Compositions and Methods for siRNA Inhibition of Angiogenesis

Abstract

RNA interference using small interfering RNAs which are specific for the vascular endothelial growth factor (VEGF) gene and the VEGF receptor genes Flt-1 and Flk-1/KDR inhibit expression of these genes. 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 the small interfering RNAs.

1 . A method of inhibiting expression of human VEGF mRNA, human Flt-1 mRNA, or human Flk-1/KDR mRNA, or an alternative splice form, mutant or cognate thereof, comprising administering to a subject an effective amount of an siRNA comprising a sense RNA strand and an antisense RNA strand, wherein the sense and an antisense RNA strands form an RNA duplex, and wherein the sense RNA strand comprises a nucleotide sequence identical to a target sequence of about 19 to about 25 contiguous nucleotides in human VEGF mRNA, human Flt-1 mRNA, or human Flk-1/KDR mRNA, or an alternative splice form, mutant or cognate thereof, is degraded. 
     
     
         2 . The method of  claim 1 , wherein the subject is a human being. 
     
     
         3 . The method of  claim 1 , wherein the effective amount of the siRNA is from about 1 nM to about 100 nM. 
     
     
         4 . The method of  claim 1 , wherein the siRNA is administered in conjunction with a delivery reagent. 
     
     
         5 . The method of  claim 4 , wherein the delivery agent is selected from the group consisting of lipofectin, lipofcctamine, cellfectin, polycations, and liposomes. 
     
     
         6 . The method of  claim 5 , wherein the delivery agent is a liposome. 
     
     
         7 . The method of  claim 6 , wherein the liposome comprises a ligand which targets the liposome to cells at or near the site of angiogenesis. 
     
     
         8 . The method of  claim 7 , wherein the ligand binds to receptors on tumor cells or vascular endothelial cells. 
     
     
         9 . The method of  claim 8 , wherein the ligand comprises a monoclonal antibody. 
     
     
         10 . The method of  claim 6 , wherein the liposome is modified with an opsonization-inhibition moiety. 
     
     
         11 . The method of  claim 10 , wherein the opsonization-inhibiting moiety comprises a PEG, PPG, or derivatives thereof. 
     
     
         12 . The method of  claim 1 , wherein the siRNA is expressed from a recombinant plasmid. 
     
     
         13 . The method of  claim 1 , wherein the siRNA is expressed from a recombinant viral vector. 
     
     
         14 . The method of  claim 13 , 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. 
     
     
         15 . The method of  claim 14 , wherein the recombinant viral vector is pseudotyped with surface proteins from vesicular stomatitis virus, rabies virus, Ebola virus, or Mokola virus. 
     
     
         16 . The method of  claim 13 , wherein the recombinant viral vector comprises an adeno-associated viral vector. 
     
     
         17 . The method of  claim 1 , wherein the siRNA is administered by an enteral administration route. 
     
     
         18 . The method of  claim 17 , wherein the enteral administration route is selected from the group consisting of oral, rectal, and intranasal. 
     
     
         19 . The method of  claim 1 , wherein the siRNA is administered by a parenteral administration route. 
     
     
         20 . The method of  claim 19 , 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 neovacularization. 
     
     
         21 . The method of  claim 20 , 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. 
     
     
         22 . The method of  claim 20 , wherein the peri- and intra-tissue injection is selected from the group consisting of peri-tumoral injection, intra-tumoral injection, intra-retinal injection, and subretinal injection. 
     
     
         23 . The method of  claim 20 , wherein the direct application at or near the site of neovascularization comprises application by catheter, retinal pellet, suppository, an implant comprising a porous material, an implant comprising a non-porous material, or an implant comprising a gelatinous material. 
     
     
         24 . A method of inhibiting angiogenesis in a subject, comprising administering to a subject an effective amount of an siRNA comprising a sense RNA strand and an antisense RNA strand, wherein the sense and an antisense RNA strands form an RNA duplex, and wherein the sense RNA strand comprises a nucleotide sequence identical to a target sequence of about 19 to about 25 contiguous nucleotides in human VEGF mRNA, human Flt-1 m RNA, or human Flt-1/KDR mRNA, or an alternative splice form, mutant or cognate thereof. 
     
     
         25 . The method of  claim 24 , wherein the angiogenesis is pathogenic. 
     
     
         26 . The method of  claim 24 , wherein the angiogenesis is non-pathogenic. 
     
     
         27 . The method of  claim 26 , wherein the non-pathogenic angiogenesis is associated with production of fatty tissues or cholesterol production. 
     
     
         28 . The method of  claim 26 , wherein the non-pathogenic angiogenesis comprises endometrial neovascularization. 
     
     
         29 . A method of treating an angiogenic disease in a subject, comprising administering to a subject in need of such treatment an effective amount of an siRNA comprising a sense RNA strand and an antisense RNA strand, wherein the sense and an antisense RNA strands form an RNA duplex, and wherein the sense RNA strand comprises a nucleotide sequence identical to a target sequence of about 19 to about 25 contiguous nucleotides in human VEGF mRNA, human Flt-1 mRNA, or human Flt-1/KDR mRNA, or an alternative splice form, mutant or cognate thereof, such that angiogenesis associated with the angiogenic disease is inhibited. 
     
     
         30 . The method of  claim 29 , wherein the angiogenic disease comprises a tumor associated with a cancer. 
     
     
         31 . The method of  claim 30 , wherein the cancer is selected from the group consisting of breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon caner, 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. 
     
     
         32 . The method of  claim 29 , wherein the angiogenic disease is selected from the group consisting of diabetic retinopathy, age-related macular degeneration, and inflammatory diseases. 
     
     
         33 . The method of  claim 32 , wherein the inflammatory disease is psoriasis or rheumatoid arthritis. 
     
     
         34 . The method of  claim 32 , wherein the angiogenic disease is age-related macular degeneration. 
     
     
         35 . The method of  claim 29 , wherein the siRNA is administered in combination with a pharmaceutical agent for treating the angiogenic disease, which pharmaceutical agent is different from the siRNA. 
     
     
         36 . The method of  claim 35 , wherein the angiogenic disease is cancer, and the pharmaceutical agent comprises a chemotherapeutic agent. 
     
     
         37 . The method of  claim 36 , wherein the chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin, and tamoxifen. 
     
     
         38 . The method of  claim 29 , wherein the siRNA is administered to a subject in combination with another therapeutic method designed to treat the angiogenic disease. 
     
     
         39 . The method of  claim 38 , wherein the angiogenic disease is cancer, and the siRNA is administered in combination with radiation therapy, chemotherapy or surgery.