Use and identification of biomarkers for gastrointestinal diseases

Abstract

The described invention relates to the identification of biomarkers for gastrointestinal diseases and provides methods utilizing the biomarkers for in drug discovery, monitoring of treatment efficacy, and diagnostics. The invention further provides methods for identifying a therapeutic target to treat ulcerative colitis, colorectal cancer, and Crohn's disease.

1 . A method for identifying a therapeutic target to treat a colon cancer in a subject in need thereof, the method comprising the steps:
 (a) isolating normal differentiable segment-specific human stem cell-like progenitor cells from normal mucosal tissue of the subject derived from at least one normal gastrointestinal segment;   (b) isolating diseased differentiable segment-specific human stem cell-like progenitor cells from diseased mucosal tissue derived from at least one segment of the subject's diseased human gastrointestinal segment;   (c) cultivating the normal differentiable segment-specific human stem cell-like progenitor cells from step (a) on a biosimilar matrix environment formed from the normal mucosal tissue derived from the at least one normal human gastrointestinal segment to produce a normal human intestinal primary epithelial cell line;   (d) cultivating the diseased differentiable segment-specific human stem cell-like progenitor cells from step (b) on a biosimilar matrix environment formed from the diseased mucosal tissue of step (b) to produce a diseased human intestinal primary epithelial cell line;   (e) exposing the normal human intestinal primary epithelial cell line of step (c) and the diseased human intestinal primary epithelial cell line of step (d) to a therapeutic agent;   (f) measuring the level of expression of a biomarker, wherein the level of expression of the biomarker is measured in the normal human intestinal primary epithelial cell line of step (e) and in the diseased human intestinal primary epithelial cell line of step (e); and   (g) identifying a therapeutic target for the treatment of colon cancer by comparing the level of expression of the biomarker in the normal human intestinal primary cell line of step (f) and in the diseased human intestinal primary epithelial cell line of step (f).   
     
     
         2 . The method according to  claim 1 , wherein the biomarker is selected from the group consisting of STX5A, GZMB, PCSK5, and SOD3. 
     
     
         3 . The method according to  claim 1 , wherein step (f) further comprises the step of performing an assay that detects at least one of (i) at least one overexpressed gene in a sample obtained from the subject and (ii) at least one underexpressed gene in a sample obtained from the subject. 
     
     
         4 . The method according to  claim 3 , wherein the assay in step (f) is a microarray hybridization assay. 
     
     
         5 . The method according to  claim 1 , wherein the cell lines of step (f) are compared by immunoassay. 
     
     
         6 . The method according to  claim 1 , wherein the gastrointestinal segment is at least one gastrointestinal segment selected from the group consisting of an esophagus segment, a stomach segment, a jejunum segment, an ileum segment, a duodenum segment, an ascending colon segment, a transverse colon segment, a sigmoid colon segment, or a rectum segment. 
     
     
         7 . The method according to  claim 1 , wherein the normal differentiable segment-specific human stem cell-like progenitor cells have a phenotype of at least cytokeratin (+) and β-1-integrin(+). 
     
     
         8 . The method according to  claim 1 , wherein the normal differentiable segment-specific human stem cell-like progenitor cells have a phenotype of cytokeratin (+), β-1-integrin(+), defensin-5(+), trefoil factor-3(+), mucin-2(+), chomogranin-A(+), intestinal alkaline phosphatase(+), and lysozyme(+). 
     
