Novel forms of the angiogenic factor vascular endothelial cell growth factor: VEGF

Abstract

Novel forms of vascular endothelial growth factor genes and the novel proteins encoded by these genes are disclosed. More particularly, novel forms of human VEGF-A which contain exon 6b and do not contain exon 6a are disclosed. Other novel forms of human VEGF-A contain exon 6b in addition to exon 6a. These novel forms of VEGF-A include VEGF-A 138 , VEGF-A 162 , and VEGF-A 182 . Such novel VEGF proteins may be used in treatment of the cardiovascular system and its diseases through effects on anatomy, conduit function, and permeability, and more particularly in the treatment of cardiovascular disease by stimulating vascular cell proliferation using a growth factor, thereby stimulating endothelial cell growth and vascular permeability. The invention also relates to nucleic acids encoding such novel VEGF proteins, cells, tissues and animals containing such nucleic acids; methods of treatment using such nucleic acids; and methods relating to all of the foregoing.

1 . A purified polypeptide comprising an amino acid sequence coded for by VEGF-A exons 1-5, 6b, and 8, or a derivative thereof.  
     
     
         2 . A purified polypeptide comprising an amino acid sequence coded for by VEGF-A exons 1-5, 6b, 7, and 8, or a derivative thereof.  
     
     
         3 . A purified polypeptide comprising an amino acid sequence coded for by VEGF-A exons 1-5, 6a, 6b, and 8, or a derivative thereof.  
     
     
         4 . The purified polypeptide of  claim 1  wherein said VEGF is human VEGF-A.  
     
     
         5 . The purified polypeptide of  claim 2  wherein said VEGF is human VEGF-A.  
     
     
         6 . The purified polypeptide of  claim 3  wherein said VEGF is human VEGF-A.  
     
     
         7 . The purified polypeptide of  claim 1  comprising the amino acid sequence of FIG. 3.  
     
     
         8 . The purified polypeptide of  claim 2  comprising the amino acid sequence of FIG. 4.  
     
     
         9 . The purified polypeptide of  claim 3  comprising the amino acid sequence of FIG. 5.  
     
     
         10 . A purified and isolated nucleic acid molecule coding for the purified polypeptide of  claim 1 .  
     
     
         11 . A purified and isolated nucleic acid molecule coding for the purified polypeptide of  claim 2 .  
     
     
         12 . A purified and isolated nucleic acid molecule coding for the purified polypeptide of  claim 3 .  
     
     
         13 . A purified and isolated nucleic acid molecule coding for the purified polypeptide of  claim 4 .  
     
     
         14 . A purified and isolated nucleic acid molecule coding for the purified polypeptide of  claim 5 .  
     
     
         15 . A purified and isolated nucleic acid molecule coding for the purified polypeptide of  claim 6 .  
     
     
         16 . A purified and isolated nucleotide molecule coding for the purified polypeptide of  claim 7 .  
     
     
         17 . A purified and isolated nucleic acid molecule coding for the purified polypeptide of  claim 8 .  
     
     
         18 . A purified and isolated nucleic acid molecule coding for the purified polypeptide of  claim 9 .  
     
     
         19 . The nucleic acid molecule of  claim 16  having the nucleotide sequence of FIG. 3.  
     
     
         20 . The nucleic acid molecule of  claim 17  having the nucleotide sequence of FIG. 4.  
     
     
         21 . The nucleic acid molecule of  claim 18 , having the nucleotide sequence of FIG. 5.  
     
     
         22 . A purified and isolated nucleic acid molecule coding for a biologically active fragment of VEGF-A 138 , or a derivative thereof.  
     
     
         23 . A purified and isolated nucleic acid molecule coding for a biologically active fragment of VEGF-A 182 , or a derivative thereof.  
     
     
         24 . A purified and isolated nucleic acid molecule coding for a biologically active fragment of VEGF-A 162 , or a derivative thereof.  
     
     
         25 . An expression vector comprising a nucleic acid molecule of any of claims  10 - 24 .  
     
     
         26 . The expression vector of  claim 25 , wherein said vector further comprises adenovirus sequences.  
     
     
         27 . The expression vector of  claim 26 , wherein said nucleic acid is operably linked to a promoter sequence that is active in vascular endothelial cells.  
     
