O-methyl rich fully stabilized oligonucleotides

Patent metadata

Field	Value
Publication number	US-11279930-B2
Application number	US-201916550076-A
Country	US
Kind code	B2
Filing date	Aug 23, 2019
Priority date	Aug 23, 2018
Publication date	Mar 22, 2022
Grant date	Mar 22, 2022

Abstract

Novel oligonucleotides that are fully chemically stabilized are provided. Methods of using oligonucleotides that are fully chemically stabilized are also provided.

First claim

What is claimed:

1. A double-stranded nucleic acid comprising an antisense strand and a sense strand, wherein:
the antisense strand comprises at least 14 contiguous nucleotides, a 5′ end and a 3′ end, and has complementarity to a target;
the sense strand comprises at least 13 contiguous nucleotides, a 5′ end and a 3′ end, and has complementarity to the antisense strand;
the antisense strand comprises at least 85% 2′-O-methyl modifications;
the nucleotides at positions 2 and 14 from the 5′ end of the antisense strand comprise a non-2′-O-methyl modification; and
the nucleotides at positions 7, 9, 10, and 11 from the 3′ end of the sense strand comprise a non-2′-O-methyl modification or the nucleotides at positions 7, 10, and 11 from the 3′ end of the sense strand comprise a non-2′-O-methyl modification.

2. The double-stranded nucleic acid of claim 1 , wherein the antisense strand comprises perfect or less than perfect complementarity to the target, optionally wherein the target comprises mammalian or viral mRNA.

3. The double-stranded nucleic acid of claim 2 , wherein the antisense strand comprises 20 contiguous nucleotides or 22 contiguous nucleotides.

4. The double-stranded nucleic acid of claim 2 , wherein the antisense strand comprises a 5′ vinyl phosphonate.

5. The double-stranded nucleic acid of claim 1 , wherein the antisense strand comprises at least 90% 2′-O-methyl modified nucleotides.

6. The double-stranded nucleic acid of claim 2 , wherein one or more nucleotide mismatches are present at positions 2, 6, and 12 from the 5′ end of sense strand.

7. The double-stranded nucleic acid of claim 1 , wherein the nucleotide at position 20 from the 5′ end of the antisense strand comprises a non-2′-O-methyl modification.

8. The double-stranded nucleic acid of claim 1 , wherein one or more nucleotides at positions 1-7 from the 3′ end of the antisense strand are connected to adjacent nucleotides via phosphorothioate linkages.

9. The double-stranded nucleic acid of claim 1 , wherein the nucleotides at positions 1-6 from the 3′ end or 1-7 from the 3′ end of the antisense strand are connected to adjacent nucleotides via phosphorothioate linkages.

10. The double-stranded nucleic acid of claim 1 , wherein the nucleotides at positions 2 and 14 from the 5′ end of the antisense strand comprise a 2′-F modification or 2′-H modification, or a 2′-OH moiety.

11. The double-stranded nucleic acid of claim 1 , wherein the antisense strand comprises between 15 and 22 contiguous nucleotides.

12. The double-stranded nucleic acid of claim 1 , wherein the sense strand comprises between 15 and 20 contiguous nucleotides.

13. The double-stranded nucleic acid of claim 1 , wherein the nucleotides at positions 1 and 2 from the 3′ end of sense strand and/or the nucleotides at positions 1 and 2 from the 5′ end of sense strand are connected to adjacent nucleotides via phosphorothioate linkages.

14. The double-stranded nucleic acid of claim 1 , wherein the sense strand comprises a hydrophobic molecule at the 3′ end of the sense strand.

15. The double-stranded nucleic acid of claim 1 , wherein the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand.

16. The double-stranded nucleic acid of claim 1 , wherein the antisense strand comprises a 5′ phosphate, a 5′-alkyl phosphonate, a 5′ alkylene phosphonate, or a 5′ alkenyl phosphonate.

17. The double-stranded nucleic acid of claim 1 , wherein the double-stranded nucleic acid comprises 4-16 phosphorothioate linkages or 8-13 phosphorothioate linkages.

