Methods for modulating expression of exogenous genes in mammalian systems, and products related thereto

Abstract

In accordance with the present invention, there are provided various methods for modulating the expression of an exogenous gene in a mammalian subject employing modified ecdysone receptors. Also provided are modified ecdysone receptors, as well as homomeric and heterodimeric receptors containing sane, nucleic acids encoding invention modified ecdysone receptors, modified ecdysone response elements, gene transfer vectors, recombinant cells, and transgenic animals containing nucleic acids encoding invention modified ecdysone receptor.

1 . A method for modulating the expression of an exogenous gene in a mammalian subject containing: 
 (i) a modified ecdysone receptor which, in the presence of a ligand therefor, and optionally in the further presence of a silent partner therefor, binds to a response element, and wherein said modified ecdysone receptor comprises: (a) a ligand binding domain that binds to an ecdysteroid, (b) a DNA-binding domain obtained from a DNA-binding protein, which binds to said response element; and (c) an activation domain of a transcription factor, wherein at least one of said DNA-binding domain or said activation domain is not obtained from a native ecdysone receptor, with the proviso that when said activation domain is derived from a glucocorticoid receptor, said DNA-binding domain is not derived from a glucocorticoid receptor or an  E. coli  LexA protein; and    (ii) a DNA construct comprising said exogenous gene under the control of said response element, wherein said response element (a) has about 12-20 base pairs, (b) binds to said modified ecdysone receptor, and (c) does not bind to farnesoid X receptor (FXR);    said method comprising providing to said subject an effective amount of one or more ligands for said modified ecdysone receptor; wherein said one or more ligands are not normally present in said subject; and wherein said one or more ligands are not toxic to said subject.    
     
     
         2 . A method of inducing the expression of an exogenous gene in a mammalian subject containing: 
 (i) DNA encoding a modified ecdysone receptor under the control of an inducible promoter, wherein said modified ecdysone receptor, in the presence of a ligand therefor, and optionally in the further presence of a silent partner therefor, binds to a response element, and wherein said modified ecdysone receptor comprises: (a) a ligand binding domain that binds to an ecdysteroid, (b) a DNA-binding domain obtained from a DNA-binding protein, which binds to said response element; and (c) an activation domain of a transcription factor, wherein at least one of said DNA-binding domain or said activation domain is not obtained from a native ecdysone receptor, with the proviso that when said activation domain is derived from a glucocorticoid receptor, said DNA-binding domain is not derived from a glucocorticoid receptor or an  E. coli  LexA protein;    (ii) a DNA construct comprising said exogenous gene under the control of said response element, wherein said response element (a) has about 12-20 base pairs, (b) binds to said modified ecdysone receptor, and (c) does not bind to famesoid X receptor (FXR); and    (iii) one or more ligands for said modified ecdysone receptor;    said method comprising subjecting said subject to conditions suitable to induce expression of said modified ecdysone receptor.    
     
     
         3 . A method of inducing expression of an exogenous gene in a mammalian subject containing a DNA construct containing said exogenous gene under the control of a response element, wherein said response element (a) has about 12-20 base pairs, (b) binds to a modified ecdysone receptor, and (c) does not bind to farnesoid X receptor (FXR), said method comprising introducing into said subject: 
 a modified ecdysone receptor, wherein said modified ecdysone receptor comprises: (a) a ligand binding domain that binds to an ecdysteroid, (b) a DNA-binding domain obtained from a DNA-binding protein, which binds to said response element; and (c) an activation domain of a transcription factor, wherein at least one of said DNA-binding domain or said activation domain is not obtained from a native ecdysone receptor, with the proviso that when said activation domain is derived from a glucocorticoid receptor, said DNA-binding domain is not derived from a glucocorticoid receptor or an  E. coli  LexA protein; and    one or more ligands for said modified ecdysone receptor,    wherein said modified ecdysone receptor, in combination with a ligand therefor, and optionally in the further presence of a silent partner therefor, binds to said response element, activating transcription therefrom.    
     
     
         4 . A method for modulating the expression of an exogenous gene in a mammalian subject containing: 
 (i) a DNA construct comprising said exogenous gene under the control of an ecdysone response element; and    (ii) a modified receptor which, in the presence of a ligand therefor, and optionally in the further presence of a silent partner therefor, binds to said ecdysone response element, wherein said modified receptor does not bind to endogenous response elements, and wherein said modified receptor comprises: (a) a ligand binding domain that binds to an ecdysteroid; (b) a DNA-binding domain derived from a DNA-binding protein, wherein said DNA-binding domain binds to said ecdysone response element but not to endogenous response elements; and (c) an activation domain of a transcription factor, with the proviso that when said activation domain is derived from a glucocorticoid receptor, said DNA-binding domain is not derived from a glucocorticoid receptor or an  E. coli  LexA protein;    said method comprising providing to said subject an effective amount of one or more ligands for said modified receptor; wherein said one or more ligands are not normally present in said subject; and wherein said one or more ligands are not toxic to said subject.    
     
     
         5 . A method of inducing the expression of an exogenous gene in a mammalian subject containing: 
 (i) a DNA construct comprising an exogenous gene under the control of an ecdysone response element,    (ii) DNA encoding a modified receptor under the control of an inducible promoter, wherein said modified receptor, in the presence of a ligand therefor, and optionally in the further presence of a silent partner therefor, binds to said ecdysone response element, wherein said modified receptor does not bind to endogenous response elements, and wherein said modified receptor comprises: (a) a ligand binding domain that binds to an ecdysteroid; (b) a DNA-binding domain derived from a DNA-binding protein, wherein said DNA-binding domain binds to said ecdysone response element but not to endogenous response elements; and (c) an activation domain of a transcription factor, with the proviso that when said activation domain is derived from a glucocorticoid receptor, said DNA-binding domain is not derived from a glucocorticoid receptor or an  E. coli  LexA protein;    (iii) one or more ligands for said modified receptor;    said method comprising subjecting said subject to conditions suitable to induce expression of said modified receptor.    
     
     
         6 . A method of inducing expression of an exogenous gene in a mammalian subject containing a DNA construct containing said exogenous gene under the control of an ecdysone response element, said method comprising introducing into said subject: 
 a modified receptor, wherein said modified receptor does not bind to endogenous response elements, and wherein said modified receptor comprises: (a) a ligand binding domain that binds to an ecdysteroid; (b) a DNA-binding domain derived from a DNA-binding protein, wherein said DNA-binding domain binds to said ecdysone response element but not to endogenous response elements; and (c) an activation domain of a transcription factor, with the proviso that when said activation domain is derived from a glucocorticoid receptor, said DNA-binding domain is not derived from a glucocorticoid receptor or an  E. coli  LexA protein; and    one or more ligands for said modified receptor,    wherein said modified receptor, in the presence of a ligand therefor, and optionally in the further presence of a silent partner therefor, binds to said ecdysone response element, activating transcription therefrom.