Modified gapmer oligomers and methods of use thereof

Abstract

The present disclosure includes antisense oligonucleotides (ASOs), including gapmer ASOs, and methods for making and using same.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119 to Provisional Application No. 63 / 321,019, filed March 17, 2022, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] The following discussion is provided to aid the reader in understanding the present disclosure and is not admitted to describing or constituting prior art.

[0003] Approximately 300 million people worldwide are chronically infected with HBV. HBsAg loss, a key aspect of "functional cure", is the goal of many new therapeutic approaches. Antisense oligonucleotides have been demonstrated to be an effective modality for reducing HBsAg in animal models, and clinical studies with these molecules are ongoing.

[0004] However, the treatment of HBV with antisense oligonucleotides still suffers from, for example, nuclease degradation and liver toxicity.Therefore, there is a need in the art to find antisense oligonucleotides with greater resistance to nuclease degradation and improved liver safety profile. Summary of the Invention

[0005] The present disclosure relates to oligonucleotide-containing compounds and compositions and their use in the prevention or treatment of diseases and conditions, such as Hepatitis B (HBV).

[0006] In one aspect, the present disclosure provides a nucleic acid sequence comprising 14 to 22 nucleotide units, (a) a central region (B') containing six or more consecutive DNA nucleotides; (b) a 5'-wing region (A') comprising 2 to 6 locked nucleotides or 2'-substituted nucleosides; (c) a 3'-wing region (C') containing 2 to 6 locked nucleotides or 2'-substituted nucleosides, The central region of the ASO is at least 80% complementary to or hybridizes to the target RNA sequence, and the ASO [ka] wherein B is a nucleobase, [ka] wherein B is a nucleobase, [ka] The present invention provides an antisense oligonucleotide (ASO) comprising at least one modified nucleotide selected from the group consisting of:

[0007] In another aspect, the present disclosure provides a nucleic acid sequence comprising 14-22 nucleotide units and (a) a central region (B') comprising six or more consecutive DNA nucleotides, at least one of which is a modified nucleotide selected from Gutb, Nmln, G-clamp, and 5prnl; (b) a 5'-wing region (A') comprising 2 to 6 locked nucleotides or 2'-substituted nucleosides; (c) a 3'-wing region (C') containing 2 to 6 locked nucleotides or 2'-substituted nucleosides, An antisense oligonucleotide (ASO) is provided, in which the central region of the ASO is at least 80% complementary to or hybridizes to a target RNA sequence.

[0008] In another aspect, the disclosure provides an antisense oligonucleotide (ASO) comprising 14-22 nucleotide units and (a) a central region (B') comprising six or more consecutive DNA nucleotides; (b) a 5'-wing region (A') comprising two to six locked nucleotides or 2' substituted nucleosides; and (c) a 3'-wing region (C') comprising two to six locked nucleotides or 2' substituted nucleosides, wherein the central region of the ASO is at least 80% complementary to or hybridizes to a target RNA sequence, and wherein (i) the central region (B') comprises modified nucleotides selected from G-clamp and 5prnl; (ii) the 5'-wing region (A') comprises modified nucleotides selected from Gutb and Nmln; (iii) the 3'-wing region (C') comprises modified nucleotides selected from Gutb and Nmln; or (iv) any combination thereof.

[0009] In some embodiments, the central region (B') comprises 2, 3, 4, 5, 6 or more modified nucleotides.

[0010] In some embodiments, the 5'-wing region (A'), the 3'-wing region (C'), or both, comprise modified nucleotides selected from Gutb, Nmln, G-clamp, and 5prnl.

[0011] In some embodiments, the ASO molecule further comprises one or more phosphorothioate (ps) internucleoside linkages, mesyl phosphoramidate (yp) internucleoside linkages, or a combination thereof.

[0012] In some embodiments, the ASO molecule further contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 phosphorothioate (ps) internucleoside linkages, mesyl phosphoramidate (yp) internucleoside linkages, or a combination thereof.

[0013] In another aspect, the present disclosure provides an antisense oligonucleotide (ASO) comprising 14 to 22 nucleotide units, the ASO comprising: (a) a central region (B') comprising six or more consecutive DNA nucleotides, at least one of which is a modified nucleotide; (b) a 5'-wing region (A') comprising 2 to 6 locked nucleotides or 2'-substituted nucleosides; (c) a 3'-wing region (C') comprising 2 to 6 locked nucleotides or 2'-substituted nucleosides; Antisense oligonucleotides (ASOs) are provided, wherein a central region of the ASO is at least 80% complementary to or hybridizes to a target RNA sequence, and the ASO contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mesyl phosphoramidate (yp) internucleoside linkages.

[0014] In some embodiments, the ASO comprises: [ka] wherein B is a nucleobase, [ka] wherein B is a nucleobase, [ka] The sequence includes at least one, at least two, at least three, at least four, or at least five or more nucleotides selected from the following:

[0015] In some embodiments, the ASO molecule further comprises one or more phosphorothioate (ps) internucleoside linkages.

[0016] In some embodiments of any of the aforementioned aspects, (i) at least one mesyl phosphoramidate (yp) internucleotide linkage is between nucleoside positions 3 and 4 from the 5' end of the ASO molecule; (ii) at least one mesyl phosphoramidate (yp) internucleotide linkage is between nucleoside positions 5 and 6 from the 5' end of the ASO molecule; or (iii) at least one mesyl phosphoramidate (yp) internucleotide linkage is between nucleoside positions 6 and 7 from the 5' end of the ASO molecule. (iv) at least one mesyl phosphoramidate (yp) internucleotide linkage is between nucleoside positions 7 and 8 from the 5' end of the ASO molecule; (v) at least one mesyl phosphoramidate (yp) internucleotide linkage is between nucleoside positions 8 and 9 from the 5' end of the ASO molecule; (vi) at least one mesyl phosphoramidate (yp) internucleotide linkage is between nucleoside positions 9 and 10 from the 5' end of the ASO molecule; or (vii) a combination thereof.

[0017] In some embodiments of any of the aforementioned aspects, the 5'-wing region (A'), the 3'-wing region (C'), or both, comprise at least one mesyl phosphoramidate (yp) internucleotide linkage.

[0018] In some embodiments of any of the aforementioned aspects, the ASO molecule further comprises a galactosamine. In some embodiments, the galactosamine is represented by formula (VI): [ka] N-acetylgalactosamine (GalNAc) of the formula: m is 1, 2, 3, 4, or 5; each n is independently 1 or 2; p is 0 or 1; each R is independently H; each Y is independently selected from -OP(=O)(SH)-, -OP(=O)(O)-, -OP(=O)(OH)-, and -OP(S)S-; Z is H or a second protecting group; either L is a linker or L and Y together are a linker; A is H, OH, a third protecting group, an activating group, or an oligonucleotide.

[0019] In some embodiments, the galactosamine has formula (VII): [ka] N-acetylgalactosamine (GalNAc) of the formula z is OH or SH, and each n is independently 1 or 2.

[0020] In some embodiments of any of the foregoing aspects, (i) the target RNA sequence is a viral gene, (ii) the target RNA sequence is a gene from a DNA virus, (iii) the target RNA sequence is a gene from a double-stranded DNA (dsDNA) virus, (iv) the target RNA sequence is a gene from a hepadnavirus, (v) the target RNA sequence is a gene from Hepatitis B Virus (HBV), (vi) the target RNA sequence is a gene from any one of HBV genotypes A-J, or (vii) the target RNA sequence is selected from the S gene or X gene of HBV.

[0021] In some embodiments of any of the aforementioned aspects, the target RNA sequence is selected from a gene encoding a methylation control J protein (MCJ protein), a gene encoding TAZ, a gene encoding angiopoietin-like 3 (ANGPTL3), a gene encoding diacylglycerol acyltransferase 2 (DGAT2), and a gene encoding hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13).

[0022] In some embodiments of any of the foregoing aspects, (i) the 5'-wing region of the ASO comprises from 2 to 6 phosphorothioate-linked locked nucleosides, (ii) the 3'-wing region of the ASO comprises from 2 to 6 phosphorothioate-linked locked nucleosides, or (iii) a combination thereof. In some embodiments, the locked nucleosides are LNA, ScpBNA, AmNA, AmNA(N-Me), GuNA, GuNA(NR 11 ), wherein R 11 is selected from Me, Et, i-Pr, t-Bu, and combinations thereof.

[0023] In some embodiments of any of the aforementioned aspects, the central region of the ASO comprises at least five consecutive phosphorothioate linked DNA nucleotides, at least five consecutive mesyl phosphoramidate linked DNA nucleotides, or at least five consecutive DNA nucleotides linked by one or more phosphorothioate internucleoside linkages and one or more mesyl phosphoramidate internucleoside linkages.

[0024] In some embodiments of any of the aforementioned aspects, the central region of the ASO comprises 8-10 contiguous phosphorothioate linked DNA nucleotides, 8-10 contiguous mesyl phosphoramidate linked DNA nucleotides, or 8-10 DNA nucleotides linked by one or more phosphorothioate internucleoside linkages and one or more mesyl phosphoramidate internucleoside linkages.

[0025] In some embodiments of any of the aforementioned aspects, the ASO comprises at least one modified nucleotide having the structure: [ka] During the ceremony, R is halogen or R'-C≡C-; R' is C 6-12 Aryl, 5-12 membered heteroaryl, hydroxy-C1-6 Alkyl or C 1-7 It is an alkanoyloxy.

[0026] In some embodiments of any of the aforementioned aspects, the ASO comprises at least one modified nucleotide having the structure: [ka] During the ceremony, W is independently O, N, or S; R1, R2, and R5 are independently H or D; R3 is H or F; R4 is F or OCH3; The base is [ka] and During the ceremony, R is halogen or R'-C≡C-; R' is C 6-12 Aryl, 5-12 membered heteroaryl, hydroxy-C 1-6 Alkyl or C 1-7 It represents alkanoyloxy.

[0027] In another aspect, the present disclosure provides ASO molecules as shown in Table 1.

[0028] In another aspect, the disclosure provides a pharmaceutical composition comprising an ASO molecule disclosed herein (e.g., any of the foregoing aspects or embodiments) and a pharma- ceutically acceptable excipient.

[0029] In some embodiments, the pharmaceutical composition may further comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ASO molecules disclosed herein.

[0030] In some embodiments, the pharmaceutical composition may further comprise an additional therapeutic agent, in some embodiments, the additional therapeutic agent is selected from a nucleotide analog, a nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, a small molecule immunomodulatory molecule, and an oligonucleotide therapy, the oligonucleotide therapy being optionally selected from an additional antisense oligonucleotide (ASO), a short interfering nucleic acid (siNA), a NAP, or STOPS™.

[0031] In another aspect, the disclosure provides a method of treating a subject having a Hepatitis B virus (HBV) infection, comprising administering to a subject having HBV an ASO or a pharmaceutical composition disclosed herein (e.g., any of the preceding aspects or embodiments).