     
         9 . A method for identifying at least one therapeutic target to treat ulcerative colitis in a subject in need thereof, the method comprising the steps:
 (a) isolating normal differentiable segment-specific human stem cell-like progenitor cells from normal mucosal tissue of the subject derived from at least one normal gastrointestinal segment;   (b) isolating diseased differentiable segment-specific human stem cell-like progenitor cells from diseased mucosal tissue derived from at least one segment of the subject's diseased human gastrointestinal segment;   (c) cultivating the normal differentiable segment-specific human stem cell-like progenitor cells from step (a) on a biosimilar matrix environment formed from the normal mucosal tissue derived from the at least one normal human gastrointestinal segment to produce a normal human intestinal primary epithelial cell line;   (d) cultivating the diseased differentiable segment-specific human stem cell-like progenitor cells from step (b) on a biosimilar matrix environment formed from the diseased mucosal tissue of step (b) to produce a diseased human intestinal primary epithelial cell line;   (e) exposing the normal human intestinal primary epithelial cell line of step (c) and the diseased human intestinal primary epithelial cell line of step (d) to a therapeutic agent;   (f) measuring the level of expression of a biomarker, wherein the level of expression of the biomarker is measured in the normal human intestinal primary epithelial cell line of step (e) and in the diseased human intestinal primary epithelial cell line of step (e); and   (g) identifying a therapeutic target for the treatment of ulcerative colitis by comparing the level of expression of the biomarker within the normal human intestinal primary cell line of step (f) and in the diseased human intestinal primary epithelial cell line of step (f).   
     
     
         10 . The method according to  claim 9 , wherein the biomarker is selected from the group consisting of BST2, MMP1, CACNA1E, PCSK5, GSTT1, SOD3, IL1RL1, ARH1 and IFIT156. 
     
     
         11 . The method according to  claim 9 , wherein step (f) further comprises the step of performing an assay that detects at least one of (i) at least one overexpressed gene in a sample obtained from the subject or (ii) at least one underexpressed gene in a sample obtained from the subject. 
     
     
         12 . The method according to  claim 11 , wherein the assay in step (f) is a microarray hybridization assay. 
     
     
         13 . The method according to  claim 9 , wherein in step (d), the biomarker is in a regulated state. 
     
     
         14 . The method according to  claim 9 , wherein the cell lines of step (f) are compared by an immunoassay. 
     
     
         15 . The method according to  claim 9 , wherein the gastrointestinal segment is an esophagus segment, a stomach segment, a duodenum segment, a jejunum segment, an ileum segment, an ascending colon segment, a transverse colon segment, a sigmoid colon segment, or a rectum segment. 
     
     
         16 . The method according to  claim 9 , wherein the normal differentiable segment-specific human stem cell-like progenitor cells have a phenotype of at least cytokeratin (+) and β-1-integrin(+). 
     
     
         17 . The method according to  claim 9 , wherein the normal differentiable segment-specific human stem cell-like progenitor cells have a phenotype of cytokeratin (+), β-1-integrin(+), defensin-5(+), trefoil factor-3(+), mucin-2(+), chomogranin-A(+), intestinal alkaline phosphatase(+), and lysozyme(+). 
     
     
         18 . A method for identifying at least one therapeutic target to treat Crohn's disease in a subject in need thereof, the method comprising the steps:
 (a) isolating normal differentiable segment-specific human stem cell-like progenitor cells from normal mucosal tissue of the subject derived from at least one normal gastrointestinal segment;   (b) isolating diseased differentiable segment-specific human stem cell-like progenitor cells from diseased mucosal tissue derived from at least one segment of the subject's diseased human gastrointestinal segment;   (c) cultivating the normal differentiable segment-specific human stem cell-like progenitor cells from step (a) on a biosimilar matrix environment formed from the normal mucosal tissue derived from the at least one normal human gastrointestinal segment to produce a normal human intestinal primary epithelial cell line;   (d) cultivating the diseased differentiable segment-specific human stem cell-like progenitor cells from step (b) on a biosimilar matrix environment formed from the diseased mucosal tissue of step (b) to produce a diseased human intestinal primary epithelial cell line;   (e) exposing the normal human intestinal primary epithelial cell line of step (c) and the diseased human intestinal primary epithelial cell line of step (d) to a therapeutic agent;   (f) measuring the level of expression of a biomarker, wherein the level of expression of the biomarker is measured in the normal human intestinal primary epithelial cell line of step (e) and in the diseased human intestinal primary epithelial cell line of step (e); and   (g) identifying a therapeutic target for the treatment of Crohn's disease by comparing the level of expression of the biomarker in the normal human intestinal primary cell line of step (f) and in the diseased human intestinal primary epithelial cell line of step (f).   
     
