Methods for identifying patterns of ifn induced expression and use in diagnosis, monitoring and therapy

Abstract

The present inventors identified a subpopulation of genes induced by type I and type II IFNs in a human submandibular gland (HSG) epithelial cell line. Unexpectedly, it was found that the majority of genes that are highly up-regulated by IFN-α are also highly induced by IFN-γ. In contrast, there was a substantial group of genes that are highly induced by IFN-γ only. In target tissues, this identified subpopulation of genes and probes allow different IFN patterns to be discerned, enabling more precise molecular classification of patient subpopulations. The identified gene probes are useful for selecting and monitoring therapy, and for defining efficacy of novel agents in the autoimmune rheumatic diseases.

1 . (canceled) 
     
     
         2 . A method for treating a subject suspected of having a rheumatic disease comprising:
 a) preparing a protein lysate from a tissue sample of the subject;   b) analyzing the tissue lysate of a) for the presence of at least one protein which is a marker of type I IFN activity selected from the group consisting of MDA5 and IFIT3, and at least one protein which is a marker of type II IFN activity selected from the group consisting of: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5, plus a loading control protein (e.g. β-actin or vinculin), wherein the detection of the amount and/or the concentration of at least one protein is achieved by exposing the tissue lysate of a) to one or more antibodies specific to the one or more proteins which are markers of type I IFN activity and type II IFN activity;   c) normalizing the protein expression levels of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 proteins in the subject sample to the expression of β-actin or vinculin proteins in the same sample;   d) comparing the normalized protein expression levels of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 proteins in the patient sample against the expression levels of the same proteins in a control sample;   e) determining that the subject has evidence of type I or type II IFN activity and a rheumatic disease when the expression levels of one or more of the proteins are increased 3 standard deviations over the levels in the control sample; and   f) identifying the appropriate immunological therapy for the rheumatic disease in the subject.   
     
     
         3 . (canceled) 
     
     
         4 . A method for treating a subject suspected of having a rheumatic disease comprising:
 a) obtaining mRNA from a tissue sample of the subject;   b) analyzing the mRNA of a) for the presence of at least one mRNA which is a marker of type I IFN activity selected from the group consisting of MDA5 and IFIT3, and at least one mRNA which is a marker of type II IFN activity selected from the group consisting of: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5, wherein the detection of the amount of at least one mRNA is achieved by analyzing the mRNA extracted using a chip array;   c) comparing the levels of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 mRNA in the subject sample to the levels of mRNA in a control sample;   d) determining that the subject has evidence of type I or type II IFN activity and a rheumatic disease when the expression levels of one or more of the mRNAs of interest are increased over the levels in the control sample; and   f) identifying the appropriate immunological therapy for the rheumatic disease in the subject.   
     
     
         5 . The method of  claim 4 , wherein at step b) the analyzing the amount at least one mRNA which is a marker of type I IFN activity (e.g. MDA5 or IFIT3), and at least one mRNA which is a marker of type II IFN activity: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5 is achieved using a method selected from the group consisting of PCR, qPCR, centrifugation, sequencing, mutation analyses, and polymorphism-analyses. 
     
     
         6 . The method of  claim 2 , wherein the detection of the amount and/or the concentration of at least one protein is achieved by means of an immunotherapeutic agent. 
     
     
         7 . A kit for establishing the IFN expression profile of a tissue from a subject, comprising: one or more ligand(s) for determining the levels or concentration of at least one protein which is a marker of type I IFN activity selected from the group consisting MDA5 and IFIT3, and at least one protein which is a marker of type II IFN activity selected from the group consisting of: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 in a tissue sample from a subject. 
     
     
         8 - 9 . (canceled) 
     
     
         10 . A device adapted for carrying out the method of  claim 1 , comprising:
 a) an analyzing unit comprising one or more antibodies binding at least one protein which is a marker of type I IFN selected from the group consisting of MDA5 and IFIT3, and at least one protein which is a marker of type II IFN activity selected from the group consisting of: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5, the unit being adapted for determining the level of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 proteins in a first and second sample from an individual; and   b) an evaluation unit for comparing the determined level in the first sample with the level in the second sample whereby a rheumatic disease can be diagnosed, the unit comprising a database with the levels of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 proteins in the first sample to the second sample, and a computer-implemented algorithm for carrying out a comparison step.   
     
     
         11 . A method for monitoring the treatment a subject having a rheumatic disease comprising:
 a) preparing a protein lysate from a first tissue sample of the subject;   b) analyzing the tissue lysate of a) for the presence of at least one protein which is a marker of type I IFN activity selected from the group consisting of MDA5 and IFIT3, and at least one protein which is a marker of type II IFN activity selected from the group consisting of: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5, plus a loading control protein (e.g. β-actin or vinculin), wherein the detection of the amount and/or the concentration of at least one protein is achieved by exposing the tissue lysate of a) to one or more antibodies specific to the one or more proteins which are markers of type I IFN activity and type II IFN activity;   c) normalizing the protein expression levels of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 proteins in the subject sample to the expression of β-actin or vinculin proteins in the same sample;   d) administering to the subject an immunological therapy for the rheumatic disease in the subject;   e) preparing a protein lysate from a second tissue sample of the subject;   f) comparing the normalized protein expression levels of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 proteins in the first tissue sample against the expression levels of the same proteins in the tissue sample;   g) determining that the immunological therapy in the subject is effective, when the expression levels of one or more of the proteins in the second sample are decreased when compared to the levels of the proteins in the first sample; and/or   h) determining that the immunological therapy in the subject is not effective, when the expression levels of one or more of the proteins in the second sample are the same or increased when compared to the levels of the proteins in the first sample.   
     
     
         12 . A method for monitoring the treatment a subject having a rheumatic disease comprising:
 a) obtaining mRNA from a tissue lysate from a first tissue sample of the subject;   b) analyzing the mRNA of a) for the presence of at least one mRNA which is a marker of type I IFN activity selected from the group consisting of MDA5 and IFIT, and at least one mRNA which is a marker of type II IFN activity selected from the group consisting of: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5, wherein the detection of the amount of at least one mRNA is achieved by analyzing the mRNA extracted using a chip array;   d) administering to the subject an immunological therapy for the rheumatic disease in the subject;   e) obtaining mRNA from a tissue lysate from a second tissue sample of the subject;   f) comparing the levels of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 mRNA in the second tissue sample to the levels of mRNA in the first tissue sample;   g) determining that the immunological therapy in the subject is effective, when the mRNA expression levels of one or more of the proteins in the second sample are decreased when compared to the levels of the proteins in the first sample; and/or   h) determining that the immunological therapy in the subject is not effective, when the mRNA expression levels of one or more of the proteins in the second sample are the same or increased when compared to the levels of the proteins in the first sample.