WO2001062780A1 - Gene expression system based on chimeric receptors - Google Patents
Gene expression system based on chimeric receptors Download PDFInfo
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- WO2001062780A1 WO2001062780A1 PCT/US2001/005750 US0105750W WO0162780A1 WO 2001062780 A1 WO2001062780 A1 WO 2001062780A1 US 0105750 W US0105750 W US 0105750W WO 0162780 A1 WO0162780 A1 WO 0162780A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
Definitions
- the present invention relates to methods in the field of recombinant DNA technology, and products related thereto.
- the invention relates to methods for modulating the expression of genes in mammalian or non-mammalian systems, and products useful therefor.
- genes introduced into cells and organisms are important in many areas of biology and medicine. For instance, methods that allow the intentional manipulation of gene expression would facilitate the analysis of genes whose production cannot be tolerated constitutively or at certain stages of development. They would also be valuable for clinical applications such as gene therapy protocols, where the expression of a therapeutic gene must be regulated in accordance with the needs of the patient.
- such systems will preferably be indifferent to endogenous factors and will preferably be activated by non-endogenous nontoxic drugs.
- the components of the system preferably will not interfere with endogenous cellular pathways.
- inducibility is a desirable feature, but requires that the system have a minimal basal activity in the inactive state, while rapidly achieving high levels of expression of the target gene upon activation. Additional desirable features of such a system are rapid return to the inactive state, e.g., by clearance of an inducing molecule from the system of the subject, and a response that is proportional to the concentration of the inducing molecule so that quantitative as well as qualitative problems can be addressed.
- Steroid/thyroid hormone nuclear receptors control gene expression and have been used in the field of genetic engineering as a tool for manipulating and controlling development and other physiological processes.
- applications for regulated gene expression in mammalian systems include inducible gene targeting, overexpression of toxic and teratogenic genes, anti-sense RNA expression, and gene therapy (see, for example, R. Jaenisch, Science 240:1468-1474. 1988).
- glucocorticoids and other steroids have been used to induce the expression of a desired gene.
- an inducible tetracycline regulated system has been devised and utilized in transgenic mice, whereby gene activity is induced in the absence of tetracycline and repressed in its presence (see, e.g, Gossen et al. PNAS 89:5547-5551,1992; Gossen et al, TIBS 18:471- 475, 1993; Furth et al, PNAS 91 :9302-9306, 1994; and Shockett et al, PNAS
- inducible tetracycline system includes the requirement for continuous administration of tetracycline to repress expression and the slow clearance of antibiotic from bone, a side-effect that interferes with regulation of gene expression. While this system has been improved by the recent identification of a mutant tetracycline repressor that acts conversely as an inducible activator, the pharmacokinetics of tetracycline may hinder its use during development when a precise and efficient "on-off ' switch is essential (see, e.g., Gossen et al, Science 268:1766-1769, 1995).
- EcR Drosophila melanogaster ecdysone receptor
- Usp ultraspiracle
- RXR mammalian retinoid X receptor
- Usp and RXR share a significant degree of sequence homology and some functional similarities; however, in formation of heterodimers with EcR, RXR interacts differently than Usp.
- One primary difference is that formation of EcR+RXR heterodimers is more highly stimulated by the ecdysteroid ligand muristerone A (murA) than by 20-hydroxyecdysone (20-Ec), while formation of EcR+Usp heterodimers is potently stimulated by 20-hydroxyecdysone (K. S. Christopherson et al, Proc Natl Acad Sci USA 89:6314-6318, 1982; H. E. Thomas et al, Nature 362:471-475, 1993).
- EcR+Usp and EcR+RXR heterodimer complexes concomitant binding to ecdysone response elements (EcREs).
- MurA stimulates EcR+Usp binding of EcREs approximately 3 to 7-fold over levels without ligand, but EcR+RXR complexes are completely dependent on ligand for heterodimerization.
- EcR+RXR complexes bind to EcREs at only 10- 40% the level of EcR+Usp complexes (Christopherson et al, supra 1982; Thomas et al, supra 1993; Yao et al, supra, 1992 & 1993).
- EcR for its natural dimer partner, Usp
- RXR affinity for RXR
- a number of additional ecdysone receptors are known in the art as being responsive to an applied non-endogenous chemical inducer enabling external control of expression of the gene controlled by the receptor (See, for example, PCT/GB96/01195 and PCT/US98/17723).
- chimeric receptor systems for modulating the expression of a target gene in a subject.
- Invention systems are based on the discovery that a functional chimeric transcription factor for ligand-dependent modulation of gene expression can be constituted in vivo by protein- protein interaction of separate chimeric proteins that each contain the dimerization domain of a dimerizing protein, specifically a member of the steroid/thyroid hormone nuclear receptor superfamily, and further comprise one of two components necessary to form a transcription factor, i.e., a transcription modulation domain and a DNA binding domain.
- association of the dimerization domains in the two chimeric proteins, and, hence, "formation" of the transcription factor by juxtaposition of its two component parts, optionally in the presence of a non-endogenous ligand for the system, will allow the system to operate in subjects without interference from endogenous receptors and ligands.
- inventions for modulating the expression of a target gene associated with a defined response element in a subject.
- Invention systems comprise 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 the response element to said DNA binding domain modulates expression of the target gene.
- the isolated nucleic acid jointly encodes the first and second chimeric proteins, for example with an internal ribosomal entry site located between the nucleotides encoding the first and second chimeric proteins.
- systems for modulating the expression of a target gene associated with a defined response element in a subject 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 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.
- Response of the response element to the DNA binding domain in the functional entity modulates expression of the target gene in the subject.
- invention method(s) for modulating the expression of one or more target genes in a subject that contains one or more inducible DNA constructs encoding an invention system and DNA encoding one or more target gene products under the control of a response element to which the DNA binding domain in the invention system is responsive.
- invention method(s) comprises subjecting the subject to conditions suitable to induce expression of the system, thereby modulating expression of the target gene(s).
- method(s) for modulating the expression of one or more target genes in a subject containing an invention system and DNA encoding one or more target gene products under the control of a response element to which the DNA binding domain in the invention system is responsive comprises administering to the subject an effective amount of ligand for the system, thereby modulating expression of the target gene(s).
- invention system(s) for modulating the expression of a target gene associated with a defined response element in a subject comprises 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 one of the receptors is non-mammalian and the first and second chimeric proteins associate to form a functional entity under substantially physiological conditions in the presence of a non-mammalian ligand.
- Response of the response element associated with the target gene to DNA binding domain in invention system(s) modulates expression of the target gene(s).
- Figure 1 is a schematic of the chimeric proteins which comprise the invention modulation system.
- Figure 2 is a schematic of how the components of the invention modulation system interact in the absence and presence of ligand, wherein the black bars represent a response element, the open bars represent a first chimeric protein
- the darkly shaded bars represent a second chimeric protein (comprising a dimerization domain and a transcription modulating domain), and the small circles represent molecules of ligand.
- the first and second chimera do not associate, whereas (as shown on the right hand side of the figure) in the presence of ligand, the first and second chimera associate, thereby promoting transcription of the gene(s) associated with the response element.
- FIG. 3 presents a schematic of VHB, GR and GU constructs.
- BE numbers refers to amino acids from the Bombyx ecdysone receptor hinge domain ("h") and hormone binding domain (HBD) used in construction;
- R numbers refer to amino acids from the human RXR alpha protein used in construction of GR; and
- U numbers refer to amino acids from the Drosophila Usp protein used in construction of GU.
- VP16 sequences are a 77 amino acid region of the HSV VP16 protein transactivation domain;
- GAL4 sequences are a 95 amino acid domain of the GAL4 protein capable of tightly and specifically binding its cognate operator DNA response element of 17 base-pairs.
