Patent Application
20200385735
About 300 million people are chronically infected with HBV worldwide. HBsAg loss, a key aspect of “functional cure” is the goal of many new therapies. Antisense oligonucleotides have been demonstrated to be an effective modality in reducing HBsAg in animal models and clinical studies with these molecules are ongoing.
However, the treatments of HBV with antisense oligonucleotides still suffer from, e.g., nuclease degradation and liver toxicity. Thus, there is a need in the art to discover antisense oligonucleotides having greater resistance to nuclease degradation and improved liver safety profiles.
The present disclosure relates to compounds and compositions containing oligonucleotides and their use in preventing or treating diseases and conditions, e.g., hepatitis B (HBV).
Some embodiments include 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.
In some embodiments, a method of treating a subject having a Hepatitis B virus (HBV) infection comprises administering to the subject a first antisense oligonucleotide (ASO), wherein the first ASO contains 14-22 nucleotide units, and the first ASO contains: (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. In some embodiments, the method further comprises administering to the subject a second ASO, wherein the second ASO contains 14-22 nucleotide units, and the second ASO contains: (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, 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.
In some embodiments, the 5′-wing region of at least one of the first and second ASO comprises 2 to 6 phosphorothioate-linked locked nucleosides. In some embodiments, the 5′-wing region of at least one of the first and second ASO comprises 2 to 6 phosphorothioate-linked 2′ substituted nucleosides. In some embodiments, the 5′-wing region of at least one of the first and second ASO 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 of at least one of the first and second ASO 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 of at least one of the first and second ASO are linked by a phosphorothioate linker. In some embodiments, at least 2, 3, 4, 5, or 6 nucleosides of the 5′-wing region of at least one of the first and second ASO are linked by a phosphorothioate linker. In some embodiments, the 3′-wing region of at least one of the first and second ASO comprises 2 to 6 phosphorothioate-linked locked nucleosides. In some embodiments, the 3′-wing region of at least one of the first and second ASO comprises 2 to 6 2′ phosphorothioate-linked substituted nucleosides. In some embodiments, the 3′-wing region of at least one of the first and second ASO 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 of at least one of the first and second ASO 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 of at least one of the first and second ASO are linked by a phosphorothioate linker. In some embodiments, at least 2, 3, 4, 5, or 6 nucleosides of the 3′-wing region of at least one of the first and second ASO are linked by a phosphorothioate linker. In some embodiments, the central region of at least one of the first and second 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 of at least one of the first and second ASO are linked by a phosphorothioate linker. In some embodiments, a DNA nucleoside of central region of at least one of the first and second ASO is linked to a nucleoside of a 5′-wing region of at least one of the first and second ASO by a phosphorothioate linker. In some embodiments, a DNA nucleoside of central region of at least one of the first and second ASO is linked to a nucleoside of a 3′-wing region of at least one of the first and second ASO by a phosphorothioate linker. In some embodiments, the central region of at least one of the first and second ASO each independently comprises 8 to 10 contiguous phosphorothioate-linked DNA nucleosides. In some embodiments, the locked nucleosides are selected from LNA, scpBNA, AmNA (N—H), AmNA (N-Me), GuNA, GuNA (N—R) where R is selected from Me, Et, i-Pr, t-Bu and combinations thereof. In some embodiments, the second ASO is complementary or hybridizes to the viral target RNA sequence in the second X region of HBV. In some embodiments, the second ASO is complementary or hybridizes to the viral target RNA sequence in the S region of HBV. In some embodiments, the first and/or second ASO further comprises a targeting group. In some embodiments, the targeting group comprises a GalNAc moiety. In some embodiments, the first and second ASO each independently contain 14-18 nucleotide units. In some embodiments, the first and second ASO are administered concurrently. In some embodiments, the first and second ASO are administered consecutively. In some embodiments, the subject is a mammal. In some embodiments, the mammal is an adult human. In some embodiments, the treatment comprises reducing a viral load of HBV in the subject. In some embodiments, the treatment comprises reducing a level of a virus antigen in the subject. In some embodiments, the first 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 second ASO comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the sequences in Table 1, 2A, 3, 4, 7, 8, 9, 10, 11, 13, 14, or 19. In some embodiments, the first 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 second 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 first ASO is ASO 120 or ASO 121. In some embodiments, the second ASO is ASO 120 or ASO 121.