     
         19 . The method according to  claim 18 , wherein the biomarker is selected from the group consisting of CACNA1E, GSTT1, PCSK5, COL15A1, EDIL3 and MM2. 
     
     
         20 . The method according to  claim 18 , wherein step (f) further comprises the step of performing an assay that detects at least one of (i) at least one overexpressed gene in a sample obtained from the subject or (ii) at least one underexpressed gene in a sample obtained from the subject. 
     
     
         21 . The method according to  claim 20 , wherein the assay in step (f) is a microarray hybridization assay. 
     
     
         22 . The method according to  claim 18 , wherein in step (d), the biomarker is in a regulated state. 
     
     
         23 . The method according to  claim 18 , wherein the cell lines of step (f) are compared by an immunoassay. 
     
     
         24 . The method according to  claim 18 , wherein the gastrointestinal segment is an esophagus segment, a stomach segment, a jejunum segment, an ileum segment, a duodenum segment, an ascending colon segment, a transverse colon segment, a sigmoid colon segment, or a rectum segment. 
     
     
         25 . The method according to  claim 18 , wherein the normal differentiable segment-specific human stem cell-like progenitor cells have a phenotype of at least cytokeratin (+) and β-1-integrin(+). 
     
     
         26 . The method according to  claim 18 , wherein the normal differentiable segment-specific human stem cell-like progenitor cells have a phenotype of cytokeratin (+), β-1-integrin(+), defensin-5(+), trefoil factor-3(+), mucin-2(+), chomogranin-A(+), intestinal alkaline phosphatase(+), and lysozyme(+). 
     
     
         27 . A method for identifying at least one therapeutic target to treat a gastrointestinal disease in a subject in need thereof, the method comprising the steps:
 (a) isolating normal differentiable segment-specific human stem cell-like progenitor cells from normal mucosal tissue of the subject derived from at least one normal gastrointestinal segment;   (b) isolating diseased differentiable segment-specific human stem cell-like progenitor cells from diseased mucosal tissue derived from at least one segment of the subject's diseased human gastrointestinal segment;   (c) cultivating the normal differentiable segment-specific human stem cell-like progenitor cells from step (a) on a biosimilar matrix environment formed from the normal mucosal tissue derived from the at least one normal human gastrointestinal segment to produce a normal human intestinal primary epithelial cell line;   (d) cultivating the diseased differentiable segment-specific human stem cell-like progenitor cells from step (b) on a biosimilar matrix environment formed from the diseased mucosal tissue of step (b) to produce a diseased human intestinal primary epithelial cell line;   (e) exposing the normal human intestinal primary epithelial cell line of step (c) and the diseased human intestinal primary epithelial cell line of step (d) to a therapeutic agent; and   (f) identifying at least one biomarker as a therapeutic target for treating a gastrointestinal disease by comparing the normal human intestinal primary epithelial cell line and the diseased human intestinal primary epithelial cell line of step (e).   
     
     
         28 . The method according to  claim 27 , wherein the gastrointestinal disease is selected from the group consisting of achalasia, Barrett's oesophagus, colorectal cancer, gastric cancer, oesophageal cancer, coeliac disease, colitis, Crohn's disease, diverticulosis, diverticulitis, gastritis, inflammatory bowel disease, ulcerative colitis, irritable bowel syndrome, microscopic colitis, collagenous colitis, lymphocytic colitis, pancreatitis, reflux oesophagitis, and ulcerative colitis. 
     
     
         29 . The method according to  claim 27 , wherein in step (f) further comprises the step of performing an assay that detects at least one of (i) at least one overexpressed gene in a sample obtained from the subject and (ii) at least one underexpressed gene in a sample obtained from the subject. 
     
     
         30 . The method according to  claim 29 , wherein the assay in step (f) is a microarray hybridization assay. 
     
     
         31 . The method according to  claim 27 , wherein step (f) further comprises the step of comparing telomerase activity of the normal human intestinal primary epithelial cell line of step (c) and the cancerous human intestinal primary epithelial cell line of step (d) after exposing the normal human intestinal primary epithelial cell line of step (c) and the cancerous human intestinal primary epithelial cell line of step (d) to a therapeutic agent. 
     
