Assay for drug discovery based on in vitro differentiated cells

Abstract

Provided are assay systems for determining the therapeutic or toxic effect of a putative drug based on assaying its activity in cells which have been differentiated in vitro from stem cells, and induced to display a phenotype that resembles a disease to be treated.

1 . An in vitro method for screening, identifying, or determining the therapeutic and toxic effects of a candidate drug substance suspected of being capable of ameliorating cardiac hypertrophy, comprising:
 inducing cardiac hypertrophy in a cardiomyocyte differentiated in vitro from a primate or murine pluripotent stem cell, wherein the cardiac hypertrophic phenotype is induced through genetic modification,   wherein the hypertrophic cardiomyocyte displays a cardiac hypertrophic phenotype comprising activation of an atrial natriuretic factor (ANF) and/or a brain natriuretic peptide (BNP) gene;   contacting the hypertrophic cardiomyocyte with the candidate drug substance to generate a treated hypertrophic cardiomyocyte;   comparing the treated hypertrophic cardiomyocyte to untreated hypertrophic cardiomyocytes or hypertrophic cardiomyocytes contacted with an inert compound; and   determining a responsive change in the cardiac hypertrophic phenotype of the treated hypertrophic cardiomyocyte relative to the untreated hypertrophic cardiomyocytes or hypertrophic cardiomyocytes contacted with an inert compound,   wherein the responsive change of delaying the progression of the cardiac hypertrophic phenotype in the treated hypertrophic cardiomyocyte relative to the untreated hypertrophic cardiomyocytes or hypertrophic cardiomyocytes contacted with the inert compound is indicative of a useful drug substance for the treatment of cardiac hypertrophy, or the responsive change of enhancing the progression of the cardiac hypertrophic phenotype in the treated hypertrophic cardiomyocyte relative to the untreated hypertrophic cardiomyocytes or hypertrophic cardiomyocytes contacted with the inert compound is indicative of a toxic drug sub stance.   
     
     
         2 . The method of  claim 1 , wherein the cardiomyocyte further comprises a selectable marker gene operably linked to a cell-type specific regulatory sequence. 
     
     
         3 . The method of  claim 1 , wherein the cardiomyocyte further comprises a reporter gene operably linked to a cell-type specific regulatory sequence. 
     
     
         4 . The method of  claim 3 , wherein the cell-type specific regulatory sequence is atrial and/or ventricular specific. 
     
     
         5 . The method of  claim 2 , wherein the cardiomyocyte further comprises a reporter gene operably linked to a cell-type specific regulatory sequence. 
     
     
         6 . The method of  claim 1 , wherein the displayed cardiac hypertrophic phenotype relates in addition to the activation of the ANF and/or the BNP gene to the expression of at least one indicator gene selected from the group consisting of atrial natriuretic factor (ANF), b-type natriuretic peptide (BNP), β-myosin heavy chain, α-skeletal actin, cardiac troponin T (cTnT), Nkx-2.5, c-FOS, c-JUN, c-MYC, early growth response genes, heat shock protein 70, alpha-myosin heavy chain, collagen III, preproendothelin-1, myosin light chain 2, Na+/H+ exchanger, cardiac alpha-actin, Na+/Ca2+ exchanger, phosphatidylinositol-3 receptor, angiotensin-converting enzyme, collagen I, collagen XV, sarcoplasmic reticulum Ca-ATPase-2 alpha, beta-adrenoreceptor, protein kinase C, TGF beta, cardiac myosin binding protein C (MyBPC), and phospholamban. 
     
     
         7 . The method of  claim 1 , wherein the displayed cardiac hypertrophic phenotype relates in addition to the activation of the ANF and/or the BNP gene to the activity of at least one indicator gene product selected from the group consisting of atrial natriuretic factor (ANF), b-type natriuretic peptide (BNP), a β-myosin heavy chain, a-skeletal actin, cardiac troponin T (cTnT), Nkx-2.5, c-FOS, c-JUN, c-MYC, early growth response genes, heat shock protein 70, alpha-myosin heavy chain, collagen III, preproendothelin-1, myosin light chain 2, Na+/H+ exchanger, cardiac alpha-actin, Na+/Ca2+ exchanger, phosphatidylinositol-3 receptor, angiotensin-converting enzyme, collagen I, collagen XV, sarcoplasmic reticulum Ca-ATPase-2 alpha, beta-adrenoreceptor, protein kinase C, TGF beta, cardiac myosin binding protein C (MyBPC), and phospholamban. 
     
     
         8 . The method of  claim 1 , wherein the pluripotent stem cell is human. 
     
     
         9 . The method of  claim 1 , wherein the hypertrophic cardiomyocyte comprises a mutation of a gene, wherein the gene encodes a protein selected from the group consisting of myosin heavy chain, cardiac myosin binding protein C, cardiac troponin T, cardiac troponin I, alpha-tropomyosin, cardiac actin, phospholamban, beta-1-adrenergic receptor, Gs alpha, calcineurin, calcium calmodulin dependent kinase IV, and potassium channel subunit Kir2. 
     
