MODIFIED GAPMER OLIGONUCLEOTIDES AND METHODS OF USE
20200385735 ยท 2020-12-10
Assignee
Inventors
- Leonid Beigelman (South San Francisco, CA, US)
- Rajendra K. Pandey (South San Francisco, CA, US)
- Vivek Kumar Rajwanshi (South San Fancisco, CA, US)
- David Bernard SMITH (South San Francisco, CA, US)
- Jin Hong (South San Francisco, CA, US)
Cpc classification
A61K31/7088
HUMAN NECESSITIES
A61K31/7125
HUMAN NECESSITIES
C12N2310/3231
CHEMISTRY; METALLURGY
C12N2320/32
CHEMISTRY; METALLURGY
C12N2310/3231
CHEMISTRY; METALLURGY
International classification
C12N15/113
CHEMISTRY; METALLURGY
A61K31/7125
HUMAN NECESSITIES
Abstract
The disclosure includes antisense oligonucleotides, including gapmer antisense oligonucleotides, along with methods of making and use, e.g., against HBV.
Claims
1. A method of treating a subject having a Hepatitis B virus (HBV) infection, comprising administering to the subject a first antisense oligonucleotide (ASO) and a second ASO, wherein the first and second ASO each independently contain 14-22 nucleotide units, and the first and second ASO each independently contain: (a) a central region (B) comprising 6 or more contiguous DNA nucleosides, (b) a 5-wing region (A) comprising 2 to 6 locked nucleosides or 2 substituted nucleosides, and (c) a 3-wing region (C) comprising 2 to 6 locked nucleosides or 2 substituted nucleosides, wherein the first ASO is complementary or hybridizes to a viral target RNA sequence in a first X region or a first S region of HBV, and the second ASO is complementary or hybridizes to a viral target RNA sequence in a second X region or a second S region of HBV.
2. The method of claim 1, wherein the 5-wing region or the 3-wing region of at least one of the first and second ASO comprises 2 to 6 phosphorothioate-linked locked nucleosides, or wherein the central region of at least one of the first and second ASO comprises at least 5 contiguous phosphorothioate-linked DNA nucleosides.
3. (canceled)
4. (canceled)
5. (canceled)
6. The method of claim 1, wherein the locked nucleosides are selected from LNA, scpBNA, AmNA (NH), AmNA (N-Me), GuNA, GuNA (NR) where R is selected from Me, Et, i-Pr, t-Bu and combinations thereof.
7. (canceled)
8. (canceled)
9. The method of claim 1, wherein the first and/or second ASO further comprises a targeting group, wherein the targeting group comprises a GalNAc moiety.
10.-17. (canceled)
18. The method of claim 1, wherein the first ASO or the second ASO independently comprise a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the sequences listed in Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19.
19. (canceled)
20. A pharmaceutical composition comprising a first antisense oligonucleotide (ASO) and a second ASO, wherein the first and second ASO each independently contain 14-22 nucleotide units, and the first and second ASO each independently contain: (a) a central region (B) comprising 6 or more contiguous DNA nucleosides, (b) a 5-wing region (A) comprising 2 to 6 locked nucleosides or 2 substituted nucleosides, and (c) a 3-wing region (C) comprising 2 to 6 locked nucleosides or 2 substituted nucleosides, wherein the first ASO is complementary or hybridizes to a viral target RNA sequence in a first X region of HBV, and the second ASO is complementary or hybridizes to a viral target RNA sequence in a second X region or an S region of HBV.
21. An antisense oligonucleotide (ASO) comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the sequences listed in Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19.
22.-25. (canceled)
26. A method of treating a subject having a Hepatitis B virus (HBV) infection, comprising administering to the subject a therapeutically effective amount of a first ASO and a second ASO, wherein the first and second ASO are independently selected from an ASO comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the sequences listed in Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19.
27.-45. (canceled)
46. The method of claim 1, wherein the first ASO and/or second ASO are administered in a particle or viral vector.
47. The method of claim 46, wherein the viral vector is a vector of adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus, lentivirus, measles virus, picornavirus, poxvirus, retrovirus, or rhabdovirus.
48.-52. (canceled)
53. An antisense oligonucleotide (ASO) comprising 14-22 nucleotide units, wherein the ASO comprises: (a) a central region (B) comprising 6 or more contiguous DNA nucleosides, wherein at least one of the contiguous DNA nucleosides is a modified nucleotide, (b) a 5-wing region (A) comprising 2 to 6 locked nucleosides or 2 substituted nucleosides, and (c) a 3-wing region (C) comprising 2 to 6 locked nucleosides or 2 substituted nucleosides, wherein the central region of the ASO is complementary or hybridizes to a viral target RNA sequence in an X region or an S region of HBV.
54. The ASO of claim 53, wherein the modified nucleotide occurs at the nucleotide at position 3 from the 5 end of the central region.
55. The ASO of claim 53, wherein the modified nucleotide is a nucleotide having a protected or unprotected version of ##STR00022## wherein: R is a halogen or RCC; and R is C.sub.6-12 aryl, 5- to 12-membered heteroaryl, hydroxy-C.sub.1-6 alkyl, or C.sub.1-7 alkanoyloxy.
56. The ASO of claim 53, wherein the ASO comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the sequences listed in Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19.
57. A method of treating a subject having a Hepatitis B virus (HBV) infection, comprising administering to the subject a therapeutically effective amount of the ASO of claim 21.
58. The method of claim 57, wherein the ASO is ASO 120.
59. (canceled)
60. The method of claim 58, further comprising administering a second ASO, wherein the second ASO is ASO 121.
61.-67. (canceled)
68. The ASO of claim 53, wherein the modified nucleotide is a nucleotide having the structure of: ##STR00023## wherein: W is independently O, N, or S; R.sub.1, R.sub.2, and R.sub.5 are independently H or D; R.sub.3 is H or F; R.sub.4 is F or OCH.sub.3; and Base is ##STR00024## wherein: R is a halogen or RCC; and R represents C.sub.6-12 aryl, 5- to 12-membered heteroaryl, hydroxy-C.sub.1-6 alkyl, or C.sub.1-7 alkanoyloxy.
69. A method of treating a subject having a Hepatitis B virus (HBV) infection, comprising administering to the subject a therapeutically effective amount of the ASO of claim 53.
70. The ASO of claim 53, wherein the 5-wing region or the 3-wing region comprises 2 to 6 phosphorothioate-linked locked nucleosides, or wherein the central region comprises at least 5 contiguous phosphorothioate-linked DNA nucleosides.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0058] The present disclosure is directed to modified antisense oligonucleotides and pharmaceutical compositions of modified antisense oligonucleotides. The present disclosure is also directed to methods of using and preparing the antisense oligonucleotides and pharmaceutical compositions.
[0059] Compounds of the Present Disclosure
[0060] Compounds of the present disclosure include modified antisense oligonucleotides (ASO). In some embodiments, the ASO comprises 14-22 nucleotide units, e.g., 14, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotide units. In some embodiments, the ASO is a gapmer that comprises three regions: a 5-wing region (A) comprising modified nucleotides; a central region (B) comprising nucleotides of a different type from the wings, e.g., nucleotides capable of inducing RNase H cleavage; and a 3-wing region (C) comprising modified nucleotides.
[0061] In some embodiments, the 5-wing region and the 3-wing region comprise 2-6 nucleotides, e.g., 2, 3, 4, 5, or 6 nucleotides. One or more of these nucleotides is modified (e.g., 1, 2, 3, 4, 5, or 6 of the nucleotides is modified). On the other hand, the central region may comprise 6 or more contiguous DNA nucleosides, linked by phosphodiester or thiophosphate (ps) internucleotide linkages. In other embodiments, the central region includes one or more modified nucleotide. For example, the central region may include one or more modified nucleotide where the central region is capable of inducing RNase H cleavage. In some embodiments, the central region includes one or more modified nucleotide having a modified nucleobase. In some embodiments, the central region comprises 6, 7, 8, 9, 10, or 11 contiguous 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.
[0062] Thus, in some aspects, the gapmer ASO compounds of the disclosure include compounds of formula (I):
A-B-C,
wherein A and C each independently comprise 2-6 nucleotides, with one or more being a modified nucleotide, B comprises 6 or more contiguous DNA nucleosides linked by phosphodiester or thiophosphate internucleotide linkages. In some embodiments, B comprises one or more modified DNA nucleosides. In some embodiments, the modified nucleotide is selected from locked nucleosides or 2-substituted nucleosides. In some embodiments, the modified DNA nucleoside is selected from locked nucleosides or 2-substituted nucleosides.
[0063] In certain aspects the number of nucleotides and/or nucleosides in A, B, and C are 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), (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), (5:6:5).
[0064] 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. The locked nucleoside can contain a bridge between the 4 and the 2 position of the sugar wherein the bridges comprises 2 to 4 optionally substituted atoms. For example, LNA nucleoside is:
##STR00001##
Other exemplary locked nucleosides include the following:
##STR00002##
where R is H or alkyl (or AmNA(N-Me)) when R is alkyl);
##STR00003##
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, 5-wing region are all LNA and the 3-wing region contains LNA and one or two nucleosides selected from ScpBNA, AmNA, and GuNA. Other nucleotides are included 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.
[0065] In some embodiments, the 5-wing region of an ASO comprises 2 to 6 phosphorothioate-linked locked nucleosides. In some embodiments, the 5-wing region comprises 2 to 6 phosphorothioate-linked 2 substituted nucleosides. 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 phosphorothiate linker. In some embodiments, the 5-wing region further comprises a RNA nucleoside or DNA nucleoside, wherein the RNA nucleoside and 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. In some embodiments, at least 2, 3, 4, 5, or 6 nucleosides of the 5-wing region are linked by a phosphorothioate linker.
[0066] In some embodiments, the 3-wing region of an ASO comprises 2 to 6 phosphorothioate-linked locked nucleosides. In some embodiments, the 3-wing region comprises 2 to 6 2 phosphorothioate-linked substituted nucleosides. 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 phosphorothiate linker. In some embodiments, the 3-wing region further comprises a RNA nucleoside or DNA nucleoside, wherein the RNA nucleoside and 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. In some embodiments, at least 2, 3, 4, 5, or 6 nucleosides of the 3-wing region are linked by a phosphorothioate linker.
[0067] In certain embodiments, one or more of the nucleotides in the 5-wing region and/or the 3-wing region comprises a thiophosphate internucleotide linkage. In certain embodiments, all nucleotides in the 5-wing region comprises a thiophosphate internucleotide linkage. In some embodiments, all nucleotides in the 3-wing region comprises a thiophosphate internucleotide linkage.
[0068] In some embodiments, the central region includes one or more modified nucleotide having a modified nucleobase. For example, the central region can include one or more modified nucleotide having a protected or unprotected version of the following:
##STR00004##
where R is a halogen or RCC; and R is C.sub.6-12 aryl, 5- to 12-membered heteroaryl, hydroxy-C.sub.1-6 alkyl, or C.sub.1-7 alkanoyloxy. In some embodiments, the central region includes one modified nucleotide (e.g., (2s)T or (5OH)C) at the 1.sup.st, 2.sup.nd, 3.sup.rd or 4.sup.th gap nucleoside position (from the 5 end). In some embodiments, the modified nucleotide is at the 3.sup.rd gap nucleoside position (from the 5 end). In some embodiments, the modified nucleotide is a nucleotide having the structure of:
##STR00005##
wherein:
W is independently O, N, or S;
R.sub.1, R.sub.2, and R.sub.5 are independently H or D;
R.SUB.3 .is H or F;
[0069] R.sub.4 is F or OCH.sub.3; and
Base is
[0070] ##STR00006##
wherein:
R is a halogen or RCC; and
R represents C.sub.6-12 aryl, 5- to 12-membered heteroaryl, hydroxy-C.sub.1-6 alkyl, or C.sub.1-7 alkanoyloxy. In some embodiments, C.sub.1-7 alkanoyl includes, but is not limited to. formyl, acetyl, ethyl carbonyl, n-propyl carbonyl, isopropyl carbonyl, n-butyl carbonyl, isobutyl carbonyl, t-butyl carbonyl, n-pentyl carbonyl, and n-hexyl carbonyl. Other modified nucleotides include those in PCT/JP2018/006061, which is incorporated by reference in its entirety.
[0071] As used herein, unless otherwise indicated, aryl refers to a carbocyclic (all carbon) ring that has a fully delocalized pi-electron system. The aryl group can be made up of two or more fused rings (rings that share two adjacent carbon atoms). When the aryl is a fused ring system, then the ring that is connected to the rest of the molecule has a fully delocalized pi-electron system. The other ring(s) in the fused ring system may or may not have a fully delocalized pi-electron system. Examples of aryl groups include, without limitation, the radicals of benzene, naphthalene and azulene.
[0072] As used herein, unless otherwise indicated, heteroaryl refers to a ring that has a fully delocalized pi-electron system and contains one or more heteroatoms (e.g., one to three heteroatoms, or one to four heteroatoms, or one to five heteroatoms) independently selected from the group consisting of nitrogen, oxygen, and sulfur in the ring. The heteroaryl group can be made up of two or more fused rings (rings that share two adjacent carbon atoms). When the heteroaryl is a fused ring system, then the ring that is connected to the rest of the molecule has a fully delocalized pi-electron system. The other ring(s) in the fused ring system may or may not have a fully delocalized pi-electron system. Examples of heteroaryl rings include, without limitation, furan, thiophene, pyrrole, oxazole, thiazole, imidazole, pyrazole, isoxazole, isothiazole, triazole, thiadiazole, pyridine, pyridazine, pyrimidine, pyrazine and triazine.
[0073] In some embodiments, the central region of an ASO comprises at least 5 contiguous phosphorothioate-linked DNA nucleosides. In some embodiments, at least 2, 3, 4, 5, or 6 nucleosides of the central region are linked by a phosphorothioate linker. In some embodiments, a DNA nucleoside of central region is linked to a nucleoside of a 5-wing region by a phosphorothioate linker. In some embodiments, a DNA nucleoside of central region is linked to a nucleoside of a 3-wing region by a phosphorothioate linker. In some embodiments, the central region comprises 8 to 10 contiguous phosphorothioate-linked DNA nucleosides.
[0074] In some embodiments, the ASO is complementary or hybridizes to a viral target RNA sequence that begins in an X region of HBV or in an S region of HBV. The vital target may, e.g., begin at the 5-end of target-site in acc. KC315400.1 (genotype B, gt B), or in any one of genotypes A, C, or D. The skilled person would understand the HBV position, e.g., as described in Wing-Kin Sung, et al., Nature Genetics 44:765 (2012). In some embodiments, the S region is defined as from the beginning of small S protein (in genotype B KC315400.1 isolate, position #155) to before beginning of X protein (in genotype B KC315400.1 isolate, position #1373). In some embodiments, the X region is defined as from the beginning X protein (in genotype B KC315400.1 isolate, position #1374) to end of DR2 site (in genotype B KC315400.1 isolate, position #1603).
[0075] In some embodiments, the ASO is complementary 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 contiguous nucleotides within positions 100-800 or 1050-1700 of SEQ ID NO: 1. In some embodiments, the ASO is complementary or hybridizes to a viral target RNA sequence that comprises, consists of, or consists essentially of 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 7 to 15, 7 to 14, 7 to 13, 7 to 12, or 7 to 11 contiguous nucleotides within positions 100-800 or 1050-1700 of SEQ ID NO: 1. In some embodiments, the ASO is complementary 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 contiguous nucleotides within positions 180-280, 300 to 450, 650 to 775, 1125 to 1300, or 1400 to 1650 of SEQ ID NO: 1. In some embodiments, the ASO is complementary 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 contiguous nucleotides within positions 180 to 215, 230 to 270, 350 to 420, 675 to 730, 1165 to 1210, 1245 to 1290, 1400 to 1480, or 1500 to 1630 of SEQ ID NO: 1. In some embodiments, the ASO is complementary 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 contiguous starting at position 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, 1527, 1559, 1575, 1576, 1577, 1580, 1581, 1582, or 1589 of SEQ ID NO: 1. In some embodiments, the ASO is perfectly complementary to the viral target RNA sequence. In some embodiments, there is less than or equal to 5, 4, 3, 2, or 1 mismatches between the ASO and the viral target sequence. In some embodiments, there is less than or equal to 2 mismatches between the ASO and the viral target sequence. In some embodiments, there is less than or equal to 1 mismatch between the ASO and the viral target sequence. In some embodiments, the mismatch is in the wing region of the ASO. In some embodiments, the mismatch is in the 5 wing region 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.
[0076] In some embodiments, the central region is complementary 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 contiguous nucleotides within positions 100-800 or 1050-1700 of SEQ ID NO: 1. In some embodiments, the central region is complementary or hybridizes to a viral target RNA sequence that comprises, consists of, or consists essentially of 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 7 to 15, 7 to 14, 7 to 13, 7 to 12, or 7 to 11 contiguous nucleotides within positions 100-800 or 1050-1700 of SEQ ID NO: 1. In some embodiments, the central region is complementary 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 contiguous nucleotides within positions 180-280, 300 to 450, 650 to 775, 1125 to 1300, or 1400 to 1650 of SEQ ID NO: 1. In some embodiments, the central region is complementary 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 contiguous nucleotides within positions 180 to 215, 230 to 270, 350 to 420, 675 to 730, 1165 to 1210, 1245 to 1290, 1400 to 1480, or 1500 to 1630 of SEQ ID NO: 1. In some embodiments, the central region is complementary 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 contiguous starting at position 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, 1527, 1559, 1575, 1576, 1577, 1580, 1581, 1582, or 1589 of SEQ ID NO: 1. In some embodiments, the central region is perfectly complementary to the viral target RNA sequence. In some embodiments, there is less than or equal to 5, 4, 3, 2, or 1 mismatches between the central region and the viral target sequence. In some embodiments, there is less than or equal to 2 mismatches between the central region and the viral target sequence. In some embodiments, there is less than or equal to 1 mismatch between the central region and the viral target sequence.
[0077] The following specific sequences in Table 1 are within the scope of the present disclosure. As used herein, ln=Locked nucleic acid (LNA); lnA=Locked nucleic acid (LNA) A; ln(5m)C=Locked nucleic acid (LNA)-5methyl C; lnG=Locked nucleic acid (LNA) G; lnT=Locked nucleic acid (LNA) T; (5m)C=5 methylC; mA=2-O-methoxy A; mU=2-O-methoxy U; (8nh) A=8-amino A; (8nh)G=8-amino G; (2s)T=2-thio T; (5-OH)C=5-hydroxy C; cp=scp=cyclopropyl; cpC=scpC=cyclopropyl C; cpG=scpG=cyclopropyl G; cpT=scpT=cyclopropyl T; ps=phosphorothioate linkages. The Position in HBV Genome describes the 5-end of target-site in acc. KC315400.1 (genotype B).