- Figure 4 presents data demonstrating the operability of tandem chimeric proteins as described herein using different amounts of plasmids encoding VHB (VP 16 activation domain operatively associated with the Bombyx ecdysone receptor ligand binding domain) and GR (GAL4 DNA binding domain operatively associated with the RXR ligand bonding domain) in a transient transfection assay using 1 ⁇ M tebufenozide as ligand, and measuring luciferase activity 48 hrs. after transfection and stimulation.
- the reporter plasmid is two tandem Gal4 operators coupled with a minimal Tk promoter and driving the firefly luciferase gene. Units are Relative Units.
- Figure 5 presents data illustrating the properties of RXR-USP chimeric proteins. Black indicates RXR protein regions, gray indicates Drosophila USP protein regions. R or U numbers indicate the amino acids corresponding to human RXR alpha or Drosophila Usp used in the construction of chimeras. By combining different regions of both proteins and empirically assaying, chimeric variants with novel ligand responsive properties were found. Units are Relative Units. Numbers at the end of each bar indicate fold induction (calculated by dividing the induced level by the unstimulated level for each ligand).
- GRU variant 1.2 demonstrates good response to all ligands (both 20-OH ecdysone and tebufenozide) with a relatively low baseline, however, the baseline is not down to the level of GR or GR-like chimeras with super-low uninduced expression levels.
- point mutations within the GRU1.2 hormone binding domain were made and tested (see Figure 6).
- Figure 6 presents data generated by a point mutant of GRU1.2, referred to herein as GRU1.2D-3, wherein the cysteine at position 329 (or dUsp sequence) has been converted to glycine.
- This mutant displays a significantly lower basal level of transactivation than its parent chimer, yet it retains a high level response to both 20-OH ecdysone and tebufenozide.
- the relative increase multiple transient transfection experiments routinely fall within 50-100-fold induction.
- FIG. 7 schematically illustrates exchange of the Gal4 DNA-binding domain of GRU1.2D-3 with the 260 amino acid bacterial tetracycline repressor protein DNA binding region results in a new constuct called VIRTU that allows regulation by both ecdysteroids and tetracycline analogs.
- This construct functions essentially equivalently to GRU variants with respect to induction by ecdysteroids and ecdysteroid agonists such as tebufenozide.
- two invention systems may associate to form a dimer, for example either a heterodimer or a homodimer.
- invention systems may also form dimers with native members of the steroid/thyroid hormone nuclear receptor superfamily.
- the first chimeric protein consists essentially of a DNA binding domain and a first dimerization domain that are non-endogenous to the subject to which it is administered and the second chimeric protein consists essentially of a transactivation modulating domain and a second dimerization domain.
- the DNA binding domain and first dimerization domain can be selected to be non-mammalian, for example, derived from a plant or insect.
- at least one of the DNA binding domain and the first dimerization domain can be mammalian. It is also contemplated, as one of numerous possibilities, that a non- mammalian DNA binding domain can be combined with a mammalian dimerization domain in the invention systems.
- the DNA binding domain and transactivation domains can be located anywhere within the respective chimeric proteins.
- the DNA binding domain is located at the carboxyl terminus of the first chimeric protein and the transactivation modulating domain is located at the amino terminus of the second chimeric protein.
- the transactivation modulating domain and the DNA binding domain are brought into spatial juxtaposition with one another to "form" a chimeric transcription factor functional for ligand-dependent modulation of gene expression.
- At least one dimerization domain in the invention system is optionally selected to be ligand responsive, so that interaction between the dimerization domains is preferably dependent on the presence or absence of ligand for the system.
- modulation of expression of a target gene can be achieved by inducing expression of one chimeric protein (if the other is constitutively produced), of both chimera, by introducing a non-endogenous (e.g., non-mammalian) ligand to the subject, or by removing such ligand from the subject.
- expression of target genes is preferably controlled by the addition to the system of ligand therefor or removal of ligand from the system.
- Invention chimeric proteins associate to form functional entities under a variety of conditions, including those at or near physiological conditions in the subject into which they are introduced, with the particular physiological conditions depending upon whether the subject is a plant, a mammal, an amphibian, etc. (e.g., in saline at body temperature in mammals).
- association of proteins can be influenced by manipulation of a variety of physical parameters, such as are disclosed in McPherson, Eur. J. Biochem., 189:1-23, 1990, which is incorporated herein by reference in its entirety.
- the term "functional entity" means that the associated first and second chimeric proteins (either as monomers or as dimers) possess at least some of the biological function of wild type receptors which promote transactivation of a promoter operatively associated with a suitable response element, i.e., one responsive to the invention system.
- a suitable response element i.e., one responsive to the invention system.
- two invention systems can dimerize, or an invention system can dimerize with a native member of the steroid/thyroid hormone nuclear receptor superfamily to modulate transactivation of gene(s) whose expression is controlled by a response element that responds to the two DNA binding domains in the dimer (i.e., a 2-site response element), optionally in the presence of ligand.
- the functional entity formed by association of the first and second chimeric proteins in the invention system can, as a monomer (i.e., without association with another invention system or with a member of the steroid/thyroid hormone nuclear receptor superfamily) modulate transactivation of gene(s) whose expression is controlled by a response element that is responsive to the single DNA binding domain contained therein.
- the response element is a 2-site response element, such as a hormone response element
- the invention system can, as a monomer, transactivate at least a low level of expression.
- an invention system wherein at least one chimeric protein comprises a Bombyx mori DNA binding domain can modulate the expression of a gene under the control of a Bombyx ecdysone response element.
- the invention systems function as a homodimer, or a heterodimer.
- the term "functional entity,” as applied to the invention system, means that the two chimeric proteins possess a cooperative function.
- the two dimerization domains interact with each other in a manner appropriate to substantially provide one or more of the functions in the functional entity that are present in a wild type member of the steroid/thyroid hormone nuclear receptor superfamily.
- the phrase "member(s) of the steroid/thyroid hormone nuclear receptor superfamily” refers to hormone binding proteins that operate as ligand-dependent transcription factors, including identified members of the steroid/thyroid hormone nuclear receptor superfamily for which specific ligands have not yet been identified (referred to in the art as “orphan receptors").
- Members of the steroid/thyroid hormone nuclear receptor superfamily are characterized by the presence of five domains: N- terminal or activation domain (A/B), DNA binding domain (C), hinge domain (D), ligand binding domain (E), and C-terminal domain (F) (Evans, R. Science 240:889- 895, 1988).
- Each chimeric protein in the invention system is required to contain a dimerization domain of a member of the steroid/thyroid hormone nuclear receptor superfamily.
- dimerization domain means a region derived from one or more members of the steroid/thyroid hormone nuclear receptor superfamily containing a sequence of amino acids that functions to cause dimerization of two invention chimeric proteins.
- the dimerization domain is generally located within the region of the receptor molecule that is commonly refe ⁇ ed to as including the D, E and F domains, or is referred to as the "D-E-F" domain.
- the dimerization domain therefore, includes the complete ligand binding domain (E), or a portion thereof, and may optionally include all or part of the hinge domain (D) and/or the C-terminal region (F) of a member of the steroid/thyroid nuclear receptor superfamily, or a functional equivalent thereof. In some cases the dimerization domain may include at least a portion of the DNA binding domain itself.
- the ligand binding domain employed for the preparation of invention chimeric proteins can be either endogenous or non-endogenous to a subject to whom the invention system is to be administered, with the former including ligand binding domains that are modified to be non-responsive to ligands endogenous or native to the subject.
- the ligand binding domain is derived from non- mammalian member(s) of the steroid/thyroid hormone nuclear receptor superfamily
- the ligand binding domains are preferably derived from the carboxy-terminal portion of non-mammalian members.
