Rtef-1 variants and uses thereof

Abstract

Disclosed are variant RTEF-1 polypeptides having an RTEF-1 amino acid sequence with one or more internal deletions, wherein the polypeptides reduce VEGF promoter activity. Some of the RTEF-1 polypeptides include an amino acid sequence that is at least 80% identical to the contiguous amino acids of 1) amino acids 24 to 47 of SEQ ID NO:15 and 2) each of SEQ ID NOs:16 and 17, but does not comprise the contiguous amino acids of SEQ ID NOs:8, 9, 11, or 12. Also disclosed are nucleic acids encoding the variant RTEF-1 polypeptides of the present invention. Pharmaceutical compositions that include the polypeptides and nucleic acids of the present invention are also disclosed. Methods of inducing cell contact inhibition, regulating organ size, and reducing intracellular YAP activity are also set forth, as well as methods of treating hyperproliferative diseases such as cancer using the pharmaceutical compositions of the present invention.

What is claimed is: 
     
         1 . An isolated variant RTEF-1 polypeptide that reduces VEGF promoter activity comprising an amino acid sequence that is at least 80% identical to the contiguous amino acids of 1) amino acids 24 to 47 of SEQ ID NO:15 and 2) each of SEQ ID NOs:16 and 17, but does not comprise the contiguous amino acids of SEQ ID NOs:8, 9, 11, or 12. 
     
     
         2 . The isolated variant RTEF-1 polypeptide of  claim 1 , comprising an amino acid sequence that is at least 85% identical to the contiguous amino acids of 1) amino acids 24 to 47 of SEQ ID NO:15 and 2) each of SEQ ID NOs:16 and 17. 
     
     
         3 . The isolated variant RTEF-1 polypeptide of  claim 2 , comprising an amino acid sequence that is at least 90% identical to the contiguous amino acids of 1) amino acids 24 to 47 of SEQ ID NO:15 and 2) each of SEQ ID NOs:16 and 17. 
     
     
         4 . The isolated variant RTEF-1 polypeptide of  claim 3 , comprising an amino acid sequence that is at least 95% identical to the contiguous amino acids of 1) amino acids 24 to 47 of SEQ ID NO:15 and 2) each of SEQ ID NOs:16 and 17. 
     
     
         5 . The isolated variant RTEF-1 polypeptide of  claim 4 , comprising an amino acid sequence that is at least 97% identical to the contiguous amino acids of 1) amino acids 24 to 47 of SEQ ID NO:15 and 2) each of SEQ ID NOs:16 and 17. 
     
     
         6 . The isolated variant RTEF-1 polypeptide of  claim 4 , comprising an amino acid sequence that is at least 99% identical to the contiguous amino acids of 1) amino acids 24 to 47 of SEQ ID NO:15 and 2) each of SEQ ID NOs:16 and 17. 
     
     
         7 . The isolated variant RTEF-1 polypeptide of  claim 1 , wherein the polypeptide does not comprise the contiguous amino acids of SEQ ID NOs: 8, 9, 11, and 12. 
     
     
         8 . The isolated variant RTEF-1 polypeptide of  claim 1 , wherein the polypeptide is at least 95% identical to SEQ ID NO:1. 
     
     
         9 . The isolated RTEF-1 polypeptide of  claim 8 , wherein the polypeptide is at least 97% identical to SEQ ID NO:1. 
     
     
         10 . The isolated RTEF-1 polypeptide of  claim 9 , wherein the polypeptide is at least 99% identical to SEQ ID NO:1. 
     
     
         11 . The isolated RTEF-1 polypeptide of  claim 10 , wherein the polypeptide comprises SEQ ID NO:1. 
     
     
         12 . The isolated RTEF-1 polypeptide of  claim 1 , wherein the polypeptide is at least 95% identical to SEQ ID NO:2. 
     
     
         13 . The isolated RTEF-1 polypeptide of  claim 12 , wherein the polypeptide is at least 97% identical to SEQ ID NO:2. 
     
     
         14 . The isolated RTEF-1 polypeptide of  claim 12 , wherein the polypeptide is at least 99% identical to SEQ ID NO:2. 
     