TABLE-US-00001 TABLE1 ExemplaryASOs Positionin HBV SEQ Genome_ ID ASO Length_Gapmer NO. # Structure Sequences[5to3] 2 1 1527_16mer_ 5ln(5m)CpslnGpsln(5m)CpsGpsTpsApsApsApsGpsApsGpsApsGpsln 3-10-3 GpslnTpslnG3 3 2 1559_15mer_ 5ln(5m)Cpsln(5m)CpslnGpslnGps(5m)CpsApsGpsApsTpsGpsAps 4-8-3 GpslnApslnApslnG3 4 3 1576_16mer_ 5lnApslnApslnGpsln(5m)CpsGpsApsApsGpsTpsGps(5m)CpsApsln 4-8-4 (5m)CpslnApsln(5m)CpslnG3 5 4 1432_17mer_ 5lnGpslnGpslnApslnTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m) 4-10-3 CpsGpsApsln(5m)CpslnGpslnG3 6 5 1582_16mer_ 5lnGpslnApslnGpsGpsTpsGpsApsApsGps(5m)CpsGpsApsApslnGps 3-10-3 lnTpslnG3 7 6 1522_15mer_ 5lnApslnApslnGpsApsGpsApsGpsGpsTpsGps(5m)CpslnGpsln(5m) 3-8-4 Cpsln(5m)Cpsln(5m)C3 8 7 1432_16mer_ 5lnGpslnApslnTpslnTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m) 4-9-3 CpsGpsApsln(5m)CpslnGpslnG3 9 8 1527_17mer_ 5ln(5m)Cpsln(5m)CpslnGpsln(5m)CpsGpsTpsApsApsApsGpsApsGps 4-10-3 ApsGpslnGpslnTpslnG3 10 9 1431_17mer_ 5lnGpslnApslnTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)Cps 3-10-4 GpsApsln(5m)CpslnGpslnGpslnG3 11 10 1580_15mer_ 5lnGpslnTpslnGpsApsApsGps(5m)CpsGpsApsApsGpsTpslnGpsln 3-9-3 (5m)CpslnA3 12 11 1589_15mer_ 5ln(5m)CpslnGpslnTpsGps(5m)CpsApsGpsApsGpsGpsTpsGpslnApsln 2-10-3 ApslnG3 13 12 1435_15mer_ 5lnGpslnGpslnGpsApsTpsTps(5m)CpsApsGps(5m)CpsGpsln(5m) 3-8-4 Cpsln(5m)CpslnGpslnA3 14 13 1432_17mer_ 5lnGpslnGpslnApslnTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps 4-8-5 (5m)CpslnGpslnApsln(5m)CpslnGpslnG3 15 14 1431_15mer_ 5lnTpslnTpsln(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsAps 3-9-3 (5m)CpslnGpslnGpslnG3 16 15 1432_17mer_ 5lnGpslnGpslnApslnTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m) 4-9-4 CpsGpslnApsln(5m)CpslnGpslnG3 17 16 1527_16mer_ 5ln(5m)CpslnGpsln(5m)CpsGpsTpsApsApsApsGpsApsGpsApsln 3-9-4 GpslnGpslnTpslnG3 18 17 1513_15mer_ 5lnGpsln(5m)CpslnGps(5m)Cps(5m)Cps(5m)Cps(5m)CpsGpsTpsGps 3-9-3 GpsTpsln(5m)CpslnGpslnG3 19 18 245_17mer_ 5ln(5m)CpslnApsln(5m)Cpsln(5m)CpsAps(5m)CpsGpsApsGpsTps 4-10-3 (5m)CpsTpsApsGpslnAps(5m)CpslnT3 20 19 1426_15mer_ 5ln(5m)CpslnGps(5m)CpsGpsAps(5m)CpsGpsGpsGpsApsln(5m)Cps 3-8-4 lnGpslnTpslnA3 21 20 377_17mer_ 5lnApslnApslnApsln(5m)CpsGps(5m)Cps(5m)CpsGps(5m)CpsAps 4-8-5 GpsApsln(5m)CpslnApsln(5m)CpslnApslnT3 22 21 1516_15mer_ 5lnGpslnGpslnTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cps(5m) 3-10-2 CpsGpsTpsGpslnGpslnT3 23 22 1575_16mer_ 5lnApslnGpsln(5m)CpsGpsApsApsGpsTpsGps(5m)CpsAps(5m)Cps 3-10-3 Apsln(5m)CpslnGpslnG3 24 23 1580_16mer_ 5lnGpslnGpslnTpsGpsApsApsGps(5m)CpsGpsApsApsGpslnTpslnGps 3-9-4 ln(5m)CpslnA3 25 24 1261_15mer_ 5ln(5m)CpslnGpsln(5m)CpsApsGpsTpsApsTpsGpsGpsApslnTpsln 3-8-4 (5m)CpslnGpslnG3 26 25 1519_16mer_ 5lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps 3-10-3 (5m)Cpsln(5m)CpslnGpslnT3 27 26 1433_17mer_ 5lnGpslnGpslnGpslnApslnTpsTps(5m)CpsApsGps(5m)CpsGps(5m) 5-8-4 Cps(5m)CpslnGpslnApsln(5m)CpslnG3 28 27 1433_15mer_ 5lnGpslnApslnTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)Cps 3-8-4 lnGpslnAps(5m)CpslnG3 29 28 1431_17mer_ 5lnGpslnApslnTpslnTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m) 4-10-3 CpsGpsAps(5m)CpslnGpslnGpslnG3 30 29 1518_16mer_ 5lnApslnGpslnApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m) 3-10-3 Cps(5m)CpslnGpslnTpslnG3 31 30 1431_17mer_ 5lnGpslnApslnTpslnTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m) 4-9-4 CpsGpsApsln(5m)CpslnGpslnGpslnG3 32 31 1520_15mer_ 5lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cpsln(5m) 3-8-4 Cpsln(5m)Cpsln(5m)CpslnG3 33 32 1519_17mer_ 5lnApslnGpslnApsGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m) 3-10-4 Cpsln(5m)Cpsln(5m)CpslnGpslnT3 34 33 1581_16mer_ 5lnApslnGpslnGpsTpsGpsApsApsGps(5m)CpsGpsApsApsGpslnTps 3-10-3 lnGpsln(5m)C3 35 34 1575_15mer_ 5lnGpsln(5m)CpslnGpsApsApsGpsTpsGps(5m)CpsAps(5m)CpsAps 3-10-2 (5m)CpslnGpslnG3 36 35 1438_15mer_ 5ln(5m)CpslnGps(5m)CpsGpsGpsGpsApsTpsTps(5m)CpsApsGpsln 3-9-3 (5m)CpslnGpsln(5m)C3 37 36 1520_15mer_ 5lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps 3-10-2 (5m)Cpsln(5m)CpslnG3 38 37 1520_17mer_ 5lnApslnApslnGpslnApsGpsApsGpsGpsTpsGps(5m)CpsGps(5m) 4-10-3 Cps(5m)Cpsln(5m)Cpsln(5m)CpslnG3 39 38 1517_15mer_ 5lnApslnGpslnGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cps(5m) 3-9-3 CpsGpslnTpslnGpslnG3 40 39 1262_15mer_ 5ln(5m)Cpsln(5m)CpslnGps(5m)CpsApsGpsTpsApsTpsGpsGpslnAps 3-8-4 lnTpsln(5m)CpslnG3 41 40 246_17mer_ 5ln(5m)Cpsln(5m)CpslnAps(5m)Cps(5m)CpsAps(5m)CpsGpsAps 3-10-4 GpsTps(5m)CpsTpslnApslnGpslnApsln(5m)C3 42 41 191_16mer_ 5ln(5m)Cpsln(5m)CpslnGps(5m)Cps(5m)CpsTpsGpsTpsApsAps(5m) 3-9-4 CpsApsln(5m)CpslnGpslnApslnG3 43 42 1441_15mer_ 5lnGpslnTpsln(5m)Cps(5m)CpsGps(5m)CpsGpsGpsGpsApsTpslnTps 3-8-4 ln(5m)CpslnApslnG3 44 43 1443_17mer_ 5lnGpslnGpslnTps(5m)CpsGpsTps(5m)Cps(5m)CpsGps(5m)CpsGps 3-10-4 GpsGpslnApslnTpslnTpsln(5m)C3 45 44 1408_17mer_ 5lnApsln(5m)CpslnApslnApsApsGpsGpsAps(5m)CpsGpsTps(5m) 4-10-3 Cps(5m)Cps(5m)CpslnGpsln(5m)CpslnG3 46 45 1433_16mer_ 5lnGpslnGpslnApslnTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m) 4-9-3 CpsGpslnApsln(5m)CpslnG3 47 46 1432_17mer_ 5lnGpslnGpslnApsTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m) 3-10-4 CpsGpslnApsln(5m)CpslnGpslnG3 48 47 1433_16mer_ 5lnGpslnGpslnApsTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m) 3-10-3 CpsGpslnApsln(5m)CpslnG3 49 48 246_17mer_ 5ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)CpsAps(5m)CpsGpsAps 4-10-3 GpsTps(5m)CpsTpsApslnGpslnApsln(5m)C3 50 49 1575_16mer_ 5lnApslnGpsln(5m)CpslnGpsApsApsGpsTpsGps(5m)CpsAps(5m) 4-9-3 CpsApsln(5m)CpslnGpslnG3 51 50 1576_15mer_ 5lnApslnGpsln(5m)CpslnGpsApsApsGpsTpsGps(5m)CpsAps(5m) 4-8-3 CpslnApsln(5m)CpslnG3 52 51 1580_16mer_ 5lnGpslnGpslnTpsGpsApsApsGps(5m)CpsGpsApsApsGpsTpslnGps 3-10-3 ln(5m)CpslnA3 53 52 1576_15mer_ 5lnApslnGpsln(5m)CpsGpsApsApsGpsTpsGps(5m)CpsAps(5m)Cps 3-10-2 Apsln(5m)CpslnG3 54 53 191_16mer_ 5ln(5m)Cpsln(5m)CpslnGpsln(5m)Cps(5m)CpsTpsGpsTpsApsAps 4-8-4 (5m)CpsApsln(5m)CpslnGpslnApslnG3 55 54 1435_15mer_ 5lnGpslnGpslnGpsApsTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps 3-9-3 ln(5m)CpslnGpslnA3 56 55 1518_15mer_ 5lnGpslnApslnGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cps 3-9-3 (5m)CpslnGpslnTpslnG3 57 56 1581_16mer_ 5lnApslnGpslnGpslnTpsGpsApsApsGps(5m)CpsGpsApsApsGpslnTps 4-9-3 lnGpsln(5m)C3 58 57 694_17mer_ 5lnGpsln(5m)Cpsln(5m)Cpsln(5m)CpsTpsAps(5m)CpsGpsApsAps 4-9-4 (5m)Cps(5m)CpsApsln(5m)CpslnTpslnGpslnA3 59 58 377_15mer_ 5lnApsln(5m)CpslnGpsln(5m)Cps(5m)CpsGps(5m)CpsApsGpsAps 4-8-3 (5m)CpsApsln(5m)CpsApslnT3 60 59 383_17mer_ 5lnApslnTpslnGpslnApsTpsApsApsApsAps(5m)CpsGps(5m)Cps(5m) 4-10-3 CpsGpsln(5m)CpslnApslnG3 61 60 1432_15mer_ 5lnApslnTpslnTpsln(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)Cps 4-8-3 GpsApsln(5m)CpslnGpslnG3 62 61 1408_15mer_ 5lnApslnApsApsGpsGpsAps(5m)CpsGpsTps(5m)Cps(5m)Cps(5m) 2-10-3 CpslnGpsln(5m)CpslnG3 63 62 1522_15mer_ 5lnApslnApslnGpsApsGpsApsGpsGpsTpsGps(5m)CpsGpsln(5m) 3-9-3 Cpsln(5m)Cpsln(5m)Cps3 64 63 1432_15mer_ 5lnApslnTpslnTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGp 3-8-4 slnApsln(5m)CpslnGpslnG3 65 64 383_17mer_ 5lnApslnTpslnGpslnApsTpsApsApsApsAps(5m)CpsGps(5m)Cpsln 4-8-5 (5m)CpslnGpsln(5m)CpslnApslnG3 66 65 1410_15mer_ 5lnApsln(5m)CpslnApslnApsApsGpsGpsAps(5m)CpsGpsTps(5m) 4-8-3 Cpsln(5m)Cpsln(5m)CpslnG3 67 66 1581_15mer_ 5lnGpslnGpslnTpsGpsApsApsGps(5m)CpsGpsApsApsGpsTpslnGps 3-10-2 ln(5m)C3 68 67 376_17mer_ 5lnApslnApsln(5m)CpslnGps(5m)Cps(5m)CpsGps(5m)CpsApsGps 4-10-3 Aps(5m)CpsAps(5m)CpslnApslnTpsln(5m)C3 69 68 377_17mer_ 5lnApslnApslnApsln(5m)CpsGps(5m)Cps(5m)CpsGps(5m)CpsAps 4-9-4 GpsAps(5m)CpslnApsln(5m)CpslnApslnT3 70 69 377_15mer_ 5lnApsln(5m)CpslnGps(5m)Cps(5m)CpsGps(5m)CpsApsGpsAps 3-8-4 (5m)CpslnApsln(5m)CpslnApslnT3 71 70 1582_15mer_ 5lnApslnGpslnGpsTpsGpsApsApsGps(5m)CpsGpsApsApsGpslnTps 3-10-2 lnG3 72 71 377_16mer_ 5lnApslnApsln(5m)CpsGps(5m)Cps(5m)CpsGps(5m)CpsApsGpsAps 3-10-3 (5m)CpsApsln(5m)CpslnApslnT3 73 72 1576_16mer_ 5lnApslnApslnGpsln(5m)CpsGpsApsApsGpsTpsGps(5m)CpsAps 4-9-3 (5m)CpslnApsln(5m)CpslnG3 74 73 381_17mer_ 5lnGpslnApslnTpslnApslnApsApsAps(5m)CpsGps(5m)Cps(5m)Cps 5-8-4 Gps(5m)CpslnApslnGpslnApsln(5m)C3 75 74 1580_16mer_ 5lnGpslnGpslnTpslnGpsApsApsGps(5m)CpsGpsApsApsGpsTpslnGps 4-9-3 ln(5m)CpslnA3 76 75 694_17mer_ 5lnGpsln(5m)Cpsln(5m)Cps(5m)CpsTpsAps(5m)CpsGpsApsAps 3-10-4 (5m)Cps(5m)CpsApsln(5m)CpslnTpslnGpslnA3 77 76 1261_15mer_ 5ln(5m)CpslnGpsln(5m)CpsApsGpsTpsApsTpsGpsGpsApsTps(5m) 3-10-2 CpslnGpslnG3 78 77 1518_15mer_ 5lnGpslnApslnGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cps 3-10-2 (5m)CpsGpslnTpslnG3 79 78 383_17mer_ 5lnApslnTpslnGpslnApsTpsApsApsApsAps(5m)CpsGps(5m)Cps 4-9-4 (5m)CpslnGpsln(5m)CpslnApslnG3 80 79 383_17mer_ 5lnApslnTpslnGpsApsTpsApsApsApsAps(5m)CpsGps(5m)Cps(5m) 3-10-4 CpslnGpsln(5m)CpslnApslnG3 81 80 377_17mer_ 5lnApslnApslnApsln(5m)CpsGps(5m)Cps(5m)CpsGps(5m)CpsAps 4-10-3 GpsAps(5m)CpsApsln(5m)CpslnApslnT3 82 81 1521_16mer_ 5lnApslnApslnGpslnApsGpsApsGpsGpsTpsGps(5m)CpsGps(5m) 4-9-3 Cpsln(5m)Cpsln(5m)Cpsln(5m)C3 83 82 1577_15mer_ 5lnApslnApslnGps(5m)CpsGpsApsApsGpsTpsGps(5m)CpsAps(5m) 3-10-2 CpslnApsln(5m)C3 84 83 1182_15mer_ 5lnGpslnTpslnTpsGps(5m)CpsGpsTps(5m)CpsApsGps(5m)CpslnAps 3-8-4 lnApslnApsln(5m)C3 85 84 700_17mer_ 5lnGpslnGpslnGpsApsApsApsGps(5m)Cps(5m)Cps(5m)CpsTpsAps 3-10-4 (5m)CpslnGpslnApslnApsln(5m)C3 86 85 383_17mer_ 5lnApslnTpslnGpslnApslnTpsApsApsApsAps(5m)CpsGps(5m)Cps 5-8-4 (5m)CpslnGpsln(5m)CpslnApslnG3 87 86 1576_16mer_ 5lnApslnApslnGps(5m)AmCpsGpsApsApsGpsTpsGps(5m)CpsAps 4-8-4 ln(5m)CpslnApsln(5m)CpslnG3 88 87 1576_16mer_ 5lnApslnApslnGpsln(5m)CpsGpsApsApsGpsTpsGps(5m)CpsApsln 4-8-4 (5m)CpsAmApsln(5m)CpslnG3 89 88 1576_16mer_ 5lnApslnApslnGpsln(5m)CpsGpsApsApsGpsTpsGps(5m)CpsAps 4-8-4 (5m)AmCpslnApsln(5m)CpslnG3 90 89 1575_16mer_ 5ScpApslnGpsln(5m)CpsGpsApsApsGpsTpsGps(5m)CpsAps(5m) 3-10-3 CpsApsln(5m)CpslnGpslnG3 91 90 1575_16mer_ 5lnApslnGpsln(5m)CpsGpsApsApsGpsTpsGps(5m)CpsAps(5m) 3-10-3 CpsApsln(5m)CpslnGpsscpG3 92 91 383_17mer_ 5ScpApslnTpslnGpslnApsTpsApsApsApsAps(5m)CpsGps(5m)Cps 4-8-5 Gpsln(5m)CpslnGpsln(5m)CpslnApslnG3 93 92 383_17mer_ 5lnApslnTpslnGpslnApsTpsApsApsApsAps(5m)CpsGps(5m)Cps 4-8-5 Gpsln(5m)CpslnGpsln(5m)CpslnApsScpG3 94 93 383_17mer_ 5lnApsScpTpslnGpslnApsTpsApsApsApsAps(5m)CpsGps(5m)Cpsln 4-8-5 (5m)CpslnGpsln(5m)CpslnApslnG3 95 94 1527_16mer_ 5ln(5m)CpslnGpsln(5m)CpsGpsTpsApsApsApsGpsApsGpsApsAps 3-10-3 lnGpsScpTpslnG3 96 9A 1431_17mer_ 5lnGpslnApsscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps(5m)Cps 3-10-4 (5m)CpsGpsApsln(5m)CpslnGpslnGpslnG3 97 9B 1431_17mer_ 5mU-lnGpslnApsscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps(5m) 3-10-4 Cps(5m)CpsGpsApsln(5m)CpslnGpslnGpslnG3 98 9C 1431_17mer_ 5mU- 3-10-4 lnGpslnApslnTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)Cps GpsApsln(5m)CpslnGpslnGpslnG3 99 25A 1519_16mer_ 5lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps 3-10-3 (5m)Cpsln(5m)CpslnGpslnT3 100 25B 1519_16mer_ 5lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps 3-10-3 (5m)Cpsln(5m)CpslnGpsAmT3 101 25C 1519_16mer_ 5lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps 3-10-3 (5m)CpsAm(5m)CpslnGpslnT3 102 25D 1519_16mer_ 5lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps 3-10-3 (5m)Cpsln(5m)CpslnGpsscpT3 103 25E 1519_16mer_ 5lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps 3-10-3 (5m)Cps(5m)scpCpslnGpslnT3 104 25F 1519_16mer_ 5mU- 3-10-3 lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps (5m)Cpsln(5m)CpslnGpslnT3 105 25G 1519_16mer_ 5mU- 3-10-3 lnGpslnApslnGpsAps(8nh)GpsGpsTpsGps(5m)CpsGps(5m)Cps(5m) Cps(5m)Cpsscp(5m)CpslnGpslnT3 106 47A 1433_16mer_ 5lnGpslnGpslnAps(2s)TpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps 3-10-3 (5m)CpsGpslnApsln(5m)CpslnG3 107 47B 1433_16mer_ 5lnGpslnGpslnApsTps(2s)Tps(5m)CpsApsGps(5m)CpsGps(5m)Cps 3-10-3 (5m)CpsGpslnApsln(5m)CpslnG3 108 47C 1433_16mer_ 5lnGpslnGpslnApsTpsTps(5oh)CpsApsGps(5m)CpsGps(5m)Cps 3-10-3 (5m)CpsGpslnApsln(5m)CpslnG3 109 47D 1433_16mer_ 5mU-lnGpslnGpslnApsTps(2s)Tps(5m)CpsApsGps(5m)CpsGps(5m) 3-10-3 Cps(5m)CpsGpslnApsln(5m)CpslnG3 110 47E 1433_16mer_ 5mU-lnGpslnGpslnApsTps(2s)Tps(5m)CpsApsGps(5m)CpsGps(5m) 3-10-3 Cps(5m)CpsGpslnApsscp(5m)CpslnG3 111 47F 1433_16mer_ 5mU- 3-10-3 lnGpslnGpslnApsTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m) CpsGpslnApsln(5m)CpslnG3 112 73A 381_17mer_ 5lnGpslnApslnTpslnApslnApsApsAps(5m)CpsGps(5m)Cps(5m)Cps 5-8-4 Gps(5m)CpslnApslnGpslnApsscp(5m)C3 113 73B 381_17mer_ 5mA-lnGpslnApslnTpslnApslnApsApsAps(5m)CpsGps(5m)Cps(5m) 5-8-4 CpsGps(5m)CpslnApslnGpslnApsscp(5m)C3 114 73C 381_17mer_ 5mA-lnGpslnApslnTpslnApslnAps(8nh)ApsAps(5m)CpsGps(5m) 5-8-4 Cps(5m)CpsGps(5m)CpslnApslnGpslnApsscp(5m)C3 115 34A 1575_15mer_ 5mU- 3-10-2 lnGpln(5m)CpsscpGpsAps(8nh)ApsGpsTpsGps(5m)CpsAps(5m)Cps Aps(5m)CpslnGpslnG3 116 40A 246_17mer_ 5mA- 3-10-4 ln(5m)Cpsscp(5m)CpslnApsln(5m)Cps(5m)CpsAps(5m)CpsGpsAps GpsTps(5m)CpsTpsApslnGpslnApsln(5m)C3 117 23A 1580_16mer_ 5mU- 3-9-4 lnGpslnGpsscpTpsGpsApsApsGps(5m)CpsGpsApsApsGpslnTpsln Gpsln(5m)CpslnA3
[0078] In some embodiments, the ASO comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the sequences listed in Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19. In some embodiments, the ASO is ASO 120 or ASO 121.
[0079] In some embodiments, the ASOs of the disclosure have a sequence that differs from an ASO of Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19 by one nucleoside. In other embodiments, the ASO has a sequence that differs from an ASO of Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19 by 1, 2, 3 or 4 nucleosides. In some embodiments, the nucleotide sequence is at least 90% identical to a nucleotide sequence selected from Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19. In some embodiments, the ASOs of the disclosure have a sequence of Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19, but one T in the central region is replaced by (2s)T, one C in the central region is replaced by (5OH)C, and/or one A is replaced by (8NH)A in the central region. In some embodiments, the ASOs of the disclosure have a sequence of Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19, but with one or two ScpBNA, AmNA, or GuNA in the 5 wing portion. In some embodiments, the ASOs of the disclosure have a sequence of Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19, but with one or two ScpBNA, AmNA, or GuNA in the 3 wing portion. In some embodiments, the ASOs of the disclosure have a sequence of Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19, but with a mA or mU appended to the 5 end of the sequence. In some embodiments, the ASOs of the disclosure have a sequence of Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19, but with a mA or mU appended to the 5 end of the sequence that links to a GalNAc derivative (e.g., GalNAc4, such as GalNAc4-(PS)2-p-, or GalNAc6, such as GalNAc6-(PS)2-p-), as detailed herein.