- Exemplary members that are not normally present in mammalian cells include insect, avian, amphibian, reptilian, fish, plant, bacteria, viral and fungal (including yeast) members of the steroid/thyroid hormone nuclear receptor superfamily, and the like.
- the ligand binding domain can also be derived from other mammalian systems, such as DXR or SXR.
- steroid receptors such as glucocorticoid receptor (GR), mineralocorticoid receptor (MR), estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), vitamin D 3 receptor (VDR), and the like; plus retinoid receptors, such as the various isoforms of retinoic acid receptor (e.g., RAR ⁇ , RAR ⁇ or RAR ⁇ , the various isoforms of retinoid X (or 9-cis retinoic acid) receptor (e.g., RXR ⁇ , RXR ⁇ , or RXR ⁇ , various isoforms of peroxisome proliferator-activated receptors (e.g., PPAR ⁇ , PPAR ⁇ , PPAR ⁇ ) and the like; thyroid hormone receptor (T3R), such as TR ⁇ , TR ⁇ , and the like; steroid and xenobiotic receptor (SXR
- GR glucocorticoid receptor
- MR mineralocorticoid receptor
- orphan receptors contemplated for use herein include HNF4 (see, for example, Sladek et al., Genes & Development 4:2353-2365 (1990)), the COUP family of receptors (see, for example, Miyajima et al., in Nucleic Acids Research 16:11057-11074 (1988), and Wang et al., Nature 340:163-166 (1989)), COUP-like receptors and COUP homologs, such as those described by Mlodzik et al., Cell 60:211-224 (1990) and Ladias et al., Science 251 :561-565 (1991), orphan receptor (OR1; see, e.g., Feltkamp et al, JBiol Chem (1999) 274(15): 10421-9), the insect derived knirps and knirps-related receptors, short heterodimer partner (SHP; see, e.g., Se
- one of the dimerization domains is derived from an ecdysone receptor (EcR), for example, a Drosophila EcR (DEcR) or a Bombyx EcR (BEcR).
- EcR ecdysone receptor
- DEcR Drosophila EcR
- BEcR Bombyx EcR
- one of the chimeric proteins contains the dimerization domain from an ecdysone receptor
- the other chimeric protein contain a dimerization domain from a retinoid X receptor or an Ultraspiracle receptor.
- Additional, exemplary ecdysone receptors for use in invention systems include those derived from dipteran species such as Drosophila melanogaster (M.R. Koelle, 1995), and the like, and those derived from lepidopteran species such as Bombyx mori (Swevers et al, Insect Biochem. Molec. Biol, 25(7):857-866, 1995), Choristoneura fumiferana (Palli et al, Insect Biochem. Molec. Biol, 26(5):485-499, 1996), Manduca sexta (Fujiwara et al, Insect Biochem. Molec.
- At least one of the chimeric proteins comprises a dimerization affinity enhancing domain in addition to the dimerization domain.
- the presently preferred affinity enhancing domain is the hinge region (D) i.e., the fragment bounded by the ligand binding domain (E) and DNA binding domain (C) of the native Bombyx mori receptor (BEcR), specifically, about 27 amino acid residues (i.e. amino acid residues 283-309 of BEcR), which are sufficient to confer high affinity for complex formation with an endogenous dimer partner (see U.S. Patent Application Serial No. 08/891 ,298, filed July 10, 1997, copending herewith, which is incorporated herein by reference in its entirety).
- the first chimeric protein in the invention system contains a DNA binding- domain.
- DNA-binding domains contemplated for use in the preparation of invention chimeric proteins are well known in the art and are typically obtained from DNA- binding proteins (e.g., transcription factors).
- the term "DNA-binding domain” is understood in the art to refer to an amino acid sequence that is able to bind to DNA (A. Klug, Ann N YAcad Sci, 758: 143-160, 1995).
- DNA-binding domain encompasses a minimal peptide sequence of a DNA-binding protein up to the entire length of a DNA-binding protein, so long as the DNA-binding domain functions to associate with a particular response element, as a monomer, homomultimer, or a heteromultimer, preferably a monomer or homodimer.
- the DNA-binding domain can be positioned at either the carboxyl or amino terminus of the first chimeric protein in the invention system.
- DNA-binding domains are known to function heterologously in combination with other functional domains while maintaining the ability to bind the natural DNA recognition sequence (see, e.g., Brent and Ptashne, Cell, 43:729-736, 1985).
- DNA-binding domains are interchangeable, thereby providing numerous chimeric receptor proteins (see, e.g., U.S. Patent 4,981,784; and R. Evans, Science, 240:889-895, 1988).
- "DNA- binding protein(s)" contemplated for use herein belong to the well-known class of proteins that are able to directly bind DNA and facilitate initiation or repression of transcription.
- DNA-binding proteins contemplated for use herein include transcription control proteins (e.g., transcription factors and the like; see, for example, Conaway and Conaway, Transcription Mechanisms and Regulation, Raven Press Series on Molecular and Cellular Biology, Vol. 3, Raven Press, Ltd., New York, NY, 1994; T. Boulikas, Critical Reviews in Eukaryotic Gene Expression, 4(2&3):117-321, 1994; A. Klug, Gene 135:83-92, 1993; W. M. Krajewska, Int. J. Biochem., 24:1885-1898, 1992.)
- transcription control proteins e.g., transcription factors and the like; see, for example, Conaway and Conaway, Transcription Mechanisms and Regulation, Raven Press Series on Molecular and Cellular Biology, Vol. 3, Raven Press, Ltd., New York, NY, 1994; T. Boulikas, Critical Reviews in Eukaryotic Gene Expression, 4(2&3):117-321, 1994; A. Klug, Gene 135:83-92, 1993; W
- Transcription factors contemplated for use herein as a source of such DNA binding domains include, e.g., homeobox proteins, zinc finger proteins, hormone receptors, helix-turn-helix proteins, helix-loop-helix proteins, basic-Zip proteins (bZip), ⁇ -ribbon factors, and the like. See, for example, S. Harrison, "A Structural Taxonomy of DNA-binding Domains," Nature, 353:715-719.
- Homeobox DNA-binding proteins suitable for use herein include, for example, HOX, STF-1 (Leonard et al, Mol. Endo., 7:1275-1283, 1993), Antp, Mat ⁇ -2, INV, and the like.
- the DNA-binding domain(s) used in the invention chimeric proteins can be obtained from a member of the steroid/thyroid hormone nuclear receptor superfamily, or are substantially the same as those obtained from a member of the superfamily.
- the DNA-binding domains of substantially all members of the steroid/thyroid hormone nuclear receptor superfamily are related. Such domains consist of 66-68 amino acid residues, and possess about 20 invariant amino acid residues, including nine cysteines. Members of the superfamily are characterized as proteins which contain these 20 invariant amino acid residues.
- the highly conserved amino acids of the DNA-binding domain of members of the superfamily are as follows:
- the second chimeric protein (which lacks a DNA binding domain) cannot interact productively with a member of the steroid/thyroid hormone nuclear receptor superfamily (e.g., an endogenous one) to complete the 2-half site requirement for DNA binding. Consequently, in this scenario, the invention system produces a functional entity that complexes with only one half of a 2-half site DNA recognition site.
- a preferred DNA binding domain is the GAL4 DNA binding domain, which does not interact with a 2-half site DNA recognition site.
- the DNA binding domain of the yeast GAL4 protein comprises at least the first 74 amino terminal amino acids thereof (see, for example, Keegan et al, Science 231 :699-704, 1986).
- the first 90 or more amino terminal amino acids of the GAL4 protein will be used, for example, the 147 amino terminal amino acid residues of yeast GAL4.
- the GAL4 DNA binding domain is particularly effective when combined in the first chimeric protein with the ligand binding domain of a retinoid X receptor or an Ultraspiracle receptor because a dimer partner containing this combination cannot functionally interact with endogenous proteins as can the native retinoid X receptor.
