A system for modulating the expression of a target gene associated with a defined response element in a subject, said system comprising:
    a first chimeric protein comprising at least one dimerization domain of a first member of the steroid/thyroid hormone nuclear receptor superfamily and at least one DNA binding domain, and
    a second chimeric protein comprising at least one dimerization domain of a second member of the steroid/thyroid hormone nuclear receptor superfamily and at least one transcription modulating domain,  wherein the first and second chimeric proteins associate to form a functional entity under substantially physiological conditions, and wherein response of said response element to said DNA binding domain modulates expression of said target gene.

Claim 27
A system for modulating the expression of a target gene associated with a defined response element in a subject, said system comprising:
    a first chimeric protein comprising at least one dimerization domain of a first member of the steroid/thyroid hormone nuclear receptor superfamily and at least one DNA binding domain, and
    a second chimeric protein comprising at least one dimerization domain of a second member of the steroid/thyroid hormone nuclear receptor superfamily and at least one transcription modulating domain, and wherein, at least one of the receptors is non-endogenous to said subject and the first and second chimeric proteins associate to form a functional entity under substantially physiological conditions in the presence or absence of a non-endogenous ligand, and wherein response of said response element to said DNA binding domain modulates expression of said target gene.

Claim 46
A system for modulating the expression of a target gene target gene associated with a defined response element in a subject, said system comprising:
    a first chimeric protein consisting of a DNA binding domain and a dimerization domain of a first member of the steroid/thyroid hormone nuclear receptor superfamily, and
    a second chimeric protein consisting of a transcription modulating domain and a dimerization domain of a second member of the steroid/thyroid hormone nuclear receptor superfamily, wherein at least one of the receptors is non-endogenous and the first and second chimeric proteins associate to form a functional entity under substantially physiological conditions in the presence of a non-endogenous ligand, and wherein association of said response element with said DNA binding domain modulates expression of said target gene.

A method for modulating the expression of an exogenous gene in an isolated cell 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, 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 clement; 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 a 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) is a modified response element which comprises, in any order, a first half-site and a second half-site separated by a spacer of 0-5 nucleotides; wherein said first half-site has the sequence: EQU --RGBNNM--, wherein each R is independently selected from A or G; each B is independently selected from G, C, or T; each N is independently selected from A, T, C, or G; and each M is independently selected from A or C; with the proviso that at least 4 nucleotides of each --RGBNNM--group of nucleotides are identical with the nucleotides at comparable positions of the sequence --AGGTCA--; and wherein said second half-site is obtained from a glucocorticoid receptor subfamily response element,
        (b) binds to said modified ecdysone receptor, and
        (c) does not bind to farnesoid X receptor (FXR); said method comprising providing to the cell an effective amount of one or more ligands for said modified ecdysone receptor; wherein said one or more ligands are not normally present in the cell; and wherein said one or more ligands are not toxic to said cell.

A method of inducing the expression of an exogenous gene in an isolated cell 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 elements;
        (a) is a modified response element which comprises, in any order, a first half-site and a second half-site separated by a spacer of 0-5 nucleotides; wherein said first half-site has the sequence: EQU --RGBNNM--, wherein each R is independently selected from A or G; each B is independently selected from G, C, or T; each N Is independently selected from A, T, C, or G; and each M is independently selected from A or C; with the proviso that at least 4 nucleotides of each --RGBNNM--group of nucleotides are identical with the nucleotides at comparable positions of the sequence --AGGTCA--; and wherein said second half-site is obtained from a glucocorticoid receptor subfamily response element,
        (b) binds to said modified ecdysone receptor, and
        (c) does not bind to farnesoid X receptor (FXR); and (iii) one or more ligands for said modified ecdysone receptor; said method comprising subjecting said cell to conditions suitable to induce expression of said modified ecdysone receptor.

A method of inducing expression of an exogenous gene in an isolated cell containing a DNA construct containing said exogenous gene under the control of a response element, wherein said response element:
    (a) is a modified response element which comprises, in any order, a first half-site and a second half-site separated by a spacer of 0-5 nucleotides; wherein said first half-site has the sequence; EQU --RGBNNM--, wherein each R is independently selected from A or G; each B is independently selected from G, C, or T; each N is independently selected from A, T, C, or G; and each M is independently selected front A or C; with thy proviso that at least 4 nucleotides of each --RGBNNM--group of nucleotides are identical with the nucleotides at comparable position of the sequence --AGGTCA--; and wherein said second half-site is obtained from a glucocorticoid receptor subfamily response element,
    (b) binds to said modified ecdysone receptor, and
    (c) does not bind to farnesoid X receptor (FXR), said method comprising introducing into said cell 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 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.