[0080] In some embodiments, the ASO comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of any one of SEQ ID NOs: 2-428. In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO: 400 or 404.
[0081] In some embodiments, the ASOs of the disclosure have a sequence that differs from any of the nucleotides of SEQ ID NOs: 2-428 by one nucleoside. In other embodiments, the ASO has a sequence that differs from any of the nucleotides of SEQ ID NOs: 2-428 by 1, 2, 3 or 4 nucleosides. In some embodiments, the nucleotide sequence is at least 90% identical to a nucleotide sequence of any one of SEQ ID NOs: 2-428. In some embodiments, the ASOs of the disclosure have a sequence of any one of SEQ ID NOs: 2-428, but one T in the central region is replaced by (2s)T, one C in the central region is replaced by (5OH)C, and/or one A is replaced by (8NH)A in the central region. In some embodiments, the ASOs of the disclosure have a sequence of any one of SEQ ID NOs: 2-428, but with one or two ScpBNA, AmNA, or GuNA in the 5 wing portion. In some embodiments, the ASOs of the disclosure have a sequence of any one of SEQ ID NOs: 2-428, but with one or two ScpBNA, AmNA, or GuNA in the 3 wing portion. In some embodiments, the ASOs of the disclosure have a sequence of any one of SEQ ID NOs: 2-428, but with a mA or mU appended to the 5 end of the sequence. In some embodiments, the ASOs of the disclosure have a sequence of any one of SEQ ID NOs: 2-428, but with a mA or mU appended to the 5 end of the sequence that links to a GalNAc derivative (e.g., GalNAc4, such as GalNAc4-(PS)2-p-, or GalNAc6, such as GalNAc6-(PS)2-p-), as detailed herein.
[0082] The present disclosure is also directed to additional components conjugated to the ASO such as targeting moieties and oligonucleotides modified at one or more end.
[0083] In some embodiments, the targeting moiety may comprise a carbohydrate, such as a monosaccharide, for example N-acetylgalactosamine (GalNAc), di saccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides. In certain embodiments, the targeting moiety one or more GalNAc derivatives, such as two or three GalNAc derivatives attached to the ASO through one or more linkers, optionally in a consecutive structure. In certain embodiments, the targeting moiety comprises three consecutive GalNAc moieties attached through linkers, such as:
##STR00007##
[0084] In some embodiments, the ASO contains a targeting moiety at the 5-end, the 3-end, or both ends of the ASO.
[0085] In certain embodiments, the ASO is modified at one or more end by a vinyl phosphonate moiety, such as a 5-vinyl phosphonate moiety.
[0086] Compositions
[0087] The present disclosure also encompasses pharmaceutical compositions comprising ASOs of the present disclosure. One embodiment is a pharmaceutical composition comprising one or more ASO of the present disclosure, and a pharmaceutically acceptable diluent or carrier.
[0088] In some embodiments, the pharmaceutical composition containing the ASO of the present disclosure is formulated for systemic administration via parenteral delivery. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; also subdermal administration, e.g., via an implanted device. In a preferred embodiment, the pharmaceutical composition containing the ASO of the present disclosure is formulated for subcutaneous (SC) or intravenous (IV) delivery. Formulations for parenteral administration may include sterile aqueous solutions, which may also contain buffers, diluents and other pharmaceutically acceptable additives as understood by the skilled artisan. For intravenous use, the total concentration of solutes may be controlled to render the preparation isotonic.
[0089] The pharmaceutical compositions containing the ASO of the present disclosure are useful for treating a disease or disorder, e.g., associated with the expression or activity of an HBV gene.
[0090] 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, and a second ASO of the present disclosure that is complementary or hybridizes to a viral target RNA sequence in a second X region or an S region of HBV, and a pharmaceutically acceptable diluent or carrier. When the pharmaceutical composition comprises two or more ASOs, the ASOs may be present in varying amounts. For example, in some embodiments, the weight ratio of first ASO to 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 first ASO to 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.
[0091] Methods of Use
[0092] One aspect of the present technology includes methods for treating a subject diagnosed as having, suspected as having, or at risk of having an HBV infection and/or an HBV-associated disorder. In therapeutic applications, compositions comprising one or more ASO of the present technology are administered to a subject suspected of, or already suffering from such a disease (such as, e.g., persistence of HBV cccDNA, presence of an HBV antigen (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, the symptoms of the disease, including its complications and intermediate pathological phenotypes in development of the disease.
[0093] Subjects suffering from an HBV infection and/or an HBV-associated disorder 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 an HBV-associated disorder include, but are not limited to the presence of liver HBV cccDNA, the presence of serum and/or liver HBV antigen (e.g., HBsAg and/or HBeAg), elevated ALT, elevated AST, the absence or low level of anti-HBV antibodies, liver injury, 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, fatigability, disordered gustatory acuity and smell sensations (aversion to food and cigarettes), right upper quadrant and epigastric pain (intermittent, mild to moderate), hepatic encephalopathy, somnolence, disturbances in sleep pattern, mental confusion, coma, ascites, gastrointestinal bleeding, coagulopathy, jaundice, hepatomegaly (mildly enlarged, soft liver), splenomegaly, palmar erythema, spider nevi, muscle wasting, spider angiomas, vasculitis, variceal bleeding, peripheral edema, gynecomastia, testicular atrophy, abdominal collateral veins (caput medusa), high levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (within a range of 1000-2000 IU/mL), ALT levels higher than AST levels, elevated gamma-glutamyl transpeptidase (GGT) and/or alkaline phosphatase (ALP) levels, decreased albumin levels, elevated serum iron levels, leukopenia (i.e granulocytopenia), lymphocytosis, increased erythrocyte sedimentation rate (ESR), shortened red blood cell survival, hemolysis, thrombocytopenia, a prolongation of the international normalized ratio (INR), the presence of serum HBV DNA, elevation of the aminotransferases (<5 times the ULN), increased bilirubin levels, prolonged prothrombin time (PT), hyperglobulinemia, the presence of tissue-nonspecific antibodies, such as anti-smooth muscle antibodies (ASMAs) or antinuclear antibodies (ANAs), the presence of tissue-specific antibodies, such as antibodies against the thyroid gland, elevated levels of rheumatoid factor (RF), hyperbilirubinemia, low platelet and white blood cell counts, AST levels higher than ALT levels, lobular inflammation accompanied by degenerative and regenerative hepatocellular changes, and predominantly centrilobular necrosis.
[0094] In some embodiments, subjects treated with the ASO composition of the present technology will show amelioration or elimination of one or more of the following conditions or symptoms: presence of liver HBV cccDNA, the presence of serum and/or liver HBV antigen (e.g., HBsAg and/or HBeAg), the absence or low level of anti-HBV antibodies, liver injury, 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, fatigability, disordered gustatory acuity and smell sensations (aversion to food and cigarettes), right upper quadrant and epigastric pain (intermittent, mild to moderate), hepatic encephalopathy, somnolence, disturbances in sleep pattern, mental confusion, coma, ascites, gastrointestinal bleeding, coagulopathy, jaundice, hepatomegaly (mildly enlarged, soft liver), splenomegaly, palmar erythema, spider nevi, muscle wasting, spider angiomas, vasculitis, variceal bleeding, peripheral edema, gynecomastia, testicular atrophy, abdominal collateral veins (caput medusa), ALT levels higher than AST levels, leukopenia (i.e granulocytopenia), decreased albumin levels, elevated serum iron levels, lymphocytosis, increased erythrocyte sedimentation rate (ESR), shortened red blood cell survival, hemolysis, thrombocytopenia, a prolongation of the international normalized ratio (INR), the presence of serum HBV DNA, prolonged prothrombin time (PT), hyperglobulinemia, the presence of tissue-nonspecific antibodies, such as anti-smooth muscle antibodies (ASMAs) or antinuclear antibodies (ANAs), the presence of tissue-specific antibodies, such as antibodies against the thyroid gland, hyperbilirubinemia, low platelet and white blood cell counts, AST levels higher than ALT levels, lobular inflammation accompanied by degenerative and regenerative hepatocellular changes, and predominantly centrilobular necrosis.
[0095] The present disclosure provides a method for treating a subject diagnosed as having, or suspected as having an HBV infection and/or an HBV-associated disorder comprising administering to the subject an effective amount of an ASO composition of the present technology. 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 hybridizes to a viral target RNA sequence in a first X region of HBV, and the second ASO is complementary or hybridizes to a viral target RNA sequence in a second X region or an S region of HBV. In some embodiments, the second ASO is complementary or hybridizes to the viral target RNA sequence in the second X region of HBV. In other embodiments, the second ASO is complementary or hybridizes to the viral target RNA sequence in the S region of HBV
[0096] The ASOs of the present disclosure may be used to treat a disease in a subject in need thereof. In some embodiments, a method of treating a disease in a subject in need thereof comprises administering to the subject any of the ASOs disclosed herein. In some embodiments, a method of treating a disease in a subject in need thereof comprises administering to the subject any of the compositions disclosed herein.
[0097] Administration of the ASO may be conducted by methods known in the art. In some embodiments, the ASO is administered by subcutaneous (SC) or intravenous (IV) delivery. The preparations (e.g., ASOs or compositions) of the present disclosure may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. In some embodiments, subcutaneous administration is preferred.
[0098] The phrases parenteral administration and administered parenterally as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
[0099] The phrases systemic administration, administered systemically, peripheral administration and administered peripherally as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
[0100] These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
[0101] Regardless of the route of administration selected, the compounds (e.g., ASOs) of the present disclosure, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present disclosure, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
[0102] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
[0103] The selected dosage level will depend upon a variety of factors including the activity of the particular compound (e.g., ASO) of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
[0104] 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 could start doses of the compounds (e.g., ASOs) of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
[0105] In general, a suitable daily dose of a compound (e.g., ASO) of the disclosure is the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose generally depends upon the factors described above. Preferably, the compounds are administered at about 0.01 mg/kg to about 200 mg/kg, more preferably at about 0.1 mg/kg to about 100 mg/kg, even more preferably at 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, the compound is administered at a dose equal to or greater than 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. In some embodiments, the compound is administered at a dose equal to or greater than 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. In some embodiments, the compound is administered at a dose equal to or less than 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. In some embodiments, the total daily dose of the compound is equal to or greater than 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.
[0106] If desired, the effective daily dose of the active compound (e.g., ASO) may be administered as two, three, four, five, six, seven, eight, nine, ten or more doses or sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. 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. Preferred dosing is one administration per 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 a 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 a 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 every month. 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 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, 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 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 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 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 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 months. In some embodiments, the compound is administered at least once 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 weeks. In some embodiments, the compound is administered at least once 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 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 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 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 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.
[0107] The subject of the described methods may be a mammal, and it includes humans and non-human mammals. In some embodiments, the subject is a human, such as an adult human.
[0108] Some embodiments include a method for treating an HBV virus in a subject infected with the virus comprising administering a therapeutically effective amount of one or more ASO of the present disclosure or a composition of the present disclosure to the subject in need thereof thereby reducing the viral load of the virus in the subject and/or reducing a level of a virus antigen in the subject. The ASO may be complementary or hybridize to a portion of the target RNA in the virus, e.g., a second X region and/or an S region of HBV.
[0109] In some embodiments, a modified oligonucleotide as described herein can be used in combination with one or more additional agent(s) for treating and/or inhibiting replication HBV and/or HDV. When the compounds (e.g., ASOs) described herein are co-administered with an additional agent, the effective amount may be less than when the compound is used alone. Additional agents include, but are not limited to, an interferon, nucleoside/nucleotide analogs, a capsid assembly modulator (CAM), siRNA, other ASOs, Nucleic Acid Polymers or S-Antigen Transport-inhibiting Oligonucleotide Polymers (NAPs or STOPS), an entry inhibitor and/or a small molecule immunomodulator. Examples of additional agents include ALG-010133, ALG-000184, recombinant interferon alpha 2b, IFN-, PEG-IFN--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-H0731, 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 STOPS. Exemplary STOPS are described in International Publication No. WO2020/097342 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. Exemplary CAMs are described in 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 Ser. Nos. 16/789,298, 16/837,515, and 16/849,851, each which is incorporated by reference in its entirety. 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 administering the additional agent. In some embodiments, the ASO is administered after administering the additional agent.
Definitions
[0110] Certain ranges are presented herein with numerical values being preceded by the term about. The term about is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
[0111] Unless defined otherwise, 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 illustrative methods and materials are now described.
[0112] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that 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 specifically 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.
[0113] This disclosure is not limited to particular embodiments described, as such may vary. 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, since the scope of the present invention will be limited only by the appended claims.
[0114] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
[0115] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference 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 dates of publication provided may be different from the actual publication dates that may need to be independently confirmed.
EXAMPLES
[0116] The following examples illustrate certain embodiments of the present disclosure to aid the skilled person in practicing the disclosure. Accordingly, the examples are in no way considered to limit the scope of the disclosure.
Example 1: ASO Synthesis
[0117] Gapmer ASO Sequences
[0118] The DNA, 2-O-Me, 2-MOE and LNA phosphoramidite monomers were procured from commercially available sources (Hongene Biotech USA Inc.). All the monomers were dried in vacuum desiccator with desiccants (P.sub.2O.sub.5, RT 24h). Universal solid supports (CPG) attached were obtained from ChemGenes corporation. The chemicals and solvents for synthesis workflow were purchased from VWR/Sigma commercially available sources and used without any purification or treatment. Solvent (acetonitrile) and solutions (amidite and activator) were stored over molecular sieves during synthesis.
[0119] The control and target oligonucleotide sequences were synthesized on an Expedite 8909 synthesizer using the standard cycle written by the manufacturer with modifications to a few wait steps and modified coupling steps. The solid support was controlled pore glass and the monomers contained standard protecting groups. Each chimeric oligonucleotide was individually synthesized using commercially available 5-O-(4,4-dimethoxytrityl)-3-O-(2-cyanoethyl-N, N-diisopropyl) DNA, 2-OMe, 2-MOE and or LNA phosphoramidite monomers of 6-N-benzoyladenosine (A.sup.Bz), 4-N-acetylcytidine (C.sup.Ac), 2-N-isobutyrylguanosine (G.sup.iBu), and Uridine (U) or Thymidine (T), according to standard solid phase Phosphoramidite synthesis protocols. The 2-O-Me-2,6-diaminopurine phosphoramidite was purchased from Glen Research. The phosphoramidites were prepared as 0.1 M solutions in anhydrous acetonitrile. 5-Ethylthiotetrazole was used as activator, 3% dichloroacetic acid in dichloromethane was used to detritylate, acetic anhydride in THF and 16% N-methylimidazole in THF were used to cap, and DDTT ((dimethylamino-methylidene) amino)-3H-1,2,4-dithiazaoline-3-thione was used as the sulfur-transfer agent for the synthesis of oligoribonucleotide phosphorothioates. An extended coupling of 0.1M solution of phosphoramidite in CH.sub.3CN in the presence of 5-(ethylthio)-1H-tetrazole activator to a solid bound oligonucleotide followed by extended capping, oxidation and deprotection afforded modified oligonucleotides. The stepwise coupling efficiency of all modified phosphoramidites was more than 98.5%.
[0120] Deprotection and cleavage from the solid support was achieved with mixture of ammonia methylamine (1:1, AMA) for 15 min at 65 C., when the universal linker was used, the deprotection was left for 90 min at 65 C. or solid supports were heated with aqueous ammonia (28%) solution at 55 C. for 8 h to deprotect the base labile protecting groups.
[0121] After filtering to remove the solid support, the deprotection solution was removed under vacuum in a GeneVac centrifugal evaporator.
##STR00008## ##STR00009## ##STR00010##
The 2-MOE phosphoramidites
##STR00011## ##STR00012##
The Locked Nucleic Acid (LNA) Phosphoramidite
[0122] ##STR00013##
[0123] Modified Gapmer Sequences
[0124] The AmNA (N-Me)-T, AmNA (N-Me)-4-N-benzoyl (5m) cytidine ((5m) C.sup.Bz), AmNA (N-Me)-4-N-benzoylcytidine (A.sup.Bz), and AmNA (N-Me)-2-N-pac (G.sup.pac), were purchased from Luxna Biotech, whereas scp-BNA-T, scp-BNA-6-N-benzoyladenosine (A.sup.Bz), scp-BNA-4-N-benzoyl-5 methyl cytidine ((5m) C.sup.Bz), scp-BNA-2-N-iguanosine (G.sup.iBu) phosphoramidite monomers synthesized by following the procedure described in 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 the monomers were dried in a vacuum desiccator with desiccants (KOH and P.sub.2O.sub.5, at room temperature for 24 hours). In the case of AmNA(N-Me)-PS-DNA-PS and scp-BNA-PS-DNA-PS, modifications the synthesis was carried out on a 1 M scale in a 3 to 5 direction with the phosphoramidite monomers diluted to a concentration of 0.12 M in anhydrous CH.sub.3CN in the presence of 0.3 M 5-(benzylthio)-1H-tetrazole activator (coupling time 16 min) to a solid bound oligonucleotide followed by modified capping, oxidation and deprotection afforded modified oligonucleotides. The stepwise coupling efficiency of all modified phosphoramidites was more than 97%. The DDTT (dimethylamino-methylidene) amino)-3H-1,2,4-dithiazaoline-3-thione was used as the sulfur-transfer agent for the synthesis of oligoribonucleotide phosphorothioates. Oligonucleotide-bearing solid supports were washed with 20% DEA solution in acetonitrile for 15 min then column was washed thoroughly with MeCN. The support was heated at 65 C. with diisopropylamine:water:methanol (1:1:2) for 8 h in heat block to cleavage from support and deprotect the base labile protecting groups.
AmNA (N-Me) Monomers
[0125] ##STR00014## ##STR00015##
scp-BNA Monomers
##STR00016## ##STR00017##
[0126] 5 and 3-GalNAc conjugated oligonucleotides were synthesized with various length GalNAc moieties, e.g., as described below. The GalNAc3, GalNAc4, GalNAc5 and GalNAc6 were conjugated to oligonucleotides during synthesis with 1 2, or 3 moieties in the same manner as described below. Further GalNAc moieties, such as GalNAc-1 and GalNAc-2, which are described previously herein, are also used to form 5 and 3-GalNAc using post synthesis conjugation.
GalNAc Phosphoramidites
[0127]
TABLE-US-00002 After Attachment to GalNAc building blocks Oligos (Nomenclature) GalNAc-3 phosphoramidite (GalNAc3-(PS)2-p)
[0128] Quantitation of Crude Oligomer or Raw Analysis
[0129] Samples were dissolved in deionized water (1.0 mL) and quantitated as follows: Blanking was first performed with water alone on Nanodrop UV spectrophotometer. Nano Drop instruments can measure a wide concentration range of nucleic acids through use of multiple path lengths. The most accurate quantification results can be achieved by measuring diluted oligonucleotides with an absorbance at 260 nm. The crude material is stored at 20 C.
[0130] Crude HPLC/LC-MS Analysis
[0131] The 0.1 OD of the crude samples were used for crude MS analysis. After confirming the crude LC-MS data, then the purification step was performed.
[0132] HPLC Purification
[0133] The Phosphodiester (PO), Phosphorothioate (PS) and chimeric modified oligonucleotides were purified by anion-exchange HPLC. The buffers were 20 mM sodium phosphate in 10% CH.sub.3CN, pH 8.5 (buffer A) and 20 mM sodium phosphate in 10% CH.sub.3CN, 1.8 M NaBr, pH 8.5 (buffer B). Fractions containing full-length oligonucleotides were pooled, desalted and lyophilized.
[0134] The lipid conjugated oligonucleotides were purified by an in-house packed RPC-Source15 reverse-phase column. The buffers were 20 mM sodium acetate in 10% CH.sub.3CN, (buffer A) and CH.sub.3CN (buffer B). Fractions containing full-length oligonucleotides were pooled, desalted and lyophilized.
[0135] Desalting of Purified Oligomer
[0136] The purified dry oligomer was then desalted using Sephadex G-25 M (Amersham Biosciences). The cartridge was conditioned with 10 mL of deionized water thrice. The purified oligonucleotide dissolved thoroughly in 2.5 mL deionized water was applied to the cartridge with very slow drop wise elution. The salt free oligomer was eluted with 3.5 ml deionized water directly into a screw cap vial.
[0137] Final HPLC and Electrospray LC/MS Analysis
[0138] Approximately 0.10 OD of oligomer is dissolved in water and then pipetted in special vials for IEX-HPLC and LC/MS analysis. Analytical HPLC and ES LC-MS established the integrity of the chimeric oligonucleotides.
[0139] Post-Synthesis Conjugation of GalNAc Esters to Oligonucleotides
[0140] 5-C6-Amino Precursor Synthesis
[0141] The sequences were synthesized at 10 mol scale using universal support (Loading 65 mol/g). At the 5-terminal to introduce C6-NH.sub.2 linker the 6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N, N-diisopropyl)-phosphoramidite in 0.1 M Acetonitrile was used with coupling time 10 min. The Oligonucleotide-bearing solid supports were heated at room temperature with aqueous ammonia/Methylamine (1:1) solution for 3 h in shaker to cleavage from support and deprotect the base labile protecting groups. After IEX purification and desalting the C6-NH.sub.2 modified ASO's was used to perform post synthesis conjugation.