[0032] In some embodiments, the method may further comprise administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from a nucleotide analog, a nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, an immunomodulatory small molecule, and an oligonucleotide therapy, the oligonucleotide therapy being optionally selected from an additional antisense oligonucleotide (ASO), a short interfering nucleic acid (siNA), a NAP, or STOPS™. In some embodiments, the additional therapeutic agent is ALG-010133, ALG-000184, recombinant interferon alpha 2b, IFN-a, PEG-IFN-a-2a, lamivudine, telbivudine, adefovir dipivoxil, clevudine, entecavir, tenofovir alafenamide, tenofovir disoproxil, NVR3-778, BAY41-4109, JNJ-632, JNJ- 3989 (ARO-HBV), RG6004, GSK3228836, REP-2139, REP-2165, AB-729, VIR-2218, DCR-HBVS, JNJ-6379, GLS4, ABI-HO731, JNJ-440, NZ-4, RG7907, EDP-514, AB-423, AB-506, ABI-H03733, and ABI-H2158. In some embodiments, the ASO and the additional therapeutic agent are administered simultaneously or sequentially.

[0033] In some embodiments, the treatment comprises reducing the HBV viral load in the subject, reducing the level of viral antigens in the subject, or a combination thereof.

[0034] In another aspect, the present disclosure provides a method for reducing the expression of a target gene in a subject, comprising administering to an ASO or pharmaceutical composition disclosed herein. In some embodiments, the target gene is a gene that is endogenous to the subject, or the target gene is not endogenous to the subject. In some embodiments, the subject has a disease selected from Hepatitis B virus (HBV), coronavirus infection, and liver disease, and the liver disease is optionally selected from non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and hepatocellular carcinoma (HCC).

[0035] In some embodiments of the methods of the present disclosure, the subject is a mammal, optionally an adult human.

[0036] In some embodiments of the methods of the disclosure, the ASO is administered at a dose of at least 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, or 15 mg / kg.

[0037] In some embodiments of the disclosed methods, the ASO is administered at a dose of 0.5 mg / kg to 50 mg / kg, 0.5 mg / kg to 40 mg / kg, 0.5 mg / kg to 30 mg / kg, 1 mg / kg to 50 mg / kg, 1 mg / kg to 40 mg / kg, 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 3 mg / kg to 50 mg / kg, 3 mg / kg to 40 mg / kg, 3 mg / kg to 30 mg / kg, 3 mg / kg to 20 mg / kg, 3 mg / kg to 15 mg / kg g, 3 mg / kg to 10 mg / kg, 4 mg / kg to 50 mg / kg, 4 mg / kg to 40 mg / kg, 4 mg / kg to 30 mg / kg, 4 mg / kg to 20 mg / kg, 4 mg / kg to 15 mg / kg, 4 mg / kg to 10 mg / kg, 5 mg / kg to 50 mg / kg, 5 mg / kg to 40 mg / kg, 5 mg / kg to 30 mg / kg, 5 mg / kg to 20 mg / kg, 5 mg / kg to 15 mg / kg, or 5 mg / kg to 10 mg / kg.

[0038] In some embodiments of the methods of the disclosure, the ASO is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0039] In some embodiments of the disclosed methods, the ASO is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per week, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per month.

[0040] In some embodiments of the methods of the disclosure, the ASO is administered at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.

[0041] In some embodiments of the methods of the disclosure, the ASO is administered for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or for a period of at least 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, 35, 40, 45, 50, 51, 52, 53, 54, or 55 weeks.

[0042] The foregoing summary and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the present disclosure as claimed. Other objects, advantages, and novel features will become readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the present disclosure. [Brief description of the drawings]

[0043] [Figure 1]Figure 1 (drawing) shows the improved in vivo potency of selected ASOs over their LNA-DNA-LNA parents. G01 is vehicle administered at 5 mL / kg SC, G02 is ASO59 administered once at 5 mg / kg SC on day 0, and G-04 is ASO84 administered once at 5 mg / kg SC on day 0. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The present disclosure is directed to modified antisense oligonucleotides and pharmaceutical compositions of the modified antisense oligonucleotides. The present disclosure is also directed to methods of using and preparing the antisense oligonucleotides and pharmaceutical compositions.

[0045] Antisense Oligonucleotides (ASOs) The compounds of the present disclosure include modified antisense oligonucleotides (ASOs). In some embodiments, the ASOs include 14-22 nucleotide units, e.g., 14, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotide units. In some embodiments, the ASOs are gapmers that include three regions: a 5'-wing region (A') that includes modified nucleotides, a central region (B') that includes nucleotides of a different type than the wings, e.g., nucleotides that can induce RNase H cleavage, and a 3'-wing region (C') that includes modified nucleotides.

[0046] The ASOs of the disclosure comprise (i) modified nucleotides, such as Gutb, Nmln, 5prnl, G-clamp, or combinations thereof; (ii) at least one mesyl phosphoramidate internucleoside linkage (R a is a methyl group, referred to as "yp" in the sequence, or (iii) a combination thereof. The structures of Gutb, Nmln, 5prnl, and G-clamp are shown below, and the structure of the mesyl phosphoramidate linker is [ka] where Ra is C1-C6 alkyl 、 C 6-12 It is aryl, or 5- to 12-membered heteroaryl. In some embodiments, the structure of the mesyl phosphoramidate linker is: [ka] It is.

[0047] For example, the 5'-wing region and the 3'-wing region can each independently comprise 2 to 6 nucleotides, e.g., 2, 3, 4, 5, or 6 nucleotides. One or more of these nucleotides can be modified (e.g., 1, 2, 3, 4, 5, or 6 of the nucleotides are modified). At least one of the modified nucleotides has the following structure: [ka] It may include, wherein B is a nucleobase. Additionally or alternatively, at least one of the modified nucleotides has the following structure: [ka] (5(Me)-propyl (5prnl)). Thus, the 5'-wing region and the 3'-wing region can each independently comprise one or more of Gutb, Nmln, or both. Similarly, the 5'-wing region and the 3'-wing region can each independently comprise one or more of G-clamp, 5prnl, or both. However, G-clamp and 5prnl are similarly suitable for inclusion in the central region. For example, in some embodiments, (i) the central region (B') comprises modified nucleotides selected from G-clamp and 5prnl, (ii) the 5'-wing region (A') comprises modified nucleotides selected from Gutb and Nmln, (iii) the 3'-wing region (C') comprises modified nucleotides selected from Gutb and Nmln, or (iv) any combination thereof.

[0048] Additionally or alternatively, the central region may comprise 1, 2, 3, 4, 5 or more consecutive DNA nucleosides linked by phosphodiester internucleoside linkages or thiophosphate ("ps") internucleoside linkages. In other embodiments, the central region comprises one or more modified nucleotides, mesyl phosphoramidate (yp) internucleoside linkages, or combinations thereof. Additionally, the central region may comprise one or more modified nucleotides that are capable of inducing RNase H cleavage in the central region. In some embodiments, the central region comprises one or more modified nucleotides having modified nucleobases. In some embodiments, the central region comprises 6, 7, 8, 9, 10, or 11 consecutive DNA nucleosides. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the DNA nucleosides in the central region are modified. At least one of the modified nucleotides has the following structure: [ka] It may include, wherein B is a nucleobase. Additionally or alternatively, at least one of the modified nucleotides has the following structure: [ka] may include.

[0049] For purposes of this disclosure, the ASO of the present disclosure may comprise at least one, at least two, at least three, at least four, or at least five or more of Gutb, Nmln, 5prnl, G-clamp, or a combination thereof. Gutb, Nmln, 5prnl, G-clamp, or a combination thereof may be incorporated in the central region, the wing region, or both. In general, modified locked nucleotides (Gutb and Nmln) are preferred for inclusion in the wing region, while 5prnl and G-clamp are preferred for inclusion throughout the ASO or specifically in the central region. Additionally or alternatively, the ASO of the present disclosure may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mesyl phosphoramidate (yp) internucleoside linkages.

[0050] In some embodiments, the gapmer ASO compound of the disclosure has formula (I): A'-B'-C', wherein A' and C' each independently comprise 2-6 nucleotides, one or more of which are modified nucleotides, and B' comprises 6 or more contiguous DNA nucleosides linked by phosphodiester or thiophosphate internucleoside linkages. In some embodiments, B' comprises one or more modified DNA nucleosides. In some embodiments, the modified nucleotides are selected from locked nucleosides or 2' substituted nucleosides. In some embodiments, the modified DNA nucleosides are selected from locked nucleosides or 2' substituted nucleosides.

[0051] The number of nucleotides and / or nucleosides in A', B', and C' may be selected from the following group (A':B':C'): (2:10:2), (2:10:3), (2:10:4), (2:10:5), (3:10:2), (3:10:3), (3:10:4), (3:10:5), (4:10:2), (4:10:3), (4:10:4), (4:10:5), (5:10:2), (5:10:3), (5:10:4), (5:10:5), (5:10:2), (5:10:5 ... 0:3), (5:10:4), (5:10:5), (2:9:2), (2:9:3), (2:9:4), (2:9:5), (3:9:2), (3:9:3), (3:9:4), (3:9:5), (4:9:2), (4:9:3), (4:9:4), (4:9:5), (5:9:2), (5:9:3), (5:9:4), (5:9:5), (2:8:2), (2:8:3), (2:8:4), (2:8 :5), (3:8:2), (3:8:3), (3:8:4), (3:8:5), (4:8:2), (4:8:3), (4:8:4), (4:8:5), (5:8:2), (5:8:3), (5:8:4), (5:8:5), (2:7:2), (2:7:3), (2:7:4), (2:7:5), (3:7:2), (3:7:3), (3:7:4), (3:7:5), (4:7:2), (4:7:3) , (4:7:4), (4:7:5), (5:7:2), (5:7:3), (5:7:4), (5:7:5), (2:6:2), (2:6:3), (2:6:4), (2:6:5), (3:6:2), (3:6:3), (3:6:4), (3:6:5), (4:6:2), (4:6:3), (4:6:4), (4:6:5), (5:6:2), (5:6:3), (5:6:4), and (5:6:5).