- Another DNA binding domain preferred for use in the practice of the present invention is the Tet operon.
- the tetracycline inducible system is well-known in the art (see, e.g, Gossen et al. (1992) Proc. Natl. Acad. Sci. 89, 5547-5551; Gossen et al.(1993) TIBS 18, 471-475; Furth et al. (1994) Proc. Natl. Acad. Sci. 91, 9302-9306; and Shockett et al. (1995) Proc. Natl. Acad. Sci. 92, 6522-6526) .
- Use of the Tet operon as DNA binding domain in the first chimeric protein is particularly effective when combined with the ligand binding domain of a Bombyx ecdysone receptor.
- the second chimeric protein in the invention system comprises a transcription modulating domain in addition to a dimerization domain.
- the second chimeric protein and not the first chimeric protein comprises a transcription modulating domain.
- Transcription modulating domains are of two types, those that activate transcription of a gene sequence operatively associated with a response element that is responsive to the invention system (i.e., transcription activation domains) and those that repress or de-activate transcription of a gene sequence operatively associated with a response element that is responsive to the invention system (i.e., transcription repression domains).
- the transcription modulating domain can be located anywhere within the second chimeric protein, but is preferably located at the amino terminus thereof.
- transcription activation domains contemplated for use in the practice of the present invention are well known in the art and can readily be identified by those of skill in the art. Such transcription activation domains are typically derived from transcription factors and comprise a contiguous sequence that functions to activate gene expression when associated with a suitable DNA-binding domain and, optionally, a suitable ligand binding domain.
- Suitable activation domains can be obtained from a variety of sources, e.g., from the N-terminal region of members of the steroid/thyroid hormone nuclear receptor superfamily, from transcription factor activation domains, such as, for example, VP16, GAL4, NF-kB or BP64 activation domains, and the like (See, for example, M. Manteuffel-Cymborowska, Acta Biochim Pol. 46(11:77-89. 1999; T. Tagami et al., Biochem Biophys Res. Commun. 253(2):358-63, 1998, W. Westin, Adv Pharmacol, 47:89-112, 2000).
- the activation domain presently preferred for use in the practice of the present invention is obtained from the C-terminal region of the VP16 protein, and is known as VP16 ⁇ .
- the second chimeric protein comprises the VP16 ⁇ activation domain and both the hinge domain and the ligand binding domain of a Bombyx ecdysone receptor as the second dimerization domain.
- Transcription repressor domains specifically include those that repress transactivation of gene expression by nuclear receptors by affecting the interaction of the ligand and/or functional entity with a response element that is otherwise responsive to the functional entity, either as a monomer or as a dimer, as well as those that "de-activate" by inhibiting association of the first and second chimeric proteins.
- Exemplary transcription repressor domains suitable for use as the transcription modulating domain in the second chimeric protein include RAFT, CREM, MECP-2, SMRT, NcoR, mSin3 A, RAR, TR, SMRTR, and the like.
- one or more isolated nucleic acid(s) encoding invention chimeric protein(s) and cells containing such one or more isolated nucleic acids can be either mammalian or non-mammalian, for example, plant, bacterial, viral and fungal (including yeast) cells, and the like.
- invention methods comprise administering to the subject an effective amount of ligand for the invention system.
- the ligand for use in the practice of the present invention is preferably non-endogenous to the subject treated for modulation of expression of one or more target genes, i.e., for enhancing or repressing expression of the respective gene product(s).
- the target gene is generally non- endogenous, but may also be endogenous if either increased or decreased production of an endogenous gene product is desired.
- one of the dimerization domains in the functional entity formed by association of the two chimeric proteins is from an insect species, and the preferred ligand for use in the invention method is an insect hormone, non-mammalian analog, or mimic thereof.
- the dimerization domain in one invention chimeric protein is derived from a diptera species (e.g., the Drosophila ecdysone receptor) or a lepidoptera species (e.g., the Bombyx ecdysone receptor), and the dimerization domain contained in the other invention chimeric protein is derived from either the ultraspiracle protein or a retinoid X receptor (or a chimeric combination of the two). Invention systems containing either one of these combinations will complex with an ecdysone response element in the presence of such a ligand for the insect receptor.
- the invention method comprises administering to the subject an effective amount of an inducer for the invention system.
- modulate and “modulating” refer to the ability of a given functional entity or a dimer of such functional entities to activate/deactivate and/or up-regulate/down-regulate transcription of target nucleic acid(s) (e.g., non- endogenous nucleic acid(s)), relative to transcription of target nucleic acid in the absence of the functional entity or dimer of such functional entities.
- target nucleic acid(s) e.g., non- endogenous nucleic acid(s)
- an invention functional entity or dimer thereof on the transcription of non-endogenous or endogenous nucleic acids will vary depending on the particular combination of transcription modulating domain, dimerization domain(s) and/or specific members of the steroid/thyroid hormone nuclear receptor superfamily in the two chimeric proteins, on the presence or absence of specific ligand for the ligand binding domain(s) employed in the chimeric proteins, on the regulatory element (e.g., response element) with which the DNA binding domain and/or the functional entity interacts, on the presence or absence of additional components of a receptor complex (e.g., co-activators, co-repressors), and the like. It is specifically contemplated within the scope of the present invention that modulation includes repression of expression of one or more genes.
- the cell line contains:
- the invention method comprises administering to the cell line an effective amount of a non-endogenous ligand for the invention system to either enhance or repress expression of the target gene.
- a non-endogenous ligand for the invention system to either enhance or repress expression of the target gene.
- This embodiment of the invention can be practiced either in vitro or in vivo.
- the invention method in this situation comprises introducing to the subject an invention system containing the first and second chimeric proteins, wherein the functional entity formed by the chimeric proteins interacts with the endogenous response element, thereby modulating expression of the gene(s) dependent on the presence of ligand therefor.
- the ligand will not be endogenous to the subject, thereby providing control over expression of the gene product(s).
- the modulating can further comprise inducing expression of the nucleic acid(s) encoding the components of the invention system.
- the invention method comprises introducing to the subject an invention system that interacts with the endogenous ligand and with the endogenous response element, thereby modulating expression of the one or more gene(s).
- the invention system is encoded by an inducible DNA construct, the modulation will be controlled by subjecting the subject to conditions suitable to cause expression of the first and second chimeric proteins.
- the subject can be administered an effective amount of an inducer for the DNA construct.
- This embodiment of the invention is especially useful for controlling expression of a non-endogenous gene that is under the control of an endogenous response element when the ligand for the invention functional entity is also endogenous.
- Response elements contemplated for use in the practice of the present invention include elements responsive to the invention functional entity and/or to the DNA binding domain contained therein. In a preferred embodiment of the present invention, such elements are non-endogenous response elements not normally present in the cells of the host.
- One class of non-endogenous response elements contemplated for use herein includes hormone response elements that modulate transcription of non-endogenous nucleic acid when associated with the DNA binding domain of an invention receptor peptide. Such response elements are referred to herein as being “defined” or “responsive to" the DNA binding domain in one or more invention systems. Response elements useful in conjunction with invention functional entities are well known in the art.
- retinoic acid receptor response elements are composed of at least one direct repeat of two or more defined half sites separated by a spacer of five nucleotides.
- the spacer nucleotides can independently be selected from any one of A, C, G or T.
- Each half site of the 2-site response elements contemplated for use in the practice of the invention comprises the sequence:
- R is selected from A or G
- M is selected from A or C
- thyroid hormone receptor response elements can be composed of the same half site repeats, with a spacer of four nucleotides.
- palindromic constructs as have been described in the art are also functional as thyroid receptor response elements.
- the response element selected for use in the invention system is a GAL4 response element.
