Molecular switch for regulating mammalian gene expression

Abstract

Expression vectors are disclosed that are comprised of (a) one or more silencer elements and conditionally inducible elements to form silencer-inducible regions and (b) promoters in operative linkage upstream of at least one expressed region. The expression vector thereby regulates expression of at least one downstream region by conditional silencing in which an expressed DNA region of a gene is transcribed to produce RNA transcripts, which may or may not be translated to produce polypeptides. Genetically engineered mammalian cells and non-human mammals can be made using such expression vectors through transfection and transgenic techniques. Moreover, processes of making and using the aforementioned products are disclosed (e.g., the expression vector may be used diagnostically, therapeutically, or prophylactically).

1 . An isolated expression vector comprising: (a) a silencer element, (b) an inducible element and (c) a promoter upstream of at least one nucleotide sequence and in operable linkage with and regulated by the silencer element and the inducible element, 
 wherein the silencer element reversibly represses expression of the at least one nucleotide sequence when the vector is incorporated in a eukaryotic cell.    
     
     
         2 . An isolated expression vector comprising (a) a silencer-inducible region comprising at least one hypoxia response enhancer element and at least one silencer element and (b) a promoter upstream of at least one nucleotide sequence and in operable linkage with and regulated by the silencer-inducible region, 
 wherein expression of the at least one nucleotide sequence is silenced when the vector is incorporated in a non-hypoxic cell and the silenced expression is reversed when the cell is made hypoxic.    
     
     
         3 . The isolated expression vector of  claim 2 , wherein the at least one silencer element is from the human B29 gene.  
     
     
         4 . The isolated expression vector of  claim 3 , wherein the at least one silencer element is a TOAD element.  
     
     
         5 . The isolated expression vector of  claim 3 , wherein the at least one silencer element is a FROG element.  
     
     
         6 . The isolated expression vector of  claim 2 , wherein the silencer-inducible region comprises at least one TOAD element, at least one FROG element, and at least one neuron restrictive silencer element.  
     
     
         7 . The isolated expression vector of  claim 2 , wherein the promoter is a viral promoter.  
     
     
         8 . The isolated expression vector of  claim 2 , wherein the promoter is a mammalian promoter.  
     
     
         9 . The isolated expression vector of  claim 2 , wherein the promoter is a cell type-specific promoter.  
     
     
         10 . The isolated expression vector of  claim 2 , wherein the nucleotide sequence is a functional coding region of a gene selected from the group consisting of monocyte chemoattractant protein-1 (MCP-1), stromal cell-derived factor 1 (SDF-1), eotaxin, Myo D, adenosine deaminase, angiopoietin, apoptosis inhibitor protein, angiostatin, B-cell CLL/lymphoma, catalase, deoxyribonuclease, DT-diaphorase, endostatin, erythropoeitin, fibroblast growth factor, fumagillin, β-globin, glutathione peroxidase, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, heat shock transcription factor, hepatocyte growth factor, interferon, tissue metalloproteinase inhibitor, nitric oxide synthase, platelet derived growth factor, proliferin, somatomedin C, superoxide dismutase, survivin, thymidine kinase, tissue plasminogen activator, tumor protein p53, urokinase, and vascular endothelial growth factor.  
     
     
         11 . The isolated expression vector of  claim 2 , wherein the expression vector further comprises one or more sequences selected from the group consisting of a Kozak sequence, a transcription termination sequence, a polyadenylation sequence and a mRNA degradation sequence.  
     
     
         12 . The isolated expression vector of  claim 2 , wherein the expression vector is encapsulated within a particle selected from the group consisting of Adeno-Associated Virus, lentivirus, retrovirus, and Adenovirus.  
     
     
         13 . An isolated expression vector comprising (a) a silencer-inducible region comprising at least one hypoxia response enhancer element and at least one silencer element that comprises the sequence of SEQ ID NO: 17 and (b) a promoter upstream of at least one nucleotide sequence and in operable linkage with and regulated by the silencer-inducible region, 
 wherein expression of the at least one nucleotide sequence is silenced when the vector is incorporated in a non-hypoxic cell and the silenced expression is reversed when the cell is made hypoxic.    
     
     
         14 . An isolated expression vector comprising (a) a silencer-inducible region comprising at least one hypoxia response enhancer element and at least one silencer element that comprises the sequence of SEQ ID NO: 18 and (b) a promoter upstream of at least one nucleotide sequence and in operable linkage with and regulated by the silencer-inducible region, 
 wherein expression of the at least one nucleotide sequence is silenced when the vector is incorporated in a non-hypoxic cell and the silenced expression is reversed when the cell is made hypoxic.    
     
     
         15 . An isolated expression vector comprising (a) a silencer-inducible region comprising at least one NF-KB responsive element and at least one silencer element and (b) a promoter upstream of at least one nucleotide sequence and in operable linkage with and regulated by the silencer-inducible region, 
 wherein expression of the at least one nucleotide sequence is silenced when the vector is incorporated in a cell subjected to a condition that results in NF-κB binding to the NF-κB responsive element, and the silenced expression is reversed when the condition is removed from the cell.