[0052] In some embodiments, the 5'-wing region comprises one or more locked nucleosides or 2'-substituted nucleosides. In some embodiments, the 3'-wing region comprises one or more locked nucleosides or 2'-substituted nucleosides. In some embodiments, the central region comprises one or more locked nucleosides or 2'-substituted nucleosides. In some embodiments, the 5'-wing region, the 3'-wing region, the central region, or a combination thereof comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) locked nucleosides or 2'-substituted nucleosides. A locked nucleoside can comprise a bridge between the 4' and 2' sugar positions, the bridge comprising 2 to 4 optionally substituted atoms. For example, an LNA nucleoside can be [ka] Other exemplary locked nucleosides include: [ka] (R 10 AmNA, where R is H 10 AmNA(N-Me) where R is CH3 10 AmNA(N-alkyl) where is C1-C6 alkyl; [ka] where B is a nucleobase and R 10 is H or C1-C6 alkyl, R 11is C1-C6 alkyl. In certain embodiments, all nucleosides in the 5'-wing region are locked nucleosides. In some embodiments, all nucleosides in the 3'-wing region are locked nucleosides. In some embodiments, the 3'-wing region comprises LNA and one or two nucleosides selected from ScpBNA, AmNA, and GuNA. In some embodiments, the 5'-wing region is all LNA and the 3'-wing region contains LNA and one or two nucleosides selected from ScpBNA, AmNA, and GuNA. Other nucleotides are listed in PCT / JP2010 / 068409, PCT / JP2013 / 075370, PCT / JP2015 / 054308, PCT / JP2018 / 006061, and / or PCT / JP2018 / 006062, which are incorporated by reference in their entirety. Gutb and Nmln are additional examples of locked nucleotides that can be included in the 5'-wing region or the 3'-wing region or both.

[0053] In some embodiments, the 5'-wing region of the ASO comprises 2-6 phosphorothioate-linked locked nucleosides, mesyl phosphoramidate-linked locked nucleosides, or a combination thereof. In some embodiments, the 5'-wing region comprises 2-6 phosphorothioate-linked 2'-substituted nucleosides, mesyl phosphoramidate-linked 2'-substituted nucleosides, or a combination thereof. In some embodiments, the 5'-wing region comprises at least one locked nucleoside and at least one 2'-substituted nucleoside, wherein the locked nucleoside and the 2'-substituted nucleoside are linked by a phosphorothioate linker or a mesyl phosphoramidate linker. In some embodiments, the 5'-wing region further comprises an RNA nucleoside or a DNA nucleoside, wherein the RNA nucleoside and the DNA nucleoside are not locked nucleosides or 2'-substituted nucleosides. In some embodiments, at least two nucleosides of the 5'-wing region are linked by a phosphorothioate linker or a mesyl phosphoramidate linker, in some embodiments, at least 2, 3, 4, 5, or 6 nucleosides of the 5'-wing region are linked by a phosphorothioate linker, a mesyl phosphoramidate linker, or a combination thereof.

[0054] In some embodiments, the 3'-wing region of the ASO comprises 2-6 phosphorothioate-linked locked nucleosides, mesyl phosphoramidate-linked locked nucleosides, or a combination thereof. In some embodiments, the 3'-wing region comprises 2-6 phosphorothioate-linked substituted nucleosides, mesyl phosphoramidate-linked substituted nucleosides, or a combination thereof. In some embodiments, the 3'-wing region comprises at least one locked nucleoside and at least one 2'-substituted nucleoside, wherein the locked nucleoside and the 2'-substituted nucleoside are linked by a phosphorothioate linker or a mesyl phosphoramidate linker. In some embodiments, the 3'-wing region further comprises an RNA nucleoside or a DNA nucleoside, wherein the RNA nucleoside and the DNA nucleoside are not locked nucleosides or 2'-substituted nucleosides. In some embodiments, at least two nucleosides of the 3'-wing region are linked by a phosphorothioate linker or a mesyl phosphoramidate linker. In some embodiments, at least 2, 3, 4, 5, or 6 nucleosides of the 3'-wing region are linked by a phosphorothioate linker, a mesyl phosphoramidate linker, or a combination thereof.

[0055] In some embodiments, one or more nucleotides in the 5'-wing region and / or the 3'-wing region comprise a thiophosphate internucleoside linkage or a mesyl phosphoramidate internucleoside linkage. In some embodiments, all nucleotides in the 5'-wing region comprise a thiophosphate internucleoside linkage. In some embodiments, all nucleotides in the 3'-wing region comprise a thiophosphate internucleoside linkage. In some embodiments, all nucleotides in the 5'-wing region comprise a mesyl phosphoramidate internucleoside linkage. In some embodiments, all nucleotides in the 3'-wing region comprise a mesyl phosphoramidate internucleoside linkage.

[0056] In some embodiments, the central region comprises one or more modified nucleotides having modified nucleobases. For example, the central region can comprise at least one, at least two, at least three, at least four, or at least five or more of Gutb, Nmln, 5prnl, G-clamp, or a combination thereof. In some embodiments, the central region comprises at least one, at least two, at least three, at least four, or at least five or more of 5prnl, G-clamp, or a combination thereof. Additionally or alternatively, the central region can comprise the following structure: [ka] wherein R is a halogen or R'-C≡C- and R' is C 6-12 Aryl, 5-12 membered heteroaryl, hydroxy-C 1-6 Alkyl or C 1-7 In some embodiments, the central region comprises one modified nucleotide (e.g., (2s)T or (5OH)C) at the first, second, third, or fourth gap nucleoside position (from the 5' end). In some embodiments, the modified nucleotide is at the third gap nucleoside position (from the 5' end). In some embodiments, the modified nucleotide is a nucleotide having the structure: [ka] During the ceremony, W is independently O, N, or S; R1, R2, and R5 are independently H or D; R3 is H or F; R4 is F or OCH3; The base is [ka] and During the ceremony, R is halogen or R'-C≡C-; R' is C6-12 Aryl, 5-12 membered heteroaryl, hydroxy-C 1-6 Alkyl or C 1-7 In some embodiments, C represents alkanoyloxy. 1-7 Alkanoyl includes, but is not limited to, formyl, acetyl, ethylcarbonyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, isobutylcarbonyl, t-butylcarbonyl, n-pentylcarbonyl, and n-hexylcarbonyl. Other modified nucleotides include those in PCT / JP2018 / 006061, the entirety of which is incorporated by reference.

[0057] As used herein, unless otherwise indicated, "aryl" refers to a carbocyclic (all carbon) ring with a fully delocalized pi-electron system. An "aryl" group may be composed of two or more fused rings (rings that share two adjacent carbon atoms). When an aryl is a fused ring system, the ring that is connected to the rest of the molecule has a fully delocalized pi-electron system. The other ring in the fused ring system may or may not have a fully delocalized pi-electron system. Examples of aryl groups include, but are not limited to, radicals of benzene, naphthalene, and azulene.

[0058] As used herein, unless otherwise indicated, "heteroaryl" refers to a ring having a fully delocalized pi-electron system and containing one or more heteroatoms (e.g., 1-3 heteroatoms, or 1-4 heteroatoms, or 1-5 heteroatoms) independently selected from the group consisting of nitrogen, oxygen, and sulfur in the ring. A "heteroaryl" group may be composed of two or more fused rings (rings that share two adjacent carbon atoms). When a heteroaryl is a fused ring system, the ring that is connected to the rest of the molecule has a fully delocalized pi-electron system. The other ring in the fused ring system may or may not have a fully delocalized pi-electron system. Examples of heteroaryl rings include, but are not limited to, furan, thiophene, pyrrole, oxazole, thiazole, imidazole, pyrazole, isoxazole, isothiazole, triazole, thiadiazole, pyridine, pyridazine, pyrimidine, pyrazine, and triazine.

[0059] In some embodiments, the central region of the ASO comprises at least 5 consecutive phosphorothioate-linked DNA nucleosides, at least 5 consecutive mesyl phosphoramidate-linked DNA nucleosides, or a combination thereof. In some embodiments, at least 2, 3, 4, 5, or 6 nucleosides of the central region are linked by phosphorothioate linkers, mesyl phosphoramidate linkers, or a combination thereof. In some embodiments, the DNA nucleosides of the central region are linked to the nucleosides of the 5'-wing region by phosphorothioate linkers or mesyl phosphoramidate linkers. In some embodiments, the DNA nucleosides of the central region are linked to the nucleosides of the 3'-wing region by phosphorothioate linkers or mesyl phosphoramidate linkers. In some embodiments, the central region comprises 8-10 contiguous phosphorothioate-linked DNA nucleosides, 8-10 contiguous mesyl phosphoramidate-linked DNA nucleosides, or a combination thereof.

[0060] In some embodiments, the ASO is complementary to or hybridizes to a viral target RNA sequence starting from the X region of HBV or the S region of HBV. The vital target may start, for example, from the 5' end of the target site in acc.KC315400.1 (genotype B, "gt B") or in any one of the A, C, or D genotypes. One of skill in the art will understand HBV locations, for example, as described in Wing-Kin Sung, et al., Nature Genetics 44:765 (2012). In some embodiments, the S region is defined as from the start of the small S protein (genotype BKC315400.1 isolate, position 155) to before the start of the X protein (genotype BKC315400.1 isolate, position 1373). In some embodiments, the X region is defined as from the beginning of the X protein (genotype BKC315400.1 isolate, position number 1374) to the end of the DR2 site (genotype BKC315400.1 isolate, position number 1603).

[0061] In some embodiments, the ASO is complementary to or hybridizes to a viral target RNA sequence that comprises, consists of, or consists essentially of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive nucleotides within positions 100-800 or 1050-1700 of SEQ ID NO:89. In some embodiments, the ASO is complementary to or hybridizes to a viral target RNA sequence comprising, consisting of, or consisting essentially of the following consecutive nucleotides: positions 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 7-15, 7-14, 7-13, 7-12, or 7-11 within positions 100-800 or 1050-1700 of SEQ ID NO:89. In some embodiments, the ASO is complementary to or hybridizes to a viral target RNA sequence comprising, consisting of, or consisting essentially of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive nucleotides within positions 180-280, 300-450, 650-775, 1125-1300, or 1400-1650 of SEQ ID NO:89. In some embodiments, the ASO is complementary to or hybridizes to a viral target RNA sequence comprising, consisting of, or consisting essentially of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive nucleotides within positions 180-215, 230-270, 350-420, 675-730, 1165-1210, 1245-1290, 1400-1480, or 1500-1630 of SEQ ID NO:89.In some embodiments, the ASO comprises 191, 245, 246, 276, 376, 377, 381, 383, 694, 700, 1182, 1261, 1262, 1408, 1410, 1426, 1431, 1432, 1433, 1435, 1438, 1441, 1443, 1513, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1535, 1538, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 15 , 1521, 1522, 1527, 1559, 1575, 1576, 1577, 1580, 1581, 1582, or 1589. In some embodiments, the ASO is fully complementary to the viral target RNA sequence. In some embodiments, there are no more than 5, 4, 3, 2, or 1 mismatches between the ASO and the viral target sequence. In some embodiments, there are no more than 2 mismatches between the ASO and the viral target sequence. In some embodiments, there is no more than 1 mismatch between the ASO and the viral target sequence. In some embodiments, the mismatches are in the wing regions of the ASO. In some embodiments, the mismatches are in the 5' wing regions of the ASO. In some embodiments, the mismatch is in the 3' wing region of the ASO. In some embodiments, the mismatch is in the central region of the ASO.