##STR00019##
GalNAc Ester for Conjugation
[0142] ##STR00020##
[0143] Post Synthesis Conjugation of 5-GalNAc Synthesis
[0144] The 5-C6-NH.sub.2 modified sequences were dissolved in 0.2 M Sodium bicarbonate buffer, pH 8.5 (0.015 mM) and 5-7 mol equivalent of GalNAc ester dissolved in DMSO was added. The reaction mixture was stirred at room temperature for 4 h. The sample was analyzed to confirm if any unreacted amino modified ASO's is present. To this aqueous ammonia (28 wt. %) was added (5 reaction volume) and stirred at room temperature for 2-3 h. Reaction mixture concentrated under reduced pressure and residue dissolved in water and purified by HPLC on a strong anion exchange column.
Example 2. HBsAg Release Assay Protocol (HepG2.2.15)
[0145] HepG2.2.15 cells (a stable cell line with four integrated HBV genomes) were maintained in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, 1% Glutamine, 1% non-essential amino acids, 1% Sodium Pyruvate and 250 m/ml G418. Cells were maintained at 37 C. in a 5% CO.sub.2 atmosphere. For HBsAg release assay, assay medium was made: DMEM with 5% FBS, 1% penicillin/streptomycin, 1% Glutamine and 1% DMSO. The day before assay, trypsinize HepG2.2.15 cells were washed with Assay Medium once, spun at 250 g5 min, resuspended with Assay Medium, and seed cells at 50,000/well in assay medium in collagen coated 96 well plates. On the next day, ASOs were diluted with Opti-MEM, 9-pt, 3-fold dilution and Lipofectamine RNAiMAX (Invitrogen) was diluted according manufacturer's manual. The ASO dilution and RNAiMAX dilution was mixed, left at room temperature for 5 minutes and 15 l was added to each well of 96 well plate. The plates were left at 37 C., 5% CO.sub.2 in an incubator for 5 days. After incubation, the supernatant was harvested and measured for HBsAg with ELISA kit (Diasino). The cell viability was measured with CellTiter-Glo (Promega). The EC.sub.50, the concentration of the drug required for reducing HBsAg secretion by 50% in relation to the untreated cell control was calculated using the Prism Graphpad. The CC.sub.50, the concentration of the drug required for reducing cell viability by 50% in relation to the untreated cell control was calculated with the same software.
[0146] The resulting EC.sub.50 and CC.sub.50 for the compounds in Table 1 are presented in the following Table 2. The EC.sub.50 values are as follows: A: <0.1 nM, B: 0.1 nM-1 nM, C: 1-10 nM.
TABLE-US-00003 TABLE 2 HBsAg Release Assay HepG2.2.15 HepG2.2.15 Cell HBsAg Release Viability CC.sub.50 ASO # Assay EC.sub.50 nM 1 A >10 2 A >10 3 A >10 4 A >10 5 B >10 6 B >10 7 B >10 8 B >10 9 B >10 10 B >10 11 B >10 12 B >10 13 B >10 14 B >10 15 B >10 16 B >10 17 B >10 18 B >10 19 B >10 20 B >10 21 B >10 22 B >10 23 B >10 24 B >10 25 B >10 26 B >10 27 B >10 28 B >10 29 B >10 30 B >10 31 B >10 32 B >10 33 B >10 34 B >10 35 B >10 36 B >10 37 B >10 38 B >10 39 B >10 40 B >10 41 B >10 42 B >10 43 B >10 44 B >10 45 B >10 46 B >10 47 B >10 48 B >10 49 B >10 50 B >10 51 B >10 52 B >10 53 C >10 54 C >10 55 C >10 56 C >10 57 C >10 58 C >10 59 C >10 60 C >10 61 C >10 62 C >10 63 C >10 64 C >10 65 C >10 66 C >10 67 C >10 68 C >10 69 C >10 70 C >10 71 C >10 72 C >10 73 C >10 74 C >10 75 C >10 76 C >10 77 C >10 78 C >10 79 C >10 80 C >10 81 C >10 82 C >10 83 C >10 84 C >10 85 C >10 86 C >10 87 C >10 88 C >10 89 B >10 90 C >10 91 C >10 92 B >10 93 C >10 94 C >10
TABLE-US-00004 TABLE2A SerumHBsAgLogReduction(nadir)with1x5mg/kg SEQ Location, ID ASO length& Serum NO. # Sequence5to3 structure HBsAg 118 116 5lnGpslnApslnTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps 1432_17mer_ B (5m)CpsGpsApsln(5m)CpslnGpslnGpslnG3 3-10-4 119 117 5lnGpslnGpslnApsTpsTps(5m)Cps 1580_16mer_ B ApsGps(5m)CpsGps(5m)Cps(5m)CpsGpslnApsln(5m)CpslnG 3-10-3 3 120 118 5ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)CpsAps(5m)Cps 1576_17mer_ B GpsApsGpsTps(5m)CpsTpsApslnGpslnApsln(5m)C3 4-10-3 121 119 5lnGpslnApslnTpslnApslnApsApsApsAps(5m)CpsGps(5m) 1431_17mer_ C Cps(5m)CpsGps(5m)CpsCpslnApslnGpslnApsln(5m)C3 5-8-4
[0147] In Table 2A, the bold nucleosides contain the following modifications:
##STR00021##
Example 3. GalNac ASO Testing in AAV-HBV Mouse Model
[0148] AAV/HBV is a recombinant AAV carrying replicable HBV genome. Taking advantage of the highly hepatotropic feature of genotype 8 AAV, the HBV genome can be efficiently delivered to the mouse liver cells. Infection of immune competent mouse with AAV/HBV can result in long term HBV viremia, which mimics chronic HBV infection in patients. The AAV/HBV model was used to evaluate the in vivo activity of various types of anti-HBV agents. Mice were infected with AAV-HBV on day 28 of the study. The test articles or negative control (PBS) were dosed subcutaneously (unless specified otherwise) as a single dose on day 0 at 5 mg/kg, or single dose on day 0 at 10 mg/kg; or 310 mg/kg once a week (QW); or 310 mg/kg every 3 days (Q3D); or 510 mg/kg Q3D; or 6 doses of 3 mg/kg on days 0, 3, 7, 14, 21, 28; or 6 doses of 10 mg/kg on days 0, 3, 7, 14, 21, 28. Serial blood collections were taken every 5 days on day 0, 5, 10 and 15; or longer duration depending each study design. Serum HBV S antigen (HBsAg),E antigen (HBeAg) and ALT were assayed through the following methods:
TABLE-US-00005 Parameters Equipment Reagent HBsAg ARCHITECT i2000 (Abbott HBsAg Reagent Kit (Abbott Ireland Laboratories, Lake Bluff, IL, Diagnostics Division, Finisklin Business USA) Park Sligo, IRL) Catalog: 6C36 / 08P08 HBeAg ARCHITECT i2000 (Abbott HBeAg Reagent Kit (Abbott GmbH & Laboratories, Lake Bluff, IL, USA) Co. KG, Wiesbaden, GER) Catalog: 6C32 / 07P64 Alanine Roche Cobas 6000 c501 Chemistry Alanine Aminotransferase acc. to IFCC Aminotransferase Analyzer (Roche Diagnostics, (Roche Diagnostics, Mannheim, GER) (ALT) Mannheim, GER) Catalog: ACN 685
[0149] The resulting nadir log.sub.10 reduction in serum HBsAg during the study are presented in the following table, where A1 log.sub.10 reduction in HBsAg, B is 0.5-1 log.sub.10 reduction in HBsAg, and C is <0.5 log.sub.10 reduction in HBsAg
TABLE-US-00006 TABLE3 SerumHBsAgLogReduction(nadir)formicetreatedwith1x5mg/kgASO SEQ Location, ID ASO length& Serum No. # Sequence5to3 structure HBsAg 122 95 5-GalNAc1-C6-p- 1432_16mer_ B lnGpslnApslnTpslnTps(5m)CpsApsGps(5m)CpsGps(5m) 4-9-3 Cps(5m)CpsGpsApsln(5m)CpslnGpslnG3 123 96 5-GalNAc3-(PS)2-p- 1580_15mer_ C lnGpslnTpslnGpsApsApsGps(5m)CpsGpsApsApsGpsTpsln 3+9+3 Gpsln(5m)CpslnA3 124 97 5-GalNAcl-C6-p- 1576_16mer_ B lnApslnApslnGpsln(5m)CpsGpsApsApsGpsTpsGps(5m)m 4-8-4 CpsApsln(5m)CpslnApsln(5m)CpslnG3 125 98 5-GalNAc3-(PS)2-p- 1431_17mer_ A lnGpslnApslnTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps 3-10-4 (5m)CpsGpsApsln(5m)CpslnGpslnGpslnG3 126 99 5-GalNAc3-(PS)2-p- 1582_16mer_ B lnGpslnApslnGpsGpsTpsGpsApsApsGps(5m)CpsGpsApsAps 3-10-3 lnGpslnTpslnG3 127 100 5-GalNAc3-(PS)2-p- 246_17mer_ A ln(5m)Cpsln(5m)CpslnAps(5m)Cps(5m)CpsAps(5m) 3-10-4 CpsGpsApsGpsTps(5m)CpsTpslnApslnGpslnApsln(5m)C 3 128 101 5-GalNAc3-(PS)2-p- 1575_15mer_ B lnGpsln(5m)CpslnGpsApsApsGpsTpsGps(5m)CpsAps(5m) 3-10-2 CpsAps(5m)CpslnGpslnG3 129 102 5-GalNAc3-(PS)2-p- 1432_17mer_ C lnGpslnGpslnApslnTpsTps(5m)CpsApsGps(5m)Cps 4-9-4 Gps(5m)Cps(5m)CpsGpslnApsln(5m)CpslnGpslnG3 130 103 5-GalNAc3-(PS)2-p- 1581_15mer_ B lnGpslnGpslnTpsGpsApsApsGps(5m)CpsGpsAps 3-10-2 ApsGpsTpslnGpsln(5m)C3 131 104 5-GalNAc3-(PS)2-p- 1576_15mer_ B lnApslnGpsln(5m)CpsGpsApsApsGpsTpsGps(5m)CpsAps 3-10-2 (5m)CpsApsln(5m)CpslnG3 132 105 5-GalNAc3-(PS)2-p-- 1582_15mer_ A lnApslnGpslnGpsTpsGpsApsApsGps(5m)CpsGpsApsApsG 3-10-2 pslnTpslnG3 133 106 5-GalNAc5-(PS)2-p-po- 1517_15mer_ A lnApslnGpslnGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m) 3-9-3 Cps(5m)CpsGpslnTpslnGpslnG3 134 107 5-GalNAcl-C6-p-CA- 1433_16mer_ A lnGpslnGpslnApsTpsTps(5m)CpsApsGps(5m)CpsGps(5m) 3-10-3 Cps(5m)CpsGpslnApsln(5m)CpslnG3 135 108 5-GalNAcl-C6-p-CA- 1519_16mer_ A lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps 3-10-3 (5m)Cps(5m)Cpsln(5m)CpslnGpslnT3 136 109 5-GalNAcl-C6-p-CA- 1581_16mer_ A lnApslnGpslnGpslnTpsGpsApsApsGps(5m)CpsGpsApsAps 4-9-3 GpslnTpslnGpsln(5m)C3 137 110 5-GalNAcl-C6-p-CA- 1580_16mer_ A lnGpslnGpslnTpsGpsApsApsGps(5m)CpsGpsApsApsGpsln 3-9-4 TpslnGpsln(5m)CpslnA3 138 111 5-GalNAcl-C6-p-CA- 191_16mer_ A ln(5m)Cpsln(5m)CpslnGpsln(5m)Cps(5m)CpsTpsGpsTps 4-8-4 ApsAps(5m)CpsApsln(5m)CpslnGpslnApslnG3 139 112 5-GalNAcl-C6-p-CA- 381_17mer_ A lnGpslnApslnTpslnApslnApsApsAps(5m)CpsGps(5m)Cps 5-8-4 (5m)CpsGps(5m)CpslnApslnGpslnApsln(5m)C3 140 113 5-GalNAcl-C6-p-CA- 246_17mer_ A ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)CpsAps(5m)Cps 4-10-3 GpsApsGpsTps(5m)CpsTpsApslnGpslnApsln(5m)C3 141 114 5-GalNAcl-C6-p-CA- 245_17mer_ A ln(5m)CpslnApsln(5m)Cpsln(5m)CpsAps(5m)CpsGpsApsG 4-10-3 psTps(5m)CpsTpsApsGpslnApsln(5m)CpslnT3 142 115 5-GalNAcl-C6-p-CA- 191_16mer_ A ln(5m)Cpsln(5m)CpslnGps(5m)Cps(5m)CpsTpsGpsTpsAps 3-9-4 Aps(5m)CpsApsln(5m)CpslnGpslnApslnG3 143 108A 5-GalNAc5-(PS)2-p- 1519_16mer_ lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps 3-10-3 (5m)Cps(5m)Cpsln(5m)CpslnGpslnT3 144 108B 5-GalNAc5-(PS)2-p- 1519_16mer_ lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps 3-10-3 (5m)Cps(5m)Cpsln(5m)CpslnGpsAmT3 145 108C 5-GalNAc5-(PS)2-p- 1519_16mer_ lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps 3-10-3 (5m)Cps(5m)CpsAm(5m)CpslnGpslnT3 146 108D 5-GalNAc5-(PS)2-p- 1519_16mer_ lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps 3-10-3 (5m)Cps(5m)Cpsln(5m)CpslnGpsscpT3 147 108E 5-GalNAc5-(PS)2-p- 1519_16mer_ lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps 3-10-3 (5m)Cps(5m)Cps(5m)scpCpslnGpslnT3 148 107A 5-GalNAc6-(PS)2-p- 1433_16mer_ lnGpslnGpslnApsTpsTps(5m)CpsApsGps(5m)CpsGps(5m) 3-10-3 Cps(5m)CpsGpslnApsln(5m)CpslnG3 149 107B 5-GalNAc6-(PS)2-p- 1433_16mer_ lnGpslnGpslnAps(2s)TpsTps(5m)CpsApsGps(5m)CpsGps 3-10-3 (5m)Cps(5m)CpsGpslnApsln(5m)CpslnG3 150 107C 5-GalNAc6-(PS)2-p- 1433_16mer_ lnGpslnGpslnApsTps(2s)Tps(5m)CpsApsGps(5m)CpsGps 3-10-3 (5m)Cps(5m)CpsGpslnApsln(5m)CpslnG3 151 107D 5-GalNAc6-(PS)2-p- 1433_16mer_ lnGpslnGpslnApsTpsTps(5oh)CpsApsGps(5m)CpsGps(5m) 3-10-3 Cps(5m)CpsGpslnApsln(5m)CpslnG3 152 107E 5-GalNAc4-(PS)2-p-mU-po- 1433_16mer_ lnGpslnGpslnApsTps(2s)Tps(5m)CpsApsGps(5m)CpsGps 3-10-3 (5m)Cps(5m)CpsGpslnApsln(5m)CpslnG3 153 107F 5-GalNAc4-(PS)2-p-mU-po- 1433_16mer_ lnGpslnGpslnApsTps(2s)Tps(5m)CpsApsGps(5m)CpsGps 3-10-3 (5m)Cps(5m)CpsGpslnApsscp(5m)CpslnG-3 154 73B- 5-GalNAc4-(PS)2-p-mA-po- 381_17mer_ G lnGpslnApslnTpslnApslnApsApsAps(5m)CpsGps(5m)Cps 5-8-4 (5m)CpsGps(5m)CpslnApslnGpslnApsscp(5m)C-3 155 73C- 5-GalNAc4-(PS)2-p-mA-po- 381_17mer_ G lnGpslnApslnTpslnApslnAps(8nh)ApsAps(5m)CpsGps(5m) 5-8-4 Cps(5m)CpsGps(5m)CpslnApslnGpslnApsscp(5m)C-3 156 121 5-GalNAc4-(PS)2-p-mU- 1431_17mer_ lnGpslnApsscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps 3-10-4 (5m)Cps(5m)CpsGpsApsln(5m)CpslnGpslnGpslnG-3 157 9C-G 5-GalNAc4-(PS)2-p-mU-po- 1431_17mer_ lnGpslnApslnTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps 3-10-4 (5m)CpsGpsApsln(5m)CpslnGpslnGpslnG-3 158 34A- 5-GalNAc4-(PS)2-p-mU-po- 1575_15mer_ G lnGpin(5m)CpsscpGpsAps(8nh)ApsGpsTpsGps(5m)CpsAps 3-10-2 (5m)CpsAps(5m)CpslnGpslnG-3 159 23A- 5-GalNAc4-(PS)2-p-mU-po- 1580_16mer_ G lnGpslnGpsscpTpsGpsApsApsGps(5m)CpsGpsApsApsGpsln 3-9-4 TpslnGpsln(5m)CpslnA-3 160 25F- 5-GalNAc4-(PS)2-p-mU-po- 1519_16mer_ G lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps 3-10-3 (5m)Cps(5m)Cpsln(5m)CpslnGpslnT-3 161 25G- 5-GalNAc4-(PS)2-p-mU-po- 1519_16mer_ G lnGpslnApslnGpsAps(8nh)GpsGpsTpsGps(5m)CpsGps(5m) 3-10-3 Cps(5m)Cps(5m)Cpsscp(5m)CpslnGpslnT3 162 40A- 5-GalNAc4-(PS)2-p-mA-po- 246_17mer_ G ln(5m)Cpsscp(5m)CpslnApsln(5m)Cps(5m)CpsAps(5m)Cps 3-10-4 GpsApsGpsTps(5m)CpsTpsApslnGpslnApsln(5m)C-3 163 47F- 5-GalNAc4-(PS)2-p-mU-po- 1433_16mer_ G lnGpslnGpslnApsTpsTps(5m)CpsApsGps(5m)CpsGps(5m) 3-10-3 Cps(5m)CpsGpslnApsln(5m)CpslnG-3
Example 4. Combination ASO Testing in HBsAg Release Assay Protocol
[0150] ASO combinations were tested in the HBsAg Release Assay Protocol described above in Example 2. Individual ASOs and a combination of the two ASOs were compared, and the reports are presented in Table 4. The EC.sub.50 values are as follows: A: <0.2 nM, B: 0.2 nM-0.3 nM, C: 0.3-5 nM.
TABLE-US-00007 TABLE 4 HBsAg Assay for ASO Combinations ASO # EC.sub.50 nM CC.sub.50 nM Positionlength_structure 8 C >10 1527_17mer_4-10-3 30 C >10 1431_17mer_4-9-4 50% ASO 8 + A >10 50% ASO 30 ASO # EC.sub.50 nM CC.sub.50 nM Position_length_structure 8 C >10 1527_17mer_4-10-3 7 B >10 1432_16mer_4-9-3 50% ASO 8 + A >10 50% ASO 7 ASO # EC.sub.50 nM CC.sub.50 nM Position_length_structure 8 C >10 1527_17mer_4-10-3 9 C >10 1431_17mer_3-10-4 50% ASO 8 + A >10 50% ASO 9 ASO # EC.sub.50 nM CC.sub.50 nM Position_length_structure 5 A >10 1582_16mer_4-9-3 7 B >10 1432_16mer_4-9-3 50% ASO 5 + B (lower >10 50% ASO 7 than 7 alone) ASO # EC50 nM CC.sub.50 nM Position_length_structure 40 C >10 246_17mer_3-10-4 1 A >10 1527_16mer_3-10-3 50% ASO 40 + A (lower 10 50% ASO 1 than 1 alone)
Example 5. Modified ASO Testing in AAV-HBV Mouse Model with Point Modifications
[0151] ASOs with LNA and BNA chemistries were synthesized on ABI 394 and Expedite 8909 synthesizers using standard phosphoramidite chemistry. In vitro screening of LNA ASOs was carried out in HepG2.2.15 cells using HBsAg release assay. Potent LNA-containing ASOs were chosen for N-Acetylgalactosamine (GalNac) conjugation and tested at 310 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model. BNA wing modifications were applied and compared to its all-LNA ASO. Table 5 shows HBsAg Nadir with 310 mg/kg QW compared to ASO 108. In these LNA-containing ASO in HBx region, targeting all HBV transcripts including HBx, a single replacement of a 5-methyl LNA C in the wing with 5-methyl spirocyclopropyl C improved the nadir for HBsAg by 0.5 Log.sub.10 IU/ml while reducing serum alanine aminotransferase (ALT) by 3-fold.
TABLE-US-00008 TABLE 5 HBsAg Nadir HBsAg Nadir with Max ALT with ASO # 3 10mg/kg QW 3 10 mg/kg QW 108A 1 log drop 611 U/L 108B 0.97 log drop 629 U/L 108C 1.25 log drop 488 U/L 108D 1.17 log drop 272 U/L 108E 1.44 log drop 243 U/L
[0152] ASOs with LNA and gap-modified chemistries were synthesized on ABI 394 and Expedite 8909 synthesizers using standard phosphoramidite chemistry. In vitro screening of LNA ASOs was carried out in HepG2.2.15 cells using HBsAg release assay. Potent LNA-containing ASOs were chosen for N-Acetylgalactosamine (GalNac) conjugation and tested at 310 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model. Nucleobase gap modifications were applied and compared to ASO 107. Table 6 shows HBsAg Nadir with 310 mg/kg QW compared to ASO 107. In these HBx region LNA ASO, a single replacement of deoxy-T in the gap with 2-thio T reduced serum ALT by 30-fold to normal levels while maintaining in vivo activity.
