Methods and Compositions for Modulating Angiogenesis

• US 20110172293A1
• Filed: 07/01/2009
• Published: 07/14/2011
• Est. Priority Date: 07/08/2008
• Status: Abandoned Application

• Current Assignee: The J. David Gladstone Institutes
• Original Assignee: The J. David Gladstone Institutes
• Inventors: Srivastava, Deepak, Fish, Jason E.
• Application Number:
  US13/001,604
• Publication Number:
  US 20110172293A1
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• 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 2310/11 Antisense
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2320/30 Special therapeutic applications

Abstract

The present invention provides compositions comprising antisense nucleic acids that reduce miR-126 levels in an endothelial cell. The present invention provides compositions comprising a target protector nucleic acid. The present invention provides methods of modulating angiogenesis in an individual, the methods generally involving administering to the individual an effective amount of an agent that increases or that decreases the level of miR-126 in endothelial cells of the individual.

Claims

1. A composition comprising an antisense nucleic acid comprising a nucleotide sequence that is complementary to a nucleotide sequence included in a miR-126 nucleic acid, wherein the antisense nucleic acid comprises a nucleotide sequence capable of forming a stable duplex with a portion of the miR-126 nucleic acid comprising a ribonuclease III cleavage site, and wherein the antisense nucleic acid has a length of from about 20 nucleotides to about 50 nucleotides.

2. The composition of claim 1, wherein the miR-126 nucleic acid comprises a nucleotide sequence having at least about 80% nucleotide sequence identity to nucleotides 15-74 of the nucleotide sequence depicted in FIG. 12A and set forth in SEQ ID NO:

1.

3. The composition of claim 1, wherein the antisense nucleic acid comprises fewer than five mismatches in complementarity with the portion of the miR-126 nucleic acid comprising a ribonuclease III cleavage site.

4. The composition of claim 1, wherein the ribonuclease III cleavage site is a Dicer cleavage site or a Drosha cleavage site.

6. The composition of claim 1, wherein the antisense nucleic acid comprises at least one non-phosphodiester internucleosidic linkage, at least one modified nucleotide, or at least one substituted sugar moiety.

5. (canceled)

7-17. -17. (canceled)

18. A method of reducing angiogenesis in a mammal, the method comprising administering to a mammal in need thereof an effective amount of an agent that decreases a level and/or activity of miR-126 in an endothelial cell in the mammal.

19. The method of claim 18, wherein the agent is:

i) an antisense nucleic acid that reduces a level of miR-126 in the cell;

ii) a nucleic acid comprising a nucleotide sequence encoding an antisense nucleic acid that reduces a level of miR-126 in the cell;

iii) a nucleic acid comprising a nucleotide sequence that is complementary to a mature miR-126 nucleic acid and that inhibits binding of a mature miR-126 to a miR-126 target;

or iv) a nucleic acid comprising a nucleotide sequence encoding a nucleic acid comprising a nucleotide sequence that is complementary to a mature miR-126 nucleic acid and that inhibits binding of a mature miR-126 to a miR-126 target.

20. The method of claim 19, wherein the antisense-encoding nucleotide sequence is operably linked to an endothelial-specific transcriptional control element, or to an inducible promoter.

22. The method of claim 18, wherein the agent is an antisense nucleic acid that reduces a level of miR-126 in the cell.

23. The method of claim 22, wherein the antisense nucleic acid forms a stable duplex with a portion of the miR-126 nucleic acid comprising a ribonuclease III cleavage site.

27. The method of claim 18, wherein the agent is a target protector nucleic acid that binds to a miR-126 target mRNA, and that does not induce cleavage or translational repression of the target mRNA, wherein the target protector nucleic acid inhibits binding of a miR-126 to the miR-126 target mRNA.

28. The method of claim 27, wherein the target mRNA is a Spred1 mRNA or a Pik3r2 mRNA.

21. (canceled)

24-26. -26. (canceled)

29-32. -32. (canceled)

33. A method of increasing angiogenesis in an individual, the method comprising administering to a mammal having a disorder that is treatable by increasing angiogenesis an effective amount of an agent that increases a level of miR-126 in an endothelial cell in the mammal, wherein said agent is a recombinant nucleic acid comprising a nucleotide sequence encoding a miR-126 nucleic acid.

35. The method of claim 33, wherein said miR-126-encoding nucleotide sequence is operably linked to an endothelial cell-specific promoter.

37. The method of claim 33, wherein said miR-126 nucleic acid comprises a nucleotide sequence having at least about 75% nucleotide sequence identity to nucleotides 15-41 of the nucleotide sequence depicted in FIG. 12A and set forth in SEQ ID NO:

1.

38. The method of claim 33, wherein said administering is via delivery to a local site, or via system administration.

34. (canceled)

36. (canceled)

39-42. -42. (canceled)

43. A synthetic target protector nucleic acid that binds to a miR-126 target mRNA, wherein the target protector nucleic acid does not induce cleavage or translational repression of the target mRNA, and wherein the target protector nucleic acid inhibits binding of a miR-126 to the miR-126 target mRNA.

44. The nucleic acid of claim 43, wherein the target mRNA is a Spred1 mRNA, a Pik3r2 mRNA, a CRK mRNA, an RGS3 mRNA, an IGFA6 mRNA, or a VCAM1 mRNA.

45. The nucleic acid of claim 43, wherein the synthetic target protector nucleic acid comprises a nucleotide sequence having at least about 85% nucleotide sequence identity to the complement of any one of the nucleotide sequences depicted in FIG. 6B.

46. The nucleic acid of claim 43, wherein the target mRNA is Spred1, and wherein the synthetic target protector nucleic acid comprises a nucleotide sequence having at least about 85% nucleotide sequence identity to 5′

TCGTACCTTACATTTAGTTAAA-3′

(SEQ ID NO;

32), or wherein the target mRNA is Pik3r2, and wherein the synthetic target protector nucleic acid comprises a nucleotide sequence having at least about 85% nucleotide sequence identity to 5′

-ACGTACCGTACAAAACCTGCCT-3′

(SEQ ID NO;

33).

48. The nucleic acid of claim 43, wherein the synthetic target protector nucleic acid has a length of from about 19 nucleotides to about 50 nucleotides, or from about 19 nucleotides to about 25 nucleotides.

54. A composition comprising:

a) the synthetic target protector nucleic acid of claim 43; and

b) a pharmaceutically acceptable carrier.

47. (canceled)

49-53. -53. (canceled)

55. (canceled)

56. A synthetic competitive inhibitor nucleic acid comprising a nucleotide sequence that is complementary to a mature miR-126 nucleic acid and that inhibits binding of a mature miR-126 to a miR-126 target.

57. The synthetic competitive inhibitor nucleic acid of claim 56, wherein the synthetic competitive inhibitor nucleic acid comprises one or more of a nuclease-resistant internucleosidic linkage, a modified nucleotide, and a covalent modification.

58. A recombinant vector comprising a nucleotide sequence encoding the synthetic competitive inhibitor nucleic acid of claim 56.

59. A composition comprising:

a) the synthetic competitive inhibitor nucleic acid of claim 56; and

b) a pharmaceutically acceptable carrier.