Stabilized polynucleotides for use in RNA interference

• US 20040198640A1
• Filed: 04/02/2003
• Published: 10/07/2004
• Est. Priority Date: 04/02/2003
• Status: Abandoned Application

• Current Assignee: Dharmacon Incorporated (Revvity, Inc.)
• Original Assignee: Dharmacon Incorporated (Revvity, Inc.)
• Inventors: Marshall, William, Khvorova, Anastasia, Reynolds, Angela, Scaringe, Stephen, Leake, Devin
• Application Number:
  US10/406,908
• Publication Number:
  US 20040198640A1
• Time in Patent Office: Days
• US Class Current: 514/44.00R
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/313 Phosphorodithioates
• 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 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/3527 Other alkyl chain
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

Abstract

Methods and compositions for performing RNA interference comprising a wide variety of stabilized polynucleotides suitable for use in serum-containing media and for in vivo applications, such as therapeutic applications, are provided. These polynucleotides permit effective and efficient applications of RNA interference to applications such as diagnostics and therapeutics through the use of one or more modifications including orthoesters, terminal conjugates, modified linkages and 2′modified nucleotides.

Claims

1. A method of performing RNA interference, said method comprising exposing a double stranded polynucleotide to a target nucleic acid, wherein said double stranded polynucleotide is comprised of a sense strand and an antisense strand, and wherein at least one of said sense strand and said antisense strand comprises at least one orthoester modified nucleotide.

2. The method according to claim 1, wherein said at least one orthoester modified nucleotide is located on said sense strand.

3. The method according to claim 2, wherein the antisense strand comprises at least one nucleotide selected from the group consisting of a 2′

halogen modified nucleotide, a 2′

amine modified nucleotide, a 2′

-O-alkyl modified nucleotide and a 2′

alkyl modified nucleotide.

4. The method according to claim 3, wherein the antisense strand comprises at least one 2′

halogen modified nucleotide and said halogen is fluorine.

5. The method according to claim 2, wherein the double stranded polynucleotide further comprises a conjugate.

6. The method according to claim 5, wherein said conjugate is selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof.

7. The method according to claim 5, wherein the conjugate is cholesterol.

8. The method according to claim 5, wherein conjugate is polyethylene glycol.

9. The method according to claim 1, wherein the double stranded polynucleotide comprises 18-30 nucleotide base pairs.

10. The method according to claim 9, wherein the double stranded polynucleotide comprises 19 nucleotide base pairs.

11. The method according to claim 1, wherein the double stranded polynucleotide has an overhang of at least one nucleotide unit on at least one of said sense strand and said antisense strand.

12. The method according to claim 1, wherein at least one strand of the double stranded polynucleotide comprises at least one modified internucleotide linkage.

13. The method according to claim 12, wherein the modified internucleotide linkage is selected from the group consisting of a phosphorothioate linkage and a phosphorodithioate linkage.

14. The method according to claim 1, wherein at least one strand of the double stranded polynucleotide is a polyribonucleotide.

15. A method of performing RNA interference, said method comprising exposing a double stranded polynucleotide to a target nucleic acid, wherein said double stranded polynucleotide is comprised of:

(i) a sense strand,

(ii) an antisense strand, and

(iii) a conjugate,

wherein at least one of said sense strand and said antisense strand comprises a 2′

modified nucleotide.

16. A double stranded polynucleotide comprising:

(a) a sense strand, wherein said sense strand comprises a polynucleotide that is comprised of at least one orthoester modified nucleotide; and

(b) an antisense strand, wherein said antisense strand comprises a polynucleotide that is comprised of at least one 2′

modified nucleotide.

17. The double stranded polynucleotide of claim 16, wherein the antisense strand comprises at least one nucleotide selected from the group consisting of a 2′

halogen modified nucleotide, a 2′

amine modified nucleotide, a 2′

-O-alkyl modified nucleotide and a 2′

alkyl modified nucleotide.

18. The double stranded polynucleotide of claim 17, wherein the 2′

modified nucleotide is a 2′

halogen modified nucleotide and said halogen is fluorine.

19. The double stranded polynucleotide of claim 16, further comprising a conjugate.

20. The double stranded polynucleotide of claim 19, wherein said conjugate is selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof.

21. The double stranded polynucleotide of claim 19, wherein said conjugate is cholesterol.

22. The double stranded polynucleotide of claim 19, wherein said conjugate is polyethylene glycol.

23. The double stranded polynucleotide of claim 16, wherein said double stranded polynucleotide is comprised of 18-30 nucleotide base pairs.

24. The double stranded polynucleotide of claim 23, wherein said double stranded polynucleotide is comprised of 19 nucleotide base pairs.

25. The double stranded polynucleotide of claim 16, further comprising an overhang of at least one nucleotide unit on at least one of said sense strand and said antisense strand.

26. The double stranded polynucleotide of claim 16, wherein at least one of said sense strand and said antisense strand comprises at least one modified internucleotide linkage.

27. The double stranded polynucleotide of claim 26, wherein the modified internucleotide linkage is selected from the group consisting of a phosphorothoate linkage and a phosphorodithioate linkage.

28. The double stranded polynucleotide of claim 16, wherein at least one of said sense strand and said antisense strand is a polyribonucleotide.

29. A double stranded polynucleotide comprising:

(a) a sense strand, wherein said sense strand comprises a polynucleotide that is comprised of at least one orthoester modified nucleotide;

(b) an antisense strand, wherein said antisense strand comprises a polynucleotide that is comprised of at least one 2′

modified nucleotide; and

(c) a conjugate.

