Compositions and methods for sirna inhibition of angiopoietin 1 and 2 and their receptor tie2

Abstract

RNA interference using small interfering RNAs which are specific for mRNA produced from the Ang1, Ang2 or Tie2 genes inhibits expression of these genes. Diseases which involve Ang1, Ang2 or Tie2 mediated angiogenesis, such as inflammatory and autoimmune diseases, diabetic retinopathy, age related macular degeneration and many types of cancer, can be treated by administering the small interfering RNAs.

We claim: 
     
         1 . An isolated 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 substantially identical to a target sequence of about 19 to about 25 contiguous nucleotides in human Ang1, Ang2 or Tie2 mRNA, or an alternative splice form, mutant or cognate thereof. 
     
     
         2 . The siRNA of  claim 1 , wherein the cognate of the human Ang1, Ang2 or Tie2 mRNA sequence is SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. 
     
     
         3 . The siRNA of  claim 1 , wherein the sense RNA strand comprises one RNA molecule, and the antisense RNA strand comprises one RNA molecule. 
     
     
         4 . The siRNA of  claim 1 , wherein the sense and antisense RNA strands forming the RNA duplex are covalently linked by a single-stranded hairpin. 
     
     
         5 . The siRNA of  claim 1 , wherein the siRNA further comprises non-nucleotide material. 
     
     
         6 . The siRNA of  claim 1 , wherein the siRNA further comprises an addition, deletion, substitution or alteration of one or more nucleotides. 
     
     
         7 . The siRNA of  claim 1 , wherein the sense and antisense RNA strands are stabilized against nuclease degradation. 
     
     
         8 . The siRNA of  claim 1 , further comprising a 3′ overhang. 
     
     
         9 . The siRNA of  claim 8 , wherein the 3′ overhang comprises from 1 to about 6 nucleotides. 
     
     
         10 . The siRNA of  claim 8 , wherein the 3′ overhang comprises about 2 nucleotides. 
     
     
         11 . The siRNA of  claim 3  wherein the sense RNA strand comprises a first 3′ overhang, and the antisense RNA strand comprises a second 3′ overhang. 
     
     
         12 . The siRNA of  claim 11 , wherein the first and second 3′ overhangs separately comprise from 1 to about 6 nucleotides. 
     
     
         13 . The siRNA of  claim 12 , wherein the first 3′ overhang comprises a dinucleotide and the second 3′ overhang comprises a dinucleotide. 
     
     
         14 . The siRNA of  claim 13 , where the dinucleotide comprising the first and second 3′ overhangs is dithymidylic acid (TT) or diuridylic acid (uu). 
     
     
         15 . The siRNA of  claim 8 , wherein the 3′ overhang is stabilized against nuclease degradation. 
     
     
         16 . A recombinant plasmid comprising nucleic acid sequences for expressing 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 substantially identical to a target sequence of about 19 to about 25 contiguous nucleotides in human Ang1, Ang2 or Tie2 mRNA, or an alternative splice form, mutant or cognate thereof. 
     
     
         17 . The recombinant plasmid of  claim 16 , wherein the nucleic acid sequences for expressing the siRNA comprise an inducible or regulatable promoter. 
     
     
         18 . The recombinant plasmid of  claim 16 , wherein the nucleic acid sequences for expressing the siRNA comprise a sense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter, and an antisense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter. 
     
     
         19 . The recombinant plasmid of  claim 16 , wherein the plasmid comprises a CMV promoter. 
     
     
         20 . A recombinant viral vector comprising nucleic acid sequences for expressing 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 substantially identical to a target sequence of about 19 to about 25 contiguous nucleotides in human Ang1, Ang2 or Tie2 mRNA, or an alternative splice form, mutant or cognate thereof. 
     
     
         21 . The recombinant viral vector of  claim 20 , wherein the nucleic acid sequences for expressing the siRNA comprise an inducible or regulatable promoter. 
     
     
         22 . The recombinant viral vector of  claim 20 , wherein the nucleic acid sequences for expressing the siRNA comprise a sense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter, and an antisense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter. 
     