TABLE-US-00009 TABLE 6 HBsAg Nadir ASO # HBsAg Nadir Max ALT 107A 0.90 log drop 596 U/L 107B 0.99 log drop 168 U/L 107C 0.90 log drop 29 U/L 107D 1.06 log drop 380 U/L 1xPBS 0.1 log drop 28 U/L
Example 6. GalNac ASO Testing in AAV-HBV Mouse Model at 15 mg/kg Single Dose
[0153] ASOs were tested at 15 mg/kg in the adeno-associated virus (AAV)-HBV mouse model. This dosing regimen is mainly to rank order in vivo potency of ASOs. Although we could eliminate a small amount of very toxic ASOs in liver with ALT elevation in 15 mg/kg, majority of ASOs needs more stringent dosing regimen to be differentiated in liver tox. The resulting nadir log.sub.10 reduction in serum HBsAg and fold-change in ALT during the study are presented in Table 7, where A1 log.sub.10 reduction in HBsAg, B is 0.5-1 log.sub.10 reduction in HBsAg, and C is <0.5 log.sub.10 reduction in HBsAg, and X3-fold of ALT of vehicle control, which is considered normal, Y is 3-fold-30-fold of ALT of vehicle control, and Z is 30-fold of ALT of vehicle control. Both Y and Z are considered to be liver toxic.
[0154] The following specific sequences in Table 7 are within the scope of the present disclosure. As used herein, ln=Locked nucleic acid (LNA); lnA=Locked nucleic acid (LNA) A; ln(5m)C=ln(5m)C=Locked nucleic acid (LNA)-5methyl C; lnG=Locked nucleic acid (LNA) G; lnT=Locked nucleic acid (LNA) T; (5m)C=5 methylC; mA=2-O-methoxy A; mU=2-O-methoxy U; (8nh)A=8-amino A; (8nh)G=8-amino G; (2s)T=2-thio T; cp=scp=cyclopropyl; cpC=scpC=cyclopropyl C; cpG=scpG=cyclopropyl G; cpT=scpT=cyclopropyl T; ps=phosphorothioate linkages.
TABLE-US-00010 TABLE7 HBsAgNadir(Log)andALTfor15mg/kg SEQ HBsAg ID ASO Nadir NO. # Sequence (Log) ALT 164 160 5-GalNAc6-(PS)2-p-ClnGpslnApslnTpsTps(5m) B X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpslnG-3 165 161 5GalNAc1-C6-NH-lnGpslnTpslnGpsApsApsGps B X (5m)CpsGpsApsApsGpsTpslnGpsln(5m)CpslnA3 166 162 5GalNAc1-C6-p-lnApslnGpsln(5m)CpsGpsAps B X ApsGpsTpsGps(5m)CpsAps(5m)CpsApsln(5m)Cps lnGpslnG3 167 163 5GalNAc3-(PS)2-p-lnGpslnTpslnGpsApsAps C X Gps(5m)CpsGpsApsApsGpsTpslnGpsln(5m)Cpsln A3 168 164 5GalNAc1-C6-p-lnApslnApslnGpsln(5m)Cps B X GpsApsApsGpsTpsGps(5m)mCpsApsln(5m)Cpsln Apsln(5m)CpslnG3 169 165 5GalNAc1-C6-p-lnGpslnApslnTpslnTps(5m) B X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnG3 170 166 5GalNAc1-C6-p-ln(5m)CpslnGpsln(5m)CpsGps B X TpsApsApsApsGpsApsGpsApslnGpslnGpslnTpsln G3 171 167 5GalNAc1-C6-p-lnGpslnGpslnApslnTpsTps C X (5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpslnGps lnApsln(5m)CpslnGpslnG3 172 168 5GalNAc3-(PS)2-p-lnGpslnApslnGpsGpsTps B Y GpsApsApsGps(5m)CpsGpsApsApslnGpslnTpsln G3 173 169 5-GalNAc3-(PS)2-p-ln(5m)Cpsln(5m)CpslnGps C X ln(5m)CpsGpsTpsApsApsApsGpsApsGpsApsGpsln GpslnTpslnG3 174 170 5GalNAc3-(PS)2-p-lnGpslnApslnTpsTps(5m) A X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpslnG3 175 171 5-GalNAc3-(PS)2-p-ln(5m)Cpsln(5m)CpslnAps A X (5m)Cps(5m)CpsAps(5m)CpsGpsApsGpsTps(5m) CpsTpslnApslnGpslnApsln(5m)C3 176 172 5-GalNAc3-(PS)2-p-lnApslnTpslnGpslnApsTps C X ApsApsApsAps(5m)CpsGps(5m)Cpsln(5m)Cpsln Gpsln(5m)CpslnApslnG3 177 173 5-GalNAc3-(PS)2-p-lnGpsln(5m)Cpsln(5m)C C X (5m)CpsTpsAps(5m)CpsGpsApsAps(5m)Cps(5m) CpsApslnCpslnTpslnGpslnA3 178 174 5-GalNAc3-(PS)2-p-lnApslnGpsln(5m)CpsGps B X ApsApsGpsTpsGps(5m)CpsAps(5m)CpsApsln(5m) CpslnGpslnG3 179 175 5-GalNAc3-(PS)2-p-ln(5m)CpslnGpsln(5m)Cps C X GpsGpsGpsApsTpsTps(5m)CpsApsGpsln(5m)Cpsln Gpsln(5m)C3 180 176 5-GalNAc3-(PS)2-p-lnGpsln(5m)CpslnGpsAps B X ApsGpsTpsGps(5m)CpsAps(5m)CpsAps(5m)Cpsln GpslnG3 181 177 5-GalNAc3-(PS)2-p-lnGpslnGpslnTpsGps(5m) C X CpsGps(5m)Cps(5m)Cps(5m)Cps(5m)CpsGpsTps GpslnGpslnG3 182 178 5-GalNAc3-(PS)2-p-lnGpslnGpslnTpsGpsAps B X ApsGps(5m)CpsGpsApsApsGpsTpslnGpsln(5m) C3 183 179 5-GalNAc3-(PS)2-p-lnGpslnGpslnApslnTpsTps C X (5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsln Apsln(5m)CpslnGpslnG3 184 180 5-lnGpslnApslnGpsGpsTpsGpsApsApsGps(5m) C X CpsGpsApsApslnGpslnTpslnG-p-(PS)2- GalNAc33 185 181 5-GalNAc3-(PS)2-p-lnApslnGpsln(5m)CpsGps B X ApsApsGpsTpsGps(5m)CpsAps(5m)CpsApsln(5m) CpslnG3 186 182 5-GalNAc3-(PS)2-p-lnApslnGpslnGpsTpsGps A Y ApsApsGps(5m)CpsGpsApsApsGpslnTpslnG3 187 183 5-GalNAc5-(PS)2-p-lnGpsln(5m)CpslnGps(5m) B X Cps(5m)Cps(5m)Cps(5m)CpsGpsTpsGpsGpsTpsln (5m)CpslnGpslnG3 188 184 5-GalNAc5-(PS)2-p-lnApslnGpslnGpsTpsGps A Y (5m)CpsGps(5m)Cps(5m)Cps(5m)Cps(5m)CpsGps lnTpslnGpslnG3 189 185 5-GalNAc3-(PS)2-p-ln(5m)CpslnGpsln(5m)Cps C X GpsTpsApsApsApsGpsApsGpsApsGpslnGpslnTpsln G3 190 186 5-GalNAc3-(PS)2-p-ln(5m)CpslnGpsln(5m)Cps C X GpsTpsApsApsApsGpsApsGpsApsGpslnGpslnTpsln G3 191 187 5-GalNAc4-(PS)2-p-ln(5m)CpslnGpsln(5m)Cps C X GpsTpsApsApsApsGpsApsGpsApsGpslnGpslnTpsln G3 192 188 5-GalNAc4-(PS)2-p-lnGpslnApslnTpsTps(5m) B X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpslnG3 193 189 5-GalNAc3-(PS)2-p-lnApscpTpslnGpslnApsTps C X ApsApsApsAps(5m)CpsGps(5m)Cpsln(5m)Cpsln Gpsln(5m)CpslnApslnG3 194 190 5-GalNAc2-C6-p-CAlnApslnTpslnGpslnApsTps C X ApsApsApsAps(5m)CpsGps(5m)Cps(5m)CpslnGps ln(5m)CpslnApslnG3 195 191 5-GalNAc2-C6-p-CAlnApslnTpslnGpsApsTpsAps C X ApsApsAps(5m)CpsGps(5m)Cps(5m)CpslnGpsln (5m)CpslnApslnG3 196 192 5-GalNAc6-(PS)2-p-lnGpslnApslnTpsTps(5m) A X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpslnG3 197 193 5-GalNAc6-(PS)2-p-ln(5m)CpslnGpsln(5m)Cps C X GpsTpsApsApsApsGpsApsGpsApsGpslnGpslnTpsln G3 198 194 5GalNAc6-(PS)2-p-CAlnGpslnApslnTpsTps(5m) B X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpslnG3 199 195 5-lnGpslnApslnTpsTps(5m)CpsApsGps(5m)Cps A X Gps(5m)Cps(5m)CpsGpsApsln(5m)CpslnGpslnGps lnGC-p-(PS)2-GalNAc6-3 200 196 5lnGpslnApslnTpsTps(5m)CpsApsGps(5m)Cps A X Gps(5m)Cps(5m)CpsGpsApsln(5m)CpslnGpslnGps lnGCA-p-(PS)2-GalNAc6-3 201 197 5GalNAc4-(PS)2-p-ClnGpslnApslnTpsTps(5m) B X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpslnG-3 202 198 5-GalNAc4-(PS)2-p-CAlnGpslnApslnTpsTps B X (5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGps Apsln(5m)CpslnGpslnGpslnG-3 203 199 5lnGpslnApslnTpsTps(5m)CpsApsGps(5m)Cps B X Gps(5m)Cps(5m)CpsGpsApsln(5m)CpslnGpslnGps lnGC-p-(PS)2-GalNAc43 204 200 5lnGpslnApslnTpsTps(5m)CpsApsGps(5m)Cps A X Gps(5m)Cps(5m)CpsGpsApsln(5m)CpslnGpslnGps lnGCA-p-(PS)2-GalNAc43 205 201 5GalNAc6-(PS)2-p-ln(5m)Cpsln(5m)CpslnAps C X ln(5m)Cps(5m)CpsAps(5m)CpsGpsApsGpsTps(5m) CpsTpsApslnGpslnApsln(5m)C3 206 202 5GalNAc4-(PS)2-p-ln(5m)Cpsln(5m)CpslnAps A X ln(5m)Cps(5m)CpsAps(5m)CpsGpsApsGpsTps(5m) CpsTpsApslnGpslnApsln(5m)C3 207 203 5-GalNAc5-(PS)2-p-lnGpsln(5m)CpslnGpsAps B X ApsGpsTpsGps(5m)CpsAps(5m)CpsAps(5m)Cpscp GpslnG3 208 204 5-GalNAc5-(PS)2-p-lnGpsln(5m)CpscpGpsAps B X ApsGpsTpsGps(5m)CpsAps(5m)CpsAps(5m)Cpsln GpslnG3 209 205 5-GalNAc5-(PS)2-p-lnGpscp(5m)CpslnGpsAps B X ApsGpsTpsGps(5m)CpsAps(5m)CpsAps(5m)Cpsln GpslnG3 210 206 5-GalNAc5-(PS)2-p-cpGpsln(5m)CpslnGpsAps B X ApsGpsTpsGps(5m)CpsAps(5m)CpsAps(5m)Cpsln GpslnG3 211 207 5-GalNAc5-(PS)2-p-lnGpscp(5m)CpslnGpsAps C X ApsGpsTpsGps(5m)CpsAps(5m)CpsAps(5m)Cpscp GpslnG3 212 208 5-GalNAc5-(PS)2-p-lnGpsln(5m)CpscpGpsAps C X ApsGpsTpsGps(5m)CpsAps(5m)CpsAps(5m)Cpscp GpslnG3 213 209 5-GalNAc5-(PS)2-p-lnGpslnApslnTpsTps(5m) A X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpsAmG 214 210 5-GalNAc5-(PS)2-p-lnGpslnApslnTpsTps(5m) A X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpsAmGpslnG 215 211 5-GalNAc5-(PS)2-p-lnGpslnApslnTpsTps(5m) B X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpsAmGpslnGpslnG 216 212 5-GalNAc5-(PS)2-p-lnGpslnApslnTpsTps(5m) B X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsam (5m)CpslnGpslnGpslnG 217 213 5-GalNAc5-(PS)2-p-lnGpslnApsamTpsTps(5m) A X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpslnG 218 214 5-GalNAc5-(PS)2-p-lnGpsAmApslnTpsTps(5m) B X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpslnG 219 215 5-GalNAc5-(PS)2-p-AmGpslnApslnTpsTps(5m) A X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpslnG 220 216 5-GalNAc2-C6-p-CAlnGpslnGpslnApslnTpsTps C X (5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGps Apsln(5m)CpslnGpslnG3 221 217 5-GalNAc2-C6-p-CAlnApslnApslnGpsApsGpsAps B X GpsGpsTpsGps(5m)CpslnGpsln(5m)Cpsln(5m)Cps ln(5m)C3 222 218 5-GalNAc5-(PS)2-p-lnGpslnApslnTpsTps(5m) A X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpscpG3 223 219 5-GalNAc5-(PS)2-p-lnGpslnApslnTpsTps(5m) B X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpscpGpslnGpslnG3 224 220 5-GalNAc5-(PS)2-p-lnGpslnApslnTpsTps(5m) B X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApscp (5m)CpslnGpslnGpslnG3 225 221 5-GalNAc5-(PS)2-p-lnGpslnApscpTpsTps(5m) A X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpslnG3 226 222 5-GalNAc5-(PS)2-p-lnGpscpApslnTpsTps(5m) B X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpslnG 227 223 5-GalNAc5-(PS)2-p-cpGpslnApslnTpsTps(5m) B X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpslnG3 228 224 5-GalNAc5-(PS)2-p-lnGpslnApscpTpsTps(5m) A X CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApscp (5m)CpslnGpslnGpslnG3 229 225 5-GalNAc5-(PS)2-p-ln(5m)Cpsln(5m)CpslnAps C X ln(5m)Cps(5m)CpsAps(5m)CpsGpsApsGpsTps(5m) CpsTpsApslnGpslnApscp(5m)C 230 226 5-GalNAc5-(PS)2-p-ln(5m)Cpsln(5m)CpslnAps B X ln(5m)Cps(5m)CpsAps(5m)CpsGpsApsGpsTps(5m) CpsTpsApscpGpslnApsln(5m)C 231 227 5-GalNAc5-(PS)2-p-ln(5m)Cpscp(5m)CpslnAps C X ln(5m)Cps(5m)CpsAps(5m)CpsGpsApsGpsTps(5m) CpsTpsApslnGpslnApsln(5m)C 232 228 5-GalNAc5-(PS)2-p-cp(5m)Cpsln(5m)CpslnAps C X ln(5m)Cps(5m)CpsAps(5m)CpsGpsApsGpsTps(5m) CpsTpsApslnGpslnApsln(5m)C 233 229 lnGpsln(5m)CpslnGpsApsApsGpsTpsGps(5m)Cps B X Aps(5m)CpsAps(5m)CpslnGpslnGA-p-(PS)2- GalNAc4-3 234 230 5lnGpsln(5m)CpslnGpsApsApsGpsTpsGps(5m) B X CpsAps(5m)CpsAps(5m)CpslnGpslnG(5m)C-p- (PS)2-GalNAc4-3 235 231 5lnGpsln(5m)CpslnGpsApsApsGpsTpsGps(5m) C X CpsAps(5m)CpsAps(5m)CpslnGpslnGG-p-(PS)2- GalNAc4-3 236 232 5lnGpsln(5m)CpslnGpsApsApsGpsTpsGps(5m) C X CpsAps(5m)CpsAps(5m)CpslnGpslnGmA-p-(PS)2- GalNAc4-3 237 233 5lnGpsln(5m)CpslnGpsApsApsGpsTpsGps(5m) C X CpsAps(5m)CpsAps(5m)CpslnGpslnGm(5m)C-p- (PS)2-GalNAc43 238 234 5lnGpsln(5m)CpslnGpsApsApsGpsTpsGps(5m) C X CpsAps(5m)CpsAps(5m)CpslnGpslnGmG-p-(PS)2- GalNAc43 239 235 5ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)Cps B X Aps(5m)CpsGpsApsGpsTps(5m)CpsTpsApslnGpsln Apsln(5m)CA-p-(PS)2-GalNAc43 240 236 5ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)Cps B X Aps(5m)CpsGpsApsGpsTps(5m)CpsTpsApslnGpsln Apsln(5m)C(5m)C-p-(PS)2-GalNAc43 241 237 5ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)Cps B X Aps(5m)CpsGpsApsGpsTps(5m)CpsTpsApslnGpsln Apsln(5m)CG-p-(PS)2-GalNAc43 242 238 5ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)Cps B X Aps(5m)CpsGpsApsGpsTps(5m)CpsTpsApslnGpsln Apsln(5m)CmA-p-(PS)2-GalNAc43 243 239 5ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)Cps B X Aps(5m)CpsGpsApsGpsTps(5m)CpsTpsApslnGpsln Apsln(5m)-p-(PS)2-GalNAc43 244 240 5lnGpslnApslnTpslnApslnApsApsAps(5m)Cps B X Gps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnAps ln(5m)CT-p-(PS)2-GalNAc43 245 241 5lnGpslnApslnTpslnApslnApsApsAps(5m)Cps B X Gps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnAps ln(5m)C(5m)C-p-(PS)2-GalNAc43 246 242 5lnGpslnApslnTpslnApslnApsApsAps(5m)Cps B X Gps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnAps ln(5m)CG-p-(PS)2-GalNAc43 247 243 5lnGpslnApslnTpslnApslnApsApsAps(5m)Cps B X Gps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnAps ln(5m)CmU-p-(PS)2-GalNAc43 248 244 5-lnGpslnApslnTpslnApslnApsApsAps(5m)Cps C X Gps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnAps ln(5m)Cm(5m)C-p-(PS)2-GalNAc43 249 245 5lnGpslnApslnTpslnApslnApsApsAps(5m)Cps B X Gps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnAps ln(5m)CmG-p-(PS)2-GalNAc43 250 246 5-GalNAc4-(PS)2-p-lnGpslnApslnTpslnApsln B X ApsApsAps(5m)CpsGps(5m)Cps(5m)CpsGps(5m) CpslnApslnGpslnApsln(5m)C 251 247 5lnGpslnGpslnTpsGpsApsApsGps(5m)CpsGps B X ApsApsGpslnTpslnGpsln(5m)CpslnAmU-p-(PS)2- GalNAc43 252 248 lnGpsln(5m)CpslnGpsApsApsGpsTpsGps(5m)Cps B X Aps(5m)CpsAps(5m)CpslnGpslnGA-p-(PS)2- GalNAc63 253 249 5-lnGpslnApslnTpslnApslnApsApsAps(5m)Cps B X Gps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnAps ln(5m)CT-3 254 250 5-GalNAc4-(PS)2-p-mUlnGpslnGpslnTpsGpsAps C X ApsGps(5m)CpsGpsApsApsGpslnTpslnGpsln(5m) CpslnA-3 255 251 5-GalNAc4-(PS)2-p-TlnGpsln(5m)CpslnGpsAps B X ApsGpsTpsGps(5m)CpsAps(5m)CpsAps(5m)Cpsln GpslnG-3 256 252 5-GalNAc4-(PS)2-p-mUlnGpsln(5m)CpslnGps B X ApsApsGpsTpsGps(5m)CpsAps(5m)CpsAps(5m)Cps lnGpslnG 257 253 5-GalNAc6-(PS)2-p-mUlnGpsln(5m)CpslnGps C X ApsApsGpsTpsGps(5m)CpsAps(5m)CpsAps(5m)Cps lnGpslnG 258 254 5-GalNAc4-(PS)2-p-AlnGpslnApslnTpslnApsln B X ApsApsAps(5m)CpsGps(5m)Cps(5m)CpsGps(5m) CpslnApslnGpslnApsln(5m)C-3 259 255 5-GalNAc6-(PS)2-p-AlnGpslnApslnTpslnApsln B X ApsApsAps(5m)CpsGps(5m)Cps(5m)CpsGps(5m) CpslnApslnGpslnApsln(5m)C-3 260 256 5-GalNAc4-(PS)2-p-Aln(5m)Cpsln(5m)Cpsln B X Apsln(5m)Cps(5m)CpsAps(5m)CpsGpsAp sGpsTps(5m)CpsTpsApslnGpslnApsln(5m)C3 261 257 lnGpslnGpslnApsTpsTps(5m)CpsApsGps(5m)Cps B X Gps(5m)Cps(5m)CpsGpslnApsln(5m)CpslnGmA-p- (PS)2-GalNAc43 262 258 5-lnGpslnApslnGpsApsGpsGpsTpsGps(5m)Cps B X Gps(5m)Cps(5m)Cps(5m)Cpsln(5m)CpslnGpsln TmA-p-(PS)2-GalNAc43 263 259 5-GalNAc4-(PS)2-p-mUlnGpslnGpslnApsTpsTps B X (5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsln Apsln(5m)CpslnG-3 264 260 5-GalNAc4-(PS)2-p-mUlnGpslnApslnGpsApsGps B X GpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cpsln (5m)CpslnGpslnT-3 265 261 5-GalNAc4-(PS)2-p-mUlnGpslnApslnTpsTps A X (5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGps Apsln(5m)CpslnGpslnGpslnG-3 266 262 5lnGpslnApslnTpsTps(5m)CpsApsGps(5m)Cps A X Gps(5m)Cps(5m)CpsGpsApsln(5m)CpslnGpslnGps lnGmU-p-(PS)2-GalNAc43
Example 7. Modified ASO Testing in AAV-HBV Mouse Model at 310 mg/kg QW
[0155] ASOs were tested at 310 mg/kg every week in the adeno-associated virus (AAV)-HBV mouse model. This dosing regimen of 310 mg/kg QW is more stringent than 15 mg/kg shown in previous section. We can further select potent ASO with least ALT elevation. The resulting nadir log.sub.10 reduction in serum HBsAg and fold-change in ALT during the study are presented in Table 8, where A1 log.sub.10 reduction in HBsAg, B is 0.5-1 log.sub.10 reduction in HBsAg, and C is <0.5 log.sub.10 reduction in HBsAg, and X3-fold of ALT of vehicle control, which is considered normal. Y is 3-fold-30-fold of ALT of vehicle control, and Z is 30-fold of ALT of vehicle control. Both Y and Z are considered to show liver toxicity with Z being more severe.
