0.1 mg / kg to 25.0 mg / kg, 0.1 mg / kg to 22.0 mg / kg, 0.1 mg / kg to 20.0 mg / kg, 0.1 mg / kg to 18.0 mg / kg, 0.1 mg / kg to 15.0 mg / kg, 0.1 mg / kg to 12.0 mg / kg, 0.1 mg / kg to 10.0 mg / kg, 0.1 mg / kg to 8.0 mg / kg, 0.1 mg / kg to 5.0 mg / kg, 0.1 mg / kg to 4.0 mg / kg, 0.1 mg / kg to 3.0 mg / kg, 0.1 mg / kg to 2.0 mg / kg, 0.1 mg / kg to 1.0 mg / kg. mg / kg, 0.1 mg / kg to 0.5 mg / kg, 1.0 mg / kg to 30.0 mg / kg, 1.0 mg / kg to 27.0 mg / kg, 1.0 mg / kg to 25.0 mg / kg, 1.0 mg / kg to 22.0 mg / kg, 1.0 mg / kg to 20.0 mg / kg, 1.0 mg / kg to 18.0 mg / kg, 1.0 mg / kg to 15.0 mg / kg, 1.0 mg / kg to 12.0 mg / kg, 1.0 mg / kg to 10.0 mg / kg, 1.0 mg / kg to 8.0 mg / kg, 1.0 mg / kg to 5.0 mg / kg, 1.0 mg / kg to 4.0 mg / kg, 1.0 mg / kg to 3.0 mg / kg, 1.0 mg / kg to 2.0 mg / kg, 5.0 mg / kg to 30.0 mg / kg, 5.0 mg / kg to 27.0 mg / kg, 5.0 mg / kg to 25.0 mg / kg, 5.0 mg / kg to 22.0 mg / kg, 5.0 mg / kg to 20.0 mg / kg, 5.0 mg / kg to 18.0 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. The dosage ranges from 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 some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered at a dose of the modified oligonucleotide ranging from 0.01 mg / kg to 30 mg / kg. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered at a dose of the modified oligonucleotide ranging from 0.1 mg / kg to 25 mg / kg. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered at a dose of the modified oligonucleotide ranging from 1.0 mg / kg to 20 mg / kg. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered at a dose of the modified oligonucleotide ranging from 5.0 mg / kg to 20 mg / kg. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered at a dose of the modified oligonucleotide ranging from 1.0 mg / kg to 15 mg / kg. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered at a dose of the modified oligonucleotide ranging from 1.0 mg / kg to 5.0 mg / kg.

[0518] In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered to the subject daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every 11 days, every 12 days, every 13 days, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 2 months, every 3 months, or every 4 months.

[0519] In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered to the subject daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every two weeks, every three weeks, or monthly.

[0520] In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered to the subject for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

[0521] In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered to the subject until a specific outcome is achieved, such as a reduction in the subject's serum levels of HBsAg and / or HBeAg. In some embodiments, administration of the modified oligonucleotide or the pharmaceutical composition comprising the modified oligonucleotide to the subject continues until, compared to before administration of the modified oligonucleotide or the pharmaceutical composition comprising the modified oligonucleotide, the subject's serum levels of HBsAg and / or HBeAg are reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.

[0522] In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered to the subject once, twice, three times, or four times daily.

[0523] In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject daily. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every two days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every three days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every four days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every five days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every six days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every seven days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every eight days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every nine days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every ten days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every eleven days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every twelve days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every thirteen days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every two weeks. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every three weeks. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject monthly. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every two months. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every three months. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every 4 months. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every 5 months. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every 6 months.In some embodiments, a modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to a subject two or more times per year. In some embodiments, a modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to a subject two or more times every two years. In some embodiments, a modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to a subject two or more times every two years or more.

[0524] In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every 1-30 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every 1-22 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every 1-15 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every 1-8 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every 1-5 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every 1-4 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every 1-3 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising said modified oligonucleotide is administered to the subject every 10-20 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered to the subject every 5-15 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered to the subject every 15-30 days.

[0525] In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 36 hours. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 48 hours. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 60 hours. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 72 hours. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 84 hours. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 96 hours. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 5 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 6 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 7 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 8-10 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 10-12 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 12-15 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 15-25 days. In some embodiments, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered at least once every 20-30 days.

[0526] In some embodiments, administration of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide includes a dosing holiday. For example, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide may be administered to the subject every three days, followed by a one-week, two-week, three-week, or one-month dosing period, after which administration may resume. Those skilled in the art will understand that this dosing holiday is exemplary. Other durations and frequencies of dosing holidays are contemplated within the scope of this disclosure. As another example, the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide may be administered to the subject until the level of HBsAg or HBeAg detected in the subject drops below a certain threshold or HBV infection is absent, followed by a dosing holiday, and administration may resume if HBsAg or HBeAg is detected again in the subject's serum.

[0527] In some embodiments, a single, one-time dose of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide is administered to a subject. In other embodiments, multiple doses of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide are administered to a subject.

[0528] In some embodiments, administration of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide may include a dosing schedule in which the modified oligonucleotide or the pharmaceutical composition comprising the modified oligonucleotide is initially administered more frequently, followed by less frequent administration. An advantage of such a dosing schedule is that it can be used to maintain a steady-state hepatic concentration of the modified oligonucleotide described herein. For example, if the hepatic half-life of the modified oligonucleotide is 3-4 weeks, a steady-state hepatic concentration of the modified oligonucleotide of this disclosure can be achieved using an initial dosing schedule comprising loading doses on days 1 and 4, followed by weekly maintenance doses starting from day 8 (e.g., days 8, 15, and 22, etc.).

[0529] In some embodiments, administration of the modified oligonucleotide or a pharmaceutical composition comprising the modified oligonucleotide includes a loading dose followed by a maintenance load dose.

[0530] The term "loading dose" refers to one or more doses of a modified oligonucleotide or pharmaceutical composition administered outside of or above the remaining dose in a dosing regimen. As used herein, "loading dose" can refer to one or more doses of a modified oligonucleotide or pharmaceutical composition having the same or lower (if administered outside of a regular dosing regimen) or higher concentrations of the modified oligonucleotide or pharmaceutical composition compared to a dose of the modified oligonucleotide or pharmaceutical composition administered as part of a dosing regimen (if administered in lieu of a regular dosing regimen). In a dosing regimen, the loading dose may also be administered more frequently than the maintenance dose.

[0531] As used herein, the term "maintenance dose" refers to repeated, routine administration of the therapeutic agent. As used herein, a maintenance dose does not include a loading dose, which in some embodiments may be administered, for example, prior to the maintenance dose.

[0532] In exemplary dosing regimens that include 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 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 4.5, or 5 times 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 at least one, two, three, four, or five times daily, every two days, every three days, every four days, or every five days, followed by maintenance doses administered weekly, every ten days, every fourteen days, etc. Those skilled in the art will understand that, if necessary, a subject who has already been dosed according to the maintenance dosing schedule may be re-administered with one or more loading doses. As another exemplary dosing schedule, a loading dose may be administered to the subject on days 1 and 4, followed by a weekly maintenance dose on day 8.