30. The double stranded polynucleotide of claim 29, wherein the conjugate is located on the sense strand.

31. The double stranded polynucleotide of claim 29, wherein the conjugate is located on the antisense strand.

32. The double stranded polynucleotide of claim 29, wherein the antisense strand comprises at least one nucleotide selected from the group consisting of a 2′

halogen modified nucleotide, a 2′

amine modified nucleotide, a 2′

-O-alkyl modified nucleotide and a 2′

alkyl modified nucleotide.

33. The double stranded polynucleotide of claim 32, wherein the sense strand is comprised of a 2′

halogen modified nucleotide and said halogen is fluorine.

34. The double stranded polynucleotide of claim 29, wherein the conjugate is selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof.

35. The double stranded polynucleotide of claim 29, wherein the conjugate is cholesterol.

36. The double stranded polynucleotide of claim 29, wherein the conjugate is polyethylene glycol.

37. The double stranded polynucleotide of claim 29, wherein said polynucleotide is comprised of 18-30 nucleotide base pairs.

38. The double stranded polynucleotide of claim 37, wherein said polynucleotide is comprised of 19 nucleotide base pairs.

39. The double stranded polynucleotide of claim 29, further comprising an overhang of at least one nucleotide unit on at least one of said sense strand and said antisense strand.

40. The double stranded polynucleotide of claim 29, wherein at least one of said sense strand and said antisense strand comprises at least one modified internucleotide linkage.

41. The double stranded polynucleotide of claim 40, wherein the modified internucleotide linkage is selected from the group consisting of a phosphorothioate linkage and a phosphorodithioate linkage.

42. The double stranded polynucleotide of claim 29, wherein at least one of said sense strand and said antisense strand is a polyribonucleotide.

43. A double stranded polynucleotide comprising:

(a) a sense strand comprised of at least one orthoester modified nucleotide;

(b) an antisense strand; and

(c) a conjugate.

44. The double stranded polynucleotide of claim 43, wherein said conjugate is located on the sense strand.

45. The double stranded polynucleotide of claim 43, wherein said is located on the antisense strand.

46. The double stranded polynucleotide of claim 43 wherein the antisense strand comprises at least one nucleotide selected from the group consisting of a 2′

halogen modified nucleotide, a 2′

amine modified nucleotide, a 2′

-O-alkyl modified nucleotide and a 2′

alkyl modified nucleotide.

47. The double stranded polynucleotide of claim 46, wherein the antisense strand is comprised of a 2′

halogen modified nucleotide and said halogen is fluorine.

48. The double stranded polynucleotide of claim 43, wherein the conjugate is selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof.

49. The double stranded polynucleotide of claim 43, wherein the conjugate is cholesterol.

50. The double stranded polynucleotide of claim 43, wherein the conjugate is polyethylene glycol.

51. The double stranded polynucleotide of claim 43, wherein the polynucleotide is comprised of 18-30 nucleotide base pairs.

52. The double stranded polynucleotide of claim 51, wherein the polynucleotide is comprised of 19 nucleotide base pairs.

53. The double stranded polynucleotide of claim 43, further comprising an overhang of at least one nucleotide unit on at least one of said sense stand and said antisense strand.

54. The double stranded polynucleotide of claim 43, wherein at least one of said sense strand and said antisense strand comprises at least one modified internucleotide linkage.

55. The double stranded polynucleotide of claim 43, wherein at least one of said sense strand and said antisense strand is a polyribonucleotide.

56. A double stranded polynucleotide comprising:

(a) a sense strand;

(b) an antisense strand; and

(c) a conjugate;

wherein the sense strand and/or the antisense strand comprises at least one 2′

modified nucleotide.

57. The double stranded polynucleotide of claim 56, wherein the 2′

modified nucleotide is selected from the group consisting of a 2′

halogen modified nucleotide, a 2′

amine modified nucleotide, a 2′

-O-alkyl modified nucleotide and a 2′

alkyl modified nucleotide.

58. The double stranded polynucleotide of claim 57, wherein the 2′

modified nucleotide is a 2′

halogen modified nucleotide and said halogen is fluorine.

59. The double stranded polynucleotide of claim 56, wherein the conjugate is selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof.

60. The double stranded polynucleotide of claim 56, wherein the conjugate is cholesterol.

61. The double stranded polynucleotide of claim 56, wherein the conjugate is polyethylene glycol.

62. The double stranded polynucleotide of claim 56, wherein said polynucleotide is comprised of 18-30 nucleotide base pairs.

63. The double stranded polynucleotide of claim 56, wherein said polynucleotide is comprised of 19 nucleotide base pairs.

64. The double stranded polynucleotide of claim 56, further comprising an overhang of at least one nucleotide unit on at least one of said sense strand and said antisense strand.

65. The double stranded polynucleotide of claim 56, wherein at least one of said sense strand and said antisense strand comprises at least one modified internucleotide linkage.

66. The double stranded polynucleotide of claim 65, wherein the modified internucleotide linkage is selected from the group consisting of a phosphorothioate linkage and a phosphorodithioate linkage.

67. The double stranded polynucleotide of claim 56, wherein at least one of said sense strand and said antisense strand is a polyribonucleotide.

68. A double stranded polyribonucleotide comprising:

(a) a sense strand, wherein said sense strand is comprised of at least one 2′

orthoester modified nucleotide;

(b) an antisense strand, wherein said antisense strand is comprised of at least one 2′

modified nucleotide selected from the group consisting of a 2′

halogen modified nucleotide, a 2′

amine modified nucleotide, a 2′

-O-alkyl modified nucleotide, and a 2′

alkyl modified nucleotide; and

(c) a conjugate selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof;

wherein said polyribonucleotide comprises between 18 and 30 nucleotide base pairs.

69. A composition comprising: