Methods and compositions for diagnosing disease

• US 9,222,121 B2
• Filed: 08/14/2014
• Issued: 12/29/2015
• Est. Priority Date: 08/23/2007
• Status: Active Grant

• Current Assignee: Intrexon Corporation
• Original Assignee: Intrexon Corporation
• Inventors: Beech, Robert P., Reed, Thomas D., Patzig, Robert
• Primary Examiner(s): Long, Scott
• Assistant Examiner(s): Hiriyanna, Kelaginamane T
• Application Number:
  US14/459,480
• Publication Number:
  US 20150056631A1
• Time in Patent Office: 502 Days
• Field of Search: 424/9.2
• US Class Current: 1/1
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2800/245 Transplantation related diseases, e.g. graft versus host 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.

Claims

1. A method of detecting kidney transplant rejection in a subject that has received a kidney transplant, comprising:

introducing into cells of said organ or tissue transplant (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic ADAM-17 switch promoter, wherein the activity of the promoter is modulated during transplant rejection, and (2) a polynucleotide encoding a CD95-ADAM8 dual reporter or a alkaline phosphatase—

c terminal CD40 reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

administering ligand to said modified cells; and

detecting reporter gene expression;

wherein expression of the CD95-ADAM8 dual reporter or the alkaline phosphatase—

c terminal CD40 reporter gene indicates that transplant rejection has been detected.

2. The method of claim 1, wherein said polynucleotides are introduced into said cells prior to transplantation.

3. The method of claim 1, wherein said polynucleotides are introduced into said cells after transplantation.

4. The method of claim 1, wherein said gene switch is an ecdysone receptor (EcR)-based gene switch.

5. The method of claim 4, wherein said ligand binds to the EcR ligand binding domain.

6. The method of claim 5, wherein said ligand is a diacylhydrazine.

7. The method of claim 6, wherein said ligand is selected from the group consisting of RG-115819, RG-115830, and RG-115932.

8. The method of claim 1, wherein one of said polynucleotides further encodes a lethal polypeptide operably linked to an inducible promoter.