[0533] Antisense compounds

[0534] Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleotide analogs, oligonucleotide mimics, antisense compounds, antisense oligonucleotides, and siRNA. Oligomeric compounds can be “antisense” to the target nucleic acid, meaning they can hybridize with the target nucleic acid via hydrogen bonding.

[0535] In some embodiments, the antisense compound has a nucleobase sequence that, when written in the 5' to 3' direction, includes the inverse complement of the target segment of the target nucleic acid it targets. In some such embodiments, the antisense oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, includes the inverse complement of the target segment of the target nucleic acid it targets.

[0536] In some embodiments, the antisense oligonucleotide targeting HBV nucleic acid can be shortened or truncated. For example, a single subunit may be deleted from the 5' end (5' truncated) or alternatively from the 3' end (3' truncated). The shortened or truncated antisense compound targeting HBV nucleic acid may have two subunits deleted from the 5' end of the antisense compound, or alternatively may have two subunits deleted from the 3' end. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, dispersed in an antisense compound having one nucleoside deleted from the 5' end and one nucleoside deleted from the 3' end.

[0537] When a single additional subunit is present in an extended antisense compound, the additional subunit may be located at the 5' or 3' end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound with two subunits, said two subunits are added to the 5' end of the antisense compound (5' addition), or alternatively added to the 3' end (3' addition). Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, dispersed in an antisense compound having one subunit added to the 5' end and one subunit added to the 3' end.

[0538] It is possible to increase or decrease the length of antisense compounds, such as antisense oligonucleotides, and / or introduce mismatched bases without eliminating activity. For example, Woolf et al. (Proceedings of the National Academy of Sciences of the United States of America, 89:7305-7309, 1992) tested the ability of a series of antisense oligonucleotides of 13–25 nucleotides in length to induce target RNA cleavage in an oocyte injection model. Antisense oligonucleotides of 25 nucleotides in length with 8 or 11 mismatched bases near their ends were able to guide specific cleavage of the target mRNA, although to a lesser extent than those without mismatches. Similarly, target-specific cleavage was achieved using 13-nucleotide antisense oligonucleotides (including oligonucleotides with 1 or 3 mismatches).

[0539] Gautschi et al. (National Cancer Institute Journal, 93:463-471, March 2001) demonstrated that an oligonucleotide with 100% complementarity to bcl-2 mRNA and three mismatches with bcl-xL mRNA could reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, the oligonucleotide exhibited potent antitumor activity in vivo.

[0540] Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested the ability of a series of tandem 14-nucleobase antisense oligonucleotides, as well as 28- and 42-nucleobase antisense oligonucleotides containing two or three of the tandem antisense oligonucleotides, respectively, in rabbit reticulocyte assays to inhibit the translation of human DHFR. Only each of the three 14-nucleobase antisense oligonucleotides was able to inhibit translation, although at a more moderate level than the 28- or 42-nucleobase antisense oligonucleotides.

[0541] Antisense compound motif

[0542] In some embodiments, the antisense compound targeting HBV nucleic acid has chemically modified subunits arranged in a pattern or motif to endow the antisense compound with properties such as enhanced inhibitory activity, increased binding affinity to the target nucleic acid, or resistance to degradation by nucleases in vivo.

[0543] Chimeric antisense compounds typically contain at least one modified region to confer 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 act as a substrate for RNase H, a cellular endonuclease that cleaves the RNA:DNA double-stranded RNA chain.

[0544] Antisense compounds with a spacer motif are considered chimeric antisense compounds. In a spacer, an inner region containing multiple nucleotides supporting RNase H cleavage is located between an outer region containing multiple nucleotides, which are chemically distinct from the nucleosides of the inner region. In the case of antisense oligonucleotides with a spacer motif, the spacer segment typically acts as a substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In some embodiments, the regions of the spacer are distinguished by the type of sugar moiety comprising each distinct region. In some embodiments, the types of sugar moiety used to distinguish the regions of the spacer may include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides may include 2'-MOE and 2'-O-CH3, etc.), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides may include bicyclic sugar-modified nucleosides with a constrained ethyl group). In some embodiments, the nucleosides in the wings may comprise several modified sugar moieties, including, for example, 2'-MOE and bicyclic sugar moieties such as constrained ethyl groups or LNA. In some embodiments, the wing may comprise several modified and unmodified sugar moieties. In some embodiments, the wing may comprise various combinations of 2'-MOE nucleotides, bicyclic sugar moieties such as restricted ethyl nucleotides or LNA nucleotides, and 2'-deoxy nucleotides.

[0545] Each distinct region may include a homogeneous sugar portion, a variant, or an alternating sugar portion. Wing-gap-wing motifs are often described as “XYZ”, where “X” represents the length of the 5' wing, “Y” represents the length of the gap, and “Z” represents the length of the 3' wing. “X” and “Z” may include homogeneous, variant, or alternating sugar portions. In some embodiments, “X” and “Y” may comprise one or more 2'-deoxynucleotides. “Y” may comprise a 2'-deoxynucleotide. As used herein, the gap body described as “XYZ” has a configuration such that the gap is located immediately adjacent to each of the 5' and 3' wings. Therefore, there is no intermediate nucleotide between the 5' wing and the gap, or between the gap and the 3' wing. Any antisense compound described herein may have a gap body motif. In some embodiments, “X” and “Z” are the same; in other embodiments, they are different. In some embodiments, “Y” is between 8 and 15 nucleotides. X, Y, or Z can be any nucleotide in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more nucleosides.

[0546] Target nucleic acid, target region and nucleotide sequence

[0547] In some embodiments, the nucleotide sequence encoding HBV includes, but is not limited to, the following: GENBANK accession number U95551.1 (incorporated herein as SEQ ID NO: 1).

[0548] It should be understood that the sequence shown in each of the examples contained herein is independent of any modification to the sugar moiety, the internucleotide bond, or the nucleobase. Therefore, an antisense compound defined by SEQ ID NO may independently include one or more modifications to the sugar moiety, the internucleotide bond, or the nucleobase.

[0549] In some 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 initiation regions, translation termination regions, or other defined nucleic acid regions. The structurally defined region of HBV can be obtained from sequence databases such as NCBI using accession numbers, and such information is incorporated herein by reference. In some 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.

[0550] The targeting comprises identifying at least one target segment to which an antisense compound hybridizes, thereby causing the desired effect. In some embodiments, the desired effect is a reduction in the level of the mRNA target nucleic acid. In some embodiments, the desired effect is a reduction in the level of the protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.

[0551] A target region may contain one or more target segments. Multiple target segments within a target region may overlap. Alternatively, they may be non-overlapping. In some embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In some embodiments, target segments within a target region are separated by multiple nucleotides on the target nucleic acid, said nucleotides being, about, no more than, no more than about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 target nucleic acids, or a range defined by any two of the foregoing values. In some embodiments, target segments within a target region are separated by no more than or no more than about 5 nucleotides on the target nucleic acid. In some embodiments, the target segments are contiguous. Target regions defined by a range having an initiating nucleic acid, said initiating nucleic acid being any of the 5' or 3' target sites listed herein are envisioned.

