Compositions and methods for selective inhibition of VEGF
Disclosed herein are siRNA compositions and methods useful for inhibiting expression of vascular endothelial growth factor (VEGF) isoforms. Such compositions and methods further involve siRNA capable of selectively targeting angiogenic VEGF isoforms while selectively sparing anti-angiogenic isoforms. Diseases which involve angiogenesis stimulated by overexpression of VEGF, such as diabetic retinopathy, age related macular degeneration and many types of cancer, can be treated by administering small interfering RNAs as disclosed.
1. An isolated siRNA comprising of a duplex of a first RNA strand and a second RNA strand, said first RNA strand comprising a nucleotide sequence identical to a target sequence of a vascular endothelial growth factor (VEGF) isoform VEGF 189 , further wherein said siRNA is non-complementary to VEGF 165b , wherein the nucleotide sequence identical to a target sequence consists of a sequence selected from the group consisting of SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118.
2. The siRNA of claim 1 , wherein the first and second RNA strands forming the RNA duplex are covalently linked by a single-stranded hairpin.
3. The siRNA of claim 1 , wherein the siRNA further comprises non-nucleotide material.
4. The siRNA of claim 1 , wherein the first and second RNA strands are stabilized against nuclease degradation.
5. The siRNA of claim 1 , further comprising a 3′ overhang.
6. The siRNA of claim 5 , wherein the 3′ overhang comprises from 1 to about 6 nucleotides.
7. The siRNA of claim 5 , wherein the 3′ overhang comprises about 2 nucleotides.
8. The siRNA of claim 1 , wherein the sense RNA strand comprises a first 3′ overhang, and the antisense RNA strand comprises a second 3′ overhang.
9. The siRNA of claim 8 , wherein the first and second 3′ overhangs each comprise from 1 to about 6 nucleotides.
10. The siRNA of claim 8 , wherein the first 3′ overhang comprises a dinucleotide and the second 3′ overhang comprises a dinucleotide.
11. The siRNA of claim 10 , where the dinucleotide comprising the first and second 3′ overhangs is dithymidylic acid (tt) or diuridylic acid (uu).
12. The siRNA of claim 5 , wherein the 3′ overhang is stabilized against nuclease degradation.
13. The siRNA of claim 1 , wherein said siRNA comprises at least one blunt end.
14. The siRNA of claim 1 , wherein said siRNA can inhibit the production or secretion of VEGF from a human cell and a rat cell.
15. The siRNA of claim 1 , wherein said siRNA can inhibit the production or secretion of VEGF from a human cell, a mouse cell, and a rat cell.
16. An isolated siRNA comprising of a duplex of a first RNA strand and a second RNA strand, said first RNA strand comprising a nucleotide sequence identical to a target sequence of a vascular endothelial growth factor (VEGF) isoform VEGF 189 , further wherein said siRNA is non-complementary to VEGF 165b , wherein the nucleotide sequence identical to a target sequence consists of a sequence selected from the group consisting of SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118, wherein the siRNA does not function to silence VEGF 165b or form a RISC complex with VEGF 165b .
17. A pharmaceutical composition comprising a siRNA and a pharmaceutically acceptable carrier, said siRNA comprised of a duplex of a first RNA strand a second RNA strand, said first RNA strand comprising a nucleotide sequence identical to a target sequence of a vascular endothelial growth factor (VEGF) isoform VEGF 189 , further wherein said siRNA is non-complementary to VEGF 165b , wherein the nucleotide sequence identical to a target sequence consists of a sequence selected from the group consisting of SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118.
18. The pharmaceutical composition of claim 17 , wherein the first and second RNA strands are stabilized against nuclease degradation.
19. The pharmaceutical composition of claim 17 , further comprising at least one 3′ overhang.
20. The pharmaceutical composition of claim 19 , wherein the at least one 3′ overhang comprises about 2 nucleotides.
21. The pharmaceutical composition of claim 19 , where the at least one 3′ overhang comprises a dithymidylic acid (tt) or diuridylic acid (uu).
22. The pharmaceutical composition of claim 17 , wherein the sense RNA strand comprises a first 3′ overhang, and the antisense RNA strand comprises a second 3′ overhang.
23. The pharmaceutical composition of claim 17 , wherein siRNA comprises at least one blunt end.
24. The siRNA of claim 17 , wherein said siRNA can inhibit the production or secretion of VEGF from a human cell and a rat cell.