Some embodiments include 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. In some embodiments, the 5′-wing region of at least one of the first and second ASO 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 2, 3, 4, 5, or 6 nucleosides of the 5′-wing region of at least one of the first and second ASO are linked by a phosphorothioate linker. In some embodiments, the 3′-wing region of at least one of the first and second ASO 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 2, 3, 4, 5, or 6 nucleosides of the 3′-wing region of at least one of the first and second ASO are linked by a phosphorothioate linker. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleosides of the central region of at least one of the first and second ASO are linked by a phosphorothioate linker. In some embodiments, a DNA nucleoside of central region of at least one of the first and second ASO is linked to a nucleoside of a 5′-wing region of at least one of the first and second ASO by a phosphorothioate linker. In some embodiments, a DNA nucleoside of central region of at least one of the first and second ASO is linked to a nucleoside of a 3′-wing region of at least one of the first and second ASO by a phosphorothioate linker.
Other embodiments include an antisense oligonucleotide 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. In some embodiments, the ASO further comprises a targeting moiety. In some embodiments, the targeting moiety comprises a GalNAc moiety. In some embodiments, the targeting moiety comprises three consecutive GalNAc moieties attached through linkers.
Other embodiments include an antisense oligonucleotide comprising 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 further comprises a targeting moiety. In some embodiments, the targeting moiety comprises a GalNAc moiety. In some embodiments, the targeting moiety comprises three consecutive GalNAc moieties attached through linkers.
Additional embodiments include a method of treating a subject having a Hepatitis B virus (HBV) infection, comprising administering to the subject a therapeutically effective amount of one or more ASO of any of the preceding embodiments.
Some embodiments include methods of any of the preceding embodiments, further comprising administering to the patient an additional HBV treatment agent, such as a nucleotide analog, a capsid assembly modulator or another oligonucleotide. In some embodiments, the additional HBV treatment agent is selected from the group consisting of include STOPS™ ALG-010133, Capsid Assembly Modulator ALG-000184, recombinant interferon alpha 2b, IFN-α, PEG-IFN-α-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, the GalNAc moiety comprises one GalNAc moiety or three consecutive GalNAc moieties attached through linkers, wherein the GalNAc moiety is GalNAc-4 or GalNAc-6.
Some embodiments include methods of any of the preceding embodiments, wherein the patient has been treated with an additional HBV treatment agent, such as a nucleotide analog, a capsid assembly modulator or another oligonucleotide.
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.
Compounds of the Present Disclosure
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.
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.
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.
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).
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:
Other exemplary locked nucleosides include the following:
where R is H or alkyl (or AmNA(N-Me)) when R is alkyl);
In certain embodiments, all nucleosides in the 5′-wing region are locked nucleosides. In some embodiments, all nucleosides in the 3′-wing region are locked nucleosides. In some embodiments, the 3′-wing region comprises LNA and one or two nucleosides selected from ScpBNA, AmNA, and GuNA. In some embodiments, 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.
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.
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.
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.
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:
where R is a halogen or R′—C≡C—; and R′ is C6-12 aryl, 5- to 12-membered heteroaryl, hydroxy-C1-6 alkyl, or C1-7 alkanoyloxy. In some embodiments, the central region includes one modified nucleotide (e.g., (2s)T or (5OH)C) at the 1st, 2nd, 3rd or 4th gap nucleoside position (from the 5′ end). In some embodiments, the modified nucleotide is at the 3rd gap nucleoside position (from the 5′ end). In some embodiments, the modified nucleotide is a nucleotide having the structure of:
wherein:
W is independently O, N, or S;
R1, R2, and R5 are independently H or D;
R4 is F or OCH3; and
wherein:
R is a halogen or R′—C≡C—; and
R′ represents C6-12 aryl, 5- to 12-membered heteroaryl, hydroxy-C1-6 alkyl, or C1-7 alkanoyloxy. In some embodiments, C1-7 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.
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.
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.
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.
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).
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.
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.
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).
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.
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.
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.
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.
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.
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:
In some embodiments, the ASO contains a targeting moiety at the 5′-end, the 3′-end, or both ends of the ASO.
In certain embodiments, the ASO is modified at one or more end by a vinyl phosphonate moiety, such as a 5′-vinyl phosphonate moiety.
Compositions
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.
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.
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.
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.
Methods of Use
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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-α, PEG-IFN-α-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.
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.
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.
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.
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.
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.
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.
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.
Gapmer ASO Sequences
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 (P2O5, 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.