[0156] The following specific sequences in Table 8 are within the scope of the present disclosure. As used herein, ln=Locked nucleic acid (LNA); lnA=Locked nucleic acid (LNA) A; =ln(5m)C=Locked nucleic acid (LNA)-5methyl C; lnG=Locked nucleic acid (LNA) G; lnT=Locked nucleic acid (LNA) T; (5m)C=5 methylC; mA=2-O-methoxy A; mU=2-O-methoxy U; (8nh)A=8-amino A; (8nh)G=8-amino G; (2s)T=2-thio T; cp=scp=cyclopropyl; cpC=scpC=cyclopropyl C; cpG=scpG=cyclopropyl G; cpT=scpT=cyclopropyl T; ps=phosphorothioate linkages; p=phosphodiester linkage.
TABLE-US-00011 TABLE8 HBsAgNadir(Log)andALTfor310mg/kgQW SEQ HBsAg ID ASO Nadir NO # Sequence (Log) ALT 267 263 5-GalNAc1-C6-p-CpsApslnGpslnApslnTpsln A X Tps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)Cps GpsApsln(5m)CpslnGpslnG3 268 264 5-GalNAc1-C6-p-CpsApslnGpslnTpslnGpsAps A Y ApsGps(5m)CpsGpsApsGpsTpslnGpsln(5m)Cps lnA3 269 265 5-GalNAc2-C6-p-CAlnGpslnApslnTpsTps(5m) A Y CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsAps ln(5m)CpslnGpslnGpslnG3 270 147 5-GalNAc2-C6-p-CAlnApslnGpsln(5m)CpsGps A Y ApsApsGpsTpsGps(5m)CpsAps(5m)CpsApsln(5m) CpslnGpslnG-3 271 266 5-GalNAc2-C6-p-CAlnApsln(5m)CpslnGpsln A Z (5m)Cps(5m)CpsGps(5m)CpsApsGpsAps(5m)Cps Apsln(5m)CpslnApslnT-3 425 132B 5-GalNAc2-C6-p-CAlnGpslnApslnGpsApsGps A Y GpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cps ln(5m)CpslnGpslnT3 426 267A 5-GalNAc2-C6-p-CAlnGpslnGpslnApsTpsTps A Y (5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGps lnApsln(5m)CpslnG3 274 268 5-GalNAc2-C6-p-CAln(5m)Cpsln(5m)Cpsln A Z Gps(5m)Cps(5m)CpsTpsGpsTpsApsAps(5m)Cps Apsln(5m)CpslnGpslnApslnG3 275 269 5-GalNAc2-C6-p-CAln(5m)Cpsln(5m)Cpsln A Z Gpsln(5m)Cps(5m)CpsTpsGpsTpsApsAps(5m) CpsApsln(5m)CpslnGpslnApslnG3 276 270 5-GalNAc2-C6-p-CAlnGpslnApslnTpslnApsln A X ApsApsAps(5m)CpsGps(5m)Cps(5m)CpsGps(5m) CpslnApslnGpslnApsln(5m)C3 277 271 5-GalNAc2-C6-p-CAln(5m)CpslnApsln(5m) A Z Cpsln(5m)CpsAps(5m)CpsGpsApsGpsTps(5m) CpsTpsApsGpslnApsln(5m)CpslnT3 278 272 5-GalNAc2-C6-p-CAlnApslnTpslnGpslnAps B X TpsApsApsApsAps(5m)CpsGps(5m)Cps(5m)Cps Gpsln(5m)CpslnApslnG3 427 273A 5-GalNAc2-C6-p-CAln(5m)Cpsln(5m)Cpsln A Y Apsln(5m)Cps(5m)CpsAps(5m)CpsGpsApsGps Tps(5m)CpsTpsApslnGpslnApsln(5m)C3 280 274 5-GalNAc2-C6-p-CAlnGpsln(5m)Cpsln(5m) B X Cpsln(5m)CpsTpsAps(5m)CpsGpsApsAps(5m) Cps(5m)CpsApsln(5m)CpslnTpslnGpslnA3 281 275 5-GalNAc2-C6-p-CAlnApslnApslnApsln(5m) A Z CpsGps(5m)Cps(5m)CpsGps(5m)CpsApsGpsAps ln(5m)CpslnAln(5m)CpslnApslnT3 282 276 5-GalNAc2-C6-p-CAlnApslnGpslnGpslnTps A Y GpsApsApsGps(5m)CpsGpsApsApsGpslnTpsln Gpsln(5m)C3 283 277 5-GalNAc2-C6-p-CAlnGpslnGpslnTpsGpsAps A X ApsGps(5m)CpsGpsApsApsGpslnTpslnGpsln(5m) CpslnA3 284 151 5-GalNAc6-(PS)2-p-lnGpslnGpslnApsTpsTps B Y (5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGps lnApsln(5m)CpslnG-3 205 201 5-GalNAc6-(PS)2-p-ln(5m)Cpsln(5m)Cpsln B X Apsln(5m)Cps(5m)CpsAps(5m)CpsGpsApsGps Tps(5m)CpsTpsApslnGpslnApsln(5m)C3 285 279 5-GalNAc2-C6-p-CAlnGpsln(5m)CpslnGpsAps A X ApsGpsTpsGps(5m)CpsApsCpsAps(5m)CpslnGps lnG3 286 152 5-GalNAc6-(PS)2-p-lnGpslnGpslnAps(2s) B Y TpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m) CpsGpslnApsln(5m)CpslnG-3 287 153 5-GalNAc6-(PS)2-p-lnGpslnGpslnApsTps A X (2s)Tps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m) CpsGpslnApsln(5m)CpslnG-3 288 154 5-GalNAc6-(PS)2-p-lnGpslnGpslnApsTpsTps A Y (5oh)CpsApsGps(5m)CpsGps(5m)Cps(5m)Cps GpslnApsln(5m)CpslnG-3 289 280 5-GalNAc6-(PS)2-p-lnGpslnApslnTpsTps(5m) A Y CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsAps ln(5m)CpslnGpslnGpslnG-3 290 281 5-GalNAc6-(PS)2-p-lnGpslnApslnTps(2s) A X Tps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)Cps GpsApsln(5m)CpslnGpslnGpslnG-3 291 282 5-GalNAc6-(PS)2-p-ln(5m)Cpsln(5m)Cpsln A X Apsln(5m)Cps(5oh)CpsAps(5m)CpsGpsApsGps Tps(5m)CpsTpsApslnGpslnApsln(5m)C-3 292 283 5-GalNAc6-(PS)2-p-ln(5m)Cpsln(5m)Cpsln B X Apsln(5m)Cps(5m)CpsAps(5oh)CpsGpsApsGps Tps(5m)CpsTpsApslnGpslnApsln(5m)C-3 293 284 ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)Cps B X Aps(5m)CpsGpsApsGpsTps(5m)Cps(2s)TpsAps lnGpslnApsln(5m)C-3 294 285 5-GalNAc2-C6-p-CApslnApslnGpsln(5m)Cps A Y GpsApsApsGpsTpsGps(5m)CpsAps(5m)CpsApsln (5m)CpslnG-3 295 286 5-lnGpslnApslnTpslnApslnApsApsAps(5m) A X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApslnGps lnApsln(5m)C-3 296 287 5-GalNAc5-(PS)2-p-lnGpslnApslnTpslnAps A X lnApsApsAps(5m)CpsGps(5m)Cps(5m)CpsGps (5m)CpslnApslnGpslnApscp(5m)C-3 297 288 5-lnGpslnApscpTpslnApslnApsApsAps(5m) A X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApsln(5m)C-3 298 289 5-GalNAc5-(PS)2-p-lnGpslnGpslnApsTpsTps A Z (5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGps lnApsln(5m)CpslnG-3 299 290 5-lnGpslnGpslnApsTpsTps(5m)CpsApsGps A Z (5m)CpsGps(5m)Cps(5m)CpsGpslnApscp(5m) CpslnG-3 300 136 5-GalNAc5-(PS)2-p-lnGpslnApslnGpsApsGps A Y GpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cps ln(5m)CpslnGpscpT-3 301 137 5-lnGpslnApslnGpsApsGpsGpsTpsGps(5m)Cps A Y Gps(5m)Cps(5m)Cps(5m)Cpscp(5m)CpslnGpsln T-3 302 133 5-GalNAc5-(PS)2-p-lnGpslnApslnGpsApsGps A Y GpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cps ln(5m)CpslnGpslnT-3 303 134 5-GalNAc5-(PS)2-p-lnGpslnApslnGpsApsGps B Y GpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cps ln(5m)CpslnGpsamT-3 304 135 5-GalNAc5-(PS)2-p-lnGpslnApslnGpsApsGps A Y GpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cps am(5m)CpslnGpslnT-3 305 291 5-GalNAc2-C6-p-CAlnApslnGpslnApsGpsAps A Y GpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cpsln CpslnCpslnGpslnT-3 306 292 5-GalNAc2-C6-p-CAlnApsln(5m)CpslnApsln A X ApsApsGpsGpsAps(5m)CpsGpsTps(5m)Cps(5m) Cps(5m)CpslnGpsln(5m)CpslnG-3 307 293 5-GalNAc2-C6-p-CAlnApslnTpslnGpslnAps B X TpsApsApsApsAps(5m)CpsGps(5m)Cpsln(5m) CpslnGpsln(5m)CpslnApslnG-3 308 294 5-GalNAc2-C6-p-CAln(5m)Cpsln(5m)Cpsln B X Gpsln(5m)CpsGpsTpsApsApsApsGpsApsGpsAps GpslnGpslnTpslnG-3
Example 8. Modified ASO Testing in AAV-HBV Mouse Model with 310 mg/kg Q3D
[0157] ASOs were tested at 310 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model. The dosing regimen of 310 mg/kg Q3D is more stringent than 310 mg/kg QW and can further select ASOs with best therapeutic indexes. The resulting nadir log.sub.10 reduction in serum HBsAg and fold-change in ALT during the study are presented in Table 9, where A1 log.sub.10 reduction in HBsAg, B is 0.5-1 log.sub.10 reduction in HBsAg, and C is <0.5 log.sub.10 reduction in HBsAg, and X3-fold of ALT of vehicle control, which is considered mormal. Y is 3-fold-30-fold of ALT of vehicle control, and Z is 30-fold of ALT of vehicle control. Both Y and Z are considered to be liver toxic with Z being more severe.
[0158] The following specific sequences in Table 9 are within the scope of the present disclosure. As used herein, ln=Locked nucleic acid (LNA); lnA=Locked nucleic acid (LNA) A; ln(5m)C=ln(5m)C=Locked nucleic acid (LNA)-5methyl C; lnG=Locked nucleic acid (LNA) G; lnT=Locked nucleic acid (LNA) T; (5m)C=5 methylC; mA=2-O-methoxy A; mU=2-O-methoxy U; (8nh)A=8-amino A; (8nh)G=8-amino G; (2s)T=2-thio T; cp=scp=cyclopropyl; cpC=scpC=cyclopropyl C; cpG=scpG=cyclopropyl G; cpT=scpT=cyclopropyl T; ps=phosphorothioate linkages. The Position in HBV Genome describes the 5-end of target-site in acc. KC315400.1 (genotype B), which corresponds to SEQ ID NO: 1.
TABLE-US-00012 TABLE9 HBsAgNadir(Log)andALTfor310mg/kgQ3D SEQ HBsAg ID ASO Nadir No. # Sequence (Log) ALT 270 147 5GalNAc2-C6-p-CAlnApslnGpsln(5m)Cps B Z GpsApsApsGpsTpsGps(5m)CpsAps(5m)CpsAps ln(5m)CpslnGpslnG3 309 295 5GalNAc6-(PS)2-p-lnGpslnApslnTpsTps B X (5oh)CpsApsGps(5m)CpsGps(5m)Cps(5m)Cps GpsApsln(5m)CpslnGpslnGpslnG3 244 240 5lnGpslnApslnTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApsln(5m)CT-p-(PS)2-GalNAc43 310 297 5lnGpslnApslnTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApscp(5m)CT-p-(PS)2-GalNAc43 311 298 5lnGpsln(5m)CpscpGpsApsApsGpsTpsGps B X (5m)CpsAps(5m)CpsAps(5m)CpslnGpslnGA- p-(PS)2-GalNAc4-p-(PS)2-GalNAc43 312 299 5lnGpscp(5m)CpslnGpsApsApsGpsTpsGps B X (5m)CpsAps(5m)CpsAps(5m)CpslnGpslnGA- p-(PS)2-GalNAc4 313 300 5cpGpsln(5m)CpslnGpsApsApsGpsTpsGps B X (5m)CpsAps(5m)CpsAps(5m)CpslnGpslnGA- p-(PS)2-GalNAc43 314 301 5lnGpsln(5m)CpslnGpsApsApsGpsTpsGps B X (5m)CpsAps(5m)CpsAps(5m)CpslnGpscpGA3 315 302 5lnGpsln(5m)CpslnGpsApsApsGpsTpsGps B X (5m)CpsAps(5m)CpsAps(5m)CpscpGpslnGA- p-(PS)2-GalNAc43 316 303 5lnGpslnApslnTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpscpApsln(5m)CT-p-(PS)2-GalNAc43 317 304 5lnGpslnApslnTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpscpApsln GpslnApsln(5m)CT-p-(PS)2-GalNAc43 318 305 5lnGpslnApslnTpslnApscpApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApsln(5m)CT-p-(PS)2-GalNAc43 319 306 5lnGpslnApscpTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApsln(5m)CT-p-(PS)2-GalNAc43 320 307 5lnGpsln(5m)CpsamGpsApsApsGpsTpsGps B X (5m)CpsAps(5m)CpsAps(5m)CpslnGpslnGA- p-(PS)2-GalNAc43 321 308 5lnGpsam(5m)CpslnGpsApsApsGpsTpsGps B X (5m)CpsAps(5m)CpsAps(5m)CpslnGpslnGA- p-(PS)2-GalNAc43 322 309 5lnGpsln(5m)CpslnGpsApsApsGpsTpsGps B X (5m)CpsAps(5m)CpsAps(5m)CpslnGpsamGA- p-(PS)2-GalNAc43 323 310 5lnGpsln(5m)CpslnGpsApsApsGpsTpsGps B X (5m)CpsAps(5m)CpsAps(5m)CpsamGpslnGA- p-(PS)2-GalNAc43 324 311 5amGpsln(5m)CpslnGpsApsApsGpsTpsGps B X (5m)CpsAps(5m)CpsAps(5m)CpslnGpslnGA- p-(PS)2-GalNAc43 325 312 5lnGpslnApslnTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApsam(5m)CT-p-(PS)2-GalNAc43 326 313 5lnGpslnApsamTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApsln(5m)CT-p-(PS)2-GalNAc43 327 314 5amGpslnApslnTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApsln(5m)CT-p-(PS)2-GalNAc43 328 315 5lnGpsamApslnTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApsln(5m)CT-p-(PS)2-GalNAc43 329 316 5lnGpslnApslnTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApscp GpslnApsln(5m)CT-p-(PS)2-GalNAc43 330 317 5ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m) B Y CpsAps(5m)CpsGpsApsGpsTps(5m)CpsTpsAps lnGpslnApscp(5m)CA-p-(PS)2-GalNAc43 331 318 5ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m) B Y CpsAps(5m)CpsGpsApsGpsTps(5m)CpsTpsAps cpGpslnApsln(5m)CA-p-(PS)2-GalNAc43 332 319 5ln(5m)Cpsln(5m)CpslnApscp(5m)Cps(5m) B Y CpsAps(5m)CpsGpsApsGpsTps(5m)CpsTpsAps lnGpslnApsln(5m)CA-p-(PS)2-GalNAc43 333 320 5ln(5m)Cpscp(5m)CpslnApsln(5m)Cps(5m) B Y CpsAps(5m)CpsGpsApsGpsTps(5m)CpsTpsAps lnGpslnApsln(5m)CA-p-(PS)2-GalNAc43 334 321 5cp(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m) B Y CpsAps(5m)CpsGpsApsGpsTps(5m)CpsTpsAps lnGpslnApsln(5m)CA-p-(PS)2-GalNAc43 335 322 5lnGpslnApslnTpslnApslnAps(8nh)ApsAps B X (5m)CpsGps(5m)Cps(5m)CpsGps(5m)CpslnAps lnGpslnApscp(5m)CT-p-(PS)2-GalNAc43 336 323 5lnGpscp(5m)CpslnGpsAps(8nh)ApsGpsTps B X Gps(5m)CpsAps(5m)CpsAps(5m)CpslnGpsln GA-p-(PS)2-GalNAc43 337 324 5cpGpslnApslnTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApsln(5m)CT-p-(PS)2-GalNAc43 338 325 5lnGpslnGpslnTpsGpsApsApsGps(5m)Cps B X GpsApsApsGpslnTpslnGpscp(5m)CpslnAA-p- (PS)2-GalNAc43 339 326 5lnGpslnGpslnTpsGpsApsApsGps(5m)Cps B X GpsApsApsGpslnTpscpGpsln(5m)CpslnAA-p- (PS)2-GalNAc43 340 327 5lnGpslnGpslnTpsGpsApsApsGps(5m)Cps B X GpsApsApsGpscpTpslnGpsln(5m)CpslnAA- p-(PS)2-GalNAc43 341 328 5lnGpslnGpscpTpsGpsApsApsGps(5m)Cps B X GpsApsApsGpslnTpslnGpsln(5m)CpslnAA 342 329 5lnGpscpGpslnTpsGpsApsApsGps(5m)Cps B X GpsApsApsGpslnTpslnGpsln(5m)CpslnAA- p-(PS)2-GalNAc43 343 330 5cpGpslnGpslnTpsGpsApsApsGps(5m)Cps B X GpsApsApsGpslnTpslnGpsln(5m)CpslnAA- p-(PS)2-GalNAc43 344 331 5lnGpslnApslnTpscpApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApsln(5m)CT-p-(PS)2-GalNAc43 345 332 5lnGpscpApslnTpslnApslnApsApsAps(5m) B Z CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApsln(5m)CT-p-(PS)2-GalNAc43 346 333 5ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m) B Y CpsAps(5m)CpsGpsApsGpsTps(5m)CpsTpsAps lnGpscpApsln(5m)CA-p-(PS)2-GalNAc43 347 334 5ln(5m)Cpsln(5m)CpscpApsln(5m)Cps(5m) B Y CpsAps(5m)CpsGpsApsGpsTps(5m)CpsTpsAps lnGpslnApsln(5m)CA-p-(PS)2-GalNAc43 348 335 5cp(5m)Cpscp(5m)CpscpApscp(5m)Cps(5m) B X CpsAps(5m)CpsGpsApsGpsTps(5m)CpsTpsAps cpGpscpApscp(5m)CA-p-(PS)2-GalNAc43 349 336 5lnGpslnApslnTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpsamApsln GpslnApsln(5m)CT-p-(PS)2-GalNAc43 350 337 5lnGpslnApslnTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnAps amGpslnApsln(5m)CT-p-(PS)2-GalNAc43 351 338 5lnGpslnApslnTpslnApsamApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApsln(5m)CT-p-(PS)2-GalNAc43 352 339 5lnGpslnGpslnTpsGpsApsApsGps(5m)Cps B X GpsApsApsGpslnTpslnGpsln(5m)CpscpAA 353 340 5lnGpslnApslnTpsamApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpslnApsln(5m)CT-p-(PS)2-GalNAc43 354 341 5lnGpslnApslnTpslnApslnApsApsAps(5m) B X CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApsln GpsamApsln(5m)CT-p-(PS)2-GalNAc43 355 342 5lnGpslnGpslnTpsGpsApsApsGps(5m)Cps B X GpsApsApsGpslnTpslnGpsam(5m)CpslnAA-p- (PS)2-GalNAc43 356 343 5lnGpslnGpslnTpsGpsApsApsGps(5m)Cps B X GpsApsApsGpslnTpsamGpsln(5m)CpslnAA- p-(PS)2-GalNAc43 357 344 5lnGpslnGpslnTpsGpsApsApsGps(5m)Cps B X GpsApsApsGpsamTpslnGpsln(5m)CpslnAA- p-(PS)2-GalNAc43 358 345 5lnGpslnGpsamTpsGpsApsApsGps(5m)Cps B X GpsApsApsGpslnTpslnGpsln(5m)CpslnAA- p-(PS)2-GalNAc43 359 346 5lnGpslnGpslnApsTpsTps(5m)CpsApsGps B Z (5m)CpsGps(5m)Cps(5m)CpsGpslnApsln(5m) CpscpGA-p-(PS)2-GalNAc43 360 347 5lnGpslnGpslnAps(2s)TpsTps(5m)CpsAps B Z Gps(5m)CpsGps(5m)Cps(5m)CpsGpslnApscp (5m)CpslnGA-p-(PS)2-GalNAc43 361 348 5lnGpslnGpslnApsTpsTps(5m)CpsApsGps B Z (5m)CpsGps(5m)Cps(5m)CpsGpscpApsln(5m) CpslnGA-p-(PS)2-GalNAc43 362 349 5lnGpslnGpscpApsTpsTps(5m)CpsApsGps B Z (5m)CpsGps(5m)Cps(5m)CpsGpslnApsln(5m) CpslnGA-p-(PS)2-GalNAc43 363 350 5lnGpscpGpslnApsTpsTps(5m)CpsApsGps B Z (5m)CpsGps(5m)Cps(5m)CpsGpslnApsln(5m) CpslnGA-p-(PS)2-GalNAc43 364 351 5cpGpslnGpslnApsTpsTps(5m)CpsApsGps B Z (5m)CpsGps(5m)Cps(5m)CpsGpslnApsln(5m) CpslnGA-p-(PS)2-GalNAc43 365 352 5lnGpslnApslnGpsAps(8nh)GpsGpsTpsGps B Y (5m)CpsGps(5m)Cps(5m)Cps(5m)Cpscp(5m) CpslnGpslnTA-p-(PS)2-GalNAc43 366 353 5lnGpslnApscpTpsTps(5oh)CpsApsGps(5m) B Y CpsGps(5m)Cps(5m)CpsGpsApsln(5m)Cpsln GpslnGpslnGT-p-(PS)2-GalNAc43 367 354 5lnGpslnApslnGps(8nh)ApsGpsGpsTpsGps B Z (5m)CpsGps(5m)Cps(5m)Cps(5m)Cpscp(5m) CpslnGpslnTA-p-(PS)2-GalNAc43 368 145 5ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m) B Y CpsAps(5m)CpsGpsApsGpsTps(5m)CpsTpsAps lnGpslnApsam(5m)CA-p-(PS)2-GalNAc43 369 143 5ln(5m)Cpsam(5m)CpslnApsln(5m)Cps(5m) B Y CpsAps(5m)CpsGpsApsGpsTps(5m)CpsTpsAps lnGpslnApsln(5m)CA-p-(PS)2-GalNAc43 370 144 5am(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m) B Y CpsAps(5m)CpsGpsApsGpsTps(5m)CpsTpsAps lnGpslnApsln(5m)CA-p-(PS)2-GalNAc43 371 146 5ln(5m)Cpsln(5m)CpslnApsam(5m)Cps(5m) B X CpsAps(5m)CpsGpsApsGpsTps(5m)CpsTpsAps lnGpslnApsln(5m)CA-p-(PS)2-GalNAc43 372 355 5amGpsam(5m)CpsamGpsApsApsGpsTpsGps B X (5m)CpsAps(5m)CpsAps(5m)CpsamGpsamGA-p- (PS)2-GalNAc43 373 356 5lnGpslnApscpTpslnApslnApsAps(8nh)Aps B X (5m)CpsGps(5m)Cps(5m)CpsGps(5m)CpslnAps lnGpslnApsln(5m)CT-p-(PS)2-GalNAc43 374 357 5lnGpsln(5m)CpscpGpsAps(8nh)ApsGpsTps B Y Gps(5m)CpsAps(5m)CpsAps(5m)CpslnGpsln GA-p-(PS)2-GalNAc43 375 358 5lnGpslnApslnGpsApsGps(8nh)GpsTpsGps B Y (5m)CpsGps(5m)Cps(5m)Cps(5m)Cpscp(5m) CpslnGpslnTA-p-(PS)2-GalNAc43 376 359 5GalNAc4-(PS)2-p-mUlnGpscp(5m)Cpsln B Y GpsAps(8nh)ApsGpsTpsGps(5m)CpsAps(5m) CpsAps(5m)CpslnGpslnG 377 156 5lnGpslnGpslnApsTpsTps(5oh)CpsApsGps B Y (5m)CpsGps(5m)Cps(5m)CpsGpslnApscp(5m) CpslnGA-p-(PS)2-GalNAc43 428 155A 5lnGpslnGpslnApsTps(2s)Tps(5m)CpsAps B X Gps(5m)CpsGps(5m)Cps(5m)CpsGpslnApscp (5m)CpslnGA-p-(PS)2-GalNAc43
Example 9. Modified ASO Testing in AAV-HBV Mouse Model
[0159] This example evaluates the therapeutic index of ASOs using a dosing regimen of 510 mg/kg. ASOs were tested at 510 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model. The resulting nadir log.sub.10 reduction in serum HBsAg and fold-change in ALT during the study are presented in Table 10, where A1 log.sub.10 reduction in HBsAg, B is 0.5-1 log.sub.10 reduction in HBsAg, and C is <0.5 log.sub.10 reduction in HBsAg, and X3-fold of ALT of vehicle control, which is considered normal. Y is 3-fold-30-fold of ALT of vehicle control, and Z is 30-fold of ALT of vehicle control. Both Y and Z showed liver tox with Z being more severe.
