Stable hypoxia inducible factor-1 alpha and method of use

Abstract

Substantially purified stable human hypoxia-inducible factor-1α (sHIF-1alpha) proteins and polynucleotides encoding stable human hypoxia-inducible factor-1α proteins are provided. A method is provided for treating a hypoxia-related tissue damage in a subject by administering to the subject a therapeutically effective amount of a sHIF-1alpha protein or a nucleic acid encoding a stable HIF-1alpha protein. Formulations are provided for the administration of stable human hypoxia inducible factor-1α (HIF-1alpha) polypeptide or a polynucleotide encoding stable human hypoxia inducible factor-1alpha (HIF-1alpha) to a patient having or at risk of having hypoxia- or ischemia-related tissue damage.

What is claimed is:  
     
         1 . An isolated nucleic acid sequence comprising: 
 a) a nucleotide sequence encoding a DNA binding domain and a dimerization domain of a hypoxia inducible factor; and    b) a nucleotide sequence encoding a transcriptional activation domain.    
     
     
         2 . The nucleic acid sequence of  claim 1 , wherein the hypoxia inducible factor is HIF-1alpha, HIF-2alpha, or HIF-3alpha.  
     
     
         3 . The nucleic acid sequence of  claim 1 , wherein the transcriptional activation domain is derived from a protein selected from the group consisting of HSV VP16, a heat shock factor, p53, fos, v-jun, factor EF-C, HIV tat, HPV E2, Ad E1A, Sp1, AP1, CTF/NF1, E2F1, HAP1, HAP2, MCM1, PHO2, GAL4, GCN4, and GAL11.  
     
     
         4 . The nucleic acid sequence of  claim 1 , wherein the hypoxia inducible factor is HIF-1alpha and the transcriptional activation domain is from the HSV VP16 protein.  
     
     
         5 . The nucleic acid sequence of  claim 1 , wherein the DNA binding domain and dimerization domain of HIF comprises amino acids 1-703 of SEQ ID NO:1.  
     
     
         6 . The nucleic acid sequence of  claim 1 , wherein the nucleotide sequence of a) encodes HIF-1alpha amino acids 1-681 of SEQ ID NO:1.  
     
     
         7 . The nucleic acid sequence of  claim 1 , wherein the nucleotide sequence of a) encodes HIF-1alpha amino acids 1-608 of SEQ ID NO:1.  
     
     
         8 . The nucleic acid sequence of  claim 1 , wherein the nucleotide sequence of a) encodes HIF-1alpha amino acids 1-391 of SEQ ID NO:1.  
     
     
         9 . An expression vector comprising a nucleic acid sequence of  claim 1 .  
     
     
         10 . The expression vector of  claim 9 , comprising an inducible or a constitutive promoter operatively linked to the nucleic acid sequence of  claim 1 .  
     
     
         11 . A host cell containing an expression vector of  claim 10 .  
     
     
         12 . A biologically active, chimeric transactivating protein encoded by a nucleic acid sequence as in any of claims  1 - 8 .  
     
     
         13 . A polypeptide selected from the group comprising: 
 amino acid residues 1-391 and 521-826 of SEQ ID NO:1;    amino acid residues 1-391 and 549-826 of SEQ ID NO:1;    amino acid residues 1-391 and 576-826 of SEQ ID NO:1;    amino acid residues 1-391 and 429-826 of SEQ ID NO:1, wherein 551 is no longer serine and 552 is not threonine;    amino acid residues 1-391 and 469-826 of SEQ ID NO:1, wherein 551 is no longer serine and 552 is not threonine;    amino acid residues 1-391 and 494-826 of SEQ ID NO:1, wherein 551 is no longer serine and 552 is not threonine;    amino acid residues 1-391 and 508-826 of SEQ ID NO:1, wherein 551 is no longer serine and 552 is not threonine;    amino acid residues 1-391 and 512-826 of SEQ ID NO:1, wherein 551 is no longer serine and 552 is not threonine; and    amino acid residues 1-391 and 517-826 of SEQ ID NO:1, wherein 551 is no longer serine and 552 is not threonine.    
     
     
         14 . A polypeptide of  claim 13 , wherein amino acid residue 551 is glycine.  
     
     
         15 . A polypeptide of  claim 13 , wherein amino acid residue 552 is alanine.  
     
     
         16 . A nucleic acid sequence encoding a polypeptide of  claim 13 .  
     
     
         17 . An expression vector comprising a nucleic acid sequence of  claim 13 .  
     
     
         18 . A method for increasing expression of a hypoxia inducible gene in a cell comprising contacting the cell with an expression vector as in  claim 9  or  17  under conditions that allow expression of the nucleic acid sequence contained in the vector thereby providing for increased expression of a hypoxia inducible gene in the cell.  
     
     
         19 . The method of  claim 18 , wherein the hypoxia inducible factor is HIF-1alpha.  
     
     
         20 . The method of  claim 18 , wherein the transcriptional activation domain is selected from the group consisting of HSV VP16, a heat shock factor, p53, fos, v-jun, factor EF-C, HIV tat, HPV E2, Ad E1A, Sp1, AP1, CTF/NF1, E2F1, HAP1, HAP2, MCM1, PHO2, GAL4, GCN4, and GAL11.  
     
     
         21 . A method for providing constitutive expression of a hypoxia inducible factor in a cell comprising contacting the cell with a nucleic acid sequence of  claim 1  or  claim 14  under conditions that allow expression of the nucleic acid sequence, thereby providing constitutive expression of a hypoxia inducible factor.  
     