[0552] Suitable target regions can be found in the 5' UTR, coding region, 3' UTR, introns, exons, or exon / intron junctions. Target regions containing start or stop codons are also suitable. Suitable target regions can specifically exclude structurally defined regions, such as start or stop codons.

[0553] Identifying suitable target regions can involve comparing the sequence of the target nucleic acid with other sequences throughout the genome. For example, the BLAST algorithm can be used to identify regions of similarity between different nucleic acids. This comparison prevents the selection of antisense compound sequences (i.e., non-target or off-target sequences) that can hybridize non-target nucleic acids in a non-specific manner.

[0554] The activity of antisense compounds within the active target region (e.g., as defined by the percentage reduction in target nucleic acid levels) may vary. In some 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. Further, phenotypic changes indicate inhibition of HBV expression. In some embodiments, reduced fatigue, reduced flu-like symptoms, increased appetite, reduced nausea, reduced joint pain, reduced jaundice, reduced abdominal pain, reduced weakness, reduced weight loss, reduced gynecomastia, reduced palmar rashes, reduced clotting difficulties, reduced cirrhosis, reduced spider veins in the skin, increased absorption of vitamins A and D, reduced tumor growth, reduced tumor volume, reduced headache, reduced fever, reduced diarrhea, reduced pain in the liver region of the body, reduced clay-colored or gray stools, reduced itching, reduced dark urine, and reduced nausea and vomiting may indicate inhibition of HBV expression. In some embodiments, improvement in symptoms associated with HBV-related conditions, diseases, and illnesses may indicate inhibition of HBV expression. In some embodiments, reduction in cirrhosis indicates inhibition of HBV expression. In some embodiments, a reduction in liver cancer markers may indicate inhibition of HBV expression.

[0555] Hybridization

[0556] In some embodiments, hybridization occurs between the antisense compound disclosed herein and HBV nucleic acid. The most common hybridization mechanism involves hydrogen bonding between complementary nucleobases of nucleic acid molecules (e.g., Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding).

[0557] Hybridization can occur under different conditions. Strict conditions are sequence-dependent and determined by the properties and composition of the nucleic acid molecules to be hybridized.

[0558] Methods for determining whether a sequence can specifically hybridize with a target nucleic acid are well known in the art. In some embodiments, the antisense compounds provided herein can specifically hybridize with HBV nucleic acids.

[0559] Complementarity

[0560] When a sufficient number of nucleobases of an antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, the antisense compound and the target nucleic acid complement each other, resulting in the desired effect (e.g., antisense inhibition of target nucleic acids such as HBV nucleic acid).

[0561] Non-complementary nucleobases between the antisense compound and HBV nucleic acid can be tolerated, provided that the antisense compound can still specifically hybridize with the target nucleic acid. Furthermore, the antisense compound can hybridize on one or more segments of the HBV nucleic acid such that intermediate or adjacent segments are not involved in hybridization events (e.g., loop structures, mismatches, or hairpin structures).

[0562] In some embodiments, the antisense compound or a designated portion thereof provided herein is complementary to HBV nucleic acid, target region, target segment or a designated 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 antisense compound and the target nucleic acid can be determined using conventional methods.

[0563] For example, an antisense compound in which 18 of the 20 nucleosides are complementary to the target region and thus specifically hybridizes will represent 90% complementarity. In this example, the remaining non-complementary nucleosides can cluster or disperse with the complementary nucleosides and do not necessarily need to be adjacent to each other or to the complementary nucleosides. Thus, an antisense compound of 18 nucleosides has four non-complementary nucleosides flanking two regions that are fully complementary to the target nucleic acid, and these non-complementary nucleosides have an overall complementarity of 77.8% with the target nucleic acid, and therefore fall within the scope of this invention. The percentage of complementarity between the antisense compound and the target nucleic acid region can be routinely determined using the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). The percentages of homology, sequence identity, or complementarity can be determined, for example, using the Gap program (University Research Park, Madison Wisconsin, Genetics Computer Group, Unix version 8, Wisconsin Sequence Analysis Package), which uses default settings and employs the Smith and Waterman algorithm (Advances in Appl. Math., 1981, 2, 482 489).

[0564] In some embodiments, the antisense compound or a designated portion thereof provided herein is completely complementary (i.e., 100% complementary) to the target nucleic acid or a designated portion thereof. For example, the antisense compound may be completely complementary to the HBV nucleic acid or its target region or target segment or target sequence. As used herein, “completely complementary” means that each nucleobase of the antisense compound is precisely complementary to the corresponding nucleobase of the target nucleic acid. For example, a 20-nucleobase antisense compound is completely complementary to a 400-nucleobase-long target sequence, provided that the corresponding 20-nucleobase portion of the target nucleic acid is completely complementary to the antisense compound. Complete complementarity may also be used with reference to designated portions of the first and / or second nucleic acids. For example, the 20-nucleobase portion of a 30-nucleobase antisense compound may be “completely complementary” to a 400-nucleobase-long target sequence. If the target sequence has a corresponding 20-nucleobase portion, then the 20-nucleobase portion of a 30-nucleobase oligonucleotide is completely complementary to the target sequence, wherein each nucleobase is complementary to the 20-nucleobase portion of the antisense compound. Meanwhile, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence, the entire 30-nucleobase antisense compound may or may not be completely complementary to the target sequence.

[0565] The non-complementary nucleobase can be located at the 5' or 3' end of the antisense compound. Alternatively, the non-complementary nucleobase and nucleobase can be located inside the antisense compound. When two or more non-complementary nucleobases are present, the nucleobases can be continuous (i.e., linked) or discontinuous. In one embodiment, the non-complementary nucleobase is located in the wing segment of the interstitial antisense oligonucleotide.

[0566] In some embodiments, antisense compounds having a length of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides relative to the target nucleic acid (such as HBV nucleic acid or a designated portion thereof) include no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleotides. In some embodiments, antisense compounds 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 nucleotides relative to the target nucleic acid (such as HBV nucleic acid or a designated portion thereof) include no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleotides.

[0567] The provided antisense compounds also comprise antisense compounds complementary to a portion of the target nucleic acid. As used herein, "portion" refers to a defined number of consecutive (i.e., linked) nucleobases within a region or segment of the target nucleic acid. "Portion" may also refer to a defined number of consecutive nucleobases of the antisense compound. In some embodiments, the antisense compound is complementary to at least 8 nucleobase portions of the target segment. In some embodiments, the antisense compound is complementary to at least 9 nucleobase portions of the target segment. In some embodiments, the antisense compound is complementary to at least 10 nucleobase portions of the target segment. In some embodiments, the antisense compound is complementary to at least 11 nucleobase portions of the target segment. In some embodiments, the antisense compound is complementary to at least 12 nucleobase portions of the target segment. In some embodiments, the antisense compound is complementary to at least 13 nucleobase portions of the target segment. In some embodiments, the antisense compound is complementary to at least 14 nucleobase portions of the target segment. In some embodiments, the antisense compound is complementary to at least 15 nucleobase portions of the target segment. Antisense compounds complementary to at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleobase moieties of the target region, or ranges defined by any two of these values, are also envisioned.