- exemplary GAL4 response elements are those containing the palindromic 17-mer:
- 5'-CGGAGGACTGTCCTCCG-3' (SEQ ID NO:5), such as, for example, 17MX, as described by Webster et al, in Cell 52:169-178 (1988), as well as derivatives thereof. Additional examples of suitable response elements include those described by Hollenberg and Evans in Cell 55:899-906 (1988); or Webster et al. in Cell 54:199-207 (1988).
- Ecdysone response element sequences are preferred for use herein with invention systems containing an ecdysone receptor DNA binding domain. Such response elements function in a position- and orientation-independent fashion.
- the native ecdysone response element has been previously described, see, e.g., Yao et al., Cell, 71:63-72, 1992.
- any DNA binding recognition function can be substituted in place of those having the 2-half-site requirement of members of the steroid/thyroid hormone nuclear receptor superfamily. For this reason, the invention system and methods can be used to transactivate endogenous target genes in a ligand specific manner.
- nucleic acid constructs contemplated for use in one aspect of the present invention include promoters and/or regulatory elements operatively associated with non- endogenous nucleic acids.
- the invention system in the presence of a ligand therefor, complexes with the response element and activates transcription of one or more non-endogenous nucleic acids, including non- endogenous reporter gene(s).
- a ligand complexes with the response element and activates transcription of one or more non-endogenous nucleic acids, including non- endogenous reporter gene(s).
- an invention system containing a DNA binding domain and/or dimerization domains derived from RXR and EcR will transactivate an ecdysone response element-containing promoter in the presence of the hormone ecdysone, or the synthetic analog, muristerone A.
- the response element is operatively associated with one or more non-endogenous target gene(s) whose expression it is desirable to control.
- operatively associated with refers to the functional relationship of DNA with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences.
- operative association of DNA with a promoter refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
- operatively associated with means that a nucleic acid construct containing the respective DNA sequences (represented by the terms “response element” and “non-endogenous or endogenous gene”) may contain additional nucleotide sequences (i.e., regulatory elements) that perform regulatory and effector functions controlling expression of a target gene.
- promoter refers to a specific nucleotide sequence recognized by RNA polymerase, the enzyme that initiates RNA synthesis.
- the promoter sequence is the site at which transcription can be specifically initiated under proper conditions.
- Promoters contemplated for controlling expression of non-endogenous nucleic acids employed in the practice of the present invention include inducible (e.g., minimal CMV promoter, minimal TK promoter, modified MMLV LTR), constitutive (e.g., chicken ⁇ -actin promoter, MMLV LTR (non-modified), DHFR), and/or tissue specific promoters.
- inducible e.g., minimal CMV promoter, minimal TK promoter, modified MMLV LTR
- constitutive e.g., chicken ⁇ -actin promoter, MMLV LTR (non-modified), DHFR
- tissue specific promoters e.g., tissue specific promoters.
- Inducible promoters contemplated for use in the practice of the present invention comprise transcription regulatory regions that function maximally to promote transcription of mRNA under inducing conditions.
- suitable inducible promoters include DNA sequences corresponding to: the E. coli lac operator responsive to IPTG (see Nakamura et al. , Cell, 18 : 1109- 1117, 1979); the metallothionein promoter metal-regulatory-elements responsive to heavy-metal (e.g., zinc) induction (see Evans et al, U.S. Patent No. 4,870,009), the phage T71ac promoter responsive to IPTG (see Studier et al, Meth. Enzymol, 185: 60-89, 1990; and U.S. Patent No. 4,952,496), the heat-shock promoter; the TK minimal promoter; the CMV minimal promoter; a synthetic promoter; and the like.
- Exemplary constitutive promoters contemplated for use in the practice of the present invention include the CMV promoter, the SV40 promoter, the DHFR promoter, the mouse mammary tumor virus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus (MMLV) promoter, elongation factor la (EFla) promoter, albumin promoter, APO Al promoter, cyclic AMP dependent kinase II (CaMKII) promoter, keratin promoter, CD3 promoter, immunoglobulin light or heavy chain promoters, neurofiliment promoter, neuron specific enolase promoter, L7 promoter, CD2 promoter, myosin light chain kinase promoter, HOX gene promoter, thymidine kinase (TK) promoter, RNA Pol II promoter, MYOD promoter, MYF5 promoter, phosphoglycerokinase (PGK) promoter, Stfl promoter
- tissue specific refers to the substantially exclusive initiation of transcription in the tissue from which a particular promoter that drives gene expression is derived (e.g., expressed only in T- cells, endothelial cells, smooth muscle cells, and the like).
- tissue specific promoters contemplated for use in the practice of the present invention include the GH promoter, the NSE promoter, the GFAP promoter, neurotransmitter promoters (e.g., tyrosine hydroxylase, TH, choline acetyltransferase, ChAT, and the like), promoters for neurotropic factors (e.g., a nerve growth factor promoter, NT-3, BDNF promoters, and the like), and so on.
- neurotransmitter promoters e.g., tyrosine hydroxylase, TH, choline acetyltransferase, ChAT, and the like
- promoters for neurotropic factors e.g., a nerve growth factor promoter, NT-3, BDNF promoters, and the like
- non-endogenous to said host refers to nucleic acids not naturally found at levels sufficient to provide a function in the particular cell where transcription is desired.
- non-endogenous nucleic acids can be either natural or synthetic nucleic acids, which are introduced into the host in the form of DNA or RNA.
- the nucleic acids of interest can be introduced into target cells (for in vitro applications), or the nucleic acids of interest can be introduced directly or indirectly into a host, for example, by the transfer of transformed cells into a host.
- endogenous nucleic acids or “endogenous genes” refers to nucleic acids naturally found at levels sufficient to provide a function in the particular cell where transcription is desired.
- Non-endogenous nucleic acids contemplated for use in the practice of the present invention include wild type and/or therapeutic nucleic acids.
- Wild type genes are those that are native to cells of a particular type.
- Exemplary wild type nucleic acids are genes which encode products the substantial absence of which leads to the occurrence of a non-normal state in a host; or a substantial excess of which leads to the occurrence of a non-normal state in a host.
- Such genes may not be expressed in biologically significant levels or may be undesirably overexpressed.
- a synthetic or natural gene coding for human insulin would be non-endogenous genetic material to a yeast cell (since yeast cells do not naturally contain insulin genes)
- a human insulin gene inserted into a human skin fibroblast cell would be a wild type gene with respect to the fibroblast since human skin fibroblasts contain genetic material encoding human insulin, although human skin fibroblasts do not express human insulin in biologically significant levels.
- Therapeutic nucleic acids contemplated for use in the practice of the present invention include those which encode products which are toxic to the cells in which they are expressed; or encode products which impart a beneficial property to a host; or those which transcribe nucleic acids which modulate transcription and/or translation of endogenous genes.
- therapeutic nucleic acids refers to nucleic acids that impart a beneficial function to the host in which such nucleic acids are transcribed.
- Therapeutic nucleic acids are those that are not naturally found in host cells. For example, synthetic or natural nucleic acids coding for wild type human insulin would be therapeutic when inserted into a skin fibroblast cell so as to be expressed in a human host, where the human host is not otherwise capable of expressing functionally active human insulin in biologically significant levels.
- therapeutic nucleic acids include nucleic acids that transcribe antisense constructs used to suppress the expression of endogenous genes. Such antisense transcripts bind endogenous nucleic acid (mRNA or DNA) and effectively cancel out the expression of the gene.
- therapeutic nucleic acids are expressed at a level that provides a therapeutically effective amount of the corresponding therapeutic protein.
- Non-endogenous nucleic acids useful in the practice of the present invention include genes that encode biologically active proteins of interest, such as, e.g., secretory proteins that can be released from a cell; enzymes that can metabolize a toxic substance to produce a non-toxic substance, or that metabolize an inactive substance to produce a useful substance; regulatory proteins; cell surface receptors; and the like.