     
         22 . A method for reducing or preventing hypoxia or ischemia-related tissue damage in a subject having or at risk of having such damage comprising administering to the subject a therapeutically effective amount of a nucleic acid sequence of  claim 1  or  claim 14  in a pharmaceutically acceptable carrier, thereby reducing the tissue damage.  
     
     
         23 . The method of  claim 22 , wherein the administering is in vivo.  
     
     
         24 . The method of  claim 22 , wherein the administering is ex vivo.  
     
     
         25 . The method of  claim 22 , wherein the hypoxia or ischemia-related tissue damage is due to a disorder of the cerebral, coronary or peripheral circulation.  
     
     
         26 . A method for providing prophylactic therapy for tissue in a subject in need thereof comprising administering to the subject an amount of a polypeptide encoded by a polynucleotide of  claim 1  or of a polypeptide of  claim 13 , such that angiogenesis is induced at levels that are greater than before administration of the polypeptide, thereby providing prophylactic therapy.  
     
     
         27 . The method of  claim 26 , wherein the subject is at risk of coronary artery disease.  
     
     
         28 . The method of  claim 26 , wherein the subject is at risk of ischemic tissue damage.  
     
     
         29 . The expression vector of  claim 10 , wherein the promoter is selected from the group consisting of metallothionein promoter, SV40 early promoter, retroviral LTR, CMV immediate early promoter, and tissue specific promoter.  
     
     
         30 . A substantially purified stable form of hypoxia-inducible factor-1alpha (HIF-1alpha), having a sequence as set forth in SEQ ID NO:1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid.  
     
     
         31 . The stable form of hypoxia-inducible factor-1alpha of  claim 30 , wherein amino acid 551 is a glycine.  
     
     
         32 . The stable form of hypoxia-inducible factor-1alpha of  claim 30 , wherein amino acid 552 is an alanine.  
     
     
         33 . The stable hypoxia-inducible factor-1alpha of  claim 30 , further comprising a deletion of amino acids 576-785, or any portion thereof.  
     
     
         34 . A nucleic acid sequence comprising a polynucleotide encoding a polynucleotide encoding the stable form of human hypoxia-inducible factor-1alpha (HIF-1alpha) of  claim 30 .  
     
     
         35 . The nucleic acid of  claim 34 , further comprising an expression control sequence operably linked thereto.  
     
     
         36 . The nucleic acid sequence of  claim 35 , wherein the expression control sequence is a promoter.  
     
     
         37 . The polynucleotide of  claim 36 , wherein the promoter is tissue specific.  
     
     
         38 . An expression vector containing the polynucleotide of  claim 34 .  
     
     
         39 . The vector of  claim 38 , wherein the vector is a plasmid.  
     
     
         40 . The vector of  claim 38 , wherein the vector is a viral vector.  
     
     
         41 . The vector of  claim 40 , wherein the vector is a retroviral vector.  
     
     
         42 . A host cell containing the vector of  claim 38 .  
     
     
         43 . A host cell of  claim 42 , wherein the cell is a eukaryotic cell.  
     
     
         44 . A host cell of  claim 42 , wherein the cell is a prokaryotic cell.  
     
     
         45 . An antibody which selectively binds to the polypeptide of  claim 30 .  
     
     
         46 . The antibody of  claim 45 , wherein the antibody is monoclonal.  
     
     
         47 . The antibody of  claim 45 , wherein the antibody is polyclonal.  
     
     
         48 . A method of treating a hypoxia-related tissue damage in a subject, comprising administering to said subject a therapeutically effective amount of a nucleotide sequence comprising an expression control sequence operatively linked to a polynucleotide encoding a polypeptide having a sequence as set forth in SEQ ID NO:1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid.  
     
     
         49 . The method of  claim 48 , wherein amino acid 551 is a glycine.  
     
     
         50 . The method of  claim 48 , wherein amino acid 552 is an alanine.  
     
     
         51 . A method of treating a hypoxia-related tissue damage in a subject, comprising administering to said subject a therapeutically effective amount of a polypeptide having a nucleotide sequence as set forth in SEQ ID NO:1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid.  
     
     
         52 . The method of  claim 51 , wherein amino acid 551 is a glycine.  
     
     
         53 . The method of  claim 51 , wherein amino acid 552 is an alanine.  
     
     
         54 . A formulation for administration of stable human hypoxia-inducible factor-1alpha (HIF-1alpha) polypeptide to a patient having hypoxia related tissue damage, comprising: 
 (a) a therapeutically effective amount of a substantially pure polypeptide having a sequence as set forth in SEQ ID NO:1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid; and    (b) a pharmaceutically acceptable carrier.    
     
     
         55 . The formulation of  claim 54 , wherein the carrier is a liposome.  
     
     
         56 . The formulation of  claim 54 , wherein amino acid 551 is a glycine.  
     
     
         57 . The formulation of  claim 54 , wherein amino acid 552 is an alanine.  
     
     
         58 . A formulation for administration of a polynucleotide encoding stable human hypoxia-inducible factor-1alpha (HIF-1alpha) to a patient having hypoxia related tissue damage, comprising: 
 (a) a therapeutically effective amount of a nucleic acid sequence comprising an expression control sequence operatively linked to a polynucleotide encoding a polypeptide having a sequence as set forth in SEQ ID NO:1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from a serine to a any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid; and    (b) a pharmaceutically acceptable carrier.    
     
     
         59 . The formulation of  claim 58 , wherein the carrier is a liposome.  
     
     
         60 . The formulation of  claim 58 , wherein amino acid 551 is a glycine.  
     
     
         61 . The formulation of  claim 58 , wherein amino acid 552 is an alanine.