     
         15 . The isolated RTEF-1 polypeptide of  claim 14 , wherein the RTEF-1 polypeptide comprises SEQ ID NO:2. 
     
     
         16 . The isolated RTEF-1 polypeptide of  claim 1 , further comprising a cell internalization moiety or a nuclear localization sequence that is a polypeptide, an antibody, an aptamer or an avimer. 
     
     
         17 . The isolated RTEF-1 polypeptide of  claim 16 , wherein the cell internalization moiety is a polypeptide comprising an HIV tat polypeptide, HSV-1 tegument protein VP22, or  Drosophila  antennopedia protein. 
     
     
         18 . The isolated RTEF-1 polypeptide of  claim 16 , wherein the cell internalization moiety is a polypeptide comprising poly-arginine, poly-methionine or poly-glycine. 
     
     
         19 . The isolated RTEF-1 polypeptide of  claim 16 , wherein the cell internalization moiety is a polypeptide comprising RMRRMRRMRR (SEQ ID NO:20). 
     
     
         20 . The isolated RTEF-1 polypeptide of  claim 16 , wherein the cell internalization moiety is an antibody that is an IgA, an IgM, an IgE, an IgG, a Fab, a F(ab′)2, a single chain antibody or a paratope peptide. 
     
     
         21 . The isolated RTEF-1 polypeptide of  claim 1 , further comprising a cell secretion signal. 
     
     
         22 . The isolated RTEF-1 polypeptide of  claim 21 , wherein the cell secretion signal comprises the human IL-2 secretion signal sequence (SEQ ID NO:23). 
     
     
         23 . A nucleic acid comprising a nucleic acid segment encoding a variant RTEF-1 polypeptide according to any of  claims 1  to  22 . 
     
     
         24 . The nucleic acid of  claim 23 , wherein the nucleic acid comprises an expression cassette comprising an antiantiogenesis gene operably coupled to a promoter. 
     
     
         25 . The nucleic acid of  claim 23 , wherein the nucleic acid is comprised in a vector. 
     
     
         26 . The nucleic acid of  claim 25 , wherein the vector is a viral vector or a liposome. 
     
     
         27 . The nucleic acid of  claim 26 , wherein the vector is a viral vector that is an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, an SV-40 virus vector, a retrovirus vector, or a vaccinia virus vector. 
     
     
         28 . The nucleic acid of  claim 27 , wherein the viral expression vector is a retrovirus vector. 
     
     
         29 . The nucleic acid of  claim 28 , wherein the retrovirus vector is a lentiviral vector. 
     
     
         30 . The nucleic acid of  claim 29 , wherein the lentiviral vector is an HIV vector. 
     
     
         31 . The nucleic acid of  claim 24 , wherein the promoter is a cell type specific promoter or inducible promoter. 
     
     
         32 . The nucleic acid of  claim 31 , wherein the inducible promoter is a hypoxia inducible promoter. 
     
     
         33 . The nucleic acid of  claim 31 , wherein the inducible promoter is an angiogenesis inducible promoter 
     
     
         34 . The nucleic acid of  claim 24 , wherein the expression cassette comprises two or more antiangiogenesis genes. 
     
     
         35 . A pharmaceutical composition comprising a) one or more variant RTEF-1 polypeptides as set forth in any of  claims 1 - 22  or one or more nucleic acids as set forth in any of  claims 23 - 34 ; and b) a pharmaceutically acceptable carrier. 
     
     
         36 . The pharmaceutical composition of  claim 35 , comprising SEQ ID NO:1 or SEQ ID NO:2, or a nucleic acid encoding SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         37 . A method of inducing cell contact inhibition in a population comprising two or more cells, comprising contacting a cell of the population with an effective amount of a variant RTEF-1 polypeptide of any of  claims 1 - 22  or a nucleic acid of any of  claims 23 - 36 , wherein cell contact inhibition in the population is induced. 
     
     
         38 . A method of regulating size of an organ, comprising contacting an organ with an effective amount of a variant RTEF-1 polypeptide of any of  claims 1 - 22  or a nucleic acid of any of  claims 23 - 36 , wherein organ size is regulated. 
     
