Other patents related to steroid-responsive promoters

There is an increasing number of patent documents related to the steroid/retinoid/thyroid receptor superfamily and their multiple applications, which are beyond of the scope of this report. The disclosure of the European application EP 1112360 A1 discusses several patent applications and patents related to this topic. As such, it is a good reference to those who would like to assess the patent landscape in this field in a much broader context.

The following list of patent documents refers to chimeric inducible receptors that combine domains from steroid-responsive promoters such as glucocorticoid, mineralocorticoid, and estrogen, among others. Some of them also combine receptors that can respond to both steroid and metal compounds making it a doubly inducible promoter system. Finally, ecdysone receptors isolated from different insects and new receptors based on the retinoic acid receptor are also included. In no way do these documents represent the total number of patents directed to this extensive topic, but they give an idea of the diverse applications in this field of technology.

Patent/application number

Title, Independent Claims and Summary of Claims

Assignee

WO 01/62780

Earliest priority – 24 February 2000
Filed – 30 August 2001

Title – Gene expression system based on chimeric receptors

Claim 1
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.

The claims are geneally directed to systems to modulate the expression of a target gene by the interaction of two chimeric proteins each having a dimerization domain member of the streoid/thyroid hormone nuclear receptor. The dimerization domain is required for functional transactivation. The chimeric proteins also contain a DNA-binding domain and a transcription-modulating domain.

The Salk Institute for Biological Studies

Remarks

Related application in the United States ( US 2004/102367) is pending, and applications in Australia (AU 2001241682) and Europe (EP 1259537) were lapsed and withdrawn, respectively.

US 6723531

Earliest priority – 5 April 1996
Filed – 16 March 1998
Granted – 20 April 2004
Expected expiry – 5 April 2016

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

Claim 1
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.

Claim 18
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 methodcomprising subjecting said cell to conditions suitable to induce expression of said modified ecdysone receptor.

Claim 19
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.

The claims are drawn to methods for modulating the expression of an exogenous gene in a mammalian cell by using modified ecdysone receptors that is paired with a silent partner of the steroid/thyroid superfamily of receptors.

Remarks

Related patent is granted in Australia (AU 734051 B2). Applications are also pending in Europe (EP 910652 A1) and Canada (CA 2251466)

US 5599904

Earliest priority – 2 December 1987
Filed – 3 March 1992
Granted – 4 February 1997
Expected expiry – 4 February 2014

Title – Chimeric steroid hormone superfamily receptor proteins

Claim 1
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.

Claim 2
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.

The claims are to chimeric receptor having component domains derived from at least two different members of the steroid hormone superfamily of receptors, with one of the domains derived from a human retinoic acid receptor alpha.

US 4981784

Earliest priority – 2 December 1987
Filed – 30 November 1988
Granted – 1 January 1991
Expected expiry – 30 November 2008

Title – Retinoic acid receptor composition and method for identifying ligands

Claim 1
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.

Claim 7
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).

The patent mainly claims for methods for identifying ligands for receptor proteins by replacing the DNA-binding domain of a putative novel receptor with the DNA-binding domain of a known receptor. The expression of the hybrid receptor depends on the presence of the new ligand.

Remarks

Related patent was also granted in Europe (EP 325849 B1).

US 5646013

Earliest priority – 9 November 1988
Filed – 28 June 1995
Granted – 8 July 1997
Expected expiry – 9 November 2008

Title – Method of producing foreign products

Claim 1
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.

Claim 2
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.

The claims of this patent are directed to methods using inducible promoter system for mammalian cells comprising a first plasmid having a mouse mammary tumor virus (MMTV) long terminal repeat (LTR) linked to a glucocorticoid receptor and a second plasmid with a MMTV-LTR linked to a gene of interest. The plasmids interact for the expression of the gene of interest in the presence of a glucocorticoid.

Daiichi Seiyaku Co. Ltd

Remarks

Related patent was also granted in Europe (EP 316717 B1).

US 5877018

Earliest priority – 20 April 1994
Filed – 16 April 1996
Granted – 2 March 1999
Expected expiry – 20 April 2014

Title – Synthetic eukaryotic promoters containing two inducible elements

Claim 1
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.

Claim 3
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.

Claim 12
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.

Claim 19
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.

Claim 23
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.

Synthetic inducible promoters containing a metal-responsive element and a glucocorticoid responsive element for driving the expression of a gene in a eukaryotic system.

Inducible eukaryotic promoters derived from a human metallothionen gene and mouse mammary tumor virus. The first one contains metal-responsive elements and the second one glucocorticoid-responsive elements.

Connaught Laboratories Ltd

US 5559027

Earliest priority – 30 March 1993
Filed – 20 October 1994
Granted – 24 September 1996
Expected expiry – 24 September 2013

Title – Synthetic eukaryotic promoters containing two inducible elements

Claim 1
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.

EP 633941 B1

Earliest priority – 30 March 1992
Filed – 30 March 1992
Granted – 15 May 2002
Expected expiry – 30 March 2012

Title – Synthetic eukaryotic promoters containing two inducible elements

Claim 1
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).

US 5514578

Earliest priority – 26 February 1990
Filed – 30 September 1992
Granted – 7 May 1996
Expected expiry – 7 May 2013

Title – Polynucleotides encoding insect steroid hormone receptor polypeptides and cells transformed with same. / Polynucleotide encoding insect ecdysone receptor

Claim 1
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.

Claim 5
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.

Trustees of Leland Stanford University

Remarks

There is a PCT application (WO 01/70816 A2) titled “Novel ecdysone receptor-based inducible gene expression system”, which was filed by Rohm and Hass Co. It discloses gene expression modulation systems with two expression cassettes, one having a DNA-binding and ligand-binding domain and in the other one a transactivation domain and a ligand-binding domain. The ligand-binding domains are derived from a retinoid X receptor, an ultraspiracle and an ecdysone receptor. The DNA-binding domain is derived from GAL4 and LexA. Mutations to the ecdysone or retinoid receptor enhance the ligand-binding activity and non-steroid binding activity. Related applications are filed in the United States (US 2002/119521 A1 and US 2004/96942 A1).

Note: Patent information on this page was last updated on 2 May 2006.

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Other patents related to steroid-responsive promoters