     
         10 . An in vitro method for screening, identifying, or determining the therapeutic and toxic effects of a candidate drug substance suspected of being capable of ameliorating cardiac hypertrophy, comprising:
 inducing cardiac hypertrophy in a cardiomyocyte differentiated in vitro from a primate or murine pluripotent stem cell, wherein the cardiac hypertrophic phenotype is induced through selection for a mutated gene,   wherein the hypertrophic cardiomyocyte displays a cardiac hypertrophic phenotype comprising activation of an atrial natriuretic factor (ANF) and/or a brain natriuretic peptide (BNP) gene;   contacting the hypertrophic cardiomyocyte with the candidate drug substance to generate a treated hypertrophic cardiomyocyte;   comparing the treated hypertrophic cardiomyocyte to untreated hypertrophic cardiomyocytes or hypertrophic cardiomyocytes contacted with an inert compound; and   determining a responsive change in the cardiac hypertrophic phenotype of the treated hypertrophic cardiomyocyte relative to the untreated hypertrophic cardiomyocytes or hypertrophic cardiomyocytes contacted with an inert compound,   wherein the responsive change of delaying the progression of the cardiac hypertrophic phenotype in the treated hypertrophic cardiomyocyte relative to the untreated hypertrophic cardiomyocytes or hypertrophic cardiomyocytes contacted with the inert compound is indicative of a useful drug substance for the treatment of cardiac hypertrophy, or the responsive change of enhancing the progression of the cardiac hypertrophic phenotype in the treated hypertrophic cardiomyocyte relative to the untreated hypertrophic cardiomyocytes or hypertrophic cardiomyocytes contacted with the inert compound is indicative of a toxic drug sub stance.   
     
     
         11 . The method of  claim 10 , wherein the cardiomyocyte further comprises a selectable marker gene operably linked to a cell-type specific regulatory sequence. 
     
     
         12 . The method of  claim 10 , wherein the cardiomyocyte further comprises a reporter gene operably linked to a cell-type specific regulatory sequence. 
     
     
         13 . The method of  claim 12 , wherein the cell-type specific regulatory sequence is atrial and/or ventricular specific. 
     
     
         14 . The method of  claim 11 , wherein the cardiomyocyte further comprises a reporter gene operably linked to a cell-type specific regulatory sequence. 
     
     
         15 . The method of  claim 10 , wherein the displayed cardiac hypertrophic phenotype relates in addition to the activation of the ANF and/or the BNP gene to the expression of at least one indicator gene selected from the group consisting of atrial natriuretic factor (ANF), b-type natriuretic peptide (BNP), a β-myosin heavy chain, a-skeletal actin, cardiac troponin T (cTnT), Nkx-2.5, c-FOS, c-JUN, c-MYC, early growth response genes, heat shock protein 70, alpha-myosin heavy chain, collagen III, preproendothelin-1, myosin light chain 2, Na+/H+ exchanger, cardiac alpha-actin, Na+/Ca2+ exchanger, phosphatidylinositol-3 receptor, angiotensin-converting enzyme, collagen I, collagen XV, sarcoplasmic reticulum Ca-ATPase-2 alpha, beta-adrenoreceptor, protein kinase C, TGF beta, cardiac myosin binding protein C (MyBPC), and phospholamban. 
     
     
         16 . The method of  claim 10 , wherein the displayed cardiac hypertrophic phenotype relates in addition to the activation of the ANF and/or the BNP gene to the activity of at least one indicator gene product selected from the group consisting of atrial natriuretic factor (ANF), b-type natriuretic peptide (BNP), β-myosin heavy chain, a-skeletal actin, cardiac troponin T (cTnT), Nkx-2.5, c-FOS, c-JUN, c-MYC, early growth response genes, heat shock protein 70, alpha-myosin heavy chain, collagen III, preproendothelin-1, myosin light chain 2, Na+/H+ exchanger, cardiac alpha-actin, Na+/Ca2+ exchanger, phosphatidylinositol-3 receptor, angiotensin-converting enzyme, collagen I, collagen XV, sarcoplasmic reticulum Ca-ATPase-2 alpha, beta-adrenoreceptor, protein kinase C, TGF beta, cardiac myosin binding protein C (MyBPC), and phospholamban. 
     
     
         17 . The method of  claim 10 , wherein the pluripotent stem cell is human. 
     
     
         18 . The method of  claim 10 , wherein the hypertrophic cardiomyocyte comprises a mutation of a gene, wherein the gene encodes a protein selected from the group consisting of myosin heavy chain, cardiac myosin binding protein C, cardiac troponin T, cardiac troponin I, alpha-tropomyosin, cardiac actin, phospholamban, beta-1-adrenergic receptor, Gs alpha, calcineurin, calcium calmodulin dependent kinase IV, and potassium channel subunit Kir2. 
     
     
         19 . The method of  claim 3 , wherein the cell-type specific regulatory sequence is ventricular specific. 
     
     
         20 . The method of  claim 12 , wherein the cell-type specific regulatory sequence is ventricular specific.