[0062] In some embodiments, the central region is complementary to or hybridizes to a viral target RNA sequence comprising, consisting of, or consisting essentially of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous nucleotides within positions 100-800 or 1050-1700 of SEQ ID NO:89. In some embodiments, the central region is complementary to or hybridizes to a viral target RNA sequence comprising, consisting of, or consisting essentially of the following consecutive nucleotides: 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 7-15, 7-14, 7-13, 7-12, or 7-11 within positions 100-800 or 1050-1700 of SEQ ID NO:89. In some embodiments, the central region is complementary to or hybridizes to a viral target RNA sequence comprising, consisting of, or consisting essentially of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous nucleotides within positions 180-280, 300-450, 650-775, 1125-1300, or 1400-1650 of SEQ ID NO:89. In some embodiments, the central region is complementary to or hybridizes to a viral target RNA sequence comprising, consisting of, or consisting essentially of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous nucleotides within positions 180-215, 230-270, 350-420, 675-730, 1165-1210, 1245-1290, 1400-1480, or 1500-1630 of SEQ ID NO:89.In some embodiments, the central region comprises 191, 245, 246, 276, 376, 377, 381, 383, 694, 700, 1182, 1261, 1262, 1408, 1410, 1426, 1431, 1432, 1433, 1435, 1438, 1441, 1443, 1513, 1516, 1517, 1518, 1519, 1520 of SEQ ID NO: 89. , 1521, 1522, 1527, 1559, 1575, 1576, 1577, 1580, 1581, 1582, or 1589. In some embodiments, the central region is fully complementary to the viral target RNA sequence. In some embodiments, there are no more than 5, 4, 3, 2, or 1 mismatches between the central region and the viral target sequence. In some embodiments, there are no more than 2 mismatches between the central region and the viral target sequence. In some embodiments, there is no more than 1 mismatch between the central region and the viral target sequence.

[0063] In some embodiments, the ASO comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to a nucleotide sequence selected from the sequences listed in Table 1.

[0064] In some embodiments, an ASO of the disclosure may have a sequence that differs by one nucleoside from an ASO in Table 1. In some embodiments, an ASO of the disclosure may have a sequence that differs by two nucleosides from an ASO in Table 1. In some embodiments, an ASO of the disclosure may have a sequence that differs by three nucleosides from an ASO in Table 1. In some embodiments, an ASO of the disclosure may have a sequence that differs by four nucleosides from an ASO in Table 1.

[0065] In some embodiments, an ASO of the disclosure may have a sequence in Table 1, but with one T in the central region replaced with a (2s)T, one C in the central region replaced with a (5OH)C, and / or one A in the central region replaced with an (8nh)A. In some embodiments, an ASO of the disclosure may have a sequence in Table 1, but with one or two ScpBNAs, AmNAs, or GuNAs in the 5' wing portion. In some embodiments, an ASO of the disclosure may have a sequence in Table 1, but with one or two ScpBNAs, AmNAs, or GuNAs in the 3' wing portion. In some embodiments, an ASO of the disclosure may have a sequence in Table 1, but with an mA or mU added to the 5' end of the sequence. In some embodiments, an ASO of the disclosure may have a sequence in Table 1, but with mA or mU added to the 5' end of the sequence that binds to a GalNAc derivative (e.g., GalNAc4, e.g., GalNAc4-(PS)2-p-, or GalNAc6, e.g., GalNAc6-(PS)2-p-), as detailed herein, the 3' end of the sequence, or both.

[0066] In some embodiments, the ASO comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs:1-88.

[0067] In some embodiments, the ASO of the present disclosure has a sequence that differs from any of the nucleotides of SEQ ID NOs: 1-88 by one nucleoside. In other embodiments, the ASO has a sequence that differs from any of the nucleotides of SEQ ID NOs: 1-88 by one, two, three, or four nucleosides. In some embodiments, the ASO of the present disclosure has a sequence of any of SEQ ID NOs: 1-88, but one T in the central region is replaced with a (2s)T, one C in the central region is replaced with a (5OH)C, and / or one A is replaced with an (8nh)A in the central region. In some embodiments, the ASO of the present disclosure has a sequence of any of SEQ ID NOs: 1-88, but has one or two ScpBNAs, AmNAs, or GuNAs in the 5' wing portion. In some embodiments, the ASO of the present disclosure has a sequence of any of SEQ ID NOs: 1-88, but has one or two ScpBNAs, AmNAs, or GuNAs in the 3' wing portion. In some embodiments, an ASO of the disclosure has a sequence of any one of SEQ ID NOs: 1-88, but with mA or mU added to the 5' end of the sequence. In some embodiments, an ASO of the disclosure has a sequence of any one of SEQ ID NOs: 1-88, but with mA or mU added to the 5' end of the sequence that binds to a GalNAc derivative (e.g., GalNAc4, e.g., GalNAc4-(PS)2-p-, or GalNAc6, e.g., GalNAc6-(PS)2-p-), as detailed herein.

[0068] Target RNA sequence The ASOs of the disclosure can reduce expression of a target RNA sequence (e.g., a target gene) by recruiting RNAse H to cleave and degrade the RNA transcript of the target RNA sequence, reducing RNA levels and thereby reducing the levels of the protein encoded by the target RNA sequence.

[0069] For purposes of this disclosure, the target RNA sequence may be any gene in a cell. In some embodiments, the target gene is a viral gene. In some embodiments, the viral gene is from a DNA virus. In some embodiments, the DNA virus is a double-stranded DNA (dsDNA) virus. In some embodiments, the dsDNA virus is a hepadnavirus. In some embodiments, the hepadnavirus is a Hepatitis B virus (HBV). In some embodiments, the HBV is selected from HBV genotypes A-J. In some embodiments, the viral disease is caused by an RNA virus. In some embodiments, the RNA virus is a single-stranded RNA virus (ssRNA virus). In some embodiments, the ssRNA virus is a positive-sense single-stranded RNA virus ((+)ssRNA virus). In some embodiments, the (+)ssRNA virus is a coronavirus. In some embodiments, the coronavirus is a β-coronavirus. In some embodiments, the β-coronavirus is selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (also known by the provisional name of 2019 novel coronavirus, or 2019-nCoV), human coronavirus OC43 (hCoV-OC43), Middle East respiratory syndrome-related coronavirus (MERS-CoV, also known by the provisional name of 2012 novel coronavirus, or 2012-nCoV), and severe acute respiratory syndrome-related coronavirus (SARS-CoV, also known as SARS-CoV-1). In some embodiments, the β-coronavirus is SARS-CoV-2, the causative agent of COVID-19. Some exemplary target genes are shown in Table 6 at the end of this specification.

[0070] In some embodiments, the target RNA sequence is selected from the S or X gene of HBV. In some embodiments, the HBV has a genomic sequence set forth in the nucleotide sequence of SEQ ID NO: 90, which corresponds to the nucleotide sequence of GenBank Accession No. U95551.1, which is incorporated by reference in its entirety.

[0071] An exemplary HBV genome sequence is set forth in SEQ ID NO:89, which corresponds to Genbank Accession No. KC315400.1, which is incorporated by reference in its entirety. Nucleotides 2307-3215, 1-1623 of SEQ ID NO:89 correspond to the polymerase / RT gene sequence encoding the polymerase protein. Nucleotides 2848-3215, 1-835 of SEQ ID NO:89 correspond to the PreS1 / S2 / S gene sequence encoding the large S protein. Nucleotides 3205-3215, 1-835 of SEQ ID NO:89 correspond to the PreS2 / S gene sequence encoding the middle S protein. Nucleotides 155-835 of SEQ ID NO:89 correspond to the S gene sequence encoding the small S protein. Nucleotides 1374-1838 of SEQ ID NO:89 correspond to the X gene sequence encoding the X protein. Nucleotides 1814-2452 of SEQ ID NO:89 correspond to the PreC / C gene sequence encoding the precore / core protein. Nucleotides 1901-2452 of SEQ ID NO:89 correspond to the C gene sequence encoding the Core protein. The HBV genome further comprises viral regulatory elements such as viral promoters (preS2, preS1, Core, and X) and enhancer elements (ENH1 and ENH2). Nucleotides 1624-1771 of SEQ ID NO:89 correspond to ENH2. Nucleotides 1742-1849 of SEQ ID NO:60 correspond to the Core promoter. Nucleotides 1818-3215, 1-1930 of SEQ ID NO:89 correspond to the pregenomic RNA (pgRNA) encoding the Core and polymerase proteins.

[0072] In some embodiments, the target RNA sequence is selected from the genome of SARS-CoV, which has a genome corresponding to the nucleotide sequence of GenBank Accession No. NC_004718.3, which is incorporated by reference in its entirety.

[0073] In some embodiments, the target RNA sequence is selected from the genome of MERS-CoV, which has a genome corresponding to the nucleotide sequence of GenBank Accession No. NC_019843.3, which is incorporated by reference in its entirety.

[0074] In some embodiments, the target RNA sequence is selected from the genome of hCoV-OC43, which has a genome corresponding to the nucleotide sequence of GenBank Accession No. NC_006213.1, the entirety of which is incorporated by reference.

[0075] In some embodiments, the target RNA sequence is selected from the genome of SARS-CoV-2, which in some embodiments has a genome sequence corresponding to the nucleotide sequence of GenBank Accession No. NC_045512.2, which is incorporated by reference in its entirety.

[0076] In some embodiments, the target RNA sequence may be any hydroxysteroid dehydrogenase gene. In any embodiment, the gene is hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13). HSD17B13 has the sequence shown in the nucleotide sequence of SEQ ID NO: 91, which corresponds to the nucleotide sequence of the coding sequence of GenBank Accession No. NM_178135.5 (nucleotides 42-944), which is incorporated by reference in its entirety.

[0077] In some embodiments, the target RNA sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a nucleotide region in SEQ ID NO:91, except that thymine (Ts) in SEQ ID NO:91 is replaced with uracil (U). In some embodiments, the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides in SEQ ID NO:91.

[0078] In some embodiments, the target RNA sequence is involved in liver metabolism. In some embodiments, the target RNA sequence is an inhibitor of the electron transport chain. In some embodiments, the target gene encodes an MCJ protein (MCJ / DnaJC15 or a methylation-regulated J protein). In some embodiments, the MCJ protein is encoded by the mRNA sequence of SEQ ID NO: 92, which corresponds to the nucleotide sequence of GenBank Accession No. NM_013238.3, which is incorporated by reference in its entirety.

[0079] In some embodiments, the target RNA sequence is TAZ. In some embodiments, TAZ comprises the nucleotide sequence of SEQ ID NO: 93, which corresponds to the nucleotide sequence of GenBank Accession No. NM_000116.5, the entirety of which is incorporated by reference.

[0080] In some embodiments, the target RNA sequence is angiopoietin-like 3 (ANGPTL3). In some embodiments, ANGPTL3 comprises the nucleotide sequence of SEQ ID NO: 94, which corresponds to the nucleotide sequence of GenBank Accession No. NM_014495.4, the entirety of which is incorporated by reference.