25. The siRNA of claim 17 , wherein said siRNA can inhibit the production or secretion of VEGF from a human cell, a mouse cell, and a rat cell.
26. A method of treating an angiogenic disease in a subject comprising administering to the subject an effective amount of a pharmaceutical composition comprising the isolated siRNA of claim 1 and a pharmaceutically acceptable carrier.
27. The method of claim 26 , wherein the angiogenic disease comprises a tumor associated with a cancer.
28. The method of claim 27 , wherein the cancer is selected from the group consisting of breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, liver cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, wilm's tumor, multiple myeloma, skin cancer, lymphoma, and blood cancer.
29. The method of claim 26 , wherein the angiogenic disease is selected from the group consisting of diabetic retinopathy, age-related macular degeneration, and inflammatory diseases.
30. The method of claim 29 , wherein the inflammatory disease is psoriasis or rheumatoid arthritis.
31. The method of claim 29 , wherein the angiogenic disease is age-related macular degeneration.
32. The method of claim 26 , wherein the pharmaceutical composition is administered in combination with a pharmaceutical agent for treating the angiogenic disease, which pharmaceutical agent is different from the short interfering ribonucleic acid (siRNA).
33. The method of claim 32 , wherein the angiogenic disease is cancer, and the pharmaceutical agent comprises a chemotherapeutic agent.
34. The method of claim 33 , wherein the chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin, and tamoxifen.
35. The method of claim 26 , wherein the pharmaceutical composition is administered to a subject in combination with another therapeutic method designed to treat the angiogenic disease.
36. The method of claim 35 , wherein the angiogenic disease is cancer, and the pharmaceutical composition is administered in combination with radiation therapy, chemotherapy or surgery.
37. A method for inhibiting expression of human vascular endothelial growth factor (VEGF) comprising administering to a subject an effective amount of a pharmaceutical composition comprising the isolated siRNA of claim 1 and a pharmaceutically acceptable carrier.
38. The method of claim 37 , wherein the effective amount comprises from about 1 nm to about 100 nm of the short interfering ribonucleic acid (siRNA).
39. The method of claim 37 , wherein the pharmaceutical composition further comprises a delivery reagent.
40. The method of claim 37 , wherein the delivery agent is selected from the group consisting of lipofectin, lipofectamine, cellfectin, polycations, and liposomes.
41. The method of claim 40 , wherein the delivery agent is a liposome.
42. The method of claim 41 , wherein the liposome comprises a ligand which targets the liposome to cells at or near the site of angiogenesis.
43. The method of claim 42 , wherein the ligand binds to receptors on tumor cells or vascular endothelial cells.
44. The method of claim 42 , wherein the ligand comprises a monoclonal antibody.
45. The method of claim 41 , wherein the liposome is modified with an opsonization-inhibition moiety.
46. The method of claim 45 , wherein the opsonization-inhibiting moiety comprises a PEG, PPG, or derivatives thereof.
47. The method of claim 37 , wherein the short interfering ribonucleic acid (siRNA) is expressed from a recombinant plasmid.
48. The method of claim 37 , wherein the short interfering ribonucleic acid (siRNA) is expressed from a recombinant viral vector.
49. The method of claim 48 , wherein the recombinant viral vector comprises an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, or a herpes virus vector.
50. The method of claim 49 , wherein the recombinant viral vector is pseudotyped with surface proteins from vesicular stomatitis virus, rabies virus, Ebola virus, or Mokola virus.
51. The method of claim 48 , wherein the recombinant viral vector comprises an adeno-associated viral vector.
52. The method of claim 37 , wherein the pharmaceutical composition is administered by an enteral administration route.
53. The method of claim 52 , wherein the enteral administration route is selected from the group consisting of oral, rectal, and intranasal.
54. The method of claim 37 , wherein the pharmaceutical composition is administered by a parenteral administration route.
55. The method of claim 54 , wherein the parenteral administration route is selected from the group consisting of intravascular administration, peri- and intra-tissue injection, subcutaneous injection or deposition, subcutaneous infusion, and direct application at or near the site of neovascularization.
56. The method of claim 55 , wherein the intravascular administration is selected from the group consisting of intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature.
57. The isolated siRNA of claim 1 , wherein the siRNA does not silence VEGF 165b or form a RISC complex with VEGF 165b .
58. The pharmaceutical composition of claim 17 , wherein the siRNA does not silence VEGF 165b or form a RISC complex with VEGF 165b .