Modified gapmer oligonucleotides and methods of use
The disclosure includes antisense oligonucleotides, including gapmer antisense oligonucleotides, along with methods of making and use, e.g., against HBV.
1. 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 the nucleotide at position 3 from the 5′ end of the central region is a modified nucleotide, and wherein at least 5 of the DNA nucleosides in the central region are connected by phosphorothioate linkages,
(b) a 5′-wing region (A′) comprising 2 to 6 locked nucleosides and at least one 2′ substituted nucleoside, and
(c) a 3′-wing region (C′) comprising 3 to 6 locked nucleosides, wherein at least 3 of the locked nucleosides in the 3′-wing region are connected by phosphorothioate linkages,
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; and
wherein at least one of the 5′ wing region and the 3′ wing region contains at least one locked nucleoside independently selected from 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,
wherein the modified nucleotide in a central region is selected from
wherein:
R is a halogen or R′—C═C—; and
R′ is C 6-12 aryl, 5- to 12-membered heteroaryl, hydroxy-C 1-6 alkyl, or C1-7 alkanoyloxy.
2. A pharmaceutical composition comprising an ASO according to claim 1 .
3. The ASO of claim 1 , wherein the 5′-wing region comprises 2 to 6 phosphorothioate-linked locked nucleosides.
4. The ASO of claim 1 , wherein the 5′ or 3′ wing region further comprises at least one locked nucleoside independently selected from locked nucleic acid A (lnA), locked nucleic acid-5methyl C (ln(5m)C); locked nucleic acid G (lnG); locked nucleic acid T (lnT), and combinations thereof.
5. An antisense oligonucleotide (ASO) selected from:
(i) 5′-mA-lnGpslnApslnTpslnApslnApsApsAps(5oh)CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnA pscp(5m)C-3′ (SEQ ID NO: 420),
(ii) 5′-GalNac4-ps2-p-mA-lnGpslnApslnTpslnApslnApsApsAps(5oh)CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnA pscp(5m)C-3′ (SEQ ID NO: 400),
(iii) 5′-lnGpslnApslnTpslnApslnApsApsAps(5oh)CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnA pscp(5m)C-3′ (SEQ ID NO: 421),
(iv) 5′-mU-lnGpslnApscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln(5m)CpslnG pslnGpslnG-3′ (SEQ ID NO: 422),
(v) 5′-GalNac4-ps2-p-mU-po-lnGpslnApscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln(5m)CpslnG pslnGpslnG-3′ (SEQ ID NO: 156), and
(vi) 5′-lnGpslnApscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln(5m)CpslnG pslnGpslnG-3′ (SEQ ID NO: 404).
6. The ASO of claim 5 , wherein the ASO is 5′-mA-lnGpslnApslnTpslnApslnApsApsAps(5oh)CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnA pscp(5m)C-3′ (SEQ ID NO: 420).
7. The ASO of claim 5 , wherein the ASO is 5′-GalNac4-ps2-p-mA-lnGpslnApslnTpslnApslnApsApsAps(5oh)CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnA pscp(5m)C-3′ (SEQ ID NO: 400).
8. The ASO of claim 5 , wherein the ASO is 5′-lnGpslnApslnTpslnApslnApsApsAps(5oh)CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnA pscp(5m)C-3′ (SEQ ID NO: 421).
9. The ASO of claim 5 , wherein the ASO is 5′-mU-lnGpslnApscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln(5m)CpslnG pslnGpslnG-3′ (SEQ ID NO: 422).
10. The ASO of claim 5 , wherein the ASO is 5′-GalNac4-ps2-p-mU-po-lnGpslnApscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln(5m)CpslnG pslnGpslnG-3′ (SEQ ID NO: 156).
11. The ASO of claim 5 , wherein the ASO is 5′ lnGpslnApscpTpsTps(5m)Cps(8nh)ApsGps(5m)CpsGps(5m)Cps(5m)CpsGpsApsln(5m)CpslnG pslnGpslnG-3′ (SEQ ID NO: 404).
12. The ASO of claim 1 further comprising a targeting group.
13. The ASO of claim 12 , wherein the targeting group comprises a GalNAc moiety.
14. The ASO of claim 5 further comprising a targeting group.
15. The ASO of claim 14 , wherein the targeting group comprises a GalNAc moiety.
16. 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 1 .