[0081] In some embodiments, the target RNA sequence is diacylglycerol acyltransferase 2 (DGAT2). In some embodiments, DGAT2 comprises the nucleotide sequence of SEQ ID NO: 95, which corresponds to the nucleotide sequence of GenBank Accession No. NM_001253891.1, which is incorporated by reference in its entirety.

[0082] Conjugation Moiety The present disclosure also relates to additional components conjugated to the ASO, such as targeting moieties and oligonucleotides modified at one or more termini. In some embodiments, the conjugated moiety is selected from galactosamine, peptides, proteins, sterols, lipids, phospholipids, biotin, phenoxazine, active drug substrates, cholesterol, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, folate, and dyes.

[0083] In some embodiments, the targeting moiety may comprise a carbohydrate, such as a monosaccharide, e.g., N-acetylgalactosamine (GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. In some embodiments, the targeting moiety is one or more GalNAc derivatives, such as two or three GalNAc derivatives, optionally in a contiguous configuration, attached to the ASO via one or more linkers. In certain embodiments, the targeting moiety is linked to a linker, e.g., [ka] It contains three consecutive GalNAc moieties linked via

[0084] In some embodiments, the conjugation moiety is galactosamine. In some embodiments, any of the ASOs disclosed herein are connected to a conjugation moiety that is galactosamine. In some embodiments, the galactosamine is N-acetylgalactosamine (GalNAc). In some embodiments, any of the ASOs disclosed herein comprise GalNAc. In some embodiments, the GalNAc has the formula (VI): [ka] wherein m is 1, 2, 3, 4, or 5, each n is independently 1 or 2, p is 0 or 1, each R is independently H or a first protecting group, each Y is independently selected from -OP(=O)(SH)-, -OP(=O)(O)-, -OP(=O)(OH)-, -OP(S)S-, and -O-, Z is H or a second protecting group, L is either a linker or L and Y in combination are a linker, and A is H, OH, a third protecting group, an activating group, or an oligonucleotide. In some embodiments, the first protecting group is acetyl. In some embodiments, the second protecting group is trimethoxytrityl (TMT). In some embodiments, the activating group is a phosphoramidite group. In some embodiments, the phosphoramidite group is a cyanoethoxy N,N-diisopropyl phosphoramidite group. In some embodiments, the linker is a C6-NH2 group. In some embodiments, A is ASO. In some embodiments, R is H, Z is H, and n is 1. In some embodiments, R is H, Z is H, and n is 2.

[0085] In some embodiments, GalNAc has formula (VII): [ka] where R zis OH or SH, and each n is independently 1 or 2. In some embodiments, the targeting ligand can be GalNAc, and the targeting ligand can include 1, 2, 3, 4, 5, or 6 GalNAc units. In some embodiments, the targeting ligand can be a GalNAc selected from GalNAc2, GalNAc3, GalNAc4, GalNAc5, and GalNAc6.

[0086] In some embodiments, GalNAc can be a GalNAc amidite, GalNAc4 CPG, GalNAc phosphoramidite, or GalNAc4-ps-GalNAc4-ps-GalNAc4. These GalNAc moieties are shown below. [Table 1]

[0087] GalNAc3, GalNAc4, GalNAc5 and GalNAc6 may be conjugated to the ASOs disclosed herein during synthesis using moieties 1, 2, or 3. Additional GalNAc moieties, such as GalNAc1 and GalNAc2, can be used to form 5'- and 3'-GalNAc using post-synthetic conjugation. [Table 2]

[0088] In some embodiments, the ASO comprises a targeting moiety at the 5' end, 3' end, or both ends of the ASO. The conjugation moiety may be connected to the ASO via one, two, three, four, or five or more linkers. In some embodiments, the one or more linkers are independently selected from the group consisting of a phosphodiester (p or po) linker, a phosphorothioate (ps) linker, a mesyl phosphoramidate linker (yp), a phosphoramidite (HEG) linker, a triethylene glycol (TEG) linker, and / or a phosphorodithioate linker. In some embodiments, the one or more linkers are independently selected from the group consisting of p-(PS)2, (PS)2-p-TEG-p, (PS)2-p-HEG-p, and (PS)2-p-(HEG-p)2.

[0089] In some embodiments, the conjugation moiety is a lipid moiety. In some embodiments, any of the ASOs disclosed herein are connected to a conjugation moiety that is a lipid moiety. Examples of lipid moieties include, but are not limited to, cholesterol moieties, thioethers such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-SH-phosphonate, polyamines or polyethylene glycol chains, adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0090] In some embodiments, the conjugated moiety is an active drug substrate. In some embodiments, any of the ASOs disclosed herein are connected to a conjugated moiety that is an active drug substrate. Examples of active drug substrates include, but are not limited to, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (5)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folic acid, benzothiadiazid, chlorothiazide, diazepines, indomethicine, barbiturates, cephalosporins, sulfa drugs, antidiabetic drugs, antibacterial agents, or antibiotics.

[0091] Exemplary ASO As described above, the ASOs disclosed herein may include modified nucleotides such as Gutb, Nmln, 5prnl, G-clamp, or combinations thereof. Additionally or alternatively, the ASOs disclosed herein may include at least one mesyl phosphoramidate internucleoside linkage. Table 1 provides some exemplary ASOs that include either modified nucleotides such as Gutb, Nmln, 5prnl, G-clamp, or combinations thereof, at least one mesyl phosphoramidate internucleoside linkage (referred to as "yp" in the sequence), or combinations thereof. [Table 3] TIFF2025509552000031.tif255170TIFF2025509552000032.tif255170TIFF2025509552000033.tif255170TIFF2025509552000034.tif74170

[0092] In Table 1, the bolded nucleosides contain one of the following modifications: [Table 4]

[0093] Pharmaceutical Compositions The disclosure also encompasses pharmaceutical compositions comprising the ASOs of the disclosure. One embodiment is a pharmaceutical composition comprising one or more ASOs of the disclosure and a pharma- ceutically acceptable diluent or carrier.

[0094] In some embodiments, pharmaceutical compositions comprising the ASO of the present disclosure are formulated for systemic administration via parenteral delivery. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, and also includes subcutaneous administration, for example, via an implanted device. In a preferred embodiment, pharmaceutical compositions comprising the ASO of the present disclosure are formulated for subcutaneous (SC) or intravenous (IV) delivery. Formulations for parenteral administration may include sterile aqueous solutions, and may also include buffers, diluents, and other pharma- ceutical acceptable additives as understood by those skilled in the art. For intravenous use, the total concentration of solutes may be controlled to make the preparation isotonic.

[0095] Pharmaceutical compositions comprising the ASOs of the present disclosure are useful, for example, for treating diseases or disorders associated with HBV gene expression or activity.

[0096] In some embodiments, the pharmaceutical composition comprises a first ASO of the present disclosure that is complementary or hybridizes to a viral target RNA sequence in a first X region of HBV, a second ASO of the present disclosure that is complementary or hybridizes to a viral target RNA sequence in a second X region or S region of HBV, and a pharma- ceutically acceptable diluent or carrier. When the pharmaceutical composition comprises more than one ASO, the ASOs may be present in various amounts. For example, in some embodiments, the weight ratio of the first ASO to the second ASO is 1:4 to 4:1, e.g., 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, or 4:1. In some embodiments, the molar ratio of the first ASO to the second ASO is 1:4 to 4:1, e.g., 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, or 4:1.

[0097] process The siNA molecules and compositions described herein may be administered to a subject to treat a disease. Also disclosed herein is the use of any of the siNA molecules or compositions disclosed herein in the manufacture of a medicament for treating a disease. Specifically, the present disclosure provides ASO for the treatment of various diseases, such as infectious diseases, including but not limited to viral diseases and liver diseases.

[0098] One aspect of the disclosure includes a method for treating a subject diagnosed with, suspected of, or at risk of having an HBV infection and / or an HBV-related disorder. In therapeutic applications, a composition comprising at least one disclosed ASO is administered to a subject suspected of or already suffering from such a disease (e.g., persistence of HBV cccDNA, the presence of HBV antigens (e.g., HBsAg and / or HBeAg) in the serum and / or liver of the subject, or elevated HBV viral load levels) in an amount sufficient to cure or at least partially arrest symptoms of the disease, including its complications and intermediate pathological phenotypes in the development of the disease.

[0099] Subjects suffering from HBV infection and / or HBV-related disorders can be identified by any or a combination of diagnostic or prognostic assays known in the art. For example, typical symptoms of HBV infection and / or HBV-related disorders include: Presence of cccDNA, presence of serum and / or liver HBV antigens (e.g., HBsAg and / or HBeAg), elevated ALT, elevated AST, absence or low levels of anti-HBV antibodies, liver damage, cirrhosis, delta hepatitis, acute hepatitis B, acute fulminant hepatitis B, chronic hepatitis B, liver fibrosis, end-stage liver disease, hepatocellular carcinoma, serum sickness-like syndrome, anorexia, nausea, vomiting, low-grade fever, myalgia, fatigue disorder, anorexia and olfaction (aversion to food and cigarettes), right upper quadrant and epigastric pain (interstitial, mild to moderate), hepatic encephalopathy, pulsatility, disturbances in sleep patterns, mental confusion, coma, ascites, gastrointestinal bleeding, coagulopathy, jaundice, hepatomegaly (mildly swollen, soft liver), splenomegaly, palmar erythema, spider rash, muscle digestion wasting, spider angiomas, vasculitis, varicose vein bleeding, peripheral edema, gynecological tumors, testicular atrophy, abdominal collateral veins (caput medusa), high levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (in the range of 1000-2000 IU / mL), elevated ALT levels higher than AST levels, elevated gamma-glutamyl transpeptidase (GGT) and / or alkaline phosphatase (ALP) levels, decreased albumin levels, elevated serum iron levels, leukemia (i.e. granulocytopenia), lymphocytosis, elevated erythrocyte sedimentation rate (ESR), shortened red blood cell survival, red blood cell hemolysis, thrombocytopenia, prolonged international normalized ratio (INR), serum HBV These include, but are not limited to, the presence of DNA, elevated aminotransferases (less than 5x ULN), elevated bilirubin levels, prolonged prothrombin time (PT), hyperglobulinemia, the presence of tissue non-specific antibodies such as anti-smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA), the presence of tissue specific antibodies such as antibodies to the thyroid gland, elevated rheumatoid factor (RF) levels, hyperbilirubinemia, low platelet and white blood cell counts, AST levels higher than ALT levels, intralobular inflammation with degenerative and regenerative hepatocellular changes, and predominance of centrilobular necrosis.