[0160]
[0161] The following specific sequences in Table 10 are within the scope of the present disclosure. As used herein, ln=Locked nucleic acid (LNA); lnA=Locked nucleic acid (LNA) A; ln(5m)C=ln(5m)C=Locked nucleic acid (LNA)-5methyl C; lnG=Locked nucleic acid (LNA) G; lnT=Locked nucleic acid (LNA) T; (5m)C=5 methylC; mA=2-O-methoxy A; mU=2-O-methoxy U; (8nh)A=8-amino A; (8nh)G=8-amino G; (2s)T=2-thio T; cp=scp=cyclopropyl; cpC=scpC=cyclopropyl C; cpG=scpG=cyclopropyl G; cpT=scpT=cyclopropyl T; ps=phosphorothioate linkages.
TABLE-US-00013 TABLE10 HBsAgNadir(Log)andALTfor5x10mg/kgQ3D SEQ HBsAg ID ASO Nadir NO. # Sequence (Log) ALT 270 147 5-GalNAc2-C6-p- A Z CAlnApslnGpsln(5m)CpsGpsApsApsGpsTpsGps(5m)CpsAps (5m)CpsApsln(5m)CpslnGpslnG-3 276 270 5-GalNAc2-C6-p- A X CAlnGpslnApslnTpslnApslnApsApsAps(5m)CpsGps(5m) Cps(5m)CpsGps(5m)CpslnApslnGpslnApsln(5m)C3 379 361 5-GalNAc2-C6-p- A X CAlnGpsln(5m)CpslnGps(8nh)ApsApsGpsTpsGps(5m)Cps Aps(5m)CpsAps(5m)CpslnGpslnG-3 380 362 5-GalNAc2-C6-p- A Y CAlnGpsln(5m)CpslnGpsApsApsGpsTpsGps(5m)CpsAps (5m)CpsAps(50h)CpslnGpslnG-3 381 363 5-GalNAc2-C6-p- A Y CAlnGpsln(5m)CpslnGpsAps(8nh)ApsGpsTpsGps(5m) CpsAps(5m)CpsAps(5m)CpslnGpslnG-3 382 364 5-GalNAc2-C6-p- A X CAlnGpslnGpslnTps(8nh)GpsApsApsGps(5m)CpsGpsAps ApsGpslnTpslnGpsln(5m)CpslnA-3 383 365 5-GalNAc2-C6-p- A Y lnGpsln(5m)CpslnGpsApsApsGpsTpsGps(5m)CpsAps(5m) CpsAps(5m)CpslnGpslnGmG-3 384 366 5-GalNAc2-C6-p- A X ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)CpsAps(5m)Cps GpsApsGpsTps(5m)CpsTpsApslnGpslnApsln(5m)CmG-3 385 367 5-GalNAc2-C6-p- A Y CAlnGpslnApslnTpslnApslnAps(8nh)ApsAps(5m)CpsGp s(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnApsln(5m)C-3 386 368 5-GalNAc2-C6-p- A X CAlnGpslnApslnTpslnApslnApsAps(8nh)Aps(5m)CpsGps (5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnApsln(5m)C-3 387 369 5-GalNAc2-C6-p- A X CAlnGpslnApslnTpslnApslnApsApsAps(50h)CpsGps(5m) Cps(5m)CpsGps(5m)CpslnApslnGpslnApsln(5m)C-3 388 370 5-GalNAc2-C6-p- A X CAln(5m)Cpsln(5m)CpslnApsln(5m)Cps(50h)CpsAps(5m) CpsGpsApsGpsTps(5m)CpsTpsApslnGpslnApsln(5m) C-3 389 371 5-GalNAc2-C6-p- A X CAln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)Cps(8nh)Ap s(5m)CpsGpsApsGpsTps(5m)CpsTpsApslnGpslnApsln(5 m)C-3 390 372 5-GalNAc2-C6-p- A Y CAln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)CpsAps(5oh) CpsGpsApsGpsTps(5m)CpsTpsApslnGpslnApsln(5m)C- 3 391 373 5- A Y ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5oh)CpsAps(5m) CpsGpsApsGpsTps(5m)CpsTpsApslnGpscpApsln(5m)CmA- p-(PS)2-GalNAc3-3 392 157 5- A Z lnGpslnApslnTpsTps(5oh)CpsApsGps(5m)CpsGps(5m) Cps(5m)CpsGpsApsln(5m)CpslnGpslnGpscpGT-p-(PS)2- GalNAc4-3 393 158 5- B Y lnGpslnApscpTps(2s)Tps(5m)CpsApsGps(5m)CpsGps(5m) Cps(5m)CpsGpsApsln(5m)CpslnGpslnGpslnGT-p-(PS)2- GalNAc4-3 394 159 5- A X lnGpslnApscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps (5m)Cps(5m)CpsGpsApsln(5m)CpslnGpslnGpslnGT-p-(PS)2- GalNAc4-3 395 374 5- A Z ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)CpsAps(5oh) CpsGpsApsGpsTps(5m)CpsTpsApslnGpscpApsln(5m)CmA- p-(PS)2-GalNAc4-3 396 128 5-GalNAc4-(PS)2-p- A X mAlnGpslnApslnTpslnApslnApsApsAps(5m)CpsGps(5m) Cps(5m)CpsGps(5m)CpslnApslnGpslnApscp(5m)C-3 397 129 5-GalNAc4-(PS)2-p- A X mAlnGpslnApslnTpslnApslnAps(8nh)ApsAps(5m)CpsGps (5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnApscp(5m)C- 3 398 130 5-GalNAc4-(PS)2-p- B X mAln(5m)Cpscp(5m)CpslnApsln(5m)Cps(5m)Cps(8nh) Aps(5m)CpsGpsApsGpsTps(5m)CpsTpsApslnGpslnApsln (5m)C-3 399 375 5-GalNAc4-(PS)2-p- A X mAln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)Cps(8nh) Aps(5m)CpsGpsApsGpsTps(5m)CpsTpsApscpGpslnApsln (5m)C-3 400 120 5-GalNAc4-(PS)2-p- A X mAlnGpslnApslnTpslnApslnApsApsAps(50h)CpsGps(5m) Cps(5m)CpsGps(5m)CpslnApslnGpslnApscp(5m)C-3 401 376 5-GalNAc4-(PS)2-p- B X mAlnGpsamApslnTpslnApslnApsApsAps(50h)CpsGps(5m) Cps(5m)CpsGps(5m)CpslnApslnGpslnApsln(5m)C-3 402 377 5-GalNAc4-(PS)2-p- A X mAlnGpslnGpscpTpsGpsAps(8nh)ApsGps(5m)CpsGpsAps ApsGpslnTpslnGpsln(5m)CpslnA-3 403 378 5- B X ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)Cps(8nh)Aps(5m) CpsGpsApsGpsTps(5m)CpsTpsApslnGpscpApsln(5m) CmA-p-(PS)2-GalNAc4-3
Example 10. Modified ASO Testing in AAV-HBV Mouse Model
[0162] ASOs were tested at 110 mg/kg in the adeno-associated virus (AAV)-HBV mouse model as single agents or in combination (S+X Triggers as well as S+S Triggers). The resulting nadir log.sub.10 reduction in serum HBsAg and fold-change in ALT during the study are presented in Table 11, where A1 log.sub.10 reduction in HBsAg, B is 0.5-1 log.sub.10 reduction in HBsAg, and C is <0.5 log.sub.10 reduction in HBsAg, and X3-fold of ALT of vehicle control, Y is 3-fold-30-fold of ALT of vehicle control, and Z is 30-fold of ALT of vehicle control. The results demonstrated when S and X Triggers ASOs were combined, they showed additive to minor synergistic effects.
[0163]
TABLE-US-00014 TABLE11 HBsAgNadir(Log)andALTfor1x10mg/kg SEQ HBsAg ID ASO Nadir NO. # Sequence (Log) ALT 418 123 5- B X moeGpsmoe(5m)CpsmoeApsmoeGpsmoeApsGpsGps TpsGpsApsApsGps(5m)CpsGpsApsmoeApsmoeGps moeTpsmoeGpsmoe(5m)C-3 423 265A 5-GalNAc-NH-C6-CA- A X lnGpslnApslnTpsTps(5m)CpsApsGps(5m)CpsGps(5m) Cps(5m)CpsGpsApsln(5m)CpslnGpslnGpslnG3 424 147A 5-GalNAc-NH-C6-CA- A X lnApslnGpsln(5m)CpsGpsApsApsGpsTpsGps(5m)Cps Aps(5m)CpsApsln(5m)CpslnGpslnG-3 273 267 5-GalNAc-NH-C6-CA- A X lnGpslnGpslnApsTpsTps(5m)CpsApsGps(5m)CpsGps (5m)Cps(5m)CpsGpslnApsln(5m)CpslnG3 279 273 5-GalNAc-NH-C6-CA- A X ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)CpsAps(5m) CpsGpsApsGpsTps(5m)CpsTpsApslnGpslnApsln (5m)C3 ASO273 A X ASO285(1: 1) ASO273 A X ASO285(2: 1) ASO273 A X ASO285(3: 1) 396 128 5-GalNAc4-(PS)2-p- A X mAlnGpslnApslnTpslnApslnApsApsAps(5m)CpsGps (5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnApscp(5m) C3 ASO128 B X ASO377(1: 1) ASO128 A X ASO377(2: 1) 397 129 5-GalNAc4-(PS)2-p- A X mAlnGpslnApslnTpslnApslnAps(8nh)ApsAps(5m) CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnApscp (5m)C3 ASO129 A X ASO267(1: 1) ASO129 A X ASO267(2: 1) ASO129 A X ASO267(3: 1) 398 130 5-GalNAc4-(PS)2-p- B X mAln(5m)Cpscp(5m)CpslnApsln(5m)Cps(5m)Cps (8nh)Aps(5m)CpsGpsApsGpsTps(5m)CpsTpsApslnGpsln Apsln(5m)C3 130+120 B X (1:1) ASO130 B X ASO121(1:1) ASO130 B X ASO121(2:1) ASO130 B X ASO121(3:1) ASO130 B X ASO131(1:1) ASO130 B X ASO131(2:1) ASO130 A X ASO131(3:1) 399 375 5-GalNAc4-(PS)2-p- C X mAln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)Cps(8nh) Aps(5m)CpsGpsApsGpsTps(5m)CpsTpsApscpGpsln Apsln(5m)C3 400 120 5-GalNAc4-(PS)2-p- B X mAlnGpslnApslnTpslnApslnApsApsAps(5oh)CpsGps (5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnApscp(5m) C3 ASO120 A X ASO121(1:1) ASO120 A X ASO121(2:1) ASO120 A X ASO121(3:1) ASO120 A X ASO131(1:1) ASO120 A X ASO131(2:1) ASO120 A X ASO131(3:1) 401 376 5-GalNAc4-(PS)2-p- C X mAlnGpsamApslnTpslnApslnApsApsAps(50h)CpsGps (5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnApsln(5m) C3 402 377 5-GalNAc4-(PS)2-p- B X mAlnGpslnGpscpTpsGpsAps(8nh)ApsGps(5m)Cps GpsApsApsGpslnTpslnGpsln(5m)CpslnA3 156 121 5-GalNAc4-(PS)2-p- A X mUlnGpslnApscpTpsTps(5m)Cps(8nh)ApsGps(5m) CpsGps(5m)Cps(5m)CpsGpsApsln(5m)CpslnGpsln GpslnG3 419 131 5-GalNAc4-(PS)2-p- A X mUlnGpslnGpslnApsTps(2s)Tps(5m)CpsApsGps(5m) CpsGps(5m)Cps(5m)CpsGpslnApscp(5m)CpslnG 3
Example 11. Modified ASO Testing in AAV-HBV Mouse Model
[0164] ASOs were tested at 6 repeat doses of 3 mg/kg or 10 mg/kg at days 0, 3, 7, 14, 21, and 28 in the adeno-associated virus (AAV)-HBV mouse model. The resulting nadir log.sub.10 reduction in serum HBsAg and fold-change in ALT during the study are presented in Table 12, where A1 log.sub.10 reduction in HBsAg, B is 0.5-1 log.sub.10 reduction in HBsAg, and C is <0.5 log.sub.10 reduction in HBsAg, and X3-fold of ALT of vehicle control, which considered normal. Y is 3-fold-30-fold of ALT of vehicle control, and Z is 30-fold of ALT of vehicle control Both Y and Z show liver toxicity. The S and X ASO Trigger in combination of (1:1 and 2:1) while the total dosing drug amounts (in mg) are constant regardless whether they are single agents or combined agents. The results showed S and X combination (1:1) has minor synergistic effect while 2:1 (S:X) mixture showed less benefit.
TABLE-US-00015 TABLE 12 HBsAg Nadir (Log) and ALT for 6 Repeat Doses at D 0, 3, 7, 14, 21, 28 HBsAg Nadir ASO #.sup.* Dose (Log) ALT 123 6 10 mg/kg B X 123 6 3 mg/kg C X 130 6 10 mg/kg B X 130 6 3 mg/kg C X 120 6 10 mg/kg A X 120 6 3 mg/kg A X ASO 120 +ASO 130 (1:1) 6 10 mg/kg A X ASO 120 +ASO 130 (1:1) 6 3 mg/kg B X ASO 120 +ASO 121 (1:1) 6 10 mg/kg A X ASO 120 +ASO 121 (1:1) 6 3 mg/kg A X ASO 120 +ASO 121 (2:1) 6 10 mg/kg A X ASO 120 +ASO 121 (2:1) 6 3 mg/kg B X ASO 121 6 10 mg/kg A X ASO 121 6 3 mg/kg B X .sup.*For combinations, (1:1) and (2:1) refer to mass ratios of the ASOs.
Example 12. ASO Dose Response Testing in HBsAg Release Assay in HepG2.2.15 HBV Cell Model
[0165] In vitro screenings of increasing doses of ASOs were carried out in HepG2.2.15 cells using HBsAg release assay. The dose response curves and resulting IC.sub.50 (nm) values for three experiments are shown in
TABLE-US-00016 TABLE13 IC.sub.50(nM)valuesforASODoseResponse IC50(nM) SEQID ASO Expt. Expt. Expt. NO. # Sequence 1 2 3 420 126 5-mA- B B B lnGpslnApslnTpslnApslnApsApsAps(50h)CpsGps(5m) Cps(5m)CpsGps(5m)CpslnApslnGpslnApscp(5m)C-3 400 120 5-GalNac4-ps2-p-mA- C C C lnGpslnApslnTpslnApslnApsApsAps(50h)CpsGps(5m) Cps(5m)CpsGps(5m)CpslnApslnGpslnApscp(5m)C-3 421 124 5- B B B lnGpslnApslnTpslnApslnApsApsAps(5oh)CpsGps(5m) Cps(5m)CpsGps(5m)CpslnApslnGpslnApscp(5m)C-3 422 127 5mU- A A A lnGpslnApscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps (5m)Cps(5m)CpsGpsApsln(5m)CpslnGpslnGpslnG-3 156 121 5GalNac4-ps2-p-mU-po- A A A lnGpslnApscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps (5m)Cps(5m)CpsGpsApsln(5m)CpslnGpslnGpslnG-3 404 125 5 A A A lnGpslnApscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps (5m)Cps(5m)CpsGpsApsln(5m)CpslnGpslnGpslnG-3
Example 13. ASO Synthesis
[0166] ASOs with LNA and/or gap-modified chemistries were synthesized on ABI 394 and Expedite 8909 synthesizers using standard phosphoramidite chemistry. LNA-containing ASOs were conjugated to N-Acetylgalactosamine (GalNac).