- biologically active proteins of interest such as, e.g., secretory proteins that can be released from a cell
- enzymes that can metabolize a toxic substance to produce a non-toxic substance, or that metabolize an inactive substance to produce a useful substance
- regulatory proteins e.g., cell surface receptors; and the like.
- Useful genes include genes that encode blood clotting factors, such as human factors VIII and IX; genes that encode hormones, such as insulin, parathyroid hormone, luteinizing hormone releasing factor (LHRH), alpha and beta seminal inhibins, and human growth hormone; genes that encode proteins, such as enzymes, the absence of which leads to the occurrence of an abnormal state; genes encoding cytokines or lymphokines such as interferons, granulocytic macrophage colony stimulating factor (GM-CSF), colony stimulating factor- 1 (CSF-1), tumor necrosis factor (TNF), and erythropoietin (EPO); genes encoding inhibitor substances such as alphai-antitrypsin; genes encoding substances that function as drugs, e.g., genes encoding the diphtheria and cholera toxins; and the like.
- blood clotting factors such as human factors VIII and IX
- genes that encode hormones such as insulin, parathyroid hormone, luteinizing hormone
- Additional nucleic acids contemplated for use in accordance with the present invention include genes that encode proteins present in dopaminergic neurons (useful, for example, for the treatment of Parkinson's disease), cholinergic neurons (useful, for example, for the treatment of Alzheimer's disease), hippocampal pyramidal neurons (also useful for the treatment of Alzheimer's disease), norepinephrine neurons (useful, for example, for the treatment of epilepsy), spinal neurons (useful, for example, for the treatment of spinal injury), glutamatergic neurons (useful, for example, for the treatment of schizophrenia), cortical neurons (useful, for example, for the treatment of stroke and brain injury), motor and sensory neurons (useful, for example, for the treatment of amyotrophic lateral sclerosis), and the like.
- nucleic acid sequence information for proteins encoded by non- endogenous nucleic acid(s) contemplated for use herein can be located in one of many public access databases, e.g., GENBANK, EMBL, Swiss-Prot, and PIR, or in related journal publications. Thus, those of skill in the art have access to sequence information for virtually all known genes. Those of skill in the art can obtain the corresponding nucleic acid molecule directly from a public depository or from the institution that published the sequence.
- nucleic acid sequence encoding a desired protein has been ascertained, the skilled artisan can employ routine methods, e.g., polymerase chain reaction (PCR) amplification, to isolate the desired nucleic acid molecule from the appropriate nucleic acid library.
- PCR polymerase chain reaction
- Additional components that can optionally be incorporated into the invention constructs include selectable marker genes and genes encoding proteins required for retroviral packaging, e.g., the pol gene, the gag gene, the env gene, and the like.
- Genes that enable cells to process metabolic intermediaries include genes which permit cells to incorporate L-histidinol, genes encoding thymidine kinase, genes encoding xanthine-guanine phosphoribosyl transferase (gpt), genes encoding dihydrofolate reductase, genes encoding asparagine synthetase, and the like.
- the subject organism can be mammalian or mammalian-derived cells or tissue.
- Exemplary mammals include: humans; domesticated animals, e.g., rat, mouse, rabbit, canine, feline, and the like; farm animals, e.g., chicken, bovine, ovine, porcine, and the like; animals of zoological interest, e.g., monkey, baboon, and the like, or a cell thereof.
- a subject organism can be non- mammalian, preferably non-insect, such as yeast, plant, fungus, bacterial, or other non- mammalian species, or a cell of such a non-mammalian species.
- a suitable ligand is administered to the host, substantially no transcription of the desired non- endogenous nucleic acids occurs.
- ecdysteroids for example, are not naturally present in mammalian, plant and fungal systems, and the like, if it is desired that transcription of a particular non-endogenous nucleic acid be under precise control of the practitioner, a first chimeric protein containing a DNA binding domain and/or dimerization domain derived from an ecdysone receptor and a second chimeric protein that is a suitable partner for associating therewith, as taught herein, is used and the non- endogenous nucleic acid is put under the control of an ecdysone response element, i.e. a response element to which an activated ecdysone receptor binds in nature.
- an ecdysone response element i.e. a response element to which an activated ecdysone receptor binds in nature.
- ligand refers to a steroidal or non-steroidal substance or compound which, in its native state (or after conversion to its "active” form), binds to at least one of the chimeric proteins, or to the dimerized invention system, thereby creating a ligand/dimer complex, which in turn can bind an appropriate response element and activate transcription therefrom.
- Ligands function to modulate transcription of nucleic acid(s) maintained under the control of a response element.
- Such ligands are well known in the art and include synthetic organic compounds, e.g., quinolines, isoquinolines (e.g., dihydroxy isoquinoline, indoloisoquinoline, and the like), hydrazines (e.g., diacyl hydrazines), plant derived steroids, insect derived steroids, synthetic steroids, and the like.
- ecdysone and "ecdysteroid” as interchangeably used herein, are employed in the generic sense (in accordance with common usage in the art), referring to a family of ligands with the appropriate binding and transactivation activity (see, for example, Cherbas et al., in Biosynthesis, metabolism and mode of action of invertebrate hormones (Ed.
- An ecdysone therefore, is a compound which acts to modulate gene transcription for a gene maintained under the control of an ecdysone response element.
- 20-Hydroxy-ecdysone (also known as ⁇ -ecdysone) is the major naturally occurring ecdysone (found, for example, in plants). Unsubstituted ecdysone (also known as ⁇ -ecdysone) is converted in peripheral tissues to ⁇ -ecdysone. Analogs of the naturally occurring ecdysones are also contemplated within the scope of the present invention. Examples of such analogs, commonly referred to as ecdysteroids, include ponasterone A, 26 iodoponasterone A, muristerone A, inokosterone, 26-mesylinokosterone, and the like.
- phytoecdysteroids are also contemplated for use in the practice of the invention as ligands of invention systems that recognize ecdysone response elements.
- Such phytoecdysteroids are known in the art (J.H. Adler et al., Lipids 30(3):257-62, 1995).
- the biological effect of phytoecdysteroids in higher animals are also known (V.N. Syrov, Eksp. Klin. Farmakol 57(5):61-6, 1994).
- Non-steroidal ligands are also contemplated for use in the practice of the present invention.
- a synthetic organic compound such as a hydrazine can be employed as the ligand.
- Hydrazines are presently preferred as they include compounds that are readily available and/or are relatively inexpensive to manufacture.
- tebufenozide is a non-steroidal ecdysone agonist which is commercially available. This compound specifically targets lepidopteran species, including Bombyx mori. Tebufenozide has undergone extensive testing in animal hosts and has proved to be of very low toxicity to mammals and other non-insect species.
- exemplary hydrazines contemplated for use herein are well known in the art, including those disclosed in U.S. Patent Application Serial No. 09/005,286, filed January 9, 1998, which is incorporated herein by reference in its entirety.
- exemplary hydrazines include 1,2-diacyl hydrazines (e.g., tebufenozide), N'-substituted- N,N'-disubstituted hydrazines, dibenzoylalkyl cyanohydrazines, N-substituted-N-alkyl- N,N-diaroyl hydrazines, N-substituted-N-acyl-N-alkyls, carbonyl hydrazines, N-aroyl- N'-alkyl-N'-aroyl hydrazines, and the like.
- 1,2-diacyl hydrazines e.g., tebufenozide
- diacyl hydrazines are neither toxic, teratogenic, nor known to affect mammalian physiology, they are ideal candidates for use as exogenous ligands (e.g. as inducers) in cultured cells and transgenic mammals according to invention methods.
- Ligands, and formulations containing them are administered in a manner compatible with the desired route of administration, the dosage formulation, and in a therapeutically effective amount.