18. The double-stranded nucleic acid of claim 1 , wherein the double-stranded nucleic acid comprises double-stranded region of 15 base pairs to 20 base pairs.

19. The double-stranded nucleic acid of claim 1 , wherein the sense strand comprises at least 80% 2′-O-methyl modified nucleotides, at least 90% 2′-O-methyl modified nucleotides, or 100% 2′-O-methyl modified nucleotides.

20. The double-stranded nucleic acid of claim 1 , wherein the sense strand comprises 15 contiguous nucleotides, 18 contiguous nucleotides, or 20 contiguous nucleotides.

21. A pharmaceutical composition comprising one or more double-stranded nucleic acids of claim 1 , and a pharmaceutically acceptable carrier.

22. A double-stranded nucleic acid comprising an antisense strand and a sense strand, wherein:
the antisense strand comprises at least 14 contiguous nucleotides, a 5′ end and a 3′ end, and has complementarity to a target;
the sense strand comprises at least 13 contiguous nucleotides, a 5′ end and a 3′ end, and has complementarity to the antisense strand;
the antisense strand comprises at least 85% 2′-O-methyl modifications;
the nucleotides at positions 2 and 14 from the 5′ end of the first oligonucleotide are modified with 2′-F;
the sense strand comprises 100% 2′-O-methyl modifications; and
the nucleotides at positions 1-6 from the 3′ end, or positions 1-7 from the 3′ end, of the antisense strand are connected to adjacent nucleotides via phosphorothioate linkages.

23. A branched oligonucleotide compound capable of mediating RNA silencing in a cell, comprising two or more double-stranded nucleic acids, wherein the nucleic acids (N) are connected to one another by one or more moieties selected from a linker (L), a spacer (S) and optionally a branching point (B), wherein:
each double-stranded nucleic acid comprises an antisense strand and a sense strand, wherein each antisense strand comprises at least 14 contiguous nucleotides, a 5′ end and a 3′ end, and at least one antisense strand comprises at least 85% 2′-O-methyl modifications;
wherein the nucleotides at positions 2 and 14 from the 5′ end of the at least one antisense strand comprise a non-2′- O-methyl modification; and
wherein one or more nucleotides at positions 1-7 from the 3′ end of at least one antisense strand are connected to adjacent nucleotides via phosphorothioate linkages.

24. The branched oligonucleotide compound of claim 23 , wherein:
each antisense strand comprises at least 85% 2′-O-methyl modifications;
the nucleotides at positions 2 and 14 from the 5′ end of each antisense strand comprise a non-2′- O-methyl modification;
one or more nucleotides at positions 1-7 from the 3′ end of each antisense strand are connected to adjacent nucleotides via phosphorothioate linkages;
the nucleotides at positions 1 and 2 from the 5′ end of the sense and antisense strands are connected to adjacent nucleotides via phosphorothioate linkages;
each double-stranded nucleic acid is independently connected to a linker, spacer or branching point at the 3′ end or at the 5′ end of the sense strand or the antisense strand;
the compound further comprises a hydrophobic moiety attached to the terminal 5′ position of the branched oligonucleotide compound;
the nucleotide at position 20 from the 5′ end of the antisense strand comprises a non-2′-O-methyl modification;
the antisense strand comprises 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides; and
the sense strand comprises 15, 16, 17, 18, 19, or 20 contiguous nucleotides.

25. The branched oligonucleotide compound of claim 24 , wherein the hydrophobic moiety comprises an alkyl, alkenyl, or aryl moiety; a vitamin or cholesterol derivative; a lipophilic amino acid; or a combination thereof.

26. The branched oligonucleotide compound of claim 23 , wherein each linker is independently selected fro

Assignees

• Univ Massachusetts

Inventors

• Khvorova Anastasia
• Alterman Julia
• Davis Sarah
• Turanov Anton

Classifications

• C12N2310/343

  having patterns, e.g. ==--==--==-- · CPC title

• C12N2310/315

  Phosphorothioates · CPC title

• C12N15/1137

  against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138) · CPC title

• C12N2310/321

  2'-O-R Modification · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title