     
         28 . The expression vector according to  claim 27 , wherein said expression vector is an adenovirus vector.  
     
     
         29 . The expression vector according to  claim 28 , wherein said vector further comprises a partial adenoviral sequence from which the E1A/E1B genes have been deleted.  
     
     
         30 . A kit for intracoronary injection of a recombinant vector expressing VEGF-A 138 , VEGF-A 162 , or VEGF 182  comprising: 
 a nucleic acid molecule encoding VEGF-A 138 , VEGF-A 162 , or VEGF-A 182  cloned into a vector suitable for expression of said polynucleotide in vivo,    a suitable container for said vector, and    instructions for injecting said vector into a patient.    
     
     
         31 . The kit according to  claim 30 , wherein said polynucleotide is cloned into an adenovirus expression vector.  
     
     
         32 . A method of treating vascular disease in a mammal comprising the step of administering to said mammal VEGF-A 138 , VEGF-A 162 , or VEGF-A 182  in a therapeutically effective amount to stimulate vascular cell proliferation.  
     
     
         33 . A method for enhancing endothelialization of diseased vessels comprising the step of administering to a mammal a therapeutically effective amount of VEGF-A 138 , VEGF-A 162 , or VEGF-A 182 .  
     
     
         34 . The method of  claim 33 , wherein said endothelialization is reendothelialization after angioplasty.  
     
     
         35 . The method of  claim 34 , wherein said reendothelialization reduces or prevents restenosis.  
     
     
         36 . The method of  claim 32  or  33 , wherein said patient is treated with a stent.  
     
     
         37 . The method of  claim 32  or  33 , wherein said patient is treated without a stent.  
     
     
         38 . The method of  claim 32  or  33 , wherein said mammal is human.  
     
     
         39 . The method of  claim 32  or  33 , wherein an inflatable balloon catheter coated with VEGF-A 138 , VEGF-A 162 , or VEGF-A 182  is employed to administer said VEGF-A 138 , VEGF-A 162 , or VEGF-A 182 .  
     
     
         40 . The method of  claim 32  or  33 , wherein said administration comprises gene therapy.  
     
     
         41 . The method according to  claim 40 , wherein an inflatable balloon catheter coated with a polynucleotide encoding VEGF-A 138 , VEGF-A 162 , or VEGF-A 182  is employed to administer said gene therapy.  
     
     
         42 . A method of enhancing drug permeation by tumors comprising administering to a patient a nucleic acid molecule coding for VEGF-A 138 , VEGF-A 162 , or VEGF 182 .  
     
     
         43 . The method of  claim 42 , wherein said VEGF-A 138 , VEGF-A 162 , or VEGF-A 182  is delivered directly into a tumor cell.  
     
     
         44 . A therapeutic composition comprising a pharmaceutically acceptable carrier and VEGF-A 138 , VEGF-A 162 , or VEGF-A 182  in a therapeutically effective amount to stimulate vascular cell proliferation.  
     
     
         45 . A filtered injectable adenovirus vector preparation, comprising: a recombinant adenoviral vector, said vector containing no wild-type virus and comprising: 
 a partial adenoviral sequence from which the E1A/E1B genes have been deleted,    and a transgene coding for a VEGF-A 138 , VEGF-A 162 , or VEGF-A 182 , driven by a promoter flanked by the partial adenoviral sequence; and    a pharmaceutically acceptable carrier.    
     
     
         46 . A method of treating cardiovascular disease in a mammal comprising the step of transfecting cells of said mammal with a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence coded for by VEGF-A exons 1-5, 6b and 8.  
     
     
         47 . A method of treating cardiovascular disease in a mammal comprising the step of transfecting cells of said mammal with a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence coded for by VEGF-A exons 1-5, 6b, 7, and 8.  
     
     
         48 . A method of treating cardiovascular disease in a mammal comprising the step of transfecting cells of said mammal with a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence coded for by VEGF-A exons 1-5, 6a, 6b, and 8.  
     
     
         49 . The method of  claim 46 , wherein said VEGF-A is human VEGF-A.  
     
     
         50 . The method of  claim 47 , wherein said VEGF-A is human VEGF-A.  
     
     
         51 . The method of  claim 48 , wherein said VEGF-A is human VEGF-A.  
     
     
         52 . The method of  claim 46 , wherein said nucleic acid molecule codes for VEGF-A 138 .  
     