[0100] In some embodiments, subjects treated with the disclosed ASOs may have a history of symptoms such as: presence of hepatic HBV cccDNA, presence of serum and / or liver HBV antigens (e.g., HBsAg and / or HBeAg), absence or low levels of anti-HBV antibodies, liver damage, cirrhosis, delta hepatitis, acute hepatitis B, acute fulminant hepatitis B, chronic hepatitis B, liver fibrosis, end stage liver disease, hepatocellular carcinoma, serum sickness-like syndrome, anorexia, nausea, vomiting, low grade fever, myalgia, fatigue, anorexia and olfaction (aversion to food and tobacco), right upper quadrant and upper abdominal pain (intermittent, mild to moderate), hepatic encephalopathy, drowsiness, disturbed sleep patterns. , mental confusion, coma, ascites, gastrointestinal bleeding, coagulopathy, jaundice, hepatomegaly (mildly enlarged, soft liver), splenomegaly, palmate erythema, spider necrosis, muscle wasting, spider angiomas, vasculitis, variceal bleeding, peripheral edema, gynecological tumors, testicular atrophy, abdominal collateral veins (caputmedusa), ALT levels higher than AST, leukopenia (i.e. granulocytopenia), decreased albumin levels, elevated serum iron levels, lymphocytosis, increased erythrocyte sedimentation rate (ESR), decreased red blood cell survival time, thrombocytopenia, prolonged international normalized ratio (INR), serum HBV. The patient is attenuated or rid of one or more of the following conditions or symptoms: presence of DNA, prolonged prothrombin time (PT), hyperglobulinemia, presence of tissue non-specific antibodies such as anti-smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA), presence of tissue specific antibodies such as antibodies to the thyroid gland, hyperbilirubinemia, low platelet and white blood cell counts, AST levels higher than ALT levels, intralobular inflammation with degenerative and regenerative hepatocellular changes, and predominance of centrilobular necrosis.

[0101] The present disclosure provides a method for treating a subject diagnosed with or suspected of having HBV infection and / or HBV-related disorder, comprising administering to the subject an effective amount of an ASO composition of the present disclosure. In some embodiments, the method comprises administering to the subject a first ASO of the present disclosure and a second ASO of the present disclosure, wherein the first ASO is complementary or hybridized to a viral target RNA sequence in a first X region of HBV, and the second ASO is complementary or hybridized to a viral target RNA sequence in a second X region or S region of HBV. In some embodiments, the second ASO is complementary or hybridized to a viral target RNA sequence in the second X region of HBV. In other embodiments, the second ASO is complementary or hybridized to a viral target RNA sequence in the S region of HBV.

[0102] In some embodiments of the methods and uses of the present disclosure, the disease is a respiratory disease. In some embodiments, the respiratory disease is a viral infection. In some embodiments, the respiratory disease is viral pneumonia. In some embodiments, the respiratory disease is an acute respiratory infection. In some embodiments, the respiratory disease is a cold. In some embodiments, the respiratory disease is Severe Acute Respiratory Syndrome (SARS). In some embodiments, the respiratory disease is Middle East Respiratory Syndrome (MERS). In some embodiments, the disease is a coronavirus disease 2019 (e.g., COVID-19). In some embodiments, the respiratory disease can include one or more symptoms selected from cough, sore throat, runny nose, sneezing, headache, fever, shortness of breath, muscle pain, abdominal pain, fatigue, difficulty breathing, persistent chest pain or pressure, difficulty waking up, loss of smell and taste, muscle or joint pain, chills, nausea or vomiting, stuffy nose, diarrhea, hemoptysis, conjunctival congestion, sputum production, chest pressure, and palpitations. In some embodiments, the respiratory disease can include a complication selected from sinusitis, otitis media, pneumonia, acute respiratory distress syndrome, disseminated intravascular coagulation, pericarditis, and renal failure, hi some embodiments, the respiratory disease is idiopathic.

[0103] In some embodiments, the disclosure provides a method for treating or preventing a coronavirus infection comprising administering a therapeutically effective amount of one or more of the ASOs or pharmaceutical compositions disclosed herein to a subject in need thereof. In some embodiments, the coronavirus infection is selected from the group consisting of Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), and COVID-19. In some embodiments, the subject is being treated with one or more additional coronavirus therapeutic agents. In some embodiments, the subject is being treated concurrently with one or more additional coronavirus therapeutic agents.

[0104] In some embodiments, the disease is a liver disease. In some embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD). In some embodiments, the NAFLD is non-alcoholic steatohepatitis (NASH). In some embodiments, the liver disease is hepatocellular carcinoma (HCC).

[0105] The ASO of the present disclosure can be used to treat disease in a subject in need of disease treatment.In some embodiments, the method of treating disease in a subject in need of disease treatment comprises administering any of the ASOs disclosed herein to the subject.In some embodiments, the method of treating disease in a subject in need of disease treatment comprises administering any of the compositions disclosed herein to the subject.

[0106] Administration of ASO can be performed by methods known in the art. In some embodiments, ASO is administered by subcutaneous (SC) or intravenous (IV) delivery. The preparations (e.g., ASO or compositions) of the present disclosure can be given orally, parenterally, topically, or rectally. They are, of course, given in a form suitable for each administration route. For example, they are administered in tablet or capsule form, administered by injection, infusion or inhalation, topically by lotion or ointment, and rectally by suppository. In some embodiments, subcutaneous administration is preferred.

[0107] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrapleural injection and infusion.

[0108] As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" refer to administration of a compound, drug, or other material other than by direct administration into the central nervous system so that it enters the patient's system and thus undergoes metabolic and other like processes, e.g., subcutaneous administration.

[0109] These compounds may be administered to humans and other animals for treatment by any suitable route of administration, including orally, nasally, e.g., by spray, rectally, vaginally, parenterally, intracisternally, and topically, e.g., by powders, ointments or drops, buccally and sublingually.

[0110] Regardless of the route of administration selected, the compounds of the present disclosure (e.g., ASOs), which may be used in a suitable hydrated form, and / or pharmaceutical compositions of the present disclosure are formulated into pharma- ceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0111] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0112] The selected dosage level will depend upon a variety of factors, including the activity of the particular compound of the disclosure (e.g., ASO), or ester, salt, or amide thereof, being used, the route of administration, the time of administration, the rate of excretion or metabolism, the rate and extent of absorption of the particular compound being used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound being used, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.

[0113] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start the dosage of the disclosed compound (e.g., ASO) used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0114] In general, a suitable daily dose of a compound (e.g., ASO) of the present disclosure is the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. Preferably, the compound is administered at about 0.01 mg / kg to about 200 mg / kg, more preferably about 0.1 mg / kg to about 100 mg / kg, and even more preferably about 0.5 mg / kg to about 50 mg / kg. In some embodiments, the compound is administered at about 1 mg / kg to about 40 mg / kg, about 1 mg / kg to about 30 mg / kg, about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 15 mg / kg, or 1 mg / kg to about 10 mg / kg. In some embodiments, a compound of the invention is administered at a dose of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 mg / kg or more. In some embodiments, the compound is administered at a dose of 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, or 30 mg / kg or more. In some embodiments, the compound is administered at a dose of 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, or 15 mg / kg or less. In some embodiments, the total daily dose of the compound is greater than or equal to 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 100 mg.

[0115] If desired, the effective daily dose of the active compound (e.g., ASO) may be administered as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses or subdoses administered separately at appropriate intervals throughout the day, optionally in unit dosage form. In some embodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times. The preferred dosage is once a day. In some embodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times per week. In some embodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times per month. In some embodiments, the compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the compound is administered every 3 days. In some embodiments, the compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks. In some embodiments, the compound is administered monthly. In some embodiments, the compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months.In some embodiments, the compound is administered at least 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 times. The vaccine is administered over a period of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days. In some embodiments, the compound comprises at least 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 5 Three doses are administered over a period of at least 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 weeks.In some embodiments, the compound comprises at least 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 The therapeutic agent is administered once or twice a day for a period of at least 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 months. In some embodiments, the compound is administered at least once per week for a period of at least 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks. In some embodiments, the compound is administered at least once per week for a period of at least 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months.In some embodiments, the compound is administered at least twice per week for a period of at least 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks. In some embodiments, the compound is administered at least twice a week for a period of at least 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months. In some embodiments, the compound is administered at least once every two weeks for a period of at least 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks.In some embodiments, the compound is administered at least once every two weeks for a period of at least 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months. In some embodiments, the compound is administered once every four weeks for a period of at least 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks. In some embodiments, the compound is administered at least once every four weeks for a period of at least 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months.

[0116] The subject of the described methods may be a mammal, including humans and non-human mammals, hi some embodiments, the subject is a human, such as an adult human.

[0117] Some embodiments include a method for treating HBV virus in a subject infected with the virus, comprising administering a therapeutically effective amount of one or more ASOs of the present disclosure or a composition of the present disclosure to a subject in need of treatment, thereby reducing the viral load of the virus in the subject and / or reducing the level of viral antigens in the subject. The ASO may be complementary to or hybridize to a portion of a target RNA in the virus, for example, the second X region and / or S region of HBV.

[0118] In some embodiments, the modified oligonucleotides described herein may be used in combination with one or more additional agents for treating and / or inhibiting HBV and / or HDV replication. When the compounds described herein (e.g., ASOs) are co-administered with additional agents, the effective amount may be less than when the compounds are used alone. Additional agents include, but are not limited to, interferons, nucleoside / nucleotide analogs, capsid assembly modulators (CAMs), siRNAs, other ASOs, nucleic acid polymers or S-antigen transport inhibitor oligonucleotide polymers (NAPs or STOPS), entry inhibitors, and / or small molecule immunomodulators. Examples of additional agents include ALG-010133, ALG-000184, recombinant interferon alpha 2b, IFN-a, PEG-IFN-a-2a, lamivudine, telbivudine, adefovir dipivoxil, clevudine, entecavir, tenofovir alafenamide, tenofovir disoproxil, NVR3-778, BAY41-4109, JNJ-632, JNJ- 3989 (ARO-HBV), RG6004, GSK3228836, REP-2139, REP-2165, AB-729, VIR-2218, DCR-HBVS, JNJ-6379, GLS4, ABI-HO731, JNJ-440, NZ-4, RG7907, EDP-514, AB-423, AB-506, ABI-H03733, and ABI-H2158. In some embodiments, any of the ASOs disclosed herein are co-administered with one of the STOPS. Exemplary STOPS are described in International Publication No. 2020 / 097342 and U.S. Publication No. 2020 / 0147124, both of which are incorporated by reference in their entirety. In some embodiments, the STOP is ALG-010133. In some embodiments, any of the ASOs disclosed herein are co-administered with tenofovir. In some embodiments, any of the ASOs disclosed herein are co-administered with a CAM.For exemplary CAMs, see Berke et al., Antimicrob Agents Chemother, 2017, 61(8):e00560-17, Klumpp, et al., Gastroenterology, 2018, 154(3):652-662.e8, International Application Nos. PCT / US2020 / 017974, PCT / US2020 / 026116, and PCT / US2020 / 028349, and U.S. Application Nos. 16 / 789,298, 16 / 837,515, and 16 / 849,851. In some embodiments, the CAM is ALG-000184, ALG-001075, ALG-001024, JNJ-632, BAY41-4109, or NVR3-778. In some embodiments, the ASO and the additional agent are administered simultaneously. In some embodiments, the ASO and the additional agent are administered sequentially. In some embodiments, the ASO is administered prior to administration of the additional agent. In some embodiments, the ASO is administered after administration of the additional agent.

