MODIFIED RNAi AGENTS

• US 20180008724A1
• Filed: 09/15/2017
• Published: 01/11/2018
• Est. Priority Date: 11/18/2011
• Status: Abandoned Application

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: RAJEEV, Kallanthottathil G., ZIMMERMANN, Tracy, MANOHARAN, Muthiah, MAIER, Martin, KUCHIMANCHI, Satyanarayana, CHARISSE, Klaus
• Application Number:
  US15/706,389
• Publication Number:
  US 20180008724A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• 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 15/85 for animal cells
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/343 having patterns, e.g. ==--==--==--
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/3533 Halogen
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

Abstract

One aspect of the present invention relates to double-stranded RNAi (dsRNA) duplex agent capable of inhibiting the expression of a target gene. The dsRNA duplex comprises one or more motifs of three identical modifications on three consecutive nucleotides in one or both strand, particularly at or near the cleavage site of the strand. Other aspects of the invention relates to pharmaceutical compositions comprising these dsRNA agents suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA agents, e.g., for the treatment of various disease conditions.

Claims

1. A double-stranded RNAi agent capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the duplex is represented by formula (III):

2. The double-stranded RNAi agent of claim 1, wherein i is 1;

j is 1;

or both i and j are 1.

3. The double-stranded RNAi agent of claim 1, wherein k is 1;

1 is 1;

or both k and l are 1.

4. The double-stranded RNAi agent of claim 1, wherein the YYY motif occurs at or near the cleavage site of the sense strand.

5. The double-stranded RNAi agent of claim 1, wherein the Y′

Y′

Y′

motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5′

-end.

6. The double-stranded RNAi agent of claim 5, wherein the Y′

is 2′

-OMe.

7. The double-stranded RNAi agent of claim 1, wherein formula (III) is represented as formula (IIIa):

8. The double-stranded RNAi agent of claim 1, wherein formula (III) is represented as formula (IIIb):

9. The double-stranded RNAi agent of claim 1, wherein formula (III) is represented as formula (IIIc):

10. The double-stranded RNAi agent of claim 1, wherein the duplex region is 17-30 nucleotide pairs in length.

11. The double-stranded RNAi agent of claim 10, wherein the duplex region is 17-19 nucleotide pairs in length.

12. The double-stranded RNAi agent of claim 10, wherein the duplex region is 27-30 nucleotide pairs in length.

13. The double-stranded RNAi agent of claim 1, wherein each strand has 17-30 nucleotides.

14. The double-stranded RNAi agent of claim 1, wherein the modifications on the nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2′

-methoxyethyl, 2′

-O-alkyl, 2′

-O-allyl, 2′

-C-allyl, 2′

-fluoro, 2′

-deoxy, and combinations thereof.

15. The double-stranded RNAi agent of claim 14, wherein the nucleotides are modified with either 2′

-OCH₃ or 2′

-F.

16. The double-stranded RNAi agent of claim 1, further comprising at least one ligand.

17. The double-stranded RNAi agent of claim 16, wherein the ligand is a one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

18. The double-stranded RNAi agent of claim 1, wherein the modifications on the nucleotides are selected from the group consisting of 2′

-O-methyl nucleotide, 2′

-deoxyfluoro nucleotide, 2′

-O—

N-methylacetamido (2′

-O-NMA) nucleotide, a 2′

-O-dimethylaminoethoxyethyl (2′

-O-DMAEOE) nucleotide, 2′

-O-aminopropyl (2′

-O-AP) nucleotide, 2′

-ara-F, and combinations thereof.

19. The double-stranded RNAi agent of claim 14, wherein the ligand is attached to the 3′

end of the sense strand.

20. The double-stranded RNAi agent of claim 1, further comprising at least one methylphosphonate internucleotide linkage.

21. The double-stranded RNAi agent of claim 1, wherein the nucleotide at the 1 position of the 5′

-end of the duplex in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT.

22. The double-stranded RNAi agent of claim 1, wherein the base pair at the 1 position of the 5′

-end of the duplex is an AU base pair.

23. The double-stranded RNAi agent of claim 1, wherein the Y nucleotides contain a 2′

-fluoro modification.

24. The double-stranded RNAi agent of claim 1, wherein the Y′

nucleotides contain a 2′

-O-methyl modification.

33. A pharmaceutical composition comprising the double-stranded RNAi agent according to claim 1 alone or in combination with a pharmaceutically acceptable carrier or excipient.

34. A method for inhibiting the expression of a target gene comprising the step of administering the double-stranded RNAi agent according to claim 1, in an amount sufficient to inhibit expression of the target gene.

35. The method of claim 34, wherein the double-stranded RNAi agent is administered through subcutaneous or intravenous administration.

36. A method for delivering a polynucleotide to a specific target of a subject, the method comprising:

delivering the dsRNA agent according to claim 1 by subcutaneous administration into the subject, such that the polynucleotide is delivered into specific target of the subject.

25. A double-stranded RNAi agent capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides,

wherein the sense strand contains at least one motif of three 2′

-F modifications on three consecutive nucleotides, one of said motifs occurring at or near the cleavage site in the strand; and

wherein the antisense strand contains at least one motif of three 2′

-O-methyl modifications on three consecutive nucleotides, one of said motifs occurring at or near the cleavage site, and

wherein the antisense strand comprises two blocks of two phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

26. The double-stranded RNAi agent of claim 25, wherein the sense strand comprises one or more motifs of three identical modifications on three consecutive nucleotides, said motifs occurring at another portion of the strand that is separated from the three 2′

-F modifications at the cleavage site by at least one nucleotide.

27. The double-stranded RNAi agent of claim 25, wherein the antisense strand comprises one or more motifs of three identical modifications on three consecutive nucleotides, said motifs occurring at another portion of the strand that is separated from the three 2′

-O-methyl modifications by at least one nucleotide.

28. The double-stranded RNAi agent of claim 25, wherein at least one of the nucleotides having a 2′

-F modification forms a base pair with one of the nucleotides having a 2′

-O-methyl modification.

29. The double-stranded RNAi agent of claim 25, wherein the duplex is 17-30 nucleotide pairs in length.

30. The double-stranded RNAi agent of claim 25, wherein the duplex is 17-19 nucleotide pairs in length.

31. The double-stranded RNAi agent of claim 25, wherein each strand has 17-23 nucleotides.

32. The double-stranded RNAi agent of claim 25, further comprising a ligand attached to the 3′

end of the sense strand.