Modified oligonucleotides for use in RNA interference

• US 20040203024A1
• Filed: 11/04/2003
• Published: 10/14/2004
• Est. Priority Date: 06/06/1996
• Status: Abandoned Application

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Bhat, Balkrishen, Griffey, Richard H., Swayze, Eric E., Baker, Brenda F., Manoharan, Muthiah, Crooke, Stanley T., Eldrup, Anne B.
• Application Number:
  US10/700,697
• Publication Number:
  US 20040203024A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• 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 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 2310/311 Phosphotriesters
• CPC Class Codes: C12N 2310/312 Phosphonates
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/316 Phosphonothioates
• CPC Class Codes: C12N 2310/318 where the PO2 is completely replaced, e.g. MMI or formacetal
• CPC Class Codes: C12N 2310/3181 Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/335 Modified T or U
• CPC Class Codes: C12N 2310/341 Gapmers, i.e. of the type ===---===
• 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 2310/3525 MOE, methoxyethoxy
• CPC Class Codes: C12N 2310/3527 Other alkyl chain
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present invention provides modified oligonucleotides for use in the RNA interference pathway of gene modulation. At least one nucleoside has a 2′-modification other than hydroxyl that gives an RNA like 3′-endo sugar conformation. The modified oligonucleotides are also provided having a 5′-phosphate group.

Claims

1. A composition comprising a first oligomer and a second oligomer, wherein:

at least a portion of said first oligomer is capable of hybridizing with at least a portion of said second oligomer,

at least a portion of said first oligomer is complementary to and capable of hybridizing with a selected target nucleic acid,

at least one of said first or said second oligomers includes at least one nucleoside having 3′

-endo conformational geometry; and

wherein said nucleoside having said 3′

-endo conformational geometry is other than a β

-D-ribofuranose nucleoside having a 2′

-OH substituent group.

2. The composition of claim 1 wherein said first and said second oligomers are a complementary pair of siRNA oligomers.

3. The composition of claim 1 wherein said first and said second oligomers are an antisense/sense pair of oligomers.

4. The composition of claim 1 wherein each of said first and second oligomers has about 10 to about 40 linked nucleosides.

5. The composition of claim 1 wherein each of said first and second oligomers has about 18 to about 30 linked nucleosides.

6. The composition of claim 1 wherein each of said first and second oligomers has about 21 to about 24 linked nucleosides.

7. The composition of claim 1 wherein said first oligomer comprises an antisense oligomer.

8. The composition of claim 7 wherein said second oligomer comprises a sense oligomer.

9. The composition of claim 7 wherein said second oligomer has a plurality of ribose nucleoside subunits.

10. The composition of claim 1 wherein said first oligomer includes a nucleoside having 3′

-endo conformational geometry.

11. The composition of claim 10 wherein said nucleoside having 3′

-endo conformational geometry is located at the 3′

-terminus of said first oligomer.

12. The composition of claim 10 wherein said nucleoside having 3′

-endo conformational geometry is located at the 5′

-terminus of said first oligomer.

13. The composition of claim 10 having at least 2 nucleosides comprising 3′

-endo conformational geometry.

14. The composition of claim 13 having at least 3 nucleosides comprising 3′

-endo conformational geometry.

15. The composition of claim 14 having at least 5 nucleosides comprising 3′

-endo conformational geometry.

16. The composition of claim 10 wherein each nucleoside of the first oligomer has 3′

-endo conformational geometry.

17. The composition of claim 1 wherein each nucleoside of the first and second oligomers has 3′

-endo conformational geometry.

18. The composition of claim 10 wherein said nucleoside having 3′

-endo conformational geometry comprises a 2′

-substitutent group that is other than H or OH.

19. The composition of claim 18 wherein said 2′

-substitutent group is —

F, —

O—

CH₂CH₂—

O—

CH₃, —

OC₁-C₁₂ alkyl, —

O—

CH₂—

CH₂—

CH₂—

NH₂, —

O—

(CH₂)₂—

O—

N(R₄₁)₂, —

O—

CH₂C(═

O)—

N(R₄₁)₂, —

O—

(CH₂)₂—

O—

(CH₂)₂—

N(R₄₁)₂, —

O—

CH₂—

CH₂—

CH₂—

NHR₄₁, —

N₃, —

O—

CH₂—

CH═

CH₂, —

NHCOR₄₁ or —

O—

CH₂—

N(H)—

C(═

NR₄₁)[N(R₄₁)₂];

wherein each R₄₁ is, independently, H, C₁-C₁₂ alkyl, a protecting group or substituted or unsubstituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, or C₂-C₁₂ alkynyl wherein the substituent groups are halogen, hydroxyl, amino, azido, cyano, haloalkyl, alkenyl, alkoxy, thioalkoxy, haloalkoxy or aryl.

