2'-F MODIFIED RNA INTERFERENCE AGENTS

• US 20110269814A1
• Filed: 03/26/2009
• Published: 11/03/2011
• Est. Priority Date: 03/26/2008
• Status: Abandoned Application

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Manoharan, Muthiah, Rajeev, Kallanthottathil G.
• Application Number:
  US12/935,083
• Publication Number:
  US 20110269814A1
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/173 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/23 Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/3525 MOE, methoxyethoxy
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

Abstract

This invention relates to a method of modulating the expression of a target gene in an organism comprising administering an iRNA agent, wherein the iRNA comprises at least one 2′-deoxy-2′-fluoro (2′-F) nucleotide in the antisense strand and at least one modified nucleotide in the sense strand. The invention also relates to compositions comprising a single-stranded oligonucleotide that contains at least one 2′-deoxy-2′-fluoro (2′-F) nucleotide. siRNA molecule containing these oligonucleotides have decreased immunogenicity.

Claims

1. A method of modulating the expression of a target gene in an organism comprising administering an iRNA agent, wherein said iRNA comprises at least one 2′

-deoxy-2′

-fluoro (2′

-F) nucleotide in the antisense strand and at least one modified nucleotide in the sense strand, wherein said modified nucleotide is selected from the group consisting of 2′

-deoxy-2′

-fluoro, 2′

-O-methyl (2′

-OMe), 2′

-methoxyethyl (2′

-MOE), and 2′

-O,4′

-C-methylene (LNA).

2. The method of claim 1, wherein said antisense strand comprises at least one 5′

-pyrimidine-purine dinucleotide wherein the pyrimidine is 2′

-deoxy-2′

-fluoro.

3. The method of claim 1, wherein the 5′

-most pyrimidines in all occurrences of sequence motif 5′

-pyrimidine-purine-3′

in the antisense strand is a 2′

-deoxy-2′

-fluoro.

4. The method of claim 1, wherein all pyrimidines are 2′

-deoxy-2′

-fluoro in the antisense strand.

5. The method of claim 1, wherein said sense strand comprises at least one 5′

-pyrimidine-purine-3′

dinucleotide wherein the pyrimidine is modified with modification chosen from a group consisting of 2′

-deoxy-2′

-fluoro, 2′

-O-methyl, 2′

-methoxyethyl, and 2′

-O,4′

-C-methylene.

6. The method of claim 1, wherein the 5′

-most pyrimidines in all occurrences of sequence motif 5′

-pyrimidine-purine-3′

in the sense strand are modified with modification selected from the group consisting of 2′

-deoxy-2′

-fluoro, 2′

-O-methyl, 2′

-methoxyethyl, and 2′

-O,4′

-C-methylene.

7. The method of claim 1, wherein all pyrimidines in the sense strand are modified with modification selected from the consisting of 2′

-deoxy-2′

-fluoro, 2′

-O-methyl, 2′

-methoxyethyl, and 2′

-O,4′

-C-methylene.

8. The method of claim 1, wherein the 5′

-most pyrimidines in all occurrences of sequence motif 5′

-pyrimidine-purine-3′

in the antisense strand is a 2′

-deoxy-2′

-fluoro and said sense strand comprises at least one 5′

-pyrimidine-purine-3′

dinucleotide wherein the pyrimidine is modified with modification selected from the group consisting of 2′

-deoxy-2′

-fluoro, 2′

-β

-methyl, 2′

-methoxyethyl, and 2′

-O,4′

-C-methylene.

9. The method of claim 1, wherein the 5′

-most pyrimidines in all occurrences of sequence motif 5′

-pyrimidine-purine-3′

in the antisense strand is a 2′

-deoxy-2′

-fluoro and the 5′

-most pyrimidines in all occurrences of sequence motif 5′

-pyrimidine-purine-3′

in the sense strand are modified with modification selected from the group consisting of 2′

-deoxy-2′

-fluoro, 2′

-β

-methyl, 2′

-methoxyethyl, and 2′

-O,4′

-C-methylene.

10. The method of claim 1, wherein the 5′

-most pyrimidines in all occurrences of sequence motif 5′

-pyrimidine-purine-3′

in the antisense strand are 2′

-deoxy-2′

-fluoro and all pyrimidines in the sense strand are modified with modification selected from the group consisting of 2′

-deoxy-2′

-fluoro, 2′

-O-methyl, 2′

-methoxyethyl, and 2′

-O,4′

-C-methylene.

11. The method of claim 1, wherein all pyrimidines are 2′

-deoxy-2′

-fluoro in the antisense strand and said sense strand comprises at least one 5′

-pyrimidine-purine-3′

dinucleotide wherein the pyrimidine is modified with modification selected from the group consisting of 2′

-deoxy-2′

-fluoro, 2′

-O-methyl, 2′

-methoxyethyl, and 2′

-O,4′

-C-methylene.