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 (ABz), 4-N-acetylcytidine (CAc), 2-N-isobutyrylguanosine (GiBu), 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 CH3CN 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%.
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.
After filtering to remove the solid support, the deprotection solution was removed under vacuum in a GeneVac centrifugal evaporator.
The 2′-MOE phosphoramidites
Modified Gapmer Sequences
The AmNA (N-Me)-T, AmNA (N-Me)-4-N-benzoyl (5m) cytidine ((5m) CBz), AmNA (N-Me)-4-N-benzoylcytidine (ABz), and AmNA (N-Me)-2-N-pac (Gpac), were purchased from Luxna Biotech, whereas scp-BNA-T, scp-BNA-6-N-benzoyladenosine (ABz), scp-BNA-4-N-benzoyl-5 methyl cytidine ((5m) CBz), scp-BNA-2-N-iguanosine (GiBu) 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 P2O5, 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 μM scale in a 3′ to 5′ direction with the phosphoramidite monomers diluted to a concentration of 0.12 M in anhydrous CH3CN in the presence of 0.3 M 5-(benzylthio)-1H-tetrazole activator (coupling time 16 min) 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.
scp-BNA Monomers
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.
Quantitation of Crude Oligomer or Raw Analysis
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.
Crude HPLC/LC-MS Analysis
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.
HPLC Purification
The Phosphodiester (PO), Phosphorothioate (PS) and chimeric modified oligonucleotides were purified by anion-exchange HPLC. The buffers were 20 mM sodium phosphate in 10% CH3CN, pH 8.5 (buffer A) and 20 mM sodium phosphate in 10% CH3CN, 1.8 M NaBr, pH 8.5 (buffer B). Fractions containing full-length oligonucleotides were pooled, desalted and lyophilized.
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% CH3CN, (buffer A) and CH3CN (buffer B). Fractions containing full-length oligonucleotides were pooled, desalted and lyophilized.
Desalting of Purified Oligomer
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.
Final HPLC and Electrospray LC/MS Analysis
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.
Post-Synthesis Conjugation of GalNAc Esters to Oligonucleotides
5′-C6-Amino Precursor Synthesis
The sequences were synthesized at 10 μmol scale using universal support (Loading 65 μmol/g). At the 5′-terminal to introduce C6-NH2 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-NH2 modified ASO's was used to perform post synthesis conjugation.
Post Synthesis Conjugation of 5′-GalNAc Synthesis
The 5′-C6-NH2 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.
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 μm/ml G418. Cells were maintained at 37° C. in a 5% CO2 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 g×5 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 μl was added to each well of 96 well plate. The plates were left at 37° C., 5% CO2 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 EC50, 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 CC50, 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.
The resulting EC50 and CC50 for the compounds in Table 1 are presented in the following Table 2. The EC50 values are as follows: A: <0.1 nM, B: 0.1 nM-1 nM, C: 1-10 nM.
Cps(5m)CpsGps(5m)CpsCpslnApslnGpslnApsln(5m)C 3′
In Table 2A, the bold nucleosides contain the following modifications:
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 3×10 mg/kg once a week (QW); or 3×10 mg/kg every 3 days (Q3D); or 5×10 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:
The resulting nadir log10 reduction in serum HBsAg during the study are presented in the following table, where A≥1 log10 reduction in HBsAg, B is 0.5-1 log10 reduction in HBsAg, and C is <0.5 log10 reduction in HBsAg
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 EC50 values are as follows: A: <0.2 nM, B: 0.2 nM-0.3 nM, C: 0.3-5 nM.
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 3×10 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 3×10 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 Log10 IU/ml while reducing serum alanine aminotransferase (ALT) by 3-fold.
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 3×10 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 3×10 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.
ASOs were tested at 1×5 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 1×5 mg/kg, majority of ASOs needs more stringent dosing regimen to be differentiated in liver tox. The resulting nadir log10 reduction in serum HBsAg and fold-change in ALT during the study are presented in Table 7, where A≥1 log10 reduction in HBsAg, B is 0.5-1 log10 reduction in HBsAg, and C is <0.5 log10 reduction in HBsAg, and X≤3-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.
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.
ASOs were tested at 3×10 mg/kg every week in the adeno-associated virus (AAV)-HBV mouse model. This dosing regimen of 3×10 mg/kg QW is more stringent than 1×5 mg/kg shown in previous section. We can further select potent ASO with least ALT elevation. The resulting nadir log10 reduction in serum HBsAg and fold-change in ALT during the study are presented in Table 8, where A≥1 log10 reduction in HBsAg, B is 0.5-1 log10 reduction in HBsAg, and C is <0.5 log10 reduction in HBsAg, and X≤3-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.
