Zinc finger-based drug-dependent gene regulation system

Abstract

The invention is based on the discovery that one can create a drug-regulated gene regulation system by creating a fusion polypeptide or chimera that includes three portions: a zinc finger (ZF) protein that recognizes and binds to a nucleic acid recognition sequence in the vicinity of a promoter of the target gene, (ii) a dimerization domain of a tetracycline repressor, which contains a drug-dependent switch, and (iii) an accessory domain to alter the expression of the target gene (such as an activation or repression domain). This chimera is administered to, or expressed in, a cell harboring the target gene to be regulated.

1 . A fusion polypeptide that regulates a target gene, comprising 
 a zinc finger monomer comprising an amino acid sequence that binds to a nucleic acid recognition sequence in the target gene;    a dimerization domain of a tetracycline repressor linked to the zinc finger monomer such that the zinc finger can bind to the recognition sequence in the target gene; and    an accessory domain that modulates a promoter operably linked with the target gene, wherein the accessory domain is linked to the zinc finger monomer such that the accessory domain is localized to the promoter when the zinc finger monomer binds to the recognition sequence.    
     
     
         2 . A nucleic acid molecule comprising a nucleic acid sequence that encodes a fusion polypeptide of  claim 1 .  
     
     
         3 . A nucleic acid vector comprising the nucleic acid molecule of  claim 2 .  
     
     
         4 . A cell comprising the fusion polypeptide of  claim 1 .  
     
     
         5 . A dimer comprising two fusion polypeptides of  claim 1 .  
     
     
         6 . A method of altering expression of a target gene in a cell, the method comprising 
 (a) providing a cell that contains the target gene; and    (b) transfecting the cell with a nucleic acid molecule of  claim 2;     wherein expression of the fusion polypeptide alters expression of the target gene in the presence of tetracycline.    
     
     
         7 . The method of  claim 6 , wherein the target gene is an endogenous gene.  
     
     
         8 . The method of  claim 6 , further comprising adding tetracycline to the cell in an amount effective to alter expression of the target gene.  
     
     
         9 . The method of  claim 8 , wherein expression of the gene is repressed.  
     
     
         10 . The method of  claim 8 , wherein expression of the gene is activated or enhanced.  
     
     
         11 . The method of  claim 6 , further comprising adding tetracycline to the cell and thereafter removing tetracycline from the cell to alter expression of the target gene.  
     
     
         12 . The method of  claim 11 , wherein expression of the gene is repressed.  
     
     
         13 . The method of  claim 11 , wherein expression of the gene is activated or enhanced.  
     
     
         14 . The method of  claim 8 , wherein the tetracycline is added at a specific time.  
     
     
         15 . The method of  claim 6 , wherein the nucleic acid molecule is episomal in the cell.  
     
     
         16 . The method of  claim 6 , wherein the nucleic acid molecule is integrated into genomic DNA of the cell.  
     
     
         17 . The method of  claim 6 , wherein the cell is in a subject.  
     
     
         18 . The method of  claim 17 , wherein the subject is a human.  
     
     
         19 . A method of regulating expression of a target gene in a subject, the method 
 comprising    administering to the subject a fusion polypeptide of  claim 1  in an amount effective to form sufficient dimers to bind to recognition sequences in the target gene thereby regulating the expression of the target gene.    
     
     
         20 . The method of  claim 19 , further comprising administering tetracycline to the subject in an amount effective to alter expression of the target gene.  
     
     
         21 . The method of  claim 20 , wherein expression of the gene is repressed.  
     
     
         22 . The method of  claim 20 , wherein expression of the gene is activated or enhanced.  
     
     
         23 . The method of  claim 19 , further comprising administering tetracycline to the subject and thereafter ceasing administration of tetracycline to the subject when the expression of the target gene is to be altered.  
     
     
         24 . The method of  claim 22 , wherein expression of the gene is (i) repressed or 
 (ii) activated or enhanced.    
     
     
         25 . A transgenic non-human animal, the nucleated cells of which comprise a transgene encoding a fusion polypeptide of  claim 1 , wherein the animal exhibits induction or repression of a target gene in the presence of tetracycline.  
     
     
         26 . The transgenic non-human animal of  claim 25 , wherein the target gene is an endogenous gene.  
     
     
         27 . The transgenic non-human animal of  claim 25 , wherein the target gene is an exogenous gene.  
     
     
         28 . The transgenic non-human animal of claim  41 , wherein the animal is a mouse.