     
         23 . The recombinant viral vector of  claim 20 , wherein the recombinant viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, and a herpes virus vector. 
     
     
         24 . The recombinant viral vector of  claim 20 , wherein the recombinant viral vector is pseudotyped with surface proteins from vesicular stomatitis virus, rabies virus, Ebola virus, or Mokola virus. 
     
     
         25 . The recombinant viral vector of  claim 23 , wherein the recombinant viral vector comprises an adeno-associated viral vector. 
     
     
         26 . A pharmaceutical composition comprising an siRNA and a pharmaceutically acceptable carrier, wherein the siRNA comprises 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 substantially identical to a target sequence of about 19 to about 25 contiguous nucleotides in human Ang1, Ang2 or Tie2 mRNA, or an alternative splice form, mutant or cognate thereof. 
     
     
         27 . The pharmaceutical composition of  claim 26 , further comprising lipofectin, lipofectamine, cellfectin, polycations, or liposomes. 
     
     
         28 . A pharmaceutical composition comprising the plasmid of  claim 16 , or a physiologically acceptable salt thereof, and a pharmaceutically acceptable carrier. 
     
     
         29 . The pharmaceutical composition of  claim 28 , further comprising lipofectin, lipofectamine, cellfectin, polycations, or liposomes. 
     
     
         30 . A pharmaceutical composition comprising the viral vector of  claim 20  and a pharmaceutically acceptable carrier. 
     
     
         31 . A method of inhibiting expression of human Ang1, Ang2 or Tie2 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 substantially identical to a target sequence of about 19 to about 25 contiguous nucleotides in human Ang1, Ang2 or Tie2 mRNA, or an alternative splice form, mutant or cognate thereof, such that the human Ang1, Ang2 or Tie2 mRNA, or an alternative splice form, mutant or cognate thereof, is degraded. 
     
     
         32 . The method of  claim 31 , wherein the subject is a human being. 
     
     
         33 . The method of  claim 31 , wherein the siRNA is administered in conjunction with a delivery reagent. 
     
     
         34 . The method of  claim 33 , wherein the delivery agent is selected from the group consisting of lipofectin, lipofectamine, cellfectin, polycations, and liposomes. 
     
     
         35 . The method of  claim 34 , wherein the delivery agent is a liposome. 
     
     
         36 . The method  claim 35 , wherein the liposome comprises a ligand which targets the liposome to cells expressing Ang1, Ang2 or Tie2. 
     
     
         37 . The method of  claim 36 , wherein the cells are endothelial cells. 
     
     
         38 . The method of  claim 37 , wherein the ligand comprises a monoclonal antibody. 
     
     
         39 . The method of  claim 35 , wherein the liposome is modified with an opsonization-inhibition moiety. 
     
     
         40 . The method of  claim 39 , wherein the opsonization-inhibiting moiety comprises a PEG, PPG, or derivatives thereof. 
     
     
         41 . The method of  claim 31 , wherein the siRNA is expressed from a recombinant plasmid. 
     
     
         42 . The method of  claim 31 , wherein the siRNA is expressed from a recombinant viral vector. 
     
     
         43 . The method of  claim 42 , 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. 
     
     
         44 . The method of  claim 43 , wherein the recombinant viral vector is pseudotyped with surface proteins from vesicular stomatitis virus, rabies virus, Ebola virus, or Mokola virus. 
     
     
         45 . The method of  claim 42 , wherein the recombinant viral vector comprises an adeno-associated viral vector. 
     
     
         46 . The method of  claim 31 , wherein two or more siRNA are administered to the subject, and wherein each siRNA comprises a nucleotide sequence which is substantially identical to a different Ang1, Ang2 or Tie2 mRNA target sequence. 
     
     
         47 . The method of  claim 31 , wherein two or more siRNA are administered to the subject, and wherein each siRNA administered comprises a nucleotide sequence which is substantially identical to a target sequence from a different target mRNA. 
     
     
         48 . The method of  claim 31 , wherein the siRNA is administered by an enteral administration route. 
     