[0568] identity

[0569] The antisense compounds described herein may also have a defined percentage of identity with a specific nucleotide sequence, SEQ ID NO, or compound or portion thereof. As used herein, an antisense compound is considered identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, RNA containing uracil instead of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence because both uracil and thymidine pair with adenine. Shortened and extended forms of the antisense compounds described herein, as well as compounds having different bases relative to the antisense compounds provided herein, are also contemplated. Different bases may be adjacent to each other or dispersed throughout the antisense compound. The percentage of identity of an antisense compound is calculated based on the number of bases with the same base pairing ability relative to the sequence with which it is being compared.

[0570] As used herein, a “complementary” polynucleotide is a polynucleotide capable of base pairing according to the standard Watson-Crick complementarity rule. Specifically, a purine will pair with a pyrimidine base to form a combination of guanine and cytosine (G:C) and, in the case of DNA, adenine and thymine (A:T), or, in the case of RNA, adenine and uracil (A:U). For example, the sequence “AGT” binds to the complementary sequence “TCA”. It should be understood that even if two polynucleotides are not perfectly complementary to each other, the two polynucleotides can still hybridize, provided that each polynucleotide has at least one region that is substantially complementary to the other.

[0571] As used herein, the terms “substantially complementary” or “partially complementary” mean that two nucleic acid sequences are complementary to each other by at least about 50%, 60%, 70%, 80%, or 90% of their nucleotides.

[0572] In some embodiments, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the nucleotides in the two nucleic acid sequences are complementary. In some embodiments, the two nucleic acid sequences may be 60% to 100% complementary, 70% to 100% complementary, 80% to 100% complementary, 90% to 100% complementary, 60% to 90% complementary, 60% to 80% complementary, 60% to 70% complementary, 70% to 90% complementary, 70% to 80% complementary, 80% to 100% complementary or 80% to 90% complementary.

[0573] The terms “substantially complementary” and “partially complementary” can also mean that two nucleic acid sequences can hybridize under highly stringent conditions, and such conditions are well known in the field.

[0574] As used herein, the term "identity" means comparing sequences to each other as follows. To determine the percentage of identity between two nucleic acid sequences, the sequences can first be aligned relative to each other to make subsequent comparisons possible. For this purpose, gaps can be inserted into the sequence of the first nucleic acid sequence, and nucleotides can be compared to corresponding positions in the second nucleic acid sequence. If a position in the first nucleic acid sequence is occupied by the same nucleotide as that at a position in the second sequence, then the two sequences are identical at that position. 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 under study.

[0575] The "identity percentage" of the alignment segments between the test sequence and the reference sequence is calculated by dividing the percentage of identical components shared by the two alignment sequences by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence.

[0576] Mathematical algorithms can be used to determine the percentage of identity between two sequences. A preferred, but not limiting, example of a mathematical algorithm that can be used to compare two sequences is the algorithm of Karlin et al., (1993), Proceedings of the National Academy of Sciences, 90:5873-5877. Such algorithms are integrated into the NBLAST procedure, which can be used to identify sequences with the desired identity to the sequences of the present invention. To obtain alignments with gaps, as described herein, a "BLAST with gaps" procedure can be used, as described in Altschul et al., (1997), Nucleic Acid Research, 25:3389-3402. If both BLAST and BLAST with gaps procedures are used, preset parameters of the specific procedure (e.g., NBLAST) can be used. Sequences can be further aligned using GAP (Global Alignment Program) version 9 of the Genetic Computing Group with a preset (BLOSUM62) matrix (values ​​-4 to +11), where the vacancy opening penalty is -12 (for the first zero of the vacancy) and the vacancy extension penalty is -4 (for each additional consecutive zero in the vacancy). After alignment, the percentage of identity is calculated by expressing the number of similarities as a percentage of the nucleic acids in the claimed sequence. If necessary, the described method for determining the percentage of identity between two nucleic acid sequences can also be used correspondingly on the encoded amino acid sequences.

[0577] Useful methods for determining sequence identity are also published in Guide to Huge Computers (edited by Martin J. Bishop, Academic Press, San Diego (1994)) and Carillo, H. and Lipton, D. (Applied Mathematics 48:1073 (1988)). More specifically, preferred computer programs for determining sequence identity include, but are not limited to, the Basic Local Alignment Search Tool (BLAST) program, which is available from the National Center for Biotechnology Information (NCBI), 20894, National Library of Medicine, National Institute of Health, Bethesda, Md.; see BLAST Manual, (Altschul et al., NCBI, NLM, NIH; Altschul et al., Journal of Molecular Biology 215:403-410 (1990)); BLAST program version 2.0 or later allows gaps (deletions and insertions) to be introduced into the alignment; for peptide sequences, BLASTX can be used to determine sequence identity; and for polynucleotide sequences, BLASTN can be used to determine sequence identity. The identity percentage can be 70% or greater, for example, 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.

[0578] Modification

[0579] Nucleosides are base-sugar combinations. The nucleobase (also called the base) portion of a nucleoside is typically a heterocyclic base portion. A nucleotide is a nucleoside that further comprises a phosphate ester group covalently linked to the sugar portion of the nucleoside. For those nucleosides containing pentofuranosyl sugars, the phosphate ester group may be linked to the 2', 3', or 5' hydroxyl portion of the sugar. Oligonucleotides are formed by covalent bonds between adjacent nucleosides to form linear polymeric oligonucleotides. Within the oligonucleotide structure, the phosphate ester group is generally referred to as the nucleoside internucleotide bond that forms the oligonucleotide.

[0580] Modification of antisense compounds encompasses the substitution or alteration of nucleoside internucleotide bonds, sugar moieties, or nucleobases. Modified antisense compounds are generally preferred over their natural forms due to desired properties such as enhanced cellular uptake, increased affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0581] Chemically modified nucleosides can also be used to increase the binding affinity of shortened or truncated antisense oligonucleotides to their target nucleic acids. Therefore, comparable results can often be obtained with shorter antisense compounds containing such chemically modified nucleosides.

[0582] Modified nucleoside interbonds

[0583] The naturally occurring nucleotide bonds in RNA and DNA are 3' to 5' phosphodiester bonds. Antisense compounds with one or more modified, i.e., non-naturally occurring nucleotide bonds are typically chosen over naturally occurring nucleotide bonds because of desired properties such as enhanced cellular uptake, increased affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0584] Oligonucleotides with modified nucleoside bonds include both phosphorus-retaining and phosphorus-free nucleoside bonds. Representative phosphorus-containing nucleoside bonds include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, amino acid phospholipids, and thiophosphates. Methods for preparing phosphorus-containing and phosphorus-free bonds are well known.

[0585] In some embodiments, the antisense compound targeting HBV nucleic acid comprises one or more modified nucleoside internucleotide bonds. In some embodiments, the modified nucleoside internucleotide bonds are phosphate thioester bonds. In some embodiments, each nucleoside internucleotide bond of the antisense compound is a phosphate thioester nucleoside internucleotide bond.

