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 . A method of diagnosing a disease or disorder in a subject, comprising:
 (a) 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;   (b) administering ligand to said modified cells; and   (c) detecting reporter gene expression;   wherein expression of said reporter gene indicates that said subject has said disease or disorder.   
     
     
         2 . The method of  claim 1 , wherein said method is carried out ex vivo in cells that have been isolated from said subject. 
     
     
         3 . The method of  claim 1 , 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. 
     
     
         4 . The method of  claim 1 , wherein said method is carried out in vivo. 
     
     
         5 . The method of  claim 1 , wherein said gene switch is an ecdysone receptor (EcR)-based gene switch. 
     
     
         6 . The method of  claim 5 , wherein said ligand binds to the EcR ligand binding domain. 
     
     
         7 . The method of  claim 6 , wherein said ligand is a diacylhydrazine. 
     
     
         8 . The method of  claim 7 , wherein said ligand is selected from the group consisting of RG-115819, RG-115830, and RG-115932. 
     
     
         9 .- 35 . (canceled) 
     
     
         36 . A method of monitoring the progression of a disease or disorder in a subject, comprising:
 (a) administering ligand to modified cells of said subject; and   (b) detecting reporter gene expression at least twice;   wherein a change in the level of expression of said reporter gene indicates progression of said disease or disorder in said subject; and   wherein said modified cells of said subject comprise 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.   
     
     
         37 . (canceled) 
     
     
         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 .- 57 . (canceled) 
     
     
         58 . A method of monitoring the effectiveness of a treatment for a disease or disorder in a subject, comprising:
 (a) introducing into non-autologous cells 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;   (b) introducing said modified cells into said subject;   (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.   
     
     
         59 . The method of  claim 58 , wherein said modified cells are surrounded by a barrier prior to introduction into said subject. 
     
     
         60 .- 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 . The method of  claim 103 , wherein said polynucleotides are introduced into said cells prior to transplantation. 
     
     
         105 . The method of  claim 103 , wherein said polynucleotides are introduced into said cells after transplantation. 
     
     
         106 . The method of  claim 103 , wherein said gene switch is an ecdysone receptor (EcR)-based gene switch. 
     
     
         107 . The method of  claim 106 , wherein said ligand binds to the EcR ligand binding domain. 
     
     
         108 . The method of  claim 107 , wherein said ligand is a diacylhydrazine. 
     
     
         109 . The method of  claim 108 , wherein said ligand is selected from the group consisting of RG-115819, RG-115830, and RG-115932. 
     
     
         110 .- 118 . (canceled) 
     
     
         119 . A method of detecting or monitoring the progression of 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 subject modified cell; and   (c) detecting reporter gene expression from said modified cells at least twice;   wherein a change in the level of expression of said reporter gene indicates rejection or progression of said transplant rejection in said subject.   
     
     
         120 . The method of  claim 119 , wherein said polynucleotides are introduced into said cells prior to transplantation. 
     
     
         121 . The method of  claim 119 , wherein said polynucleotides are introduced into said cells after transplantation. 
     
     
         122 . The method of  claim 119 , wherein said gene switch is an ecdysone receptor (EcR)-based gene switch. 
     
     
         123 . The method of  claim 122 , wherein said ligand binds to the EcR ligand binding domain. 
     
     
         124 . The method of  claim 123 , wherein said ligand is a diacylhydrazine. 
     
     
         125 . The method of  claim 124 , wherein said ligand is selected from the group consisting of RG-115819, RG-115830, and RG-115932. 
     
     
         126 .- 175 . (canceled)