20. The composition of claim 18 wherein the 2′

-substituent group is —

F, —

O—

CH₃, —

O—

CH₂CH₂—

O—

CH₃, —

O—

CH₂—

CH═

CH₂, N₃, —

O—

(CH₂)₂—

O—

N(R₄₁)₂, —

O—

CH₂C(O)—

N(R₄₁)₂, —

O—

CH₂—

CH₂—

CH₂—

NH₂, —

O—

(CH₂)₂—

O—

(CH₂)₂—

N(R₄₁)₂ or —

O—

CH₂—

N(H)—

C(═

NR₄₁)[N(R₄₁)₂];

wherein each R₄₁ is, independently, H, C₁-C₁₂ alkyl, a protecting group or substituted or unsubstituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, or C₂-C₁₂ alkynyl wherein the substituent groups are halogen, hydroxyl, amino, azido, cyano, haloalkyl, alkenyl, alkoxy, thioalkoxy, haloalkoxy or aryl.

21. The composition of claim 18 wherein the 2′

-substituent group is —

F, —

O—

CH₂CH₂—

O—

CH₃, —

O—

CH₃, —

O—

CH₂—

CH═

CH₂ or —

O—

CH₂—

CH—

CH₂—

NH(R_j) where R_j is H or C₁-C₁₀ alkyl.

22. The composition of claim 18 wherein the 2′

-substituent group is —

F, —

O—

CH₃ or —

O—

CH₂CH₂—

O—

CH₃.

23. The composition of claim 10 wherein the nucleoside having 3′

-endo conformational geometry comprises a LNA or a bicyclic sugar moiety.

24. The composition of claim 10 wherein the nucleoside having 3′

-endo conformational geometry is of the formula;

25. The composition of claim 10 wherein the nucleoside having 3′

-endo conformational geometry comprises a sugar of the formula;

45. The composition of claim 1 wherein the nucleoside having 3′

-endo conformational geometry comprises a LNA or a bicyclic sugar moiety.

46. The composition of claim 1 wherein the nucleoside having 3′

-endo conformational geometry is of the formula;

47. The composition of claim 10 wherein the nucleoside having 3′

-endo conformational geometry comprises a sugar of the formula;

54. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier.

57. A method of modulating the expression of a target nucleic acid in a cell comprising contacting said cell with a composition of claim 1.

60. A method of treating or preventing a disease or disorder associated with a target nucleic acid comprising administering to an animal having or predisposed to said disease or disorder a therapeutically effective amount of a composition of claim 1.

26. A composition comprising a first oligomer complementary to and capable of hybridizing to a selected target nucleic acid and at least one protein, said protein comprising at least a portion of a RNA-induced silencing complex (RISC), wherein

said oligomer includes at least one nucleoside having 3′

-endo conformational geometry;

wherein said nucleoside having said 3′

-endo conformational geometry is other than a β

-D-ribofuranose nucleoside having a 2′

-OH substituent group.

27. The composition of claim 26 wherein said first oligomer is an antisense oligomer.

28. The composition of claim 26 wherein said first oligomer has 10 to 40 nucleosides.

29. The composition of claim 26 wherein said first oligomer has 18 to 30 nucleosides.

30. The composition of claim 26 wherein said first oligomer has 21 to 24 nucleosides.

31. The composition of claim 26 further comprising a second oligomer, wherein said

second oligomer is complementary to said first oligomer.

32. The composition of claim 31 wherein said second oligomer is a sense oligomer.

33. The composition of claim 31 wherein said second oligomer comprises a plurality of ribose nucleoside units.

34. The composition of claim 33 wherein each nucleoside of said first oligomer has 3′

-endo conformational geometry.

35. The composition of claim 26 wherein said first oligomer comprises a nucleoside having 3′

-endo conformational geometry at the 3′

-terminus.

36. The composition of claim 26 wherein said first oligomer comprises a nucleoside having 3′

-endo conformational geometry at the 5′

-terminus.

37. The composition of claim 26 having at least 2 nucleosides comprising 3′

-endo conformational geometry.

38. The composition of claim 37 having at least 3 nucleosides comprising 3′

-endo conformational geometry.

39. The composition of claim 38 having at least 5 nucleosides comprising 3′

-endo conformational geometry.