[0586] Modified sugar portion

[0587] The antisense compounds provided herein may optionally contain one or more nucleosides, wherein the glycosyl group has been modified. Such glycomodified nucleosides can confer enhanced nuclease stability, increased binding affinity, or other beneficial biological properties to the antisense compounds. In some embodiments, the nucleoside comprises a chemically modified furanose ring moiety. Examples of chemically modified furanose rings include, but are not limited to, the addition of substituent groups (containing 5' and / or 2' substituent groups; bridging non-homogeneous ring atoms to form bicyclic nucleic acids (BNAs); using S, N(R), or C(R) 1 (R) 2 (R = H, C1-C) 12Alkyl or protecting groups) replacing the ribosyl epoxy atom; and combinations thereof. Examples of chemically modified sugars include 2'-F-5'-methyl-substituted nucleosides (for other disclosed 5',2'-disubstituted nucleosides, see PCT International Application WO 2008 / 101157 published 8 / 21 / 08), replacing the ribosyl epoxy atom with S and further substituting at the 2' position (see U.S. Patent Application US2005 / 0130923 published June 16, 2005), or alternatively, 5'-substituted BNA (see PCT International Application WO 2007 / 134181 published 11 / 22 / 07, wherein LNA is substituted, for example, with 5'-methyl or 5'-vinyl).

[0588] Examples of nucleosides having a modified sugar moiety include, but are not limited to, nucleosides comprising substituents such as 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, and 2'-O(CH2)2OCH3. The substituent at the 2' position may also be selected from: allyl, amino, azide, thio, O-allyl, O-Cl-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 ), where each R m and R n Independently, it is H or substituted or unsubstituted C1-C. 10 alkyl.

[0589] As used herein, “bicyclic nucleoside” refers to a modified nucleoside comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include, but are not limited to, nucleosides comprising a bridge between 4' and 2' ribosyl ring atoms. In some embodiments, the antisense compounds provided herein comprise one or more bicyclic nucleosides, wherein the bridge comprises a 4' to 2' bicyclic nucleoside. Examples of such 4' to 2' bicyclic nucleosides include, but are not limited to, one of the following formulas: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (cEt) and 4'-CH(CH2OCH3)-O-2' and analogs thereof (see U.S. Patent 7,399,845, issued July 15, 2008); 4'-C(CH3)(CH3)-O-2' and analogs thereof (see 20 4'-CH2-N(OCH3)-2' and its analogues (see PCT International Application WO2008 / 150729 published on December 11, 2008); 4'-CH2-ON(CH3)-2' (see U.S. Patent Application US2004 / 0171570 published on September 2, 2004); 4'-CH2-N(R)-O-2', wherein R is H, C1-C 12Alkyl groups or protecting groups (see U.S. Patent 7,427,672, issued September 23, 2008); 4'-CH2-C(H)(CH3)-2' (see Chattopadhyaya et al., Journal of Organic Chemistry, 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' and its analogues (see published PCT International Application WO 2008 / 154401, issued December 8, 2008).See also, for example: Singh et al., *Chem. Commun.*, 1998, 4, 455-456; Koshkin et al., *Tetrahedron*, 1998, 54, 3607-3630; Wahllestedt et al., *Proceedings of the National Academy of Sciences*, 2000, 97, 5633-5638; Kumar et al., *Bioorg. Med. Chem. Lett.*, 1998, 8, 2219-2222; Singh et al., *Journal of Organic Chemistry*, 1998, 63, 10035-10039; Srivastava et al., *Journal of the American Chemical Society*, 129(26) 8362-8379 (July 4, 2007); Elayadi et al., "Recent Perspectives on Drug Research (Curr. Opinion Invens. Drugs)," 2001, 2, 558-561; Braasch et al., "Chem. Biol.," 2001, 8, 1-7; Orum et al., "New Insights in Molecular Therapy (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; Publicly available PCT International Application WO 2004 / 106356, WO 94 / 14226, WO 2005 / 021570 and WO 2007 / 134181; US ​​Patent Publications Nos. US2004 / 0171570, US2007 / 0287831 and US2008 / 0039618; and US Patent Serials Nos. 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 Applications Nos. PCT / US2008 / 064591, PCT / US2008 / 066154 and PCT / US2008 / 068922.Each of the aforementioned bicyclic nucleosides can be prepared to have one or more stereochemical sugar configurations, including, for example, α-L-ribofranose and β-D-ribofranose (see PCT International Application PCT / DK98 / 00393, published on 25 March 1999 as WO 99 / 14226).

[0590] In some embodiments, the bicyclic sugar moiety of the BNA nucleotide comprises, but is not limited to, compounds having at least one bridge between the 4' and 2' positions of the pentofuranosyl sugar moiety, wherein such bridge independently comprises one or two to four independently linked groups selected from: -[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 )-;

[0591] in:

[0592] x is 0, 1, or 2;

[0593] n is 1, 2, 3 or 4;

[0594] Each R a and R b Independently, it is H, protecting group, hydroxyl group, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 alkenyl, C2-C 12 Alkyne group, substituted C2-C 12 alkynyl C5-C 20 Aryl, substituted C5-C 20 Aryl, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, C5-C7 alicyclic, substituted C5-C7 alicyclic, halogen, OJ 1 NJ 1 J 2 SJ 1 N3, COOJ 1 Acyl group (C(=O)-H), substituted acyl group, CN, sulfonyl group (S(=O)2-J) 1 ) or synergistic sulfone (S(=O)-J 1);and

[0595] Each J 1 and J 2 Independently, H, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 alkenyl, C2-C 12 Alkyne group, substituted C2-C 12 alkynyl group, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic, substituted heterocyclic, C1-C 12 Aminoalkyl, substituted C1-C 12 Aminoalkyl groups or protecting groups.

[0596] In some embodiments, the bridge of the bicyclic sugar moiety is -[C(R a (R) b )] n -、-[C(R a (R) b )] n -O-、-C(R a (R) b )-N(R)-O- or —C(R) a (R) b )-ON(R)-. In some embodiments, the bridge is 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'-, wherein each R is independently H, a protecting group, or C1-C. 12 alkyl.

[0597] In some embodiments, the bicyclic nucleoside is further defined by the isomer configuration. For example, a nucleoside including a 4'-2' methylene-oxygen bridge can be in the α-L configuration or the β-D configuration. Previously, α-L-methyleneoxy (4'-CH2-O-2')BNA has been incorporated into antisense oligonucleotides exhibiting antisense activity (Frieden et al., Nucleic Acid Research, 2003, 21, 63 656372).

[0598] In some embodiments, the bicyclic nucleoside comprises, but is not limited to, (A) α-L-methyleneoxy(4'-CH2-O-2')BNA, (B) β-D-methyleneoxy(4'-CH2-O-2')BNA, (C) vinyloxy(4'-(CH2)2-O-2')BNA, (D) aminooxy(4'-CH2-ON(R)-2')BNA, and (E) oxyamino(4'-CH2-N(R)-O-2')BNA. (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) Methylcarbocyclic(4'-CH2-CH(CH3)-2')BNA and (J) Propylenecarbocyclic(4'-(CH2)3-2')BNA, as described below.

[0599]

[0600] Where Bx is the base moiety, and R is independently H, a protecting group, or C1-C. 12 alkyl.