- the required dosage will vary with the particular treatment desired, the degree and duration of therapeutic effect desired, the judgment of the practitioner, as well as properties peculiar to each individual.
- suitable dosage ranges for systemic application depend on the route of administration. It is anticipated that dosages between about 10 micro grams and about 1 milligram per kilogram of body weight per day will be used for therapeutic treatment.
- An effective amount of ligand contemplated for use in the practice of the present invention is the amount of ligand required to achieve the desired level of transcription and/or translation of non-endogenous nucleic acid.
- a therapeutically effective amount is typically an amount of ligand or ligand precursor that, when administered in a physiologically acceptable composition, is sufficient to achieve a plasma concentration of the active ligand from about 0.1 mg/ml to about 100 mg/ml, for example, from about 1.0 mg/ml to about 50 mg/ml, and preferably at least about 2 mg/ml and usually 5 to 10 mg/ml.
- Ligand can be administered in a variety of ways, as are well-known in the art, i.e., by any means that produces contact between ligand and the invention system.
- such ligands can be administered topically, orally, intravenously, intraperitoneally, intravascularly, and the like.
- the administration can be by any conventional means available for use in conjunction with pharmaceuticals, e.g., by intravenous injection, either as individual therapeutically active ingredients or in a combination with other therapeutically active ingredients.
- Ligands contemplated for use in the practice of the present invention can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
- pharmaceutically acceptable formulations, and kits comprising at least one ligand for an invention functional entity, for example an ecdysteroid, such as ecdysone or 20-hydroxy ecdysone, and a pharmaceutically acceptable carrier are contemplated.
- pharmaceutically acceptable formulations consisting essentially of at least one ligand and a pharmaceutically acceptable carrier, are contemplated.
- compositions of the present invention can be used in the form of a solid, a solution, an emulsion, a dispersion, a micelle, a liposome, and the like, wherein the resulting formulation contains one or more of the ligands of the present invention, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for enteral or parenteral applications.
- the ligand(s) may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers suitable for administration by oral, topical, nasal, transdermal, intravenous, subcutaneous, intramuscular, intracutaneous, intraperitoneal, intravascular, and the like means. Administration in the form of creams, lotions, tablets, dispersible powders, granules, syrups, elixirs, sterile aqueous or non-aqueous solutions, suspensions or emulsions, and the like, is contemplated.
- Exemplary pharmaceutically acceptable carriers include carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.
- Such carriers which can be used include glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form.
- auxiliary, stabilizing, thickening and coloring agents and or perfumes may be used.
- the active compound e.g., ecdysteroid as described herein
- the active compound is included in the pharmaceutically acceptable formulation in an amount sufficient to produce the desired effect upon the process or condition of diseases.
- compositions containing ligand(s) as active ingredient may be in a form suitable for oral use, for example, as aqueous or oily suspensions, syrups or elixirs, tablets, troches, lozenges, dispersible powders or granules, emulsions, or hard or soft capsules.
- suitable carriers include emulsions, solutions, suspensions, syrups, and the like, optionally containing additives such as wetting agents, emulsifying and suspending agents, dispersing agents, sweetening, flavoring, coloring, preserving and perfuming agents, and the like.
- Formulations intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutically acceptable formulations.
- Tablets containing ligand(s) as active ingredient in admixture with non-toxic pharmaceutically acceptable excipients may also be manufactured by known methods.
- the excipients used may be, for example, (1) inert diluents such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents such as corn starch, potato starch or alginic acid; (3) binding agents such as gum tragacanth, corn starch, gelatin or acacia, and (4) lubricating agents such as magnesium stearate, stearic acid or talc.
- the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
- a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in the U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874, to form osmotic therapeutic tablets for controlled release.
- formulations for oral use may be in the form of hard gelatin capsules wherein the ligand is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin. They may also be in the form of soft gelatin capsules wherein the ligand is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
- the pharmaceutically acceptable formulations may be in the form of a sterile injectable suspension.
- Suitable carriers include non-toxic parenterally-acceptable sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
- This suspension may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. They can also be manufactured in the form of sterile water, or some other sterile injectable medium immediately before use. Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
- any bland fixed oil may be employed including synthetic mono- or diglycerides, fatty acids (including oleic acid), naturally occurring vegetable oils like sesame oil, coconut oil, peanut oil, cottonseed oil, etc., or synthetic fatty vehicles like ethyl oleate or the like. They may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the formulations, by irradiating the formulations, or by heating the formulations. Sterile injectable suspensions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Buffers, preservatives, antioxidants, and the like can be incorporated as required.
- Compounds contemplated for use in the practice of the present invention may also be administered in the form of suppositories for rectal administration of the drug.
- These formulations may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters of polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.
- compositions containing suitable ligand(s) are preferably administered intravenously, for example, as by injection of a unit dose.
- unit dose when used in reference to a pharmaceutically acceptable formulation of the present invention, refers to a quantity of the pharmaceutical formulation suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier, or vehicle. It may be particularly advantageous to administer such formulations in depot or long-lasting form as discussed hereinafter.
- compositions or pharmaceutically acceptable formulations containing suitable ligand are preferably administered intravenously, as by injection of a unit dose, for example.
- unit dose when used in reference to a therapeutic composition of the present invention, refers to a quantity of ligand suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier, or vehicle. It may be particularly advantageous to administer such compounds in depot or long-lasting form.
- Suitable regimes for initial administration and booster shots are variable, but are typified by an initial administration followed by repeated doses at one or more intervals, by a subsequent injection, or other administration.
- continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.
- transgenic animals capable of prolonged and regulated expression of non-endogenous nucleic acid(s).
- the invention method for producing a transgenic animal comprises introducing into early-stage embryos or stem cells of the animal:
- nucleic acid construct comprising a promoter and said non-endogenous nucleic acid(s) under the control of a response element
- nucleic acid encoding the invention system comprising first and second chimeric proteins, wherein a functional entity formed by the chimeric proteins activates or represses the response element in the presence of a ligand.
- transgenic animal refers to an animal that contains one or more expression constructs containing one or more non-endogenous nucleic acid(s) under the transcription control of an operator and/or response element as described herein.
- transgenic animals using a particular nucleic acid construct are well-known in the art.
- the first line will express, for example, the first and second chimeric proteins as described above. Tissue specificity is conferred by the selection of a tissue-specific promoter (e.g., T-cell specific) that will direct expression of the chimeric protein to appropriate tissue.
- a second line contains a nucleic acid construct comprising a promoter and non-endogenous nucleic acid under the control of a response element, for example, an endogenous response element. Crossbreeding of these two lines will provide transgenic animal(s) that expresses an invention system and the non-endogenous nucleic acid.
- an invention transgenic animal contains one or more expression constructs containing nucleic acid encoding the invention first and second chimeric proteins and non-endogenous nucleic acid under the transcription control of a response element.
- tissue specific expression of the chimeric proteins as described above and timely ligand treatment gene expression can be induced or repressed with spatial, dosage, and/or temporal specificity.
- nucleic acid construct comprising a promoter and said target nucleic acid(s) under the control of a response element
- nucleic acid(s) under the control of an inducible promoter, said nucleic acid(s) encoding an invention system comprising first and second chimeric proteins wherein the functional entity formed by the invention chimeric proteins activates or represses the response element in the presence of a ligand for the dimer;
- said method comprising introducing a ligand not normally present in the cells of the subject and subjecting the subject to conditions suitable to induce or repress expression of the invention system.
- nucleic acid construct comprising a promoter and non- endogenous nucleic acid(s) which express the recombinant product under the control of a response element that is not normally present in the cells of said organism
- chimeric proteins activate the regulatory element in the presence of a ligand for the functional entity
- Modified cells are cultivated under growth conditions (as opposed to protein expression conditions) until a desired density is achieved.