     
         49 . The method of  claim 48 , wherein the enteral administration route is selected from the group consisting of oral, rectal, and intranasal. 
     
     
         50 . The method of  claim 31 , wherein the siRNA is administered by a parenteral administration route. 
     
     
         51 . The method of  claim 50 , wherein the parenteral administration route is selected from the group consisting of intravascular administration, peri- and intra-tissue administration, subcutaneous injection or deposition, subcutaneous infusion, intraocular administration, and direct application at or near the site of neovascularization. 
     
     
         52 . The method of  claim 51 , 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. 
     
     
         53 . The method of  claim 51 , 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. 
     
     
         54 . The method of  claim 51 , wherein the intraocular administration comprises intravitreal, intraretinal, subretinal, subtenon, peri- and retro-orbital, trans-corneal or trans-scleral administration. 
     
     
         55 . The method of  claim 51 , wherein the direct application at or near the site of neovascularization comprises application by catheter, corneal pellet, eye dropper, suppository, an implant comprising a porous material, an implant comprising a non-porous material, or an implant comprising a gelatinous material. 
     
     
         56 . The method of  claim 55 , wherein the site of neovascularization is in the eye, and the direct application at or near the site of neovascularization comprises application by eyedropper. 
     
     
         57 . 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 substantially identical to a target sequence of about 19 to about 25 contiguous nucleotides in human Ang1, Ang2 or Tie2 mRNA, or an alternative splice form, mutant or cognate thereof. 
     
     
         58 . The method of  claim 57 , wherein the angiogenesis is pathogenic. 
     
     
         59 . The method of  claim 57 , wherein the angiogenesis is non-pathogenic. 
     
     
         60 . The method of  claim 59 , wherein the non-pathogenic angiogenesis is associated with production of fatty tissues or cholesterol production. 
     
     
         61 . The method of  claim 59 , wherein the non-pathogenic angiogenesis comprises endometrial neovascularization. 
     
     
         62 . 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 substantially identical to a target sequence of about 19 to about 25 contiguous nucleotides in human Ang1, Ang2 or Tie2 mRNA, or an alternative splice form, mutant or cognate thereof, such that angiogenesis associated with the angiogenic disease is inhibited. 
     
     
         63 . The method of  claim 62 , wherein the angiogenic disease comprises a tumor associated with a cancer. 
     
     
         64 . The method of  claim 63 , 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. 
     
     
         65 . The method of  claim 62 , wherein the angiogenic disease is selected from the group consisting of diabetic retinopathy and age-related macular degeneration. 
     
     
         66 . The method of  claim 65 , wherein the angiogenic disease is age-related macular degeneration. 
     
     
         67 . The method of  claim 62 , wherein the siRNA is administered in combination with a pharmaceutical agent for treating angiogenic disease, which pharmaceutical agent is different from the siRNA. 
     
     
         68 . The method of  claim 67 , wherein angiogenic disease is cancer, and the pharmaceutical agent comprises a chemotherapeutic agent. 
     
     
         69 . The method of  claim 68 , wherein the chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin, and tamoxifen. 
     
     
         70 . The method of  claim 62 , wherein the siRNA is administered to a subject in combination with another therapeutic method designed to treat the angiogenic disease. 
     
     
         71 . The method of  claim 70 , wherein the angiogenic disease is cancer, and the siRNA is administered in combination with radiation therapy, chemotherapy or surgery. 
     
     
         72 . A method of treating complications arising from type I diabetes 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 from an RNA duplex, and wherein the sense RNA strand comprises a nucleotide sequence substantially identical to a target sequence of about 19 to about 25 contiguous nucleotides in human Ang1, Ang2 or Tie2 mRNA, or an alternative splice form, mutant or cognate thereof. 
     
     
         73 . The method of  claim 72 , wherein the complications arising from type I diabetes are selected from the group consisting of diabetic retinopathy, diabetic neuropathy, diabetic nephropathy and macrovascular disease. 
     
     
         74 . The method of  claim 73 , wherein the macrovascular disease is coronary artery disease, cerebrovascular disease or peripheral vascular disease.