Claim 20
A method for the expression of a recombinant product detrimental to isolated host cells, said method comprising: transforming suitable isolated host cells with:

    (i) DNA encoding 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; 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 encoding said recombinant product under the control of a response element, wherein said response element;
        (a) is a modified response element which comprises, in any order, a first half-site and a second half-site separated by a spacer of 0-5 nucleotides; wherein maid first half-site has the sequence; EQU --RGBNNM--, wherein each R is independently selected from A or G; each B is independently selected from G, C, or G; each N is independently selected from A, T, C, or G; and each M is independently selected from A or C; with the proviso that at least 4 nucleotides of each --RGBNNM--group of nucleotides are identical with the nucleotides at comparable positions of the sequence --AGGTCA--; and wherein said second half-site is obtained from a glucocorticoid receptor subfamily response element
        (b) binds to said modified ecdysone receptor, and
        (c) does not bind to farnesoid X receptor (FXR); growing said host cells in suitable media; and inducing expression of said recombinant product by introducing into said host cells one or more ligands for said modified ecdysone receptor, and optionally a silent partner for said modified ecdysone receptor.

A chimeric receptor having at least an N-terminus domain, a DNA-binding domain, and a ligand-binding domain; wherein each of said N-terminus, DNA-binding and ligand-binding domains is obtained from a member of the steroid hormone superfamily of receptors; wherein said domains of said chimeric receptor originate from at least two different members of the steroid hormone superfamily of receptors; and wherein at least one of the domains of said chimeric receptor originates from a human retinoic acid receptor alpha.

A chimeric receptor having at least a DNA-binding domain and a ligand-binding domain; wherein each of said DNA-binding and ligand-binding domains is obtained from a member of the steroid hormone superfamily of receptors; wherein said domains of said chimeric receptor originate from at least two different members of the steroid hormone superfamily of receptors; and wherein at least one of the domains of said chimeric receptor originates from a human retinoic acid receptor alpha.

A method for identifying functional ligands for receptor proteins, said method comprising:
    (a) isolating DNA sequences having a ligand-binding domain and a DNA-binding domain;
    (b) constructing a chimeric gene by substituting operative portions of the DNA-binding domain region of the DNA sequence of step (a) with operative portions of a DNA-binding domain region from a known ligand-responsive receptor protein;
    (c) introducing into a suitable receptor-deficient host cell:
        (1) the chimeric gene from step (b), and
        (2) a reporter gene functionally linked to an operative hormone response element wherein the hormone response element is capable of being activated by the DNA-binding domain region of the receptor protein encoded by the chimeric gene of step (b);     (d) challenging the transfected host cell from step (c) with at least one compound to be evaluated for ligand bidning activity with the chimeric receptor protein encoded by the chimeric gene of step (b);
    (e) monitoring induction of the reporter gene;
    (f) identifying as a functional ligand(s) that ligand(s) which is capable of inducing production of the protein product of the reporter gene.

A method for identifying functional ligands for receptor proteins in a cell wherein said cell contains,
    (a) an expressible chimeric DNA sequence (c) comprised of operative portions of a DNA-binding domain of a first receptor sequence linked to operative portions of a ligand-binding domain of a second receptor sequence, and
    (b) a reporter nucleic acid sequence functionally linked to an operative hormone response element wherein said chimeric DNA sequence is expressed and wherein the DNA-binding domain of the chimeric receptor protein thus produced can functionally bind to and activate the hormone response element that is functionally linked to the reporter sequence, said method comprising challenging the cell with at least one compound to be evaluated for ligand binding activity wherein said compound to be evaluated is not known to be a functional ligand for the chimeric protein encoded by said chimeric DNA sequence (c).

A method of producing gene products in mammalian cells comprising:
    (1) transfecting mammalian cells with both:
        (a) a first expression plasmid, comprising a mouse mammary tumor virus (MMTV) long terminal repeat (LTR) operably linked to a glucocorticoid receptor protein gene (GRP), wherein on transfection said cells produce glucocorticoid receptor protein as a result of expression of said first expression plasmid; and
        (b) a second expression plasmid, comprising an MMTV LTR operably linked to a gene encoding a physiologically active substance, wherein on transfection said cell produces said physiologically active substance as a result of expression of said second expression plasmid; wherein on exposing the resulting transfected cells to a glucocorticoid, transfected mammalian cells that exhibit greater expression of said physiologically active substance in the presence of said glucocorticoid than in the absence thereof are selected;
    (2) propagating the thus-obtained transfected cells in the presence of said glucocorticoid to induce expression of said physiologically active substance; and
    (3) obtaining said physiologically active substance.

A method of producing foreign gene products in mammalian cells bearing, on a chromosome thereof:
    (a) a first mouse mammary tumor virus (MMTV) long terminal repeat (LTR) operably linked to a glucocorticoid receptor protein gene (GRP), wherein on transfection said cells produce glucocorticoid receptor protein as a result of expression of said first MMTV LTR operably linked to said GRP gene; and
    (b) a second MMTV LTR operably linked to a gene encoding a physiologically active substance, wherein on transfection said cells produce said physiologically active substance as a result of expression of said second MMTV LTR operably linked to said gene encoding said physiologically active substance; wherein on exposing the resulting transfected cells to a glucocorticoid, transfected mammalian cells that exhibit greater expression of said physiologically active substance in the presence of said glucocorticoid than in the absence thereof are selected, comprising:
        (1) propagating said cells in the presence of said glucocorticoid to induce expression of said physiologically active substance; and
        (2) obtaining said physiologically active substance.

