Double Strand Compositions Comprising Differentially Modified Strands for Use in Gene Modulation

• US 20080119427A1
• Filed: 06/02/2005
• Published: 05/22/2008
• Est. Priority Date: 06/06/1996
• Status: Abandoned Application

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Bhat, Balkrishen, Allerson, Charles, Griffey, Richard H., Swayze, Eric E., Prakash, Thazha P., Dande, Prasad
• Application Number:
  US11/569,931
• Publication Number:
  US 20080119427A1
• Time in Patent Office: Days
• US Class Current: 514/44
• 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 15/111 General methods applicable to biologically active non-coding 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 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/32 of the sugar
• 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 2310/341 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 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

Abstract

The present invention provides douse stranded compositions wherein each strand is modified to have a motif defined by positioning of β-D-ribonucleosides and sugar modified nucleosides. More particularly, the present compositions comprise one strand having a gapped motif and another strand having a gapped motif, a hemimer motif, a blockmer motif, a fully modified motif, a positionally modified motif or an alternating motif. At least one of the strands has complementarity to a nucleic acid target. The compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes. In some embodiments, the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA. The present invention also provides methods for modulating gene expression.

Claims

1. A composition comprising first and second chemically synthesized oligomeric compounds wherein:

at least a portion of the first oligomeric compound is complementary to and capable of hybridizing to a selected nucleic acid target;

a portion of from about 12 to about 24 nucleosides of the first oligomeric compound is complementary to the second oligomeric compound;

one of the first and second oligomeric compounds comprises nucleosides linked by internucleoside linking groups wherein the sequence of linked nucleosides defines a gapped motif;

the other of the first and second oligomeric compounds comprises nucleosides linked by internucleoside linking groups wherein the sequence of linked nucleosides defines a gapped motif, an alternating motif, a positionally modified motif or a fully modified motif;

the composition optionally further comprises one or more overhangs, phosphate moieties, conjugate groups or capping groups; and

when the first and second oligomeric compounds each independently comprise gapped motifs then at least one of the 3′

or 5′

termini of at least one of the first and second oligomeric compounds comprises modified nucleosides other than 2′

-OCH₃ modified nucleosides or at least one of the first and second oligomeric compounds comprises an asymmetric gapped motif.

2. The composition of claim 1 wherein each gapped oligomeric compound comprises a contiguous sequence of nucleosides divided into an internal region flanked by two external regions wherein the nucleosides of the internal region have different sugar groups than the nucleosides of each of the external regions and wherein the nucleosides of each of the external regions are independently selected from 2′

-modified nucleosides, 4′

-thio modified nucleosides, 4′

-thio-2′

-modified nucleosides and nucleosides having bicyclic sugar moieties.

3. The composition of claim 2 comprising at least one gapped oligomeric compound wherein the internal region is a sequence of β

-D-ribonucleosides.

4. The composition of claim 2 comprising at least one gapped oligomeric compound wherein the internal region is a sequence of sugar modified nucleosides.

5. The composition of claim 4 wherein the sugar modified nucleosides of the internal region are selected from 2′

-F modified nucleosides or 4′

-thio modified nucleosides.

6. The composition of claim 2 comprising at least one symmetric gapped oligomeric compound.

7. The composition of claim 2 comprising at least one asymmetric gapped oligomeric compound.

8. The composition of claim 7 wherein the other of the first and second oligomeric compounds comprises a symmetric gapped oligomeric compound.

9. The composition of claim 2 comprising at least one gapped oligomeric compound wherein at least one of the external regions is a sequence of 2′

-modified nucleosides.

10. The composition of claim 9 wherein each of the external regions of the at least one gapped oligomeric compound is a sequence of 2′

-modified nucleosides.

11. The composition of claim 9 wherein each of the 2′

-modifications of the at least one external region is halogen, allyl, amino, azido, —

O-allyl, —

O—

C₁-C₁₀ alkyl, —

OCF₃, —

O—

(CH₂)₂—

O—

CH₃, —

O(CH₂)₂SCH₃, —

O—

(CH₂)₂—

O—

N(R_m)(R_n) or —

O—

CH₂—

C(═

O)—

N(R_m)(R_n), where each R_m and R_n is, independently, H, an amino protecting group or substituted or unsubstituted C₁-C₁₀ alkyl.

12. The composition of claim 11 wherein each of the 2′

-modifications is —

F, —

OCH₃ or —

O—

(CH₂)₂—

O—

CH₃.