[0601] In some embodiments, the bicyclic nucleoside is prepared according to Formula I:

[0602]

[0603] in:

[0604] Bx is the heterocyclic base moiety;

[0605] -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;

[0606] R c It is C1-C 12 alkyl or amino protecting groups; and

[0607] T a and T b Each is independently an H, a hydroxyl protecting group, a conjugated group, a reactive phosphorus group, a phosphorus moiety, or covalently linked to a supporting medium.

[0608] In some embodiments, the bicyclic nucleoside has formula II:

[0609]

[0610] in:

[0611] Bx is the heterocyclic base moiety;

[0612] T a and T b Each is independently an H, a hydroxyl protecting group, a conjugated group, a reactive phosphorus group, a phosphorus moiety, or covalently linked to the supporting medium;

[0613] Z a It is a 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.

[0614] In one embodiment, each substituted group is independently monosubstituted or polysubstituted by a substituent group selected from the following: halogen, oxo, hydroxyl, OJ. c NJ c J d SJ c N3, OC(=X)J c and NJ e C(=X)NJ c J d , where each J c J d and J e Independently, it is H, C1-C6 alkyl, or substituted C1-C6 alkyl, and X is O or NJ. c .

[0615] In some embodiments, the bicyclic nucleoside has formula III:

[0616]

[0617] in:

[0618] Bx is the heterocyclic base moiety;

[0619] T a and T b Each is independently an H, a hydroxyl protecting group, a conjugated group, a reactive phosphorus group, a phosphorus moiety, or covalently linked to the supporting medium;

[0620] Z b It is a 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)-).

[0621] In some embodiments, the bicyclic nucleoside has formula IV:

[0622]

[0623] in:

[0624] Bx is the heterocyclic base moiety;

[0625] T a and T b Each is independently an H, a hydroxyl protecting group, a conjugated group, a reactive phosphorus group, a phosphorus moiety, or covalently linked to the supporting medium;

[0626] R d It is a C1-C6 alkyl, a substituted C1-C6 alkyl, a C2-C6 alkenyl, a substituted C2-C6 alkenyl, a C2-C6 ynyl or a substituted C2-C6 ynyl;

[0627] Each q a q b q c and q d Independently, it is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 ynyl or substituted C2-C6 ynyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, acyl, substituted acyl, C1-C6 aminoalkyl or substituted C1-C6 aminoalkyl;

[0628] In some embodiments, the bicyclic nucleoside has formula V:

[0629]

[0630] in:

[0631] Bx is the heterocyclic base moiety;

[0632] T a and T b Each is independently an H, a hydroxyl protecting group, a conjugated group, a reactive phosphorus group, a phosphorus moiety, or covalently linked to the supporting medium;

[0633] q a q b q e and q f Each is independently hydrogen, halogen, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 alkenyl, C2-C 12Alkyne group, substituted C2-C 12 Alkyne group, C1-C 12 Alkoxy, 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 J k Or N(H)C(=S)NJ j J k ;

[0634] or q e and q f Together is = C(q) g )(q h );

[0635] q g and q h Each is independently H, halogen, C1-C 12 Alkyl or substituted C1-C 12 alkyl.

[0636] The synthesis and preparation of methyleneoxy (4'-CH2-O-2')BNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil, as well as their oligomerization and nucleic acid recognition properties, have been described (see, for example, Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNA and its preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0637] Analogs of methyleneoxy(4'-CH2-O-2')BNA, methyleneoxy(4'-CH2-O-2')BNA, and 2'-thio-BNA have also been prepared (see, for example, Kumar et al., Bioorganic & Medicinal Chemistry Letters, 1998, 8, 2219-2222). The preparation of locked nucleoside analogs, including oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases, has also been described (see, for example, Wengel et al., WO 99 / 14226). Furthermore, the synthesis of 2'-amino-BNA, a novel conformation-restricted high-affinity oligonucleotide analog, has been described in the art (see, for example, Singh et al., Journal of Organic Chemistry, 1998, 63, 10035-10039). Additionally, 2'-amino- and 2'-methylamino-BNA have been prepared, and their thermostability with complementary RNA and DNA duplexes has been previously reported.

[0638] In some embodiments, the bicyclic nucleoside has formula VI:

[0639]

[0640] in:

[0641] Bx is the heterocyclic base moiety;

[0642] T a and T b Each is independently an H, a hydroxyl protecting group, a conjugated group, a reactive phosphorus group, a phosphorus moiety, or covalently linked to the supporting medium;

[0643] Each q i q j q k and q l Independently, it is H, halogen, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 alkenyl, C2-C 12 Alkyne group, substituted C2-C 12 Alkyne group, C1-C 12 Alkoxy, 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 Jk 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

[0644] q i and q j or q 1 and q k Together is = C(q) g )(q h ), where q g and q h Each is independently H, halogen, C1-C 12 Alkyl or substituted C1-C 12 alkyl.

[0645] A carbocyclic bicyclic nucleotide with a 4'-(CH2)3-2' bridge and an alkenyl analog bridge 4'-CH=CH-CH2-2' has been described (see, for example, Freier et al., Nucleic Acid Research, 1997, 25(22), 44294443 and Albaek et al., Journal of Organic Chemistry, 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleotides, as well as their oligomerization and biochemical studies, have also been described (see, for example, Srivastava et al., Journal of the American Chemical Society, 2007, 129(26), 8362-8379).

[0646] As used herein, "bicyclic nucleoside" refers to a nucleoside comprising a bridge connecting two carbon atoms of a sugar ring, thereby forming a bicyclic sugar moiety. In some embodiments, the bridge connects the 2' carbon of the sugar ring to another carbon.

[0647] As used herein, “4'-2' bicyclic nucleoside” or “4' to 2' bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring including a bridge connecting the 2' carbon atom and the 4' carbon atom.

[0648] As used herein, "monocyclic nucleoside" refers to a nucleoside that includes a modified sugar moiety that is not a bicyclic sugar moiety. In some embodiments, the sugar moiety of the nucleoside or a sugar moiety analogue may be modified or substituted at any position.

[0649] As used herein, “2'-modified sugar” means a furanyl sugar modified at the 2' position. In some embodiments, such modification comprises substituents selected from: halides comprising, but not limited to, substituted and unsubstituted alkoxy groups, substituted and unsubstituted thioalkyl groups, substituted and unsubstituted aminoalkyl groups, substituted and unsubstituted alkyl groups, substituted and unsubstituted allyl groups, and substituted and unsubstituted alkynyl groups. In some embodiments, the 2' modification is selected from, but not limited to, substituents including: 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 approximately 10. Other 2'-substituent groups may also be selected from: C1-C 12Alkyl; substituted alkyl; alkenyl; alkynyl; aryl; aralkyl; O-aryl or O-aralkyl; SH; SCH3; OCN; Cl; Br; CN; CF3; OCF3; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocyclic alkyl; heterocyclic aryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving group; reporter group; intercalator; group for improving pharmacokinetic properties; and group for improving the pharmacokinetic properties of antisense compounds, as well as other substituents with similar properties. In some embodiments, the modified nucleoside includes a 2'-MOE side chain (see, for example, Baker et al., Journal of Biochemistry, 1997, 272, 11944-12000). Such 2'-MOE substitutions have been described as exhibiting improved binding affinity compared to unmodified nucleosides and other modified nucleosides such as 2'-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides with 2'-MOE substituents have also been shown to be promising antisense inhibitors of gene expression with promising features for in vivo use (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).