[0119] definition As used herein, the terms "patient" and "subject" refer to an organism that is treated by the methods of the present disclosure. Such organisms are preferably mammals (e.g., marine organisms, apes, horses, cows, pigs, dogs, cats, etc.), and more preferably humans.

[0120] As used herein, the term "effective amount" refers to an amount of a compound (e.g., an ASO of the present disclosure) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration.

[0121] As used herein, the term "treating" includes any effect, e.g., alleviating, reducing, modulating, ameliorating, or eliminating, that results in the improvement of a condition, disease, disorder, or the like, or that ameliorates the symptoms thereof.

[0122] As used herein, the terms "ameliorate" and "ameliorating" refer to reducing the severity of a condition, such as reducing the severity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0123] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier that makes the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic uses.

[0124] As used herein, the term "pharmaceutical acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0125] As used herein, the term "about" when referring to measurable values ​​(e.g., weight, time, and dose) is meant to encompass the recited value and a range of + / - 10%. For example, "about 10" should be understood as both "10" and "9 to 11."

[0126] As used herein, the term "nucleobase" or "base" refers to nitrogen-containing biological compounds that form nucleosides. Examples of nucleobases include, but are not limited to, thymine, uracil, adenine, cytosine, guanine, and analogs or derivatives thereof.

[0127] Throughout this specification, when compositions are described as having, including, or comprising certain components, or processes and methods are described as having, including, or comprising certain steps, it is contemplated that there are additionally compositions of the disclosure that consist essentially of or consist of the recited components, and processes and methods according to the disclosure that consist essentially of or consist of the recited processing steps.

[0128] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described herein.

[0129] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit between the upper and lower limits of that range, and any other stated or intervening value in the stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specific excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0130] The present disclosure is not limited to the particular embodiments described, as such may vary, and the scope of the present invention will be limited only by the appended claims, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0131] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the various other embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0132] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference herein, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed. EXAMPLES

[0133] The following examples illustrate certain embodiments of the present disclosure to aid one of ordinary skill in the art in practicing the disclosure, and as such, the examples are not to be construed as limiting the scope of the disclosure in any way.

[0134] Example 1: ASO synthesis Gapmer ASO sequences: DNA, 2'-O-Me, and LNA phosphoramidite monomers were procured from commercial sources (Hongene Biotech USA Inc.). All monomers were dried in a vacuum oven with desiccant (P2O5, 24 hours at room temperature). Universal solid support (CPG) mounting was obtained from ChemGenes. Chemicals and solvents for the synthesis workflow were purchased from commercially available sources VWR / Sigma and used without any purification or treatment. Solvents (acetonitrile) and solutions (amidites and activators) were stored over molecular sieves during synthesis.

[0135] Control and target oligonucleotide sequences were synthesized on an Expedite 8909 synthesizer using standard cycles written by the manufacturer, with modifications to several waiting and coupling steps. The solid support was control open-pore glass and the monomers contained standard protecting groups. Each chimeric oligonucleotide was synthesized using commercially available 5'-O-(4,4'-dimethoxytrityl)-3'-O-(2-cyanoethyl-N,N-diisopropyl) DNA, 2'-OMe, and / or 6-N-benzoyl adenosine (A Bz ), 5-methyl-4-N-benzoylcysteine ​​(C Bz ), 2-N-isobutyrylguanosine (G iBu ), and uridine (U) or thymidine (T) LNA phosphoramidite monomers were synthesized separately following standard solid-phase phosphoramidite synthesis protocols. 2'-O-Me-2,6-diaminopurine phosphoramidite was purchased from Glen Research. Phosphoramidites were prepared as 0.1 M solutions in anhydrous acetonitrile. 5-Ethylthiotetrazole was used as the activating agent, 3% dichloroacetic acid in dichloromethane was used to detritylate, acetic anhydride in THF was used to cap, and 16% N-methylimidazole in THF was used to cap, and DDTT ((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaolin-3-thione was used as the sulfur transfer agent for the synthesis of oligoribonucleotide phosphorothioates. The modified oligonucleotides were obtained by extension coupling of 0.1 M phosphoramidite solutions in CH3CN to the solid-bound oligonucleotides in the presence of 5-(ethylthio)-1H-tetrazole activator, followed by standard capping, oxidation, and deprotection. The stepwise coupling efficiency of all modified phosphoramidites was greater than 98.5%.

[0136] Deprotection and cleavage from the solid support was achieved with a mixture of ammonia methylamine (1:1, AMA) at 65 °C for 15 min when a universal linker was used, leaving the deprotection at 65 °C for 90 min, or by heating the solid support with aqueous ammonia (28%) at 55 °C for 8 h to deprotect the base labile protecting groups.

[0137] After filtration to remove the solid support, the deprotection solution was removed under vacuum in a GeneVac centrifugal evaporator. [Table 5] TIFF2025509552000037.tif148149 [Table 6]

[0138] Example 2: Modified Gapmer Sequences AmNA(N-Me)-T, AmNA(N-Me)-4-N-benzoyl(5m)cytidine((5m)C Bz ), AmNA(N-Me)-4-N-benzoylcytidine (A Bz ), and AmNA(N-Me)-2-N-pac(G pac ) was purchased from Luxna Biotech, and scp-BNA-T, scp-BNA-6-N-benzoyl adenosine (A Bz ), scp-BNA-4-N-benzoyl-5-methylcytidine ((5m)C Bz ), scp-BNA-2-N-isobutrylguanosine (G iBu) The phosphoramidite monomers were synthesized according to the procedures described in the references (Takao Yamaguchi, Masahiko Horiba and Satoshi Obika; Chem. Commun., 2015, 51, 9737-9740; Masahiko Horiba, Takao Yamaguchi, and Satoshi Obika; Journal of Organic Chemistry, 2016, 81, 11000-11008). All monomers were dried in a vacuum desiccator using desiccants (KOH and PO, at room temperature for 24 hours). For AmNA(N-Me)-PS-DNA-PS and scp-BNA-PS-DNA-PS, the synthesis was carried out on a 1 μM scale in the 3' to 5' direction using phosphoramidite monomers diluted to a concentration of 0.12 M in anhydrous CH3CN in the presence of 0.3 M 5-(benzylthio)-1H-tetrazole activator (coupling time 16 min) on the solid-bound oligonucleotides, followed by modified capping, oxidation, and deprotection to obtain the modified oligonucleotides. The stepwise coupling efficiency of all modified phosphoramidites was greater than 97%. DDTT (Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaolin-3-thione was used as the sulfur transfer agent for the synthesis of oligoribonucleotide phosphorothioates. The oligonucleotide-containing solid support was washed with a 20% DEA solution in acetonitrile for 15 min, and then the column was thoroughly washed with MeCN. The support was heated in a heat block for 8 hours at 65° C. in diisopropylamine:water:methanol (1:1:2) to cleave from the support and deprotect the base labile protecting groups. [Table 7] [Table 8]

[0139] 5'- and 3'-GalNAc conjugated oligonucleotides were synthesized with GalNAc moieties of various lengths, for example, as described below. GalNAc3, GalNAc4, GalNAc5 and GalNAc6 were conjugated to the oligonucleotide during synthesis using 1, 2 or 3 moieties in the same manner as described below. Additional GalNAc moieties, such as GalNAc-1 and GalNAc-2, previously described herein, were also used to form 5'- and 3'-GalNAc using post-synthesis conjugation. [Table 9]

[0140] Quantification of crude oligomer or feedstock analysis Samples were dissolved in deionized water (1.0 mL) and quantified as follows: First, blanking was performed with water alone on a Nanodrop UV spectrophotometer. The Nanodrop instrument is capable of measuring a wide range of nucleic acid concentrations by using multiple path lengths. The most accurate quantitative results can be achieved by measuring diluted oligonucleotides at absorbance at 260 nm. Crude material is stored at -20°C.

[0141] Crude HPLC / LC-MS analysis 0.1 OD of crude sample was used for crude MS analysis. Purification steps were performed after review of crude LC-MS data.

[0142] HPLC purification Phosphodiester (PO), phosphorothioate (PS) and chimeric modified oligonucleotides were purified by anion exchange HPLC. The buffers were 20 mM sodium phosphate, pH 8.5 in 10% CH3CN (buffer A) and 20 mM sodium phosphate, 1.8 M NaBr, pH 8.5 in 10% CH3CN (buffer B). Fractions containing full-length oligonucleotides were pooled, desalted and lyophilized.

[0143] Lipid-conjugated oligonucleotides were purified by internally packed RPC-Source15 reversed-phase column. Buffers were 20 mM sodium acetate in 10% CH3CN (buffer A) and CH3CN (buffer B). Fractions containing full-length oligonucleotides were pooled, desalted and lyophilized.

[0144] Desalting of purified oligomers The purified, dried oligomers were then desalted using Sephadex G-25M (Amersham Biosciences). The cartridge was conditioned three times with 10 mL of deionized water. The purified oligonucleotides, completely dissolved in 2.5 mL of deionized water, were applied to the cartridge with very slow dropwise elution. The salt-free oligomers were eluted directly into a screw-cap vial with 3.5 mL of deionized water.

[0145] Final HPLC and electrospray LC / MS analysis Approximately 0.10 OD of the oligomer was dissolved in water and then dispensed into special vials for IEX-HPLC and LC / MS analysis. Analytical HPLC and ES LC-MS established the integrity of the chimeric oligonucleotides.

[0146] Post-synthetic conjugation of GalNAc esters to oligonucleotides 5'-C6-amino precursor synthesis The sequences were synthesized on a 10 μmol scale using the universal support (Loading 65 μmol / g). At the 5' end to introduce the C6-NH2 linker, 6-(4-methoxytrityramino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite in 0.1 M acetonitrile was used with a coupling time of 10 min. The oligonucleotide-containing solid support was cleaved from the support and base-labile protecting groups were deprotected by heating in aqueous ammonia / methylamine (1:1) solution in a shaker at room temperature for 3 h. After IEX purification and desalting, post-synthetic conjugation was carried out using the C6-NH2 modified ASO. [ka] [Table 10]

[0147] Post-synthetic conjugation of 5'-GalNAc The 5'-C6-NH2 modified sequence was dissolved in 0.2 M sodium bicarbonate buffer, pH 8.5 (0.015 mM) and 5-7 mol equivalents of GalNAc ester dissolved in DMSO was added. The reaction mixture was stirred at room temperature for 4 h. A sample was analyzed to determine if any unreacted amino modified ASO was present. Aqueous ammonia (28 wt%) was added (5 x reaction volume) and stirred at room temperature for 2-3 h. The reaction mixture was concentrated under reduced pressure and the residue was dissolved in water and purified by HPLC on a strong anion exchange column.