[0167] The following specific sequences in Table 14 are within the scope of the present disclosure. As used herein, ln=Locked nucleic acid (LNA); lnA=Locked nucleic acid (LNA) A; ln(5m)Cln(5m)C=Locked nucleic acid (LNA)-5methyl C; lnG=Locked nucleic acid (LNA) G; lnT=Locked nucleic acid (LNA) T; (5m)C=5 methylC; mA=2-O-methoxy A; mU=2-O-methoxy U; (8nh)A=8-amino A; (8nh)G=8-amino G; (2s)T=2-thio T; cp=scp=cyclopropyl; cpC=scpC=cyclopropyl C; cpG=scpG=cyclopropyl G; cpT=scpT=cyclopropyl T; ps=phosphorothioate linkages.
TABLE-US-00017 TABLE14 ASOSynthesis Total Final SEQID ASO Amount Amount NO. No. Sequence5.fwdarw.3 (mg) (mole) 405 380 cpGpscpApscpTpscpApscpApsApsAps(50h) 0.33 0.05 (ASO120 CpsGps(5m)Cps(5m)CpsGps(5m)CpscpApscp analog) GpscpApscp(5m)C 406 381 cpGpscpApscpTpsTps(5m)Cps(8nh)ApsGps 0.36 0.06 (ASO121 (5m)CpsGps(5m)Cps(5m)CpsGpsApscp(5m) analog) CpscpGpscpGpscpG 407 382 cp(5m)Cpscp(5m)CpscpApscp(5m)Cps(5m) 0.36 0.06 (ASO130 Cps(8nh)Aps(5m)CpsGpsApsGpsTps(5m)Cps analog) TpsApscpGpscpApscp(5m)C
[0168] HepG2.2.15 cells (a stable cell line with four integrated HBV genomes) were maintained in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, 1% Glutamine, 1% non-essential amino acids, 1% Sodium Pyruvate and 250 g/ml G418. Cells were maintained at 37 C. in a 5% CO.sub.2 atmosphere. For HBsAg release assay, assay medium was made: DMEM with 5% FBS, 1% penicillin/streptomycin, 1% Glutamine and 1% DMSO. The day before assay, trypsinize HepG2.2.15 cells were washed with Assay Medium once, spun at 250 g5 min, resuspended with Assay Medium, and seed cells at 50,000/well in assay medium in collagen coated 96 well plates. On the next day, ASOs were diluted with Opti-MEM, 9-pt, 3-fold dilution and Lipofectamine RNAiMAX (Invitrogen) was diluted according manufacturer's manual. The ASO dilution and RNAiMAX dilution was mixed, left at room temperature for 5 minutes and 15 l was added to each well of 96 well plate. The plates were left at 37 C., 5% CO.sub.2 in an incubator for 5 days. After incubation, the supernatant was harvested and measured for HBsAg with ELISA kit (Diasino). The cell viability was measured with CellTiter-Glo (Promega). The EC.sub.50, the concentration of the drug required for reducing HBsAg secretion by 50% in relation to the untreated cell control was calculated using the Prism Graphpad. The CC.sub.50, the concentration of the drug required for reducing cell viability by 50% in relation to the untreated cell control was calculated with the same software.
[0169] The resulting EC.sub.50 and CC.sub.50 for the compounds in Table 14 are presented in the following Table 15. The EC.sub.50 values are as follows: A: <0.1 nM, B: 0.1 nM-5 nM, C: >5 nM.
TABLE-US-00018 ASO No. EC.sub.50 CC.sub.50 380 C >500 381 B >500 382 C >500
Example 14. Bioinformatics of ASOs Targeting S and X Gene Regions of HBV
[0170] This example analyzes the genotypic coverage and off target profile of ASOs targeting S and X gene regions of HBV. Table 15 shows the genotypic coverage of HBV genotypes A-J for ASO 120, which targets the S gene region, and ASO 121, which targets the X gene region. The % homology (defined as fully match or with 1 mismatch) among >8000 clinical isolates is shown in Table 16.
TABLE-US-00019 TABLE 16 Genotypic Coverage: % Homology among >8000 clinical isolates Genotype A B C D E F G H I J ASO 120 (S) 98% 100% 99% 100% 100% 100% 100% 100% 100% 100% ASO 121 (X) 99% 100% 99% 100% 96% 100% 99% 100% 100% 97% ASO 120 (S) + 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% ASO 121 (X)
Example 15. ASO Combination Therapies
[0171] This example investigates combination therapies with ASO 120 and ASO 121 and other HBV therapies (e.g., S-antigen transport-inhibiting oligonucleotide polymers (STOPS), tenofovir, and capsid assembly modulators (CAMs)). For the STOPS ALG-010133 combination studies with ASOs (ASO 120:ASO121 in 2:1, 1:1 or 1:2 ratio), 35,000 HepG2.2.15 cells per well were reverse transfected in a collagen I-coated 96-well plate (Corning, Biocoat; Catalog 356698). ALG-010133 and the ASO mixture were diluted in Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific; Catalog 31985088) to 40 the desired final test concentration then serially diluted (1:3) up to 5 or 9 distinct concentrations, respectively. A 3.25-L aliquot of each diluted compound was combined in a checkerboard fashion where the ASO mixture was added to 10 columns with highest concentration at the top of the plate and ALG-010133 was added to 7 rows with the highest concentration at the furthest well on the right of the plate. This combination of compounds was mixed with 0.3 L Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Catalog 13778150) and 6.2 L of Opti-MEM I Reduced Serum Medium. After incubating for 20 minutes, the mixture was added to the HepG2.2.15 cells. Space was also allotted for titrations of each compound alone as reference controls. Cells were incubated with compounds for 3 days at 37 C. in a 5% CO.sub.2 atmosphere. Three days after initial transfection, the media was refreshed, and the cells were re-transfected following the same protocol as used for the initial transfection. After another 3 days, the supernatant were assayed for HBsAg levels and remaining cells were analyzed for cytotoxicity. For ASO mixture combinations with small molecules such as CAM or Tenofovir, the test articles, were dissolved in dimethyl sulfoxide (DMSO) stock solutions and added to cells without transfection at a final concentration of 0.5% DMSO. All the other aspects of the assay were consistent with the protocol used for the ASO+STOPS combination studies. HBV DNA in the supernatant was measured for these combinations with small molecules such as CAM or Tenofovir.
[0172] The HBsAg level was determined using the HBsAg ELISA kit (Diasino Laboratories, Ref. DS187701) according to the manufacturer's protocol. Luminescence was recorded using a Perkin Elmer multilabel counter Victor3V. HBV DNA levels were measured with real time qPCR.
[0173] For the HepG2.2.15 cell viability assay, a Promega CellTiter-Glo Luminescent Cell Viability Assay (Catalog G7572) was used. The CellTiter-Glo Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture based on quantitation of the adenosine triphosphate (ATP) present, which signals the presence of metabolically active cells. Assay plates were set up in the same format as in the anti-HBV activity assays. A 100-L aliquot of CellTiter-Glo reagent was added to each well and incubated at room temperature for 8 minutes. Luminescence was recorded using a Perkin Elmer multilabel counter Victor3V.
[0174] Each experiment was performed in triplicate (3 plates). Mean percentage inhibition of HBsAg from the three experiments was generated and analyzed using Prichard's Method (Mac Synergy II).
[0175] As shown in Table 17, the ASO combination therapy with STOPShad an additive effect in HBsAg reduction with no cytotoxicity. As shown in Table 18, the ASO combination therapy with tenofovir had a strong synergistic effect in HBsAg reduction and the ASO combination therapy with CAM had a moderate synergistic effect in HBsAg reduction.
TABLE-US-00020 TABLE 17 ASO Combination Therapy with STOPS ALG-010133 ASO 120:ASO121 Compound ratio Synergy Cytotoxity STOPS 1:2 Additive No STOPS 1:1 Additive No STOPS 2:1 Additive No
TABLE-US-00021 TABLE 18 ASO Combination (ASO 120 + ASO121 1:1) Therapy with HBV Therapeutic Agents Compound Class Synergy CC50 Tenofovir NA Strong Synergy No CAM CAM II Moderate Synergy No
Example 16. Modified ASO Testing in AAV-HBV Mouse Model
[0176] ASOs with LNA and/or Luxna wing or gap-modified chemistries were synthesized on ABI 394 and Expedite 8909 synthesizers using standard phosphoramidite chemistry. ASOs were tested at a dose of 110 mg/kg or 510 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model.
Example 17. Evaluation of ASO 130 in AAV-HBV Mouse Model
[0177] ASO 130 was synthesized on ABI 394 and Expedite 8909 synthesizers using standard phosphoramidite chemistry. ASO 130 was tested at a dose of 110 mg/kg or 510 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model.
Example 18. Evaluation of ASO ASO 131 in AAV-HBV Mouse Model
[0178] ASO 131 was synthesized on ABI 394 and Expedite 8909 synthesizers using standard phosphoramidite chemistry and conjugated to GalNac4. ASO 131 was tested at a dose of 310 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model.
Example 19. Evaluation of ASO 121 in AAV-HBV Mouse Model
[0179] ASO 121 was synthesized on ABI 394 and Expedite 8909 synthesizers using standard phosphoramidite chemistry and conjugated to GalNac4. ASO 121 was tested at a dose of 510 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model.
Example 20. Evaluation of ASO Modifications
[0180] In this example, modifications (LNA or gap-modified chemistries) of various nucleotide positions in the ASO were screened for potency and toxicity.
[0181] The following specific sequences in Table 19 are within the scope of the present disclosure. As used herein, ln=Locked nucleic acid (LNA); lnA=Locked nucleic acid (LNA) A; ln(5m)C ln(5m)C=Locked nucleic acid (LNA)-5methyl C; lnG=Locked nucleic acid (LNA) G; lnT=Locked nucleic acid (LNA) T; (5m)C=5 methylC; mA=2-O-methoxy A; mU=2-O-methoxy U; (8nh)A=8-amino A; (8nh)G=8-amino G; (2s)T=2-thio T; am=amNA; am(5m)C=AmNA-NCH.sub.3-(5m)C phosphoramidite; cp=scp=cyclopropyl; cpC=scpC=cyclopropyl C; cpG=scpG=cyclopropyl G; A=dA; G=dG, C=dC, T=Thymidine; cpT=scpT=cyclopropyl T; ps=phosphorothioate linkages; p=phosphodiester linkage
TABLE-US-00022 TABLE19 ASOModifications SEQID ASO NO. # Sequence(5.fwdarw.3) 272 132 5-GalNAc1-C6-p-CA- lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cpsln (5m)CpslnGpslnT3 302 133 5-GalNAc5-(PS)2-p- lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cpsln (5m)CpslnGpslnT3 303 134 5-GalNAc5-(PS)2-p- lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cpsln (5m)CpslnGpsAmT3 304 135 5-GalNAc5-(PS)2-p- lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cps Am(5m)CpslnGpslnT3 300 136 5-GalNAc5-(PS)2-p- lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cpsln (5m)CpslnGpscpT3 408 137A 5-GalNAc5-(PS)2-p- lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cps (5m)scpCpslnGpslnT3 409 138 5-GalNAc6-(PS)2-p- lnGpslnGpslnApsTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsln Apsln(5m)CpslnG3 410 132A 5-GalNAc5-(PS)2-p- lnGpslnApslnGpsApsGpsGpsTpsGps(5m)CpsGps(5m)Cps(5m)Cps(5m)Cpsln (5m)CpslnGpslnT3 411 140 5-GalNAc1-C6-p-CA- lnGpslnApslnTpslnApslnAps(8nh)ApsAps(5m)CpsGps(5m)Cps(5m)CpsGps (5m)CpslnApslnGpslnApsln(5m)C-3 412 141 5-GalNAc1-C6-p-CA- lnGpslnApslnTpslnApslnApsAps(8nh)Aps(5m)CpsGps(5m)Cps(5m)CpsGps (5m)CpslnApslnGpslnApsln(5m)C-3 413 142 5-GalNAc1-C6-p-CA- lnGpslnApslnTpslnApslnApsApsAps(5OH)CpsGps(5m)Cps(5m)CpsGps(5m) CpslnApslnGpslnApsln(5m)C-3 369 143 5- ln(5m)Cpsam(5m)CpslnApsln(5m)Cps(5m)CpsAps(5m)CpsGpsApsGpsTps (5m)CpsTpsApslnGpslnApsln(5m)C-A-p-(PS)2-GalNAc4-3 370 144 5- am(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)CpsAps(5m)CpsGpsApsGpsTps (5m)CpsTpsApslnGpslnApsln(5m)C-A-p-(PS)2-GalNAc4-3 414 145A 5- am(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)CpsAps(5m)CpsGpsApsGpsTps (5m)CpsTpsApslnGpslnApsln(5m)C-A-p-(PS)2-GalNAc43 371 146 5- ln(5m)Cpsln(5m)CpslnApsam(5m)Cps(5m)CpsAps(5m)CpsGpsApsGpsTps (5m)CpsTpsApslnGpslnApsln(5m)C-A-p-(PS)2-GalNAc43 270 147 5-GalNAc2-C6-p- CAlnApslnGpsln(5m)CpsGpsApsApsGpsTpsGps(5m)CpsAps(5m)CpsApsln (5m)CpslnGpslnG-3 415 148 5-GalNAc1-C6-p-CA- ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(50H)CpsAps(5m)CpsGpsApsGpsTps (5m)CpsTpsApslnGpslnApsln(5m)C3 416 149 5-GalNAc1-C6-p-CA- ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)Cps(8nh)Aps(5m)CpsGpsApsGps Tps(5m)CpsTpsApslnGpslnApsln(5m)C3 417 150 5-GalNAc1-C6-p-CA- ln(5m)Cpsln(5m)CpslnApsln(5m)Cps(5m)CpsAps(5OH)CpsGpsApsGpsTps (5m)CpsTpsApslnGpslnApsln(5m)C3 284 151 5-GalNAc6-(PS)2-p- lnGpslnGpslnApsTpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsln Apsln(5m)CpslnG3 286 152 5-GalNAc6-(PS)2-p- lnGpslnGpslnAps(2s)TpsTps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)Cps GpslnApsln(5m)CpslnG3 287 153 5-GalNAc6-(PS)2-p- lnGpslnGpslnApsTps(2s)Tps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)Cps GpslnApsln(5m)CpslnG3 288 154 5-GalNAc6-(PS)2-p- lnGpslnGpslnApsTpsTps(5oh)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGps lnApsln(5m)CpslnG3 378 155 5nGpslnGpslnApsTps(2s)Tps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m) CpsGpslnApscp(5m)CpslnG-A-p-(PS)2-GalNAc6-3 377 156 5nGpslnGpslnApsTpsTps(5Oh)CpsApsGps(5m)CpsGps(5m)Cps(5m)Cps GpslnApscp(5m)CpslnG-A-p-(PS)2-GalNAc4-3 392 157 5- lnGpslnApslnTpsTps(5OH)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln (5m)CpslnGpslnGpscpG-T-p-(PS)2-GalNAc4-3 393 158 5- lnGpslnApscpTps(2s)Tps(5m)CpsApsGps(5m)CpsGps(5m)Cps(5m)CpsGps Apsln(5m)CpslnGpslnGpslnG-T-p-(PS)2-GalNAc4-3 394 159 5- lnGpslnApscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps(5m)Cps(5m)CpsGps Apsln(5m)CpslnGpslnGpslnG-T-p-(PS)2-GalNAc4-3
[0182] HBV mice were treated with ASOs 133-136, and 137A at a dose of 310 mg/kg QW. The resulting change in serum HBsAg is shown in
[0183] HBV mice were treated with ASOs 138 or 153 at a dose of 310 mg/kg QW.
[0184] HBV mice were treated with ASOs 132A or 137A at a dose of 310 mg/kg QW.
[0185] HBV mice were treated with ASOs 140-142 a dose of 510 mg/kg Q3D.
[0186] HBV mice were treated with ASOs 143, 144, 145A, or 146 a dose of 310 mg/kg Q3D.
[0187] HBV mice were treated with ASOs 148-150 a dose of 510 mg/kg Q3D.
[0188] HBV mice were treated with ASOs 151-154 a dose of 310 mg/kg, SC, QW.
[0189] HBV mice were treated with ASOs 147, 155, or 156 a dose of 310 mg/kg, SC, Q3D.
[0190] HBV mice were treated with ASOs 157-159 a dose of 510 mg/kg, SC, Q3D.
TABLE-US-00023 SEQID NO: Description Sequence 1 HepatitisB ctccaccactaccaccaaactatcaagatcccagagtcagggccctgtactttcctgctggtggctcaagttc virus cggaacagtaaaccctgctccgactactgcctctcccatatcgtcaatcttctcgaggactggggaccctgtac (Genbank cgaatatggagagcaccacatcaggattcctaggacccctgctcgtgttacaggcggggtttttcttgttgaca Accession agaatcctcacaataccacagagtctagactcgtggtggacttctctcaattttctagggggagcacccacgtg No. tcctggccaaaatttgcagtccccaacctccaatcactcaccaacctcttgtcctccaatttgtcctggttatcgct KC315400.1) ggatgtgtctgcggcgttttatcatcttcctcttcatcctgctgctatgcctcatcttcttgttggttcttctggactac caaggtatgttgcccgtttgtcctctacttccaggaacatcaactaccagcaccggaccatgcaaaacctgcac aactactgctcaagggacctctatgtttccotcatgttgctgtacaaaacctacggacggaaactgcacctgtat tcccatcccatcatcttgggctttcgcaaaatacctatgggagtgggcctcagtccgtttctcttggctcagtttac tagtgccatttgttcagtggttcgtagggctttcccccactgtctggctttcagttatatggatgatgtggttttggg ggccaagtotgtacaacatcttgagtccctttataccgctgttaccaattttcttttatctttgggtatacatttaaacc ctcacaaaacaaaaagatggggatattccdtaacttcatgggatatgtaattgggagttggggcactttgcctc aggaacatattgtacaaaaaatcaagcaatgttttaggaaacttcctgtaaacaggcctattgattggaaagtat gtcaacraattgtgggtcttttggggtttgccgcccctttcacgcaatgtggatatcctgctttaatgcctttatatg catgtatacaagctaagcaggcttttactttctcgccaacttacaaggcctttctgtgtaaacaatatctgaaccttt accccgttgctcggcaacggtcaggtctttgccaagtgtttgctgacgcaacccccactggttggggcttggc cataggccatcagcgcatgcgtggaacctttgtggctcctctgccgatccatactgcggaactcctagcagctt gttttgctcgcagccggtctggagcaaaacttatcggcaccgacaactctgttgtcctctctcggaaatacacct cctttccatggctgctaggatgtgctgccaactggatcctgcgcgggacgtcctttgtctacgtcccgtcggcg ctgaatcccgcggacgacccatctcggggccgtttgggactctaccgtccccttctgcgtctgccgttccgcc cgaccacggggcgcacctctctttacgcggtctccccgtctgtgccttctcatctgccggaccgtgtgcacttc gcttcacctctgcacgtcgcatggagaccaccgtgaacgcccacgggaacctgcccaaggtcttgcataaga ggactcttggactttcagcaatgtcaacgaccgaccttgaggcatacttcaaagactgtgtgtttactgagtggg aggagttgggggaggaggttaggttaaaggtctttgtactaggaggctgtaggcataaattggtgtgttcacca gcaccatgcaactttttcacctctgcctaatcatctcatgttcatgtcctactgttcaagcctccaagctgtgccttg ggtggctttggggcatggacattgacccgtataaagaatttggagcttctgtggagttactctcttttttgccttct gacttctttccttctattcgagatctcctcgacaccgcctctgctctgtatcgggaggccttagagtctccggaac attgttcacctcaccatacggcactcaggcaagcaattctgtgttggggtgagttaatgaatctagccacctgg gtgggaagtaatttggaagatccagcatccagggaattagtagtcagctatgtcaacgttaatatgggcctaaa aatcagacaactattgtggtttcacatttcctgtcttacttttgggagagaaactgttcttgaatatttggtgtcttttg gagtgtggattcgcactcctcctgcatatagaccacaaaatgcccctatcttatcaacacttccggaaactactg ttgttagacgaagaggcaggtcccctagaagaagaactccctcgcctcgcagacgaaggtctcaatcgccg cgtcgcagaagatctcaatctcgggaatctcaatgttagtattccttggacacataaggtgggaaactttacgg ggctttattcttctacggtaccttgctttaatcctaaatggcaaactccttcttttcctgacattcatttgcaggagga cattgttgatagatgtaagcaatttgtggggccccttacagtaaatgaaaacaggagacttaaattaattatgcct gctaggttttatcccaatgttactaaatatttgcccttagataaagggatcaaaccgtattatccagagtatgtagtt aatcattacttccagacgcgacattatttacacactctttggaaggcggggatcttatataaaagagagtccaca cgtagcgcctcattttgcgggtcaccatattcttgggaacaagatctacagcatgggaggttggtcttccaaac ctcgaaaaggcatggggacaaatctttctgtccccaatcccctgggattcttccccgatcatcagttggaccct gcattcaaagccaactcagaaaatccagattgggacctcaacccacacaaggacaactggccggacgccaa caaggtgggagtgggagcattcgggccagggttcacccctcctcatgggggactgttggggtggagccctc aggctcagggcatattcacaacagtgccagcagctcctcctcctgcctccaccaatcggcagtcaggaaggc agcctactcccttctctccacctctaagagacactcatcctcaggccatgcagtggaa