     
         53 . The method of  claim 47 , wherein said nucleic acid molecule codes for VEGF-A 182 .  
     
     
         54 . The method of  claim 48 , wherein said nucleic acid molecule codes for VEGF-A 162 .  
     
     
         55 . The method according to any of claims  46 - 54 , wherein said nucleic acid molecule is cloned into a vector.  
     
     
         56 . The method according to  claim 55 , wherein said vector comprises adenovirus particles.  
     
     
         57 . The method of  claim 56 , wherein said adenovirus vector particles are delivered to said mammal by injection.  
     
     
         58 . The method of  claim 57 , wherein the number of said adenovirus particles is between about 10 10  to about 10 14 .  
     
     
         59 . The method of  claim 58 , wherein the number of said adenovirus particles is between about 10 11  to about 10 13 .  
     
     
         60 . The method of  claim 46 ,  47 , or  48 , wherein said transfected cells are selected from the group consisting of myoblasts, myocytes, cardiocytes, cardioblasts, and smooth muscle cells.  
     
     
         61 . The method of  claim 57  wherein said transfected cells are coronary artery cells and wherein said injection is intracoronary injection.  
     
     
         62 . The method of  claim 61 , wherein said adenovirus particles are injected at about 1 cm into the lumens of the left and right coronary arteries.  
     
     
         63 . The method according to  claim 46 ,  47 ,  48 , wherein said cells are transfected in vivo.  
     
     
         64 . The method according to  claim 46 ,  47 , or  48 , wherein said cells are transfected ex vivo.  
     
     
         65 . The method according to  claim 61 , wherein said nucleic acid molecule is introduced into said coronary artery cells by a catheter inserted into said artery.  
     
     
         66 . The method according to  claim 65 , wherein said catheter comprises an inflatable balloon having an outer surface adapted to engage the inner wall of said artery, and wherein said nucleic acid molecule is disposed upon said balloon outer surface.  
     
     
         67 . The method according to  claim 46 ,  47 , or  48  wherein said nucleic acid molecule comprises the nucleotide sequence of FIG. 3 or FIG. 4.  
     
     
         68 . The method of  claim 46 ,  47 , or  48 , wherein said mammal is human.  
     
     
         69 . A transformed or transfected host cell comprising the expression vector of  claim 25 .  
     
     
         70 . A method of producing a VEGF-A polypeptide comprising growing, under suitable conditions, a host cell transformed or transfected with the recombinant DNA expression vector of  claim 25  in a manner allowing expression of said polypeptide, and isolating said polypeptide from the host cell.  
     
     
         71 . The method of  claim 46 ,  47 , or  48 , further comprising administering a potentiating agent that potentiates the angiogenic effect of said polypeptide.  
     
     
         72 . The method of  claim 71 , wherein said potentiating agent is an angiogenic FGF.  
     
     
         73 . The method of  claim 72 , wherein said potentiating agent is selected from the group consisting of FGF-1, FGF-2, FGF-4, FGF-5, and FGF-6.  
     
     
         74 . A method of treating a patient suffering from an ischemic condition comprising administering a therapeutic amount of a pharmaceutical composition comprising VEGF-A 138 , VEGF-A 162 , or VEGF-A 182 , in a suitable carrier.  
     
     
         75 . The method of  claim 74  further comprising administering an agent that potentiates the therapeutic effect of said VEGF-A 138 , VEGF-A 162 , or VEGF-A 182 .  
     
     
         76 . The method of  claim 75  wherein said potentiating agent is selected from the group consisting of FGF-1, FGF-2, FGF-4, FGF-5, and FGF-6.  
     
     
         77 . The method of  claim 74  wherein said ischemic condition is selected from the group consisting of: cardiac infarction, chronic coronary ischemia, chronic lower limb ischemia, stroke, and peripheral vascular disease.  
     
     
         78 . A method of increasing vascular permeability comprising administering a therapeutic amount of a pharmaceutical composition comprising VEGF-A 138 , VEGF-A 162 , or VEGF-A 182  in a suitable carrier.  
     
     
         79 . A method for treating a patient suffering from a wound comprising administering a therapeutic amount of a pharmaceutical composition comprising VEGF-A 138 , VEGF-A 162 , or VEGF-A 182  in a suitable carrier.