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.
ASOs were tested at 3×10 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model. The dosing regimen of 3×10 mg/kg Q3D is more stringent than 3×10 mg/kg QW and can further select ASOs with best therapeutic indexes. The resulting nadir log10 reduction in serum HBsAg and fold-change in ALT during the study are presented in Table 9, where A≥1 log10 reduction in HBsAg, B is 0.5-1 log10 reduction in HBsAg, and C is <0.5 log10 reduction in HBsAg, and X≤3-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.
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.
This example evaluates the therapeutic index of ASOs using a dosing regimen of 5×10 mg/kg. ASOs were tested at 5×10 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model. The resulting nadir log10 reduction in serum HBsAg and fold-change in ALT during the study are presented in Table 10, where A≥1 log10 reduction in HBsAg, B is 0.5-1 log10 reduction in HBsAg, and C is <0.5 log10 reduction in HBsAg, and X≤3-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.
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.
ASOs were tested at 1×10 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 log10 reduction in serum HBsAg and fold-change in ALT during the study are presented in Table 11, where A≥1 log10 reduction in HBsAg, B is 0.5-1 log10 reduction in HBsAg, and C is <0.5 log10 reduction in HBsAg, and X≤3-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.
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 log10 reduction in serum HBsAg and fold-change in ALT during the study are presented in Table 12, where A≥1 log10 reduction in HBsAg, B is 0.5-1 log10 reduction in HBsAg, and C is <0.5 log10 reduction in HBsAg, and X≤3-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.
*For combinations, (1:1) and (2:1) refer to mass ratios of the ASOs.
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 IC50 (nm) values for three experiments are shown in
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).
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.
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 μg/ml G418. Cells were maintained at 37° C. in a 5% CO2 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 g×5 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 μl was added to each well of 96 well plate. The plates were left at 37° C., 5% CO2 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 EC50, 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 CC50, 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.
The resulting EC50 and CC50 for the compounds in Table 14 are presented in the following Table 15. The EC50 values are as follows: A: <0.1 nM, B: 0.1 nM-5 nM, C: >5 nM.
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.
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-μL 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 μL Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Catalog 13778150) and 6.2 μL 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% CO2 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.
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.
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-μL 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.
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).
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.
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 1×10 mg/kg or 5×10 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model.
ASO 130 was synthesized on ABI 394 and Expedite 8909 synthesizers using standard phosphoramidite chemistry. ASO 130 was tested at a dose of 1×10 mg/kg or 5×10 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model.
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 3×10 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model.
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 5×10 mg/kg every 3 days in the adeno-associated virus (AAV)-HBV mouse model.
In this example, modifications (LNA or gap-modified chemistries) of various nucleotide positions in the ASO were screened for potency and toxicity.
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-NCH3-(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
HBV mice were treated with ASOs 133-136, and 137A at a dose of 3×10 mg/kg QW. The resulting change in serum HBsAg is shown in
HBV mice were treated with ASOs 138 or 153 at a dose of 3×10 mg/kg QW.
HBV mice were treated with ASOs 132A or 137A at a dose of 3×10 mg/kg QW.
HBV mice were treated with ASOs 140-142 a dose of 5×10 mg/kg Q3D.
HBV mice were treated with ASOs 143, 144, 145A, or 146 a dose of 3×10 mg/kg Q3D.
HBV mice were treated with ASOs 148-150 a dose of 5×10 mg/kg Q3D.
HBV mice were treated with ASOs 151-154 a dose of 3×10 mg/kg, SC, QW.
HBV mice were treated with ASOs 147, 155, or 156 a dose of 3×10 mg/kg, SC, Q3D.
HBV mice were treated with ASOs 157-159 a dose of 5×10 mg/kg, SC, Q3D.
This application claims priority to U.S. Provisional Application No. 62/943,532, filed Dec. 4, 2019, U.S. Provisional Application No. 62/937,760, filed Nov. 19, 2019, and U.S. Provisional Application No. 62/855,793, filed May 31, 2019, the disclosures of which are hereby incorporated by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 62855793 | May 2019 | US | |
| 62937760 | Nov 2019 | US | |
| 62943532 | Dec 2019 | US |