     
         32 . The method according to  claim 27 , wherein in step (d) the biomarker is in a regulated state. 
     
     
         33 . The method according to  claim 27 , wherein the cell lines of step (f) are compared by an immunoassay. 
     
     
         34 . The method according to  claim 27 , wherein the gastrointestinal disease is colon cancer. 
     
     
         35 . The method according to  claim 27 , wherein the gastrointestinal segment is an esophagus segment, a stomach segment, a jejunum segment, an ileum segment, a duodenum segment, an ascending colon segment, a transverse colon segment, a sigmoid colon segment, or a rectum segment. 
     
     
         36 . The method according to  claim 27 , wherein the normal differentiable segment-specific human stem cell-like progenitor cells have a phenotype of at least cytokeratin (+) and β-1-integrin(+). 
     
     
         37 . The method according to  claim 27 , wherein the normal differentiable segment-specific human stem cell-like progenitor cells have a phenotype of cytokeratin (+), β-1-integrin(+), defensin-5(+), trefoil factor-3(+), mucin-2(+), chromogranin-A(+), intestinal alkaline phosphatase(+), and lysozyme(+). 
     
     
         38 . A method for monitoring the therapeutic efficacy of a therapeutic agent for treating a gastrointestinal inflammatory disease, the method comprising the steps:
 (a) isolating normal differentiable segment-specific human stem cell-like progenitor cells from normal mucosal tissue of the subject derived from at least one normal gastrointestinal segment;   (b) isolating diseased differentiable segment-specific human stem cell-like progenitor cells from diseased mucosal tissue derived from at least one segment of the subject's diseased human gastrointestinal segment;   (c) cultivating the normal differentiable segment-specific human stem cell-like progenitor cells from step (a) on a biosimilar matrix environment formed from the normal mucosal tissue derived from the at least one normal human gastrointestinal segment to produce a normal human intestinal primary epithelial cell line;   (d) cultivating the diseased differentiable segment-specific human stem cell-like progenitor cells from step (b) on a biosimilar matrix environment formed from the diseased mucosal tissue of step (b) to produce a diseased human intestinal primary epithelial cell line;   (e) exposing the normal human intestinal primary epithelial cell line of step (c) and the diseased human intestinal primary epithelial cell line of step (d) to a therapeutic agent;   (f) measuring the level of expression of a biomarker, wherein the level of expression of the biomarker is measured in the normal human intestinal primary epithelial cell line of step (e) and in the diseased human intestinal primary epithelial cell line of step (e);   (g) correlating the difference between the level of expression of the biomarker measured in the normal human intestinal primary epithelial cell line and the level of expression of the biomarker measured in the diseased human intestinal primary epithelial cell with the therapeutic efficacy of the therapeutic agent.   
     
     
         39 . The method according to  claim 38 , wherein the biomarker is selected from the group consisting of STX5A, GZMB, and PCSK5. 
     
     
         40 . The method according to  claim 38 , wherein the biomarker is selected from the group consisting of BST2, MMP1, CACNA1E, PCSK5, GSTT1, IL1RL1, ARH1 and IFIT156. 
     
     
         41 . The method according to  claim 38 , wherein the biomarker is SOD3. 
     
     
         42 . The method according to  claim 38 , wherein the gastrointestinal segment is at least one gastrointestinal segment selected from the group consisting of an esophagus segment, a stomach segment, a jejunum segment, an ileum segment, a duodenum segment, an ascending colon segment, a transverse colon segment, a sigmoid colon segment, or a rectum segment. 
     
     
         43 . The method according to  claim 38 , wherein the normal differentiable segment-specific human stem cell-like progenitor cells have a phenotype of at least cytokeratin (+) and β-1-integrin(+). 
     
     
         44 . The method according to  claim 38 , wherein the normal differentiable segment-specific human stem cell-like progenitor cells have a phenotype of cytokeratin (+), β-1-integrin(+), defensin-5(+), trefoil factor-3(+), mucin-2(+), chomogranin-A(+), intestinal alkaline phosphatase(+), and lysozyme(+). 
     