13. The composition of claim 2 comprising at least one of gapped oligomeric compound having 4′

-thio modified nucleosides in at least one of the external regions.

14. The composition of claim 2 comprising at least one gapped oligomeric compound having 4′

-thio-2′

-modified nucleosides in at least one of the external regions.

15. The composition of claim 14 wherein the 2′

-modifications of the 4′

-thio-2′

-modified nucleosides are selected from halogen, allyl, amino, azido, —

O-allyl, —

O—

C₁-C₁₀ alkyl, —

OCF₃, —

O—

(CH₂)₂—

O—

CH₃, O(CH₂)₂SCH₃, —

O—

(CH₂)₂—

O—

N(R_m)(R_n) or —

O—

CH₂—

C(═

O)—

N(R_m)(R_n), where each R_n and R_n is, independently, H, an amino protecting group or substituted or unsubstituted —

C₁-C₁₀ alkyl.

16. The composition of claim 15 wherein each of the 2′

-modifications is —

F, —

OCH₃, —

OCF₃ or —

O—

(CH₂)₂—

O—

CH₃.

17. The composition of claim 16 wherein each of the 2′

-modifications is —

OCH₃ or —

O—

(CH₂)₂—

O—

CH₃.

18. The composition of claim 2 comprising at least one gapped oligomeric compound having bicyclic sugar moieties in at least one of the external regions.

19. The composition of claim 18 wherein each of the bicyclic sugar moieties comprises a 2′

-O—

(CH₂)_n-4′

bridge wherein n is 1 or 2.

20. The composition of claim 1 wherein the first oligomeric compound is a gapped oligomeric compound.

21. The composition of claim 20 wherein each external region of the gapped oligomeric compound independently comprises 4′

-thio modified nucleosides or 2′

-modified nucleosides.

22. The composition of claim 20 wherein one of the external regions of the gapped oligomeric compound comprises 4′

-thio modified nucleosides and the other external region comprises 2′

-modified nucleosides.

23. The composition of claim 22 wherein the external region located at the 5′

-end of the first oligomeric compound comprises 2′

-OCH₃, 2′

-F or 4′

-thio modified nucleosides.

24. The composition of claim 22 wherein the 2′

-modified nucleosides are 2′

-OCH₃ or 2′

-F modified nucleosides.

25. The composition of claim 24 wherein the 2′

-modified nucleosides are 2′

-OCH₃ modified nucleosides.

26. The composition of claim 1 wherein the second oligomeric compound is a gapped oligomeric compound.

28. The composition of claim 26 wherein at least one of the external regions of the gapped oligomeric compound comprises 2′

-modified nucleosides wherein the 2′

-modification is selected from halogen, allyl, amino, azido, —

O-allyl, —

O—

C₁-C₁₀ alkyl, —

OCF₃, —

O—

(CH₂)₂—

O—

CH₃, —

O(CH₂)₂SCH₃, —

O—

(CH₂)₂—

O—

N(R_m)(R_n) and —

O—

CH₂—

C(═

O)—

N(R_m)(R_n), where each R_m and R_n is, independently, H, an amino protecting group or substituted or unsubstituted —

C₁-C₁₀ alkyl.

29. The composition of claim 28 wherein the 2′

-modification is selected from allyl, —

O-allyl, —

O—

C₂-C₁₀ alkyl, —

O—

(CH₂)₂—

O—

CH₃ and —

O(CH₂)₂SCH₃.

30. The composition of claim 29 wherein the 2′

-modification is —

O—

(CH₂)₂—

O—

CH₃.

33. The composition of claim 2 wherein each of the external regions of each gapped oligomeric compound independently comprises from about 1 to about 6 nucleosides.

34. The composition of claim 2 wherein each of the external regions of each gapped oligomeric compound independently comprises from about 1 to about 4 nucleosides.

35. The composition of claim 2 wherein each of the external regions of each gapped oligomeric compound independently comprises from 1 to about 3 nucleosides.

36. The composition of claim 1 wherein one of the first and second oligomeric compounds comprises an alternating motif having the formula:

5′

-A(-L-B-L-A)_n(-L-B)_nn-3′

wherein;

each L is, independently, an internucleoside linking group;

each A or each B is a β

-D-ribonucleoside or a sugar modified nucleoside;

the other of each A or each B is a sugar modified nucleoside;

n is from about 7 to about 11;

nn is 0 or 1; and

wherein the sugar group of each A nucleoside is identical, the sugar group of each B nucleoside, is identical and the sugar group of each A nucleoside is different than the sugar group of each B nucleoside.