[0148] Example 3: HBsAg release assay in vitro analysis HepG2.2.15 cells (a stable cell line with four integrated HBV genomes) were maintained in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, 1% glutamine, 1% non-essential amino acids, 1% sodium pyruvate and 250 μg / ml G418. Cells were maintained at 37°C in a 5% CO2 atmosphere. For HBsAg release assay, assay medium was made: DMEM containing 5% FBS, 1% penicillin / streptomycin, 1% glutamine and 1% DMSO. The day before the assay, HepG2.2.15 cells were washed once with assay medium, spun at 250g x 5 min, resuspended in assay medium and seed cells at 50,000 / well in assay medium in collagen-coated 96-well plates. The next day, ASOs were diluted in Opti-MEM, 9-pt, 3-fold dilution, and Lipofectamine RNAiMAX (Invitrogen) was diluted according to the manufacturer's manual. ASO dilutions and RNAiMAX dilutions were mixed, left at room temperature for 5 min, and 15 μl was added to each well of a 96-well plate. Plates were left in an incubator at 37°C and 5% CO2 for 5 days. After incubation, supernatants were collected and measured for HBsAg with an ELISA kit (Diasino). Cell viability was measured with CellTiter-Glo (Promega). EC, the concentration of drug required to reduce HBsAg secretion by 50% relative to untreated cell controls, was calculated. 50 was calculated using Prism Graphpad. CC, the concentration of drug required to reduce cell viability by 50% relative to untreated cell controls, was used. 50 was calculated using the same software.

[0149] Resulting EC for compounds in Table 1 50 and C.C. 50 are presented in Table 2 below. EC 50 and C.C. 50 The values ​​are as follows: A: <1 nM, B: 1-10 nM, C: 10-100 nM, D: >100 nM. [Table 11] TIFF2025509552000045.tif255151TIFF2025509552000046.tif33148

[0150] Alternatively, several ASOs were evaluated for in vitro potency in HBV-infected primary human hepatocytes (PHH). Table 3 below shows exemplary ECs from these experiments. 50 and C.C. 50 Provide data. [Table 12]

[0151] Example 4: Melting Temperature (T m ) The melting temperatures of several exemplary ASOs disclosed herein were evaluated and representative results are provided in Table 4 below. [Table 13]

[0152] Example 5: Improved in vivo efficacy ASOs with the disclosed chemistry were synthesized on ABI394 and Expedite 8909 synthesizers using standard phosphoramidite chemistry. In vitro screening of ASOs was performed in HepG2.2.15 cells using HBsAg release assay as described above. Certain ASOs were selected for N-acetylgalactosamine (GalNac) conjugation and tested at 1×5 mg / kg for a single dose in the adeno-associated virus (AAV)-HBV mouse model.

[0153] Table 5 shows exemplary HBsAg troughs of 1×5 mg / kg QW compared to ASO59. These ASOs in the HBx region target all HBV transcripts, including HBx, and improve HBsAg troughs with a single substitution of nmlnA for lnA. [Table 14]

[0154] The figure shows an exemplary side-by-side comparison of ASO59 and ASO87, the latter containing nmlnA and the former containing lnA. As can be seen in the figure, the addition of lnmnA resulted in improved efficacy. ***** [Table 15] TIFF2025509552000051.tif255168TIFF2025509552000052.tif255170TIFF20255095520 00053.tif255170TIFF2025509552000054.tif255170TIFF2025509552000055.tif255170 TIFF2025509552000056.tif255170TIFF2025509552000057.tif255170TIFF20255095520 00058.tif255170TIFF2025509552000059.tif255170TIFF2025509552000060.tif210170

Claims

1. 14-22 nucleotide units, (a) A central region (B') containing six or more consecutive DNA nucleotides, (b) A 5'-wing region (A') containing 2 to 6 lock nucleotides or 4 to 6 2'-substituted nucleosides, (c) An antisense oligonucleotide (ASO) comprising a 3'-wing region (C') containing 2 to 6 lock nucleotides or 4 to 6 2'-substituted nucleosides, The central region of the ASO is at least 80% complementary to or hybridizes with the target RNA sequence, and the ASO is 【Chemistry 1】 (In the formula, B is a nucleic acid base.) 【Chemistry 2】 (In the formula, B is a nucleic acid base.) 【Transformation 3】 An antisense oligonucleotide (ASO) comprising at least one modified nucleotide selected from the following.

2. (i) the central region (B') comprises a modified nucleotide selected from G-clamp and 5prnl; (ii) the 5'-wing region (A') comprises a modified nucleotide selected from Gutb and Nmln; (iii) the 3'-wing region (C') comprises a modified nucleotide selected from Gutb and Nmln; or (iv) any combination thereof, according to claim 1.

3. The ASO according to claim 1, wherein the central region (B') contains two, three, four, five, or six or more modified nucleotides.

4. The ASO according to claim 1, wherein the 5'-wing region (A'), the 3'-wing region (C'), or both, contain a modified nucleotide selected from Gutb, Nmln, G-clamp, and 5prnl.

5. The ASO molecule according to claim 1, further comprising one or more phosphorothioate (ps) nucleoside bonds, mesylphosphoamidate (yp) nucleoside bonds, or a combination thereof.

6. An antisense oligonucleotide (ASO) containing 14 to 22 nucleotide units, wherein the ASO is (a) A central region (B') comprising six or more consecutive DNA nucleotides, wherein at least one of the consecutive DNA nucleotides is a modified nucleotide, (b) A 5'-wing region (A') containing 2 to 6 lock nucleotides or 2'-substituted nucleosides, (c) comprising a 3'-wing region (C') containing 2 to 6 lock nucleotides or 2'-substituted nucleosides, The central region of the ASO is at least 80% complementary to or hybridizes with the target RNA sequence, and the ASO contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mesylphosphoramidate (yp) nucleoside bonds.

7. The aforementioned ASO 【Chemistry 4】 (In the formula, B is a nucleic acid base.) 【Transformation 5】 (In the formula, B is a nucleic acid base.) 【Transformation 6】 The ASO according to claim 6, comprising at least one, at least two, at least three, at least four, or at least five nucleotides selected from.

8. The ASO molecule according to claim 7, wherein the ASO molecule further comprises one or more phosphorothioate (ps) nucleoside bonds.

9. (i) At least one mesylphosphoamide (yp) nucleotide internucleotide bond is located between nucleoside positions 3 and 4 from the 5' end of the ASO molecule, (ii) At least one mesylphosphoamide (yp) internucleotide bond is located between nucleoside positions 5 and 6 from the 5' end of the ASO molecule, (iii) At least one mesylphosphoamide (yp) internucleotide bond is located between nucleoside positions 6 and 7 from the 5' end of the ASO molecule, (iv) At least one mesylphosphoamide (yp) internucleotide bond is located between nucleoside positions 7 and 8 from the 5' end of the ASO molecule, (v) At least one mesylphosphoamide (yp) internucleotide bond is located between nucleoside positions 8 and 9 from the 5' end of the ASO molecule, (vi) At least one mesylphosphoamide (yp) internucleotide bond is located between nucleoside positions 9 and 10 from the 5' end of the ASO molecule, or (vii) The ASO molecule according to claim 1, which is a combination thereof.

10. The ASO according to claim 1, wherein the 5'-wing region (A'), the 3'-wing region (C'), or both, contain at least one mesylphosphoamide (yp) internucleotide bond.

11. The ASO molecule according to claim 1, wherein the ASO molecule further comprises galactosamine.

12. The galactosamine mentioned above is given by formula (VI): 【Transformation 7】 It is N-acetylgalactosamine (GalNAc), in which, m is 1, 2, 3, 4, or 5. Each n is independently either 1 or 2. p is 0 or 1, Each R is independently H, Each Y is independently selected from -O-P(=O)(SH)-, -O-P(=O)(O)-, -O-P(=O)(OH)-, and -O-P(S)S-. Z is H or a second protecting group, Either L is a linker, or L and Y are combined to form a linker. The ASO molecule according to claim 11, wherein A is H, OH, a third protecting group, an activating group, or an oligonucleotide.

13. (i) Whether the target RNA sequence is a viral gene, (ii) Whether the target RNA sequence is a gene derived from a DNA virus, (iii) Whether the target RNA sequence is a gene derived from a double-stranded DNA (dsDNA) virus, (iv) Whether the target RNA sequence is a gene derived from hepadnavirus, (v) Whether the target RNA sequence is a gene derived from hepatitis B virus (HBV), (vi) The target RNA sequence is a gene derived from any one of HBV genotypes A to J, or (vii) The ASO molecule according to claim 1, wherein the target RNA sequence is selected from the S gene or X gene of HBV.

14. The ASO molecule according to claim 1, wherein the target RNA sequence is selected from a gene encoding methylation control J protein (MCJ protein), a gene encoding TAZ, a gene encoding angiopoietin-like 3 (ANGPTL3), a gene encoding diacylglycerol acyltransferase 2 (DGAT2), and a gene encoding hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13).

15. (i) the 5'-wing region of the ASO contains 2 to 6 phosphorothioate-bound loc nucleosides, (ii) the 3'-wing region of the ASO contains 2 to 6 phosphorothioate-bound loc nucleosides, or (iii) a combination thereof, wherein the loc nucleosides are optionally selected from LNA, ScpBNA, AmNA, AmNA(N-Me), GuNA, GuNA(N-R11), where R11 is selected from Me, Et, i-Pr, t-Bu, and combinations thereof, the ASO molecule according to claim 1.

16. The ASO molecule according to claim 1, wherein the central region of the ASO comprises at least five consecutive phosphorothioate-binding DNA nucleotides, at least five consecutive mesylphosphoamide-binding DNA nucleotides, or at least five consecutive DNA nucleotides linked by one or more phosphorothioate nucleoside bonds and one or more mesylphosphoamide nucleoside bonds.

17. The ASO molecule according to claim 6, wherein the central region of the ASO comprises 8 to 10 consecutive phosphorothioate-binding DNA nucleotides, 8 to 10 consecutive mesylphosphoamide-binding DNA nucleotides, or 8 to 10 DNA nucleotides linked by one or more phosphorothioate nucleoside bonds and one or more mesylphosphoamide nucleoside bonds.

18. A pharmaceutical composition comprising the ASO molecule described in claim 1 and a pharmaceutically acceptable excipient.

19. Use of the ASO according to claim 1 or the pharmaceutical composition according to claim 18 in the manufacture of a pharmaceutical for treating a subject having hepatitis B virus (HBV) infection.

20. The pharmaceutical composition according to claim 18 for reducing the expression of a target gene in a subject.

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