Modifications for antisense compounds

• US 9,506,057 B2
• Filed: 02/25/2013
• Issued: 11/29/2016
• Est. Priority Date: 03/26/2010
• Status: Active Grant

• Current Assignee: Integrated DNA Technologies, Inc. (Danaher Corporation)
• Original Assignee: Integrated DNA Technologies, Inc. (Danaher Corporation)
• Inventors: Behlke, Mark Aaron, Lennox, Kimberly Ann, Jacobi, Ashley Mae, Owczarzy, Richard, Walder, Joseph Alan
• Primary Examiner(s): ZARA, JANE J
• Application Number:
  US13/776,397
• Publication Number:
  US 20130236967A1
• Time in Patent Office: 1,373 Days
• Field of Search: 435/6.1, 435/91.1, 435/91.31, 435/375, 435/455, 534/772, 536/23.1, 536/24.5
• US Class Current: 1/1
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/344 Position-specific modifications, e.g. on every purine, at the 3'-end
• CPC Class Codes: C12N 2310/352 linked to the nucleic acid via a carbon atom
• CPC Class Codes: C12N 2310/3521 Methyl

Abstract

The invention pertains to modifications for antisense oligonucleotides, wherein the modifications are used to improve stability and provide protection from nuclease degradation. The modifications could also be incorporated into double-stranded nucleic acids, such as synthetic siRNAs and miRNAs.

Claims

1. An antisense oligonucleotide comprising at least one modification that is incorporated at the terminal end or between two nucleotides of the antisense oligonucleotide, wherein the modification increases binding affinity and nuclease resistance of the antisense oligonucleotide, and wherein the modification is a napthyl-azo compound.

2. The antisense oligonucleotide of claim 1, wherein the modification is located at the 5′

-end of the antisense oligonucleotide.

3. The antisense oligonucleotide of claim 1, wherein the modification is located at the 3′

-end of the antisense oligonucleotide.

4. The antisense oligonucleotide of claim 1, wherein modifications are located at both the 5′

- and 3′

-ends of the antisense oligonucleotide.

5. The antisense oligonucleotide of claim 1, wherein the modification is inserted between nucleotides and is located near the 3′

-end of the antisense oligonucleotide.

6. The antisense oligonucleotide of claim 1, wherein the modification is inserted between nucleotides and is located near the 5′

-end of the antisense oligonucleotide.

7. The antisense oligonucleotide of claim 1, wherein modifications are inserted between nucleotides and are located near the 3′

-end and the 5′

-end of the antisense oligonucleotide.

8. The antisense oligonucleotide of claim 1, wherein a modification is located at the 5′

-end and a modification is inserted between nucleotides near the 3′

-end of the antisense oligonucleotide.

9. The antisense oligonucleotide of claim 1, wherein a modification is located at the 3′

-end and a modification is inserted between nucleotides near the 5′

-end of the antisense oligonucleotide.

10. The antisense oligonucleotide of claim 1, wherein the modification has the structure:

11. The antisense oligonucleotide of claim 10, wherein R₈ is NO₂.

12. The antisense oligonucleotide of claim 1, wherein the modification has the structure:

13. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide further comprises at least one 2′

-O-methyl RNA residue.

14. The antisense oligonucleotide of claim 13, further comprising at least one napthyl-azo compound modification that is incorporated at the terminal end of the antisense oligonucleotide, wherein the antisense oligonucleotide targets miRNA.

15. The antisense oligonucleotide of claim 14, wherein the antisense oligonucleotide is the same length as the miRNA target.

16. The antisense oligonucleotide of claim 14, wherein the antisense oligonucleotide is one base shorter than the miRNA target.

17. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide further comprises one or more phosphorothioate internucleotide linkages.

18. The antisense oligonucleotide of claim 1, wherein each nucleotide of the antisense oligonucleotide is phosphorothioate modified.

19. The antisense oligonucleotide claim 18, further comprising a chemical modification, wherein the chemical modification comprises 2′

OMe RNA, 2′

F RNA, or LNA bases.

20. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide comprises a region of bases linked through phosphodiester bonds, wherein the region is flanked at one or both ends by regions containing phosphorothioate linkages.

21. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide comprises a region of bases linked through phosphorothioate bonds, wherein the region is flanked at one or both ends by regions containing phosphodiester linkages.

24. The antisense oligonucleotide of claim 13, wherein the antisense oligonucleotide contains one or more DNA molecules and wherein each 2′

OMe and nucleotide is connected by a phosphorothioate group.

25. The antisense oligonucleotide of claim 24, additionally comprising at least one napthyl-azo compound modification that is incorporated at the terminal end of the antisense oligonucleotide, wherein the modification increases stability of the antisense oligonucleotide.

