Compositions and methods for inducing gene expression

Abstract

The present invention provides recombinant nucleic acid molecules encoding a chimeric transactivator protein including a DNA binding domain of a DNA binding protein and a protein domain capable of transcriptional activation. The present invention also provides recombinant viral and non-viral vectors that are able to infect and/or transfect and sustain expression of a biologically active chimeric transactivator proteins in mammalian cells. Also provided are host cell lines and non-human transgenic animals capable of expressing biologically active chimeric transactivator proteins. In another aspect, compositions and methods for treating or preventing ischemic damage associated with hypoxia-related disorders are provided.

1 . A nucleic acid molecule encoding a biologically active chimeric transactivator protein comprising 
 (a) the DNA binding domain of a hypoxia inducible factor protein; and    (b) a protein domain capable of transcriptional activation.    
     
     
         2 . The nucleic acid molecule according to  claim 1 , wherein the hypoxia inducible factor protein is HIF-1α.  
     
     
         3 . The nucleic acid molecule according to  claim 1 , wherein the protein domain capable of transcriptional activation is derived from a protein selected from the group consisting of: HSV VP16, NFκB, 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 molecule according to  claim 1 , wherein the protein domain capable of transcriptional activation is synthetic.  
     
     
         5 . The nucleic acid molecule according to  claim 1 , wherein the hypoxia inducible factor protein is HIF-1α and the protein domain capable of transcriptional activation is a transcriptional activation domain from HSV VP16.  
     
     
         6 . The nucleic acid molecule according to  claim 1 , wherein the hypoxia inducible factor protein is HIF-1α and the protein domain capable of transcriptional activation is a transcriptional activation domain from NFκB.  
     
     
         7 . The nucleic acid molecule according to  claim 2 ,  5  or  6 , wherein the DNA binding domain of HIF-1α comprises amino acids 1-390.  
     
     
         8 . An expression vector comprising a nucleic acid molecule according to any one of claims  1 - 7  operatively linked to an expression control sequence.  
     
     
         9 . The expression vector according to  claim 8 , wherein the expression control sequence comprises an inducible promoter.  
     
     
         10 . The expression vector according to  claim 8 , wherein the expression vector is pcDNA3/HIF/VP16/Afl2.  
     
     
         11 . An expression vector according to  claim 8 , wherein the vector is an adenoviral vector.  
     
     
         12 . A host cell comprising an expression vector according to any one of claims  8 - 11 .  
     
     
         13 . A biologically active, chimeric transactivator protein encoded by a nucleic acid molecule according to any one of claims  1 - 7 .  
     
     
         14 . A pharmaceutical composition comprising an expression vector according to any one of claims  8 - 11  and a pharmaceutically acceptable carrier.  
     
     
         15 . A method for increasing the expression in a target cell of a hypoxia-inducible gene, said method comprising the steps of: 
 (a) introducing into said cell an expression vector according to any one of claims  8 - 11 ; and    (b) allowing expression of said biologically active chimeric transactivator protein encoded by said expression vector.    
     
     
         16 . A method for providing sustained expression of biologically active HIF-1α in a cell under normoxic conditions, said method comprising the step of introducing into said cell a nucleic acid molecule according to any one of claims  1 - 7 , operatively linked to an expression control sequence which directs its expression in said cell.  
     
     
         17 . A method for reducing ischemic tissue damage in a subject having a hypoxia-associated disorder comprising the steps of administering to said subject an effective amount of a pharmaceutical composition according to  claim 14 .  
     
     
         18 . A method for reducing ischemic tissue damage in a subject having a hypoxia-associated disorder comprising the steps of: 
 (a) isolating cells to be implanted into said subject    (b) introducing into said cells an expression vector according to any one of claims  8 - 11 ; and    (c) implanting said cells containing said expression vector into said subject.