     
         45 . The method according to  claim 38 , wherein the gastrointestinal disease is selected from the group consisting of achalasia, Barrett's oesophagus, colorectal cancer, gastric cancer, oesophageal cancer, coeliac disease, colitis, Crohn's disease, diverticulosis, diverticulitis, gastritis, inflammatory bowel disease, ulcerative colitis, irritable bowel syndrome, microscopic colitis, collagenous colitis, lymphocytic colitis, pancreatitis, reflux oesophagitis, and ulcerative colitis. 
     
     
         46 . A method for diagnosing a gastrointestinal disease in a subject, the method comprising the steps:
 (a) isolating normal differentiable segment-specific human stem cell-like progenitor cells from normal mucosal tissue of the subject derived from at least one normal gastrointestinal segment;   (b) isolating diseased differentiable segment-specific human stem cell-like progenitor cells from diseased mucosal tissue derived from at least one segment of the subject's diseased human gastrointestinal segment;   (c) cultivating the normal differentiable segment-specific human stem cell-like progenitor cells from step (a) on a biosimilar matrix environment formed from the normal mucosal tissue derived from the at least one normal human gastrointestinal segment to produce a normal human intestinal primary epithelial cell line;   (d) cultivating the diseased differentiable segment-specific human stem cell-like progenitor cells from step (b) on a biosimilar matrix environment formed from the diseased mucosal tissue of step (b) to produce a diseased human intestinal primary epithelial cell line;   (e) measuring the level of expression of a biomarker, wherein the level of expression of the biomarker is measured in the normal human intestinal primary epithelial cell line of step (e) and in the diseased human intestinal primary epithelial cell line of step (e);   (f) correlating the difference between the level of expression of the biomarker measured in the normal human intestinal primary epithelial cell line and the level of expression of the biomarker measured in the diseased human intestinal primary epithelial cell line to a gastrointestinal disease state.   
     
     
         47 . The method according to  claim 46 , wherein the biomarker is selected from the group consisting of STX5A, GZMB, and PCSK5. 
     
     
         48 . The method according to  claim 46 , wherein the biomarker is selected from the group consisting of BST2, MMP1, CACNA1E, PCSK5, GSTT1, IL1RL1, ARH1 and IFIT156. 
     
     
         49 . The method according to  claim 46 , wherein the biomarker is SOD3. 
     
     
         50 . The method according to  claim 46 , wherein the biomarker is SOD3 and wherein step (f) the difference between the level of expression of the biomarker measured in the normal human intestinal primary epithelial cell line and the level of expression of the biomarker measured in the diseased human intestinal primary epithelial cell line is at least about 2-fold. 
     
     
         51 . The method according to  claim 46 , wherein the gastrointestinal segment is at least one gastrointestinal segment selected from the group consisting of an esophagus segment, a stomach segment, a jejunum segment, an ileum segment, a duodenum segment, an ascending colon segment, a transverse colon segment, a sigmoid colon segment, or a rectum segment. 
     
     
         52 . The method according to  claim 46 , wherein the normal differentiable segment-specific human stem cell-like progenitor cells have a phenotype of at least cytokeratin (+) and β-1-integrin(+). 
     
     
         53 . The method according to  claim 46 , wherein the normal differentiable segment-specific human stem cell-like progenitor cells have a phenotype of cytokeratin (+), β-1-integrin(+), defensin-5(+), trefoil factor-3(+), mucin-2(+), chomogranin-A(+), intestinal alkaline phosphatase(+), and lysozyme(+). 
     
     
         54 . The method according to  claim 46 , wherein the gastrointestinal disease is selected from the group consisting of achalasia, Barrett's oesophagus, colorectal cancer, gastric cancer, oesophageal cancer, coeliac disease, colitis, Crohn's disease, diverticulosis, diverticulitis, gastritis, inflammatory bowel disease, ulcerative colitis, irritable bowel syndrome, microscopic colitis, collagenous colitis, lymphocytic colitis, pancreatitis, reflux oesophagitis, and ulcerative colitis.