DOUBLE STRAND COMPOSITIONS COMPRISING DIFFERENTIALLY MODIFIED STRANDS FOR USE IN GENE MODULATION

• US 20070173474A1
• Filed: 12/01/2006
• Published: 07/26/2007
• Est. Priority Date: 06/03/2004
• Status: Abandoned Application

• Current Assignee: Balkrishen Bhat, Charles Allerson, Eric Swayze, Prasad Dande, Richard Griffey, Thazha Prakash
• Original Assignee: Balkrishen Bhat, Charles Allerson, Eric Swayze, Prasad Dande, Richard Griffey, Thazha Prakash
• Inventors: Bhat, Balkrishen, Allerson, Charles, Dande, Prasad, Swayze, Eric, Griffey, Richard, Prakash, Thazha
• Application Number:
  US11/565,794
• Publication Number:
  US 20070173474A1
• Time in Patent Office: Days
• US Class Current: 514/44.A
• 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 double 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 is an asymmetric gapped oligomeric compound;

the other of the first and second oligomeric compounds is a fully modified oligomeric compound; and

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

2. The composition of claim 1 wherein the asymmetric gapped oligomeric compound comprises a contiguous sequence of nucleosides divided into an internal region flanked by two external regions wherein:

the sugar groups within each region are identical and the sugar groups of each region are different from the sugar groups of each other region;

the nucleosides of the internal region are 1-D-ribonucleosides or sugar modified nucleosides and the nucleosides of the external regions are sugar modified nucleosides; and

the sugar modified nucleosides are each 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 wherein the internal region of the asymmetric gapped oligomeric compound is a sequence of β

-D-ribonucleosides.

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

5. The composition of claim 4 wherein the sugar modified nucleosides are 2′

-F modified nucleosides or 4′

-thio modified nucleosides.

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

-modified nucleosides.

7. The composition of claim 6 wherein each of the external regions of the asymmetric gapped oligomeric compound is a sequence of 2′

-modified nucleosides.

8. The composition of claim 6 wherein the 2′

-modifications of each external region having a sequence of 2′

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

O-allyl, —

O—

C₁-C₁₀ alkyl, —

OCF₃, —

O—

(CH₂)₂—

OCH₃, —

O(CH₂)₂—

SCH₃, —

O—

(CH₂)₂—

ON(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.

9. The composition of claim 8 wherein the 2′

-modifications of each external region having a sequence of 2′

-modified nucleosides is independently selected from —

F, —

OCH₃ or —

O—

(CH₂)₂—

OCH₃.

10. The composition of claim 2 wherein one of the external regions of the asymmetric gapped oligomeric compound is a sequence of 4′

-thio modified nucleosides.

11. The composition of claim 2 wherein one of the external regions of the asymmetric gapped oligomeric compound is a sequence of 4′

-thio-2′

-modified nucleosides.

12. The composition of claim 11 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—

OCH₃, —

O(CH₂)₂—

SCH₃, —

O—

(CH₂)₂—

ON(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.

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

-modifications is —

F, —

OCH₃, —

OCF₃ or —

O—

(CH₂)₂—

OCH₃.

14. The composition of claim 13 wherein each of the 2′

-modifications is —

OCH₃ or —

O—

(CH₂)₂—

OCH₃.

15. The composition of claim 2 wherein one of the external regions of the asymmetric gapped oligomeric compound is a sequence of bicyclic sugar moieties.

16. The composition of claim 15 wherein each of the bicyclic sugar moieties has a 2′

-O—

(CH₂)_n-4′

bridge wherein n is 1 or 2.

17. The composition of claim 1 wherein the first oligomeric compound is the asymmetric gapped oligomeric compound.

18. The composition of claim 17 wherein one of the external regions of the asymmetric gapped oligomeric compound is a sequence of 4′

-thio modified nucleosides and the other external region is a sequence of 2′

-modified nucleosides.

19. The composition of claim 18 wherein the external region located at the 5′

-end of the asymmetric gapped oligomeric compound is a sequence of 2′

-OCH₃, 2′

-F or 4′

-thio modified nucleosides.

20. The composition of claim 18 wherein the 2′

-modified nucleosides are 2′

-OCH₃ or 2′

-F modified nucleosides.

21. The composition of claim 20 wherein the 2′

-modified nucleosides are 2′

-OCH₃ modified nucleosides.

22. The composition of claim 1 wherein the second oligomeric compound is the asymmetric gapped oligomeric compound.

23. The composition of claim 22 wherein at least one of the external regions of the asymmetric gapped oligomeric compound is a sequence of 2′

-modified nucleosides wherein the 2′

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

O-allyl, —

O—

C₁-C₁₀ alkyl, —

OCF₃, —

O—

(CH₂)₂—

OCH₃, —

O(CH₂)₂—

SCH₃, O—

(CH₂)₂—

ON(R_m)(R_n) and O—

CH₂—

C(═

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

C₁-C₁₀ alkyl.

24. The composition of claim 23 wherein the 2′

-modification is selected from allyl, —

O-allyl, —

O—

C₂-C₁₀ alkyl, —

O—

(CH₂)₂—

OCH₃ or —

O(CH₂)₂—

SCH₃.

25. The composition of claim 24 wherein the 2′

-modification is —

O—

(CH₂)₂—

OCH₃.

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

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

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

29. The composition of claim 1 wherein each nucleoside of the fully modified oligomeric compound is a sugar modified nucleoside and wherein each sugar modification is the same.

30. The composition of claim 29 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.

31. The composition of claim 30 wherein each sugar modified nucleoside is a 2′

-modified nucleoside.

32. The composition of claim 31 wherein each sugar modified nucleoside is a 2′

-OCH₃ or a 2′

-F modified nucleoside.

33. The composition of claim 32 wherein each sugar modified nucleoside is a 2′

-OCH₃ modified nucleoside.

34. The composition of claim 32 wherein each sugar modified nucleoside is a 2′

-F modified nucleoside.

35. The composition of claim 32 wherein one or both of the 3′

and 5′

-termini of the fully modified oligomeric compound is a β

-D-ribonucleoside.

36. The composition of claim 1 having at least 2 phosphorothioate internucleoside linking groups at the 3′

-end of the first oligomeric compound.

37. The composition of claim 36 having about 7 phosphorothioate internucleoside linking groups at the 3′

-end of the first oligomeric compound.

38. The composition of claim 1 wherein the first oligomeric compound further comprises a 5′

-thiophosphate group.

39. The composition of claim 1 wherein each of the internucleoside linking groups of the first and the second oligomeric compounds are independently selected from phosphodiester or phosphorothioate.

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

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

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

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

44. 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.

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