     
         39 . A method of suppressing YAP activity in a cell, comprising contacting a cell with an effective amount of a variant RTEF-1 polypeptide of any of  claims 1 - 22  or a nucleic acid of any of  claims 23 - 36 , wherein YAP activity in the cell is suppressed. 
     
     
         40 . The method of  claim 39 , wherein the cell is contacted with a variant RTEF-1 polypeptide comprising a nuclear localization sequence and activity of YAP in the nucleus of the cell is suppressed. 
     
     
         41 . The method of  claim 39 , wherein the variant RTEF-1 polypeptide does not include a nuclear localization sequence and wherein translocation of YAP into the nucleus of the cell is inhibited. 
     
     
         42 . A method for treating a subject with a disorder associated with abnormal cell growth or abnormal cell proliferation, comprising administering to a subject with a disorder associated with abnormal cell growth or abnormal cell proliferation an effective amount of a pharmaceutical composition comprising:
 a) an variant RTEF-1 polypeptide according to any one of  claims 1 - 22 ; or   b) an isolated nucleic acid according to any of  claims 23 - 34 ,   wherein the disorder is treated.   
     
     
         43 . The method of  claim 42 , wherein the disorder associated with abnormal cell growth or abnormal cell proliferation is an angiogenic disorder that is cancer, ocular neovascularization, an arterio-venous malformation, coronary restenosis, peripheral vessel restenosis, glomerulonephritis, or rheumatoid arthritis. 
     
     
         44 . The method of  claim 42 , wherein the angiogenic disorder is cancer. 
     
     
         45 . The method of  claim 44 , wherein the cancer is a cancer selected from the group consisting of cancer of breast cancer, lung cancer, prostate cancer, leukemia, lymphoma, head and neck cancer, brain cancer, stomach cancer, intestinal cancer, colorectal cancer, renal cancer, bladder cancer, testicular cancer, esophageal cancer, ocular melanoma, retinoblastoma, liver cancer, ovarian cancer, skin cancer, cancer of the tongue, cancer of the mouth, or metastatic cancer. 
     
     
         46 . The method of  claim 44 , wherein the cancer is comprised of cancer cells that demonstrate detectable YAP in the nucleus and/or cytoplasm. 
     
     
         47 . The method of  claim 44 , wherein the cancer cells demonstrate overexpression of YAP in the nucleus and/or cytoplasm compared to expression of YAP in the nucleus and/or cytoplasm of a cell of a similar tissue type that is not cancerous. 
     
     
         48 . The method of  claim 43 , wherein the angiogenic disorder is ocular neovascularization. 
     
     
         49 . The method of  claim 48 , wherein the ocular neovascularization is neovascularization due to age-related macular degeneration, neovascularization due to corneal graft rejection, neovascularization due to retinopathy of prematurity (ROP), or neovascularization due to diabetic retinopathy. 
     
     
         50 . The method of  claim 43 , further comprising administering a secondary therapy for treatment of the antiangiogenic disorder. 
     
     
         51 . The method of  claim 50 , wherein the secondary therapy is an antibody that binds to VEGF, a VEGF receptor, FGF, an FGF receptor, bevacizumab, ranibizumab, or pegaptanib sodium. 
     
     
         52 . The method of  50 , wherein the secondary therapy is an anticancer therapy that is chemotherapy, surgical therapy, immunotherapy or radiation therapy. 
     
     
         53 . The method of  claim 42 , wherein the subject is a human. 
     
     
         54 . The method of  claim 42 , wherein the composition is administered intravenously, intraarterially, epidurally, intrathecally, intraperitoneally, subcutaneously, orally, or topically. 
     
     
         55 . The method of  claim 48 , wherein the composition is administered locally to the eye by topical drops, intracameral injection, subconjunctival injection, subtenon injection, or by intravitreous injection. 
     
     
         56 . A kit comprising a predetermined quantity of a variant RTEF-1 polypeptide of any of  claims 1 - 22  or a nucleic acid of any of  claims 23 - 34  in one or more sealed vials.