Methods and Compositions for Diagnosing Disease

Abstract

The present invention relates to methods and compositions for diagnosing a disease or disorder in a subject by introducing into cells of the subject a diagnostic gene switch construct and monitoring expression of a reporter gene. The invention further relates to methods and compositions for monitoring the progression of a disease or disorder or the effectiveness of a treatment for a disease or disorder.

1 - 38 . (canceled) 
     
     
         39 . A method of monitoring the effectiveness of a treatment for a disease or disorder in a subject, comprising:
 (a) administering said treatment to said subject;   (b) introducing into cells of said subject a polynucleotide, wherein said polynucleotide encodes a gene switch, wherein said polynucleotide comprises
 (1) at least one transcription factor sequence, which is operably linked to a diagnostic switch promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, wherein the activity of said promoter is modulated during said disease or disorder of said subject, and 
 (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells; 
   (c) administering ligand to said modified cells; and   (d) detecting reporter gene expression at least twice;   
       wherein a change in the level of expression of said reporter gene indicates the effectiveness of said treatment. 
     
     
         40 . The method of  claim 39 , wherein step (b) is carried out before step (a). 
     
     
         41 . The method of  claim 40 , wherein a baseline level of reporter gene expression is determined prior to step (a). 
     
     
         42 . The method of  claim 39 , wherein said method is carried out ex vivo in cells that have been isolated from said subject. 
     
     
         43 . The method of  claim 39 , wherein said polynucleotides are introduced into cells that have been isolated from said subject to produce modified cells, and the modified cells are re-introduced into said subject. 
     
     
         44 . The method of  claim 39 , wherein said method is carried out in vivo. 
     
     
         45 . The method of  claim 39 , wherein said gene switch is an EcR-based gene switch. 
     
     
         46 . The method of  claim 45 , wherein said ligand binds to the EcR ligand binding domain. 
     
     
         47 . The method of  claim 46 , wherein said ligand is a diacylhydrazine. 
     
     
         48 . The method of  claim 47 , wherein said ligand is selected from the group consisting of RG-115819, RG-115830, and RG-115932. 
     
     
         49 . The method of  claim 46 , wherein said ligand is an amidoketone or oxadiazoline. 
     
     
         50 . The method of  claim 39 , wherein said gene switch comprises a first transcription factor sequence under the control of a first diagnostic switch promoter and a second transcription factor sequence under the control of a second diagnostic switch promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor. 
     
     
         51 . The method of  claim 50 , wherein said first diagnostic switch promoter and said second diagnostic switch promoter are different. 
     
     
         52 . The method of  claim 50 , wherein said first diagnostic switch promoter and said second diagnostic switch promoter are the same. 
     
     
         53 . The method of  claim 50 , wherein said first transcription factor sequence encodes a protein comprising a heterodimer partner and a transactivation domain. 
     
     
         54 . The method of  claim 50 , wherein said second transcription factor sequence encodes a protein comprising a DNA binding domain and a ligand-binding domain. 
     
     
         55 . The method of  claim 39 , wherein one of said polynucleotides further encodes a lethal polypeptide operably linked to an inducible promoter. 
     
     
         56 - 59 . (canceled) 
     
     
         60 . A method of monitoring the potential toxicity of an administered treatment for a disease or disorder in a subject, comprising:
 (a) administering said treatment to said subject;   (b) introducing into cells of said subject a polynucleotide, wherein said polynucleotide encodes a gene switch, wherein said polynucleotide comprises
 (1) at least one transcription factor sequence, which is operably linked to a diagnostic switch promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, wherein the activity of said promoter is modulated by factors found in cells that are being exposed to toxic conditions, and 
 (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells; 
   (c) administering ligand to said modified cells; and   (d) detecting reporter gene expression at least twice;   
       wherein a change in the level of expression of said reporter gene indicates the toxicity of said treatment. 
     
     
         61 - 80 . (canceled) 
     
     
         81 . A method of monitoring the level of a factor that is being administered to a subject for treatment for a disease or disorder, comprising:
 (a) administering said treatment to said subject;   (b) introducing into cells of said subject a polynucleotide, wherein said polynucleotide encodes a gene switch, wherein said polynucleotide comprises
 (1) at least one transcription factor sequence, which is operably linked to a diagnostic switch promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, wherein the activity of said promoter is modulated by said factor that is being administered for treatment, and 
 (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells; 
   (c) administering ligand to said modified cells; and   (d) detecting reporter gene expression;   
       wherein the level of expression of said reporter gene indicates the level of the factor being administered for treatment. 
     
     
         82 - 102 . (canceled) 
     
     
         103 . A method of detecting transplant rejection in a subject that has received an organ or tissue transplant, comprising:
 (a) introducing into cells of said organ or tissue transplant a polynucleotide, wherein said polynucleotide encodes a gene switch, wherein said polynucleotide comprises
 (1) at least one transcription factor sequence, which is operably linked to a diagnostic switch promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, wherein the activity of said promoter is modulated during transplant rejection, and 
 (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells; 
   (b) administering ligand to said modified cells; and   (c) detecting reporter gene expression;   
       wherein expression of the reporter gene indicates that transplant rejection has been detected. 
     
     
         104 - 175 . (canceled)