A synthetic inducible eukaryotic promoter for the regulation of transcription of a gene, comprising at least two different classes of inducible elements, wherein said different classes of inducible elements are selected to provide a synergistic level of expression of a gene product in a eukaryotic expression system, said promoter being derived from a native promoter containing at least one constitutive element, wherein said at least one constitutive element is functionally disabled to decrease the level of basel gene expression by said promoter and to increase the ratio of induced to basal gene expression by said promoter.

A synthetic inducible eukaryotic promoter for the regulation of transcription of a gene, comprising at least two different classes of inducible elements, wherein said different classes of inducible elements are selected to provide a synergistic level of expression of a gene product in a eukaryotic expression system, said promoter being derived from a native promoter and one of said different classes of inducible elements is a native inducible element and another of said different classes of inducible elements is a different inducible element provided in said native promoter, wherein said native inducible element is a metal-responsive element (MRE) and said different inducible element is at least one glucocorticoid-responsive element (GRE) and is provided in said native promoter by insertion.

A synthetic inducible eukaryotic promoter for the regulation of transcription of a gene, comprising at least two different classes of inducible elements, wherein said different classes of inducible elements are selected to provide a synergistic level of expression of a gene product in a eukaryotic expression system, said promoter being derived from a native promoter and one of said different classes of inducible elements is a native inducible element and another of said different classes of inducible elements is a different inducible element provided in said native promoter, wherein said native inducible element is a metal responsive element (MRE) and said different inducible element is a glucocorticoid-responsive element (GRE) and is provided in said native promoter by insertion, wherein said native promoter is the hMT-IIA promoter, wherein multiple linked GRE's are inserted into the native promoter.

A synthetic inducible eukaryotic promoter for the regulation of transcription of a gene, comprising at least two different classes of inducible elements, wherein said different classes of inducible elements are selected to provide a synergistic level of expression of a gene product in a eukaryotic expression system, said promoter being derived from a native promoter and one of said different classes of inducible elements is a native inducible element and another of said different classes of inducible elements is a different inducible element provided in said native promoter, wherein said native responsive element is a glucocorticoid-responsive element (GRE) and said different inducible element is a metal responsive element (MRE) which is provided in said native promoter by insertion.

A synthetic inducible eukaryotic promoter for the regulation of transcription of a gene, comprising at least two different classes of inducible elements, wherein said different classes of inducible elements are selected to provide a synergistic level of expression of a gene product in a eukaryotic expression system, said promoter being derived from a native promoter and one of said different classes of inducible elements is a element is a native inducible element and another of said different classes of inducible elements is a different inducible element provided in said native promoter, wherein said native responsive element is a glucocorticoid-responsive element (GRE) and said different inducible element is a metal responsive element (MRE) which is provided in said native promoter by insertion, wherein said native promoter is the MMTV-LTR promoter, wherein at least two linked MRE's are inserted into the native promoter.

A synthetic inducible eukaryotic promoter for the regulation of transcription of a gene to produce a gene product, comprising at least two inserted linked responsive elements from a different class of inducible promoter, wherein (a) the inducible promoter is selected from the group consisting of the human metallothionein gene (hMT-IIA) promoter and the mouse mammary tumor virus/long terminal repeat (MMTV-LTR)promoter, (b) the inserted responsive elements are selected from the group consisting of glucocorticoid-responsive elements (GREs) and metal-responsive elements (MREs), and (c) said different classes of inducible elements are inserted such that induction of the promoter by both classes of inducers causes a synergistic increase in expression of the gene product in a eukaryotic expression system compared to induction by either inducer alone.

A synthetic inducible eukaryotic promoter for the regulation of transcription of a gene, comprising at least two different classes of inducible elements, wherein said different classes of inducible elements are selected to provide a synergistic level of expression of a gene product in a eukaryotic expression system, characterised in that said classes of inducible elements are selected from the group consisting of hormone-responsive elements (HREs), metal-responsive elements (MREs), heat shock-responsive elements (HSREs) and interferon-responsive elements (IREs).

An isolated polynucleotide encoding a Drosophila ecdysone receptor having the 878 amino acid predicted amino acid sequence of FIG. 4 (A-C) and which has ecdysteroid-binding activity wherein said ecdysone receptor exhibits saturable binding to β-ecdysone or ecdysone analogues.

A transformed insect bacterial or mammalian host cell comprising a polynucleotide encoding a Drosophila ecdysone receptor consistinq of the 878 amino acid predicted amino acid sequence of FIG. 4 (A-C) operably linked to a promoter, wherein said promoter is not naturally associated with an ecdysone receptor gene in the germline of naturally occurring Drosophila.