37. The composition of claim 36 wherein each A or each B is a β

-D-ribonucleoside.

38. The composition of claim 36 wherein each A or each B is a 2′

-modified nucleoside wherein the 2′

-modification is selected from halogen, allyl, amino, azido, —

O-allyl, —

O—

C₁-C₁₀alkyl, —

OCF₃, —

O—

(CH₂)₂—

O—

CH₃, —

O(CH₂)₂SCH₃, —

O—

(CH₂)₂—

O—

N(R_m)(R_n) and —

O—

CH₂—

C(═

O)—

N(R_m)(R_n), where each R_m and R_n is, independently, H, an amino protecting group or substituted or unsubstituted C₁-C₁₀ alkyl.

39. The composition of claim 38 wherein the 2′

-modification is allyl, —

O-allyl, —

O—

C₁-C₁₀ alkyl, —

O—

(CH₂)₂—

O—

CH₃ or —

O(CH₂)₂SCH₃.

40. The composition of claim 39 wherein the 2′

-modification is —

O—

(CH₂)₂—

O—

CH₃.

41. The composition of claim 36 wherein each A and each B is a sugar modified nucleoside.

42. The composition of claim 41 wherein each A or each B is a 2′

-OCH₃ modified nucleoside.

43. The composition of claim 42 wherein the other of each A or each B is a 2′

-F modified nucleoside.

44. The composition of claim 37 wherein the second oligomeric compound comprises the alternating motif.

45. The composition of claim 44 wherein each A or each B is a 2′

-modified nucleoside.

46. The composition of claim 45 wherein each 2-modification is allyl, —

O-allyl, —

O—

C₁-C₁₀ alkyl, —

O—

(CH₂)₂—

O—

CH₃ or —

O(CH₂)₂SCH₃.

47. The composition of claim 46 wherein each 2′

-modification is —

O—

(CH₂)₂—

O—

CH₃.

49. The composition of claim 1 wherein one of the first and the second oligomeric compounds comprises a fully modified oligomeric compound wherein essentially each nucleoside of the fully modified oligomeric compound is a sugar modified nucleoside and wherein each sugar modification is the same.

50. The composition of claim 49 wherein each sugar modified nucleoside of the fully modified oligomeric compound is selected from 2′

-modified nucleosides, 4′

-thio modified nucleosides, 4′

-thio-2′

-modified nucleosides and nucleosides having bicyclic sugar moieties.

51. The composition of claim 50 wherein each sugar modified nucleoside is a 2′

-modified nucleoside.

52. The composition of claim 51 wherein each sugar modified nucleoside is a 2′

-OCH₃ or a 2′

-F modified nucleoside.

53. The composition of claim 52 wherein each sugar modified nucleoside is a 2′

-OCH₃ modified nucleoside.

54. The composition of claim 49 wherein one or both of the 3′

and 5′

-termini of the fully modified oligomeric compound is a β

-D-ribonucleoside.

55. The composition of claim 1 wherein one of the first and second oligomeric compounds is a positionally modified oligomeric compound.

56. The composition of claim 55 wherein the positionally modified oligomeric compound comprises a continuous sequence of from about 12 to about 30 linked nucleosides comprising from 4 to about 8 regions wherein each region is either a sequence of β

-D-ribonucleosides or a sequence of sugar modified nucleosides and wherein the regions are alternating wherein each of the β

-D-ribonucleoside regions is flanked on each side by a region of sugar modified nucleosides and each region of sugar modified nucleosides is flanked on each side by a region of β

-D-ribonucleosides with the exception of regions located at the 3′ and

5′

-termini that are only flanked on one side and wherein the sugar modified nucleosides are selected from 2′

-modified nucleosides, 4′

-thio modified nucleosides, 4′

-thio-2′

-modified nucleosides and nucleosides having bicyclic sugar moieties.

57. The composition of claim 56 wherein the positionally modified oligomeric compound comprises from 5 to 7 regions.

58. The composition of claim 56 wherein each of the regions of β

-D-ribonucleosides comprises from 2 to 8 nucleosides.

59. The composition of claim 56 wherein each of the regions of sugar modified nucleosides comprises from 1 to 4 nucleosides.

60. The composition of claim 59 wherein each of the regions of sugar modified nucleosides comprises from 2 to 3 nucleosides.