40. The composition of claim 26 wherein said nucleoside with 3′

-endo conformational geometry comprises a 2′

-substitutent group and wherein said nucleoside is other than a β

-D-ribofuranose nucleoside having a 2′

-OH substituent group

41. The composition of claim 40 wherein said 2′

-substitutent group is is —

F, —

O—

CH₂CH₂—

O—

CH₃, —

OC₁-C₁₂ alkyl, —

O—

CH₂—

CH₂—

CH₂—

NH₂, —

O—

(CH₂)₂—

O—

N(R₄₁)₂, —

O—

CH₂C(═

O)—

N(R₄₁)₂, —

O—

(CH₂)₂—

O—

(CH₂)₂—

N(R₄₁)₂, —

O—

CH₂—

CH₂—

CH₂—

NHR₄₁, —

N₃, —

O—

CH₂—

CH═

CH₂, —

NHCOR₄₁ or —

O—

CH₂—

N(H)—

C(═

NR₄₁)[N(R₄₁)₂];

wherein each R₄₁ is, independently, H, C₁-C₁₂ alkyl, a protecting group or substituted or unsubstituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, or C₂-C₁₂ alkynyl wherein the substituent groups are halogen, hydroxyl, amino, azido, cyano, haloalkyl, alkenyl, alkoxy, thioalkoxy, haloalkoxy or aryl.

42. The composition of claim 40 wherein the 2′

-substituent group is —

F, —

O—

CH₃, —

O—

CH₂CH₂—

O—

CH₃, —

O—

CH₂—

CH═

CH₂, N₃, —

O—

(CH₂)₂—

O—

N(R₄₁)₂, —

O—

CH₂C(O)—

N(R₄₁)₂, —

O—

CH₂—

CH₂—

CH₂—

NH₂, —

O—

(CH₂)₂—

O—

(CH₂)₂—

N(R₄₁)₂ or —

O—

CH₂—

N(H)—

C(═

NR₄₁)[N(R₄₁)₂];

wherein each R₄₁ is, independently, H, C₁-C₁₂ alkyl, a protecting group or substituted or unsubstituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, or C₂-C₁₂ alkynyl wherein the substituent groups are halogen, hydroxyl, amino, azido, cyano, haloalkyl, alkenyl, alkoxy, thioalkoxy, haloalkoxy or aryl.

43. The composition of claim 40 wherein the 2′

-substituent group is —

F, —

O—

CH₂CH₂—

O—

CH₃, —

O—

CH₃, —

O—

CH₂—

CH═

CH₂ or —

O—

CH₂—

CH—

CH₂—

NH(R_j) where R_j is H or C₁-C₁₀ alkyl.

44. The composition of claim 40 wherein the 2′

-substituent group is —

F, —

O—

CH₃ or —

O—

CH₂CH₂—

O—

CH₃.

55. A pharmaceutical composition comprising the composition of claim 26 and a pharmaceutically acceptable carrier.

58. A method of modulating the expression of a target nucleic acid in a cell comprising contacting said cell with a composition of claim 26.

61. A method of treating or preventing a disease or disorder associated with a target nucleic acid comprising administering to an animal having or predisposed to said disease or disorder a therapeutically effective amount of a composition of claim 26.

48. An oligomer having at least a first region and a second region, wherein:

said first region of said oligomer is complementary to and capable of hybridizing with said second region of said oligomer,

at least a portion of said oligomer is complementary to and capable of hybridizing to a selected target nucleic acid, and

said oligomer further includes at least one sugar moiety having 3′

-endo conformational geometry.

49. The oligomer of claim 48 wherein each of said first and said second regions has at least 10 nucleosides.

50. The oligomer of claim 48 wherein said first region in a 5′

to 3′

direction is complementary to said second region in a 3′

to 5′

direction.

51. The oligomer of claim 48 wherein said oligomer includes a hairpin structure.

52. The oligomer of claim 48 wherein said first region of said oligomer is spaced from said second region of said oligomer by a third region and where said third region comprises at least two nucleosides.

53. The oligomer of claim 48 wherein said first region of said oligomer is spaced from said second region of said oligomer by a third region and wherein said third region comprises a non-nucleoside region.

56. A pharmaceutical composition comprising the oligomer of claim 48 and a pharmaceutically acceptable carrier.

59. A method of modulating the expression of a target nucleic acid in a cell comprising contacting said cell with an oligomer of claim 48.

62. A method of treating or preventing a disease or disorder associated with a target nucleic acid comprising administering to an animal having or predisposed to said disease or disorder a therapeutically effective amount of an oligomer of claim 48.