12. The method of claim 1, wherein all pyrimidines are 2′

-deoxy-2′

-fluoro in the antisense strand and the 5′

-most pyrimidines in all occurrences of sequence motif 5′

-pyrimidine-purine-3′

in the sense strand are modified with modification selected from the group consisting of 2′

-deoxy-2′

-fluoro, 2′

-O-methyl, 2′

-methoxyethyl, and 2′

-O,4′

-C-methylene.

13. The method of claim 1, wherein all pyrimidines are 2′

-deoxy-2′

-fluoro in the antisense strand and all pyrimidines in the sense strand are modified with modification selected from the group consisting of 2′

-deoxy-2′

-fluoro, 2′

-O-methyl, 2′

-methoxyethyl, and 2′

-O,4′

-C-methylene.

14. A method of decreasing the immunogenicity of an iRNA agent, wherein said iRNA comprises at least one 2′

-deoxy-2′

-fluoro (2′

-F) nucleotide in the antisense strand and at least one modified nucleotide in the sense strand, and wherein said modified nucleotide is selected from the group consisting of 2′

-deoxy-2′

-fluoro, 2′

-O-methyl, 2′

-methoxyethyl, and 2′

-O,4′

-C-methylene, wherein the siRNA molecule has a decreased immunogenicity relative to an siRNA molecule having the identical sequence but comprising fewer or no 2′

-F modifications.

15. A composition, comprising a single-stranded oligonucleotide represented by formula I:

16. The composition of claim 15, wherein at least one instance of X or Y is S.

17. The composition of claim 15, wherein a plurality of instances of Y represent S.

18. The composition of claim 15, wherein Y is S;

and X is S.

19. The composition of claim 15, wherein p ranges from 14-28.

20. The composition of claim 15, wherein R represents F in a plurality of instances.

21. The composition of claim 15, wherein the phosphorothioate internucleotide linkage is attached to the 5′

-hydroxyl of a nucleoside wherein R is F.

22. The composition of claim 15, wherein the phosphorothioate internucleotide linkage is attached to the 3′

-hydroxyl of a nucleoside wherein R is F.

23. The composition of claim 15, wherein the at least one nucleotide comprising a 2′

-deoxy-2′

-fluoro modification is not a 5′

terminal nucleotide or a 3′

terminal nucleotide.

24. The composition of claim 15, wherein Q₁ represents a phosphate or phosphorothioate group;

Q₂ represents a phosphate or phosphorothioate group, or both Q₁ and Q₂ represents a phosphate or phosphorothioate group.

25. The composition of claim 15, wherein said single-stranded oligonucleotied comprises a 3′

-terminal deoxythymidine.

26. The composition of claim 15, wherein said single-stranded oligonucleotide comprises at least one nucleotide selected from the group consisting of 2′

-O-methyl nucleotides, 2′

-methoxyethoxy nucleotides, 2′

-O—

N-methylacetamido nucleotides, LNAs, and ENAs.

27. The composition of claim 15, further comprising at least one ligand covalently attached to the 5′

-terminus of the oligonucleotide and/or at least one ligand covalently attached to the 3′

-terminus of the oligonucleotide.

28. The composition of claim 27, wherein the ligand comprises a targeting group, a protein-binding agent, or an endosomal release agent.

29. The composition of claim 28, wherein the ligand comprises a targeting group;

and said targeting group is selected from the group consisting of folate, cholesterol, bile acids, steroids, β

-GalNAc, mannose, an RGD peptide, a peptide, an antibody, and an aptamer.

30. The composition of claim 28, wherein the ligand comprises a protein-binding agent;

and said protein-binding agent is selected from the group consisting of cholesterol, lipophiles, ibuprofen, naproxen, ligands capable of binding to albumin, and ligands capable of binding to lipoproteins (LDL or HDL).

31. The composition of claim 28, wherein the attachment of the ligand to the oligonucleotide is biodegradable.

32. The composition of claim 31, wherein the biodegradability is at least partially in response to intracellular pH change, is at least partially in response to intracellular reductive environment, is at least partially in response to peptidase activity, is at least partially in response to esterase activity, or a combination thereof.

33. A pharmaceutical composition, comprising the composition of claim 15, and a pharmaceutically acceptable excipient, carrier or diluent.

34. The composition of claim 15, wherein the siRNA molecule has decreased immunogenicity relative to an siRNA molecule having the identical sequence but comprising fewer or no 2′

-deoxy-2′

-fluoro modifications.

35. A method of suppressing the endogenous expression of a gene, comprising contacting a cell with an effective amount of the composition of claim 15, wherein the effective amount is an amount that partially or substantially suppresses the endogenous expression of said gene.