61. The composition of claim 56 wherein the oligomeric compound comprising a positionally modified motif has the formula:

(X₁)_j-(Y₁)_i-X₂-Y₂-X₃-Y₃-X₄

wherein;

X₁ is a sequence of from 1 to about 3 sugar modified nucleosides;

Y₁ is a sequence of from 1 to about 5 β

-D-ribonucleosides;

X₂ is a sequence of from 1 to about 3 sugar modified nucleosides;

Y₂ is a sequence of from 2 to about 7 β

-D-ribonucleosides;

X₃ is a sequence of from 1 to about 3 sugar modified nucleosides;

Y₃ is a sequence of from 4 to about 6 β

-D-ribonucleosides;

X₄ is a sequence of from 1 to about 3 sugar modified nucleosides;

i is 0 or 1; and

j is 0 or 1 when i is 1 or 0 when i is 0.

63. The composition of claim 61 wherein:

X₄ is a sequence of 3 sugar modified nucleosides;

Y₃ is a sequence of 5 β

-D-ribonucleosides;

X₃ is a sequence of 2 sugar modified nucleosides;

i is 0; and

Y₂ is a sequence of 7 β

-D-ribonucleosides.

64. The composition of claim 61 wherein:

i is 1;

j is 0;

X₄ is a sequence of 3 sugar modified nucleosides;

Y₃ is a sequence of 5 β

-D-ribonucleosides;

X₃ is a sequence of 2 sugar modified nucleosides;

Y₂ is a sequence of 2 β

-D-ribonucleosides;

X₂ is a sequence of 2 sugar modified nucleosides; and

Y₁ is a sequence of 5 β

-D-ribonucleosides.

65. The composition of claim 61 wherein:

i is 1;

j is 1;

X₄ is a sequence of 3 sugar modified nucleosides;

Y₃ is a sequence of 5 β

-D-ribonucleosides;

X₃ is a sequence of 2 sugar modified nucleosides;

Y₂ is a sequence of 2β

-D-ribonucleosides;

X₂ is a sequence of 2 sugar modified nucleosides;

Y₁ is a sequence of 3 β

-D-ribonucleosides; and

X₁ is a sequence of 2 sugar modified nucleosides.

66. The composition of claim 61 wherein each of the sugar modified nucleosides of the positionally modified oligomeric compound is a 2′

-modified nucleoside or a 4′

-thio modified nucleoside.

67. The composition of claim 55 wherein the first oligomeric compound is the positionally modified oligomeric compound.

68. The composition of claim 1 wherein each of the internucleoside linking groups of the first and the second oligomeric compounds is, independently, selected from phosphodiester and phosphorothioate.

69. The composition of claim 1 wherein each of the first and second oligomeric compounds independently comprises from about 12 to about 30 nucleosides.

70. The composition of claim 1 wherein each of the first and second oligomeric compounds independently comprises from about 17 to about 23 nucleosides.

71. The composition of claim 1 wherein each of the first and second oligomeric compounds independently comprises from about 19 to about 21 nucleosides.

72. The composition of claim 1 wherein the first and the second oligomeric compounds form a complementary antisense/sense siRNA duplex.

74. The composition of claim 39 wherein the 2′

-modification is —

O—

CH₃.

75. The composition of claim 52 wherein each sugar modified nucleoside is a 2′

-F modified nucleoside.

76. The composition of claim 63 wherein X₂ is a sequence of 2 sugar modified nucleosides.

77. The composition of claim 76 wherein X₂ comprises a sequence of 2 4′

-thio modified nucleosides, X₃ is a sequence of 2 2′

-OCH₃ modified nucleosides and X₄ is a sequence of 3 2′

-OCH₃ modified nucleosides.

78. The composition of claim 64 wherein each of the sugar modified nucleosides is a 2′

-OCH₃ modified nucleoside.

79. The composition of claim 65 wherein X₁ comprises a sequence of 2 4′

-thio modified nucleosides, X₂ is a sequence of 2 2′

-OCH₃ modified nucleosides, X₃ is a sequence of 2 2′

-OCH₃ modified nucleosides and X₄ is a sequence of 3 2′

-OCH₃ modified nucleosides.

80. The composition of claim 1 wherein the first oligomeric compound is an antisense oligomeric compound and the second oligomeric compound is a sense oligomeric compound.

81. A method of inhibiting gene expression comprising contacting one or more cells, a tissue or an animal with a composition of claim 1.

27. (canceled)

31. (canceled)

32. (canceled)

48. (canceled)

62. (canceled)

73. (canceled)