[0650] As used herein, “modified tetrahydropyranoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran “sugar” that substitutes for a pentofuranose residue in a normal nucleoside (sugar substitute). Modified THP nucleosides include, but are not limited to, nucleosides known in the art as hexitol nucleic acid (HNA), atroitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, CJ. Bioorg. & Med. Chem. (2002) 10:841-854), fluoroHNA (F-HNA), or those compounds having formula X:

[0651] For each tetrahydropyranoside analog of at least one of the formula X, independently:

[0652]

[0653] Bx is the heterocyclic base moiety;

[0654] T 3 and T 4 Each is an inter-nucleoside linker group that independently connects a tetrahydropyran nucleoside analog to an antisense compound, or T 3 and T 4 One of them is an internucleotide linking group that connects a tetrahydropyran nucleoside analog to an antisense compound, and T 3 and T 4 The other one is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3' terminal group;

[0655] q 1 q 2 q 3 q 4 q 5 q 6 and q 7 Each is independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 ynyl, or substituted C2-C6 ynyl; and

[0656] R 1 and R 2 One of them is hydrogen, and the other is selected from the following: halogen, substituted or unsubstituted alkoxy group, 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 and CN, where X is O, S or NJ 1 And each J 1 J 2 and J 3 It is independently H or C1-C6 alkyl.

[0657] In some embodiments, a modified THP nucleoside of formula X is provided, wherein q m q n q p q r q s q t and q u Each is H. In some embodiments, q m q n q p q r qs q t and q u At least one of them is not H. In some embodiments, q m q n q p q r q s q t and q u At least one of them is a methyl group. In some embodiments, a THP nucleoside of formula X is provided, wherein R 1 and R 2 One of them is F. In some embodiments, R 1 It is fluorine and R 2 It is H, R 1 It is methoxylated and R 2 It is H, and R 1 It is methoxyethoxy and R 2 It is H.

[0658] As used herein, "2'-modified nucleoside" or "2'-substituted nucleoside" refers to a nucleoside comprising a sugar having a substituent at the 2' position of the furanose ring, excluding H or OH. 2'-modified nucleosides include, but are not limited to, bicyclic nucleosides, wherein a bridge connecting the two carbon atoms of the sugar ring links the 2' carbon of the sugar ring to another carbon, and nucleosides having a non-bridged 2'-substituent, such as allyl, amino, azide, thio, O-allyl, O-Cl-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 ), where each R m and R n Independently, it is H or substituted or unsubstituted C1-C. 10 Alkyl groups. 2'-Modified nucleosides may further include other modifications, such as modifications at other positions on the sugar and / or at the nucleobase.

[0659] As used in this article, "2'-F" refers to a sugar that includes a fluorine group at the 2' position.

[0660] As used herein, “2'-OMe” or “2'-OCH3” or “2'-O-methyl” each refers to a nucleoside comprising a sugar having a -OCH3 group at the 2' position of the sugar ring.

[0661] As used herein, "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides. In some embodiments, one or more of the nucleosides are modified. In some embodiments, the oligonucleotide comprises one or more ribonucleosides (RNA) and / or deoxyribonucleosides (DNA).

[0662] Many other bicyclic and tricyclic sugar substitute ring systems are also known in the art, and these systems can be used to modify nucleosides for incorporation into antisense compounds (see, for example, review article: Leumann, JC, *Bioorganic and Medicinal Chemistry*, 2002, 10, 841-854). Such ring systems can undergo various further substitutions to enhance activity.

[0663] The methods used to prepare modified sugars are well known to those skilled in the art.

[0664] In nucleotides with modified sugar moieties, the nucleobase moieties (natural, modified, or a combination thereof) are maintained to hybridize with appropriate nucleic acid targets.

[0665] In some embodiments, the antisense compound comprises one or more nucleotides having a modified sugar moiety. In some embodiments, the modified sugar moiety is 2'-MOE. In some embodiments, the 2'-MOE-modified nucleotide is arranged within a spacer motif. In some embodiments, the modified sugar moiety is cEt. In some embodiments, the cEt-modified nucleotide is arranged throughout the entire wing of the spacer motif.

[0666] Compositions and methods for preparing pharmaceutical compositions

[0667] Antisense oligonucleotides can be mixed with pharmaceutically acceptable active or inert substances to prepare pharmaceutical compositions or formulations. The compositions and methods used to formulate pharmaceutical compositions depend on many criteria, including but not limited to the route of administration, disease severity, or dose to be administered.

[0668] Antisense compounds targeting HBV nucleic acids can be incorporated into pharmaceutical compositions by combining them with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is suitable for use in compositions intended for parenteral delivery. Therefore, in one embodiment, the pharmaceutical composition used in the methods described herein comprises an antisense compound targeting HBV nucleic acids and a pharmaceutically acceptable diluent. In some embodiments, the pharmaceutically acceptable diluent is PBS. In some embodiments, the antisense compound is an antisense oligonucleotide.

[0669] Pharmaceutical compositions including antisense compounds encompass any pharmaceutically acceptable salt, ester, or salt of such esters, or any other oligonucleotide capable of providing (directly or indirectly) a biologically active metabolite or its residues when administered to animals, including humans. Thus, for example, this disclosure is also formulated as pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0670] The prodrug may be contained at one or both ends of the antisense compound with an additional nucleoside, which is cleaved by endogenous nucleases in the body to form the active antisense compound.

[0671] This disclosure provides pharmaceutical compositions comprising the modified oligonucleotides of this disclosure and pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the modified oligonucleotide comprises any one of the sequences of SEQ ID NO: 11-666. In some embodiments, as described herein, the modified oligonucleotide comprises any one of SEQ ID NO: 11-666 and sequences of one, two, three, four, or five modifications to said modified oligonucleotide.

[0672] Modified oligonucleotides can be mixed with pharmaceutically acceptable active or inert substances to prepare pharmaceutical compositions or formulations. The compositions and methods used to formulate pharmaceutical compositions depend on many criteria, including but not limited to the route of administration, disease severity, or dose to be administered.

[0673] The pharmaceutical compositions disclosed herein may optionally include therapeutic agents, pharmaceutical preparations, carriers, adjuvants, dispersants, diluents, etc.

[0674] Antisense compounds targeting HBV nucleic acids can be incorporated into pharmaceutical compositions by combining them with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is suitable for use in compositions intended for parenteral delivery. Therefore, in one embodiment, the pharmaceutical composition used in the methods described herein comprises an antisense compound targeting HBV nucleic acids and a pharmaceutically acceptable diluent. In some embodiments, the pharmaceutically acceptable diluent is PBS. In some embodiments, the antisense compound is an antisense oligonucleotide.

[0675] Pharmaceutical compositions comprising modified oligonucleotides may encompass any pharmaceutically acceptable salt, ester, or salt of such esters, or any other oligonucleotide capable of providing (directly or indirectly) a biologically active metabolite or its residues when administered to animals, including humans. Thus, for example, this disclosure is also formulated as pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0676] Modified oligonucleotides formulated as prodrugs are intended to be within the scope of this disclosure. The prodrug may contain additional nucleosides incorporated at one or both ends of the antisense compound, said nucleosides being cleaved by endogenous nucleases in vivo to form the active antisense compound.

[0677] The pharmaceutical composition may contain any of the reagents discussed above, as well as one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0678] A “pharmaceutical composition” is a formulation comprising the modified oligonucleotides described herein, in a form suitable for administration to a subject. In some embodiments, the pharmaceutical composition is in bulk or unit dosage form. Unit dosage form is any of a variety of forms, including, for example, capsules, IV bags, tablets, disposable syringes, aerosol inhalers, or a single pump on a vial. The amount of active ingredient (e.g., a formulation of modified oligonucleotides) in a unit dose of the composition is an effective amount and varies depending on the specific treatment involved. Those skilled in the art will understand that it is sometimes necessary to routinely change the dosage according to the patient’s age and condition. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, percutaneous, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, oral, sublingual, intrapleural, intrathecal, intranasal, etc. Dosage forms for topical or percutaneous application of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalers. In some embodiments, the modified oligonucleotide is mixed under sterile conditions with a pharmaceutically acceptable carrier and any desired preservatives, buffers, or propellants.

[0679] "Pharmaceutically acceptable excipients" means excipients that can be used to prepare pharmaceutical compositions that are generally safe, non-toxic, and biologically and otherwise desirable, and includes excipients that are acceptable for both veterinary and human pharmaceutical use. As used in the product information, "pharmaceutically acceptable excipients" includes both one and more such excipients.

[0680] Pharmaceutical compositions are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), intraperitoneal (into body cavities), and transmucosal administration. Solutions or suspensions for parenteral, intradermal, intraperitoneal, or subcutaneous application may contain the following components: sterile diluents, such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citrate, or phosphate; and agents for adjusting tension, such as sodium chloride or dextran. pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral or subcutaneous preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. These formulations may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may contain suspending agents and thickeners. Formulations may be presented in single / dose or multi-dose containers, such as sealed ampoules, syringes, and vials, and may be stored under lyophilized (freeze-dried) conditions where a sterile liquid carrier, such as saline or water for injection, is only required to be added just before use.

[0681] The pharmaceutical compositions described herein can be prepared in commonly known manner, for example, by conventional processes such as mixing, dissolving, granulation, forming into sugar-coated pellets, grinding, emulsifying, encapsulating, embedding, or lyophilizing. The pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers, including excipients and / or adjuvants that facilitate the processing of the active agent into a pharmaceutically usable formulation. Of course, the appropriate formulation depends on the chosen route of administration.

[0682] Suitable pharmaceutical compositions for injectable use comprise sterile aqueous solutions (in the water-soluble case) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, and polyoxyethylene castor oil EL. TM(BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to facilitate injection. It must be stable under the conditions of preparation and storage and must be preserved against contamination by microorganisms such as bacteria and fungi. The carrier can 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. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, the inclusion of isotonic agents in the composition, such as sugars, polyols such as mannitol and sorbitol, and sodium chloride, is preferred. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0683] Oral compositions typically contain an inert diluent or an edible, pharmaceutically acceptable carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active agent may be incorporated with excipients and used in tablet, lozenge, or capsule form. Oral compositions may also be prepared using a fluid carrier used as a mouthwash, wherein the agent in the fluid carrier is administered orally and rinsed and spat out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or agents with similar properties: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; gliding agents, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavoring.

[0684] For administration by inhalation, the agent is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant (e.g., a gas such as carbon dioxide) or a sprayer.

[0685] Pharmaceutical compositions can be prepared with pharmaceutically acceptable carriers that protect modified oligonucleotides from rapid elimination from the body, such as controlled-release formulations, implants, and microencapsulated delivery systems. Biodegradable polymers and biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art, and the materials are commercially available. Liposome suspensions (including liposomes targeting infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0686] Particularly advantageous is the formulation of oral or parenteral compositions in dose-unit form for ease of administration and dosage uniformity. As used herein, dose-unit form refers to a physically discrete unit suitable for a unit dose to a subject to be treated; each unit contains a predetermined number of modified oligonucleotides calculated to produce the desired therapeutic effect associated with the desired drug delivery. The specifications of the dose-unit forms disclosed herein are determined by and directly dependent on the unique properties of the active agent and the specific therapeutic effect to be achieved.

[0687] The pharmaceutical composition may be included in a container, package, or dispenser along with the instructions for use.

[0688] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral salts or organic salts of basic residues such as amines, and basic salts or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of parent compounds formed from non-toxic inorganic or organic acids.

[0689] Techniques for preparing and administering the disclosed compositions of the ...

Claims

1. A compound comprising a modified oligonucleotide, said modified oligonucleotide comprising, from 5' to 3': 5' W1 - G1 - S1 - G2 - W2 3' (Formula I) in: W1 is the 5' wing section; W2 is the 3' wing section; G1 is the first gap section; S1 is the first dividing sub-segment; G2 is the second gap section; - is a nucleoside internucleotide bond; and At least one nucleoside of the oligonucleotide is modified.

2. The compound of claim 1, wherein the modified oligonucleotide from 5' to 3' comprises: 5' W1 - G1 - S1 - G2 - S2 - G3 - W2 3' (Formula II) in: S2 is the second dividing sub-segment; and G3 is the third gap section.

3. The compound of claim 2, wherein the modified oligonucleotide from 5' to 3' comprises: 5' W1 - G1 - S1 - G2 - S2 - G3 - S3 - G4 - W2 3' (Formula III) in: S3 is the third dividing sub-segment; and G4 is the fourth gap section.

4. The compound of claim 3, wherein the modified oligonucleotide from 5' to 3' comprises: 5' W1 – G1 – S1 – G2 – S2 – G3 – S3 – G4 – S4 – G5 – W2 3’ (Formula IV) in: S4 is the fourth dividing sub-segment; and G5 is the fifth gap section.

5. The compound of claim 4, wherein the modified oligonucleotide from 5' to 3' comprises: 5' W1 – G1 – S1 – G2 – S2 – G3 – S3 – G4 – S4 – G5 – S5 – G6 – W2 3’ (Formula V) in: S5 is the fifth dividing sub-segment; and G6 is the sixth gap section.

6. The compound of claim 5, wherein the modified oligonucleotide from 5' to 3' comprises: 5' W1 – G1 – S1 – G2 – S2 – G3 – S3 – G4 – S4 – G5 – S5 – G6 – S6 – G7 –W2 3’ (Formula VI) in: S6 is the sixth dividing sub-segment; and G7 is the seventh gap section.

7. The compound according to any one of claims 1 to 6, wherein W1 comprises one or more linked deoxynucleosides.

8. The compound according to any one of claims 1 to 7, wherein W2 comprises one or more linked deoxynucleosides.

9. The compound according to any one of claims 1 to 8, wherein W1 comprises 2 to 25 linked nucleosides.

10. The compound according to any one of claims 1 to 9, wherein W2 comprises 2 to 35 linked nucleosides.

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