Methods and reagents for identifying synthetic genetic elements

Abstract

The present invention relates to a selection method that allows fast recovery and identification of functional gene fragments which selectively inhibit growth, e.g., are cytostatic or cytotoxic, of particular cell-types, such as transformed cells. The strategy relies, in part, on the ability of small gene fragments to encode dominant-acting synthetic genetic elements (SGEs), e.g., molecules which interfere with the function of genes from which they are derived. SGEs which can be identified by the subject method include, but are not limited to, inhibitory antisense RNA molecules, ribozymes, nucleic acid decoys, and small peptides.

1 . A method for identifying agents which selectively confer a desired phenotype on a target cell, comprising: 
 (i) transfecting subtractive cells with a library of expression vectors comprising a variegated population of coding sequences for potential synthetic genetic elements (SGEs);    (ii) isolating, from the subtractive cells, those SGE vectors of the SGE library which do interfere with the detection of the desired phenotype in the target cell;    (iii) transfecting target cells with the sub-population of SGE vectors isolated in step (ii); and    (iv) isolating those SGE vectors which confer the desired phenotype to the target cell.    
     
     
         2 . A method for identifying agents with selective antiproliferative activity for a target cell, comprising: 
 (i) transfecting subtractive cells with a library of expression vectors comprising a variegated population of coding sequences for potential synthetic genetic elements (SGEs);    (ii) isolating those SGE vectors of the SGE library that are not antiproliferative to the subtractive cells;    (iii) transfecting target cells with the non-antiproliferative SGE vectors isolated in step (ii); and    (iv) isolating those SGE vectors that are antiproliferative to the target cell.    
     
     
         3 . The method of  claim 2 , wherein the target and subtractive cells are eukaryotic cells.  
     
     
         4 . The method of  claim 3 , wherein the target and subtractive cells are mammalian cells.  
     
     
         5 . The method of  claim 3 , wherein at least one of the target and subtractive cells are human cells.  
     
     
         6 . The method of  claim 2 , wherein the target cell is a transformed cell, and subtractive cell is an untransformed cell.  
     
     
         7 . The method of  claim 2 , wherein the target cell is infected by a virus, and subtractive cell is a cell not infected with the virus.  
     
     
         8 . The method of  claim 2 , wherein the expression vectors are viral vectors.  
     
     
         9 . The method of  claim 8 , wherein the viral vectors are retroviral vectors.  
     
     
         10 . The method of  claim 2 , wherein the SGE library is generated from a normalized cDNA library.  
     
     
         11 . The method of  claim 2 , wherein the SGE library is generated from a subtractive cDNA library.  
     
     
         12 . The method of  claim 2 , wherein the SGE is a sense oriented sequence encoding a peptide.  
     
     
         13 . The method of  claim 2 , wherein the SGE is an antisense-oriented sequence encoding an antisense RNA.  
     
     
         14 . An isolated SGE identified according to the method of  claim 1 .  
     
     
         15 . A synthetic oligonucleotide having a nucleotide sequence from about 12 nucleotides to all of the nucleotide sequence of an antisense RNA encoded by an SGE identified according to the method of  claim 13 .  
     
     
         16 . An isolated peptide encoded by an SGE identified according to the method of  claim 12 .  
     
     
         17 . An peptidomimetic corresponding to a peptide encoded by an SGE identified according to the method of  claim 12 .  
     
     
         18 . The method of  claim 1 , comprising the further step of formulating, in a pharmaceutical preparation, one or more SGEs that are identified as lethal to the target cell.  
     
     
         19 . A method for identifying a compound having a selective biological effect on a target cell comprising, 
 (i) providing a drug screening assay for detecting agents which inhibit or potentiate the function of a target gene or target gene product corresponding to a SGE identified by the method according to  claim 1;  and    (ii) testing, in the drug screening assay, the ability of a test compound to modulate the function of the target gene or target gene product.    
     
     
         20 . The method of  claim 19 , comprising the further step of formulating a pharmaceutical preparation comprising one or more agents identified as able to modulate the function of the target gene or target gene product  
     
     
         21 . A method for modulating a phenotype of a target cell comprising contacting the target cells with an agent identified by the method of  claim 19  able to modulate the function of the target gene or target gene product.  
     
     
         22 . The method of  claim 2 , wherein the antiproliferative activity is the result of cell death.