In Vitro Cell-based Methods for Biological Validation and Pharmacological Screening of Chemical Entities and Biologicals

Abstract

This patent describes a novel in vitro cell-based method for biological validation and pharmacological screening of drugs, new chemical entities (NCEs) and biologics, which is predictive of in vivo testing for efficacy and adverse events in patients, as occurs in clinical trials. The same method can be used to create an in vitro cell-based assay to identify the ‘right marketed medication for the right patient’ (personalized medicine), and to identify responders and non-responders in ongoing clinical trials with NCEs.

1 . A method for detecting changes in gene expression in peripheral blood mononuclear cells, comprising: (i) transducing the peripheral blood mononuclear cells with a zinc finger protein that will turn on genes encoding known targets for a chemical entity; (ii) exposing the peripheral blood mononuclear cells to the chemical entity; (iii) isolating RNA from the peripheral blood mononuclear cells; (iv) constructing a cDNA library from the isolated RNA; (v) using subtraction hybridization to eliminate all cDNAs expressed in unexposed peripheral blood mononuclear cells; and (vi) assaying for the remaining over- and under-expressed cDNAs, wherein the detection of a remaining over- or under-expressed cDNA indicates a change in gene expression in the peripheral blood mononuclear cells.  
   
   
       2 . The method of  claim 1 , wherein step (v) further comprises using subtraction hybridization to eliminate all cDNAs expressed in clinical non-responders.  
   
   
       3 . The method of  claim 1 , wherein peripheral blood mononuclear cells are selected from the group consisting of T-lymphocytes, B-lymphocytes, monocytes, natural killer cells, and peripheral blood stem cells.  
   
   
       4 . The method of  claim 1 , wherein the patient's cells are divided into two or more portions and each portion is tested on a different chemical entity.  
   
   
       5 . The method of  claim 1 , wherein the chemical entity is selected from the group consisting of a registered chemical entity, a novel chemical entity, an environmental reagent, and a biological.  
   
   
       6 . A method for detecting changes in gene expression in peripheral blood mononuclear cells, comprising: (i) transducing the peripheral blood mononuclear cells with a zinc finger protein that will turn on genes encoding known targets for a chemical entity; (ii) exposing the peripheral blood mononuclear cells to the chemical entity; (iii) isolating RNA from the peripheral blood mononuclear cells; (iv) constructing a cDNA library from the isolated RNA; (v) using subtraction hybridization to eliminate all cDNAs expressed in clinical non-responders; and (vi) assaying for the remaining over- and under-expressed cDNAs, wherein the detection of a remaining over- or under-expressed cDNA indicates a change in gene expression in the peripheral blood mononuclear cells.  
   
   
       7 . The method of  claim 6 , wherein step (v) further comprises using subtraction hybridization to eliminate all cDNAs expressed in unexposed peripheral blood mononuclear cells.  
   
   
       8 . The method of  claim 6 , wherein peripheral blood mononuclear cells are selected from the group consisting of T-lymphocytes, B-lymphocytes, monocytes, natural killer cells, and peripheral blood stem cells.  
   
   
       9 . The method of  claim 6 , wherein the patient's cells are divided into two or more portions and each portion is tested on a different chemical entity.  
   
   
       10 . The method of claim  20 , wherein the chemical entity is a member of the group consisting of a registered chemical entity, a novel chemical entity, an environmental reagent, and a biological.