26. The antisense oligonucleotide of claim 24, wherein the length of each 2′

OMe portion is 2-5 nucleotides.

27. The antisense oligonucleotide of claim 26, wherein the length of each 2′

OMe portion is 5 nucleotides.

28. The antisense oligonucleotide of claim 27 having the formula:

zM*M*M*M*M*D*D*D*D*D*D*D*D*D*D*M*M*M*M*Mz

wherein z is a napthyl-azo compound, M is 2′

O methyl RNA, D is DNA and * is a phosphorothioate linkage.

29. The antisense oligonucleotide of claim 13 having the formula:

zMMMMM*D*D*D*D*D*D*D*D*D*D*MMMMMz

wherein z is a napthyl-azo compound, M is 2′

O methyl RNA, D is DNA and * is a phosphorothioate linkage.

30. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide contains one or more LNA molecules and wherein the LNA molecules optionally contain phosphorothioate linkage groups.

31. The antisense oligonucleotide of claim 30, wherein the modification is a napthyl-azo compound that is incorporated at the terminal end of the antisense oligonucleotide, wherein the modification increases stability of the antisense oligonucleotide.

32. The antisense oligonucleotide of claim 31, wherein the length of each LNA portion is 2-5 nucleotides.

33. The antisense oligonucleotide of claim 32, wherein the length of each LNA portion is 5 nucleotides.

34. The antisense oligonucleotide of claim 27 having the formula:

zL*L*L*L*L*D*D*D*D*D*D*D*D*D*D*L*L*L*L*Lz

wherein z is a napthyl-azo compound, D is DNA, L is LNA, and * is a phosphorothioate linkage.

35. The antisense oligonucleotide of claim 31, wherein the antisense oligonucleotide has a reduced phosphorothioate group content.

36. The antisense oligonucleotide of claim 27 having the formula:

zLLLLL*D*D*D*D*D*D*D*D*D*D*LLLLLz

wherein z is a napthyl-azo compound, D is DNA, L is LNA, and * is a phosphorothioate linkage.

37. The antisense oligonucleotide of claim 1 having the formula:

z_nM_m*D_y*M_m*Mz_n

where M=2′

OMe or LNA, D=DNA or a DNA analogue and z=a napthyl-azo compound,

wherein at least one n=1 and a second n=0 or 1, where m=2-5 and where y=7-12.

40. The antisense oligonucleotide of claim 13 having the formula:

MzMMMMMMMMMMMMMMMMMMMMz

wherein z is a napthyl-azo compound and M is 2′

O methyl RNA.

41. The antisense oligonucleotide of claim 40, wherein the napthyl-azo compound has the formula:

42. The antisense oligonucleotide of claim 40, wherein all internucleotide linkages are phosphodiester internucleotide linkages.

22. An oligonucleotide having the structure:

5′

-X₁-Z_n-X₂-X₃-X₄-Z_n-X₅-3′

wherein X₁ and X₅ are independently 0-3 nucleotides wherein the internucleotide linkages are optionally phosphorothioate;

Z is a napthyl-azo compound;

at least one n is 1, and the other n is 0 or 1;

X₂ and X₄ are independently 1-5 nucleotides wherein the internucleotide linkages are optionally phosphorothioate; and

X₃ is 10-25 nucleotides, wherein the linkages are optionally phosphorothiaote.

23. An oligonucleotide complementary to a target mRNA comprising:

(a) a modified 3′

-terminal internucleotide phosphodiester linkage, which modified 3′

-terminal internucleotide phosphodiester linkage is resistant to 3′

to 5′

exonuclease degradation;

(b) modifications on the 3′

-terminus and the 5′

-terminus of the oligonucleotide, wherein the modifications increase binding affinity of the oligonucleotide to the target mRNA;

(c) one or more additional modifications, which additional modification(s) facilitate(s) intracellular transport of said oligodeoxynucleotide; and

(d) a continuous stretch of at least five nucleotide residues having four internucleotide phosphodiester linkages which are unmodified,

wherein said oligodeoxynucleotide, when mixed with an RNA molecule for which it has complementarity under conditions in which an RNaseH is active, hybridizes to the RNA and forms a substrate that can be cleaved by the RNase H; and

wherein at least one of the modifications comprises a napthyl-azo compound.

38. An anti-miRNA oligonucleotide (AMO) comprising,

(a) at least one 2′

-O-methyl RNA (2′

OMe), and

(b) at least one napthyl-azo compound modification that is incorporated at the terminal end of the AMO, wherein the modification increases stability of the AMO.

39. An RNase H antisense oligonucleotide (ASO) comprising, at least one napthyl-azo compound modification that is incorporated at the terminal end of the ASO, wherein each nucleotide is connected by a phosphorothioate group (PS) and wherein the modification increases stability of the ASO.