- stably transfected mammalian cells may be prepared by transfecting cells with an expression vector having a selectable marker gene (such as, for example, the gene for thymidine kinase, dihydrofolate reductase, neomycin resistance, and the like), and growing the transfected cells under conditions selective for cells expressing the marker gene.
- a selectable marker gene such as, for example, the gene for thymidine kinase, dihydrofolate reductase, neomycin resistance, and the like
- mammalian cells are transfected with a non-endogenous reporter gene (such as the E. coli ⁇ -galactosidase gene) to monitor transfection efficiency.
- Selectable marker genes are typically not included in the transient transfections because the transfectants are typically not grown under selective conditions, and are usually analyzed within a few
- Recombinant products detrimental to a host organism contemplated for expression in accordance with the present invention include any gene product that functions to confer a toxic effect on the organism.
- tissue specific inducible expression of a toxin such as the diphtheria toxin, would allow for specific ablation of tissue (Ross et al. Genes and Development 7:1318-1324 (1993)), for example to create a new phenotype in the transgenic animal.
- tissue specific inducible expression of a toxin such as the diphtheria toxin
- tissue specific inducible expression of a toxin such as the diphtheria toxin
- the numerous gene products known to induce apoptosis in cells expressing such products are contemplated for use herein (see, e.g, Apoptosis, The Molecular Basis of Cell Death, Current Communications In Cell & Molecular Biology, Cold Spring Harbor Laboratory Press, 1991).
- nucleic acid construct comprising a promoter and non- endogenous nucleic acid(s) which express the recombinant product under the control of a response element that is not normally present in the cells of said organism
- the invention chimeric proteins activate the regulatory element in the presence of a ligand for the functional entity; (b) growing said cells to the desired level in the presence of the ligand; which, in combination with said dimer, binds to said response element and promotes transcription therefrom, and
- modified cells are cultivated in the presence of ligand until a desired stage of development is achieved, then administration of ligand is discontinued. Absence of ligand causes expression of the recombinant product to cease.
- inducible expression of proteins necessary for development is provided as needed during development, then discontinued at a desired stage of development, thereby providing knock-out models of isolated cells and animals containing same, wherein the subject cell or animal would not survive if expression of the "deleted" protein were prematurely terminated.
- nucleic acid construct comprising a promoter and nucleic acid(s) encoding therapeutic protein(s) under the control of a response element responsive to the DNA binding domain in the invention system
- the functional entity formed by association of the invention chimeric proteins activates or represses the response element in the presence of a ligand for the functional entity, and administering, to said host, an effective amount of ligand for the invention system.
- the cells can be obtained from the host, modified as above, and then reintroduced into the host organism.
- the non-endogenous nucleic acid can be introduced directly into cells obtained from a donor (host or separate donor) and the modified cells then can be implanted within the host organism.
- the transplanted cells are autologous with respect to the host. "Autologous" means that the donor and recipient of the cells are one and the same.
- Cells can be modified by "in vivo delivery" of biological materials by such routes of administration as oral, intravenous, subcutaneous, intraperitoneal, intrathecal, intramuscular, intracranial, inhalational, topical, transdermal, suppository (rectal), pessary (vaginal), and the like.
- the non-endogenous nucleic acid may be stably incorporated into cells or may be transiently expressed using methods known in the art.
- gene replacement therapy involves the introduction of functionally active "wild type” or “therapeutic” nucleic acids into the somatic cells of an affected host to correct a gene defect or deficiency.
- gene replacement therapy it must be possible to control the time and location at which gene expression occurs.
- Genes that encode useful "gene therapy” proteins that are not normally transported outside the cell can be used in the invention if such genes are “functionally appended” to, or operatively associated with, a signal sequence that can "transport” the encoded product across the cell membrane.
- a variety of such signal sequences are known and can be used by those skilled in the art without undue experimentation.
- Gene transfer vectors contemplated for use herein are recombinant nucleic acid molecules that are used to transport nucleic acid into host cells for expression and/or replication thereof.
- Expression vectors may be either circular or linear, and are capable of incorporating a variety of nucleic acid constructs therein.
- Expression vectors typically come in the form of a plasmid that, upon introduction into an appropriate host cell, results in expression of the inserted nucleic acid.
- Suitable expression vectors for use herein are well known to those of skill in the art and include recombinant DNA or RNA construct(s), such as plasmids, phage, recombinant virus or other vectors that, upon introduction into an appropriate host cell, result(s) in expression of the inserted DNA.
- Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome. Expression vectors typically further contain other functionally important nucleic acid sequences encoding antibiotic resistance proteins, and the like.
- the amount of non-endogenous nucleic acid introduced into a host organism, cell or cellular system can be varied by those of skill in the art.
- the amount of nucleic acid introduced can be varied by varying the amount of p_laque forming units (PFU) of the viral vector.
- PFU p_laque forming units
- transcription regulatory region refers to that portion of a nucleic acid or gene construct that controls the initiation of mRNA transcription.
- Regulatory regions contemplated for use herein typically comprise at least a minimal promoter in combination with a regulatory element responsive to the complex of the invention system with the ligand.
- a minimal promoter when combined with a regulatory element, functions to initiate mRNA transcription in response to such a complex. However, transcription will not occur unless the required inducer (ligand therefor) is present.
- certain of the invention systems activate or repress mRNA transcription even in the absence of ligand for the invention system.
- the transcription regulatory region further comprises a binding site for ubiquitous transcription factor(s).
- binding sites are preferably positioned between the promoter and the regulatory element.
- Suitable ubiquitous transcription factors for use herein are well-known in the art and include, for example, Spl.
- Exemplary eukaryotic expression vectors include eukaryotic constructs, such as the pSV-2 gpt system (Mulligan et al, (1979) Nature, 277:108-114); pBlueSkript (Stratagene, La Jolla, CA), the expression cloning vector described by Genetics Institute (Science, (1985) 228:810-815), and the like. Each of these plasmid vectors is capable of promoting expression of the chimeric proteins of interest.
- Suitable means for introducing (transducing) expression vectors containing invention nucleic acid constructs into host cells to produce transduced recombinant cells are well-known in the art (see, for review, Friedmann, Science, 244:1275-1281, 1989; Mulligan, Science, 260:926- 932. 1993, each of which are incorporated herein by reference in their entirety).
- transduction examples include, e.g., infection employing viral vectors (see, e.g., U.S. Patent 4,405,712 and 4,650,764), calcium phosphate transfection (U.S. Patents 4,399,216 and 4,634,665), dextran sulfate transfection, electroporation, lipofection (see, e.g., U.S. Patents 4,394,448 and 4,619,794), cytofection, particle bead bombardment, and the like.
- the transduced nucleic acid can optionally include sequences which allow for its extrachromosomal (i.e., episomal) maintenance, or the transduced nucleic acid can be donor nucleic acid that integrates into the genome of the host.
- a gene transfer vector contemplated for use herein is a viral vector, such as Adenovirus, adeno-associated virus, a herpes-simplex virus based vector, a synthetic vector for gene therapy, and the like (see, e.g., Suhr et al, Arch, of Neurol 50:1252-1268, 1993).
- a gene transfer vector employed herein is a retroviral vector.
- Retroviral vectors contemplated for use herein are gene transfer plasmids that have an expression construct containing an non-endogenous nucleic acid residing between two retroviral LTRs.
- Retroviral vectors typically contain appropriate packaging signals that enable the retroviral vector, or RNA transcribed using the retroviral vector as a template, to be packaged into a viral virion in an appropriate packaging cell line (see, e.g., U.S. Patent 4,650,764).
- retroviral vectors for use herein are described, for example, in U.S. Patents 5,399,346 and 5,252,479; and in WTPO publications WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829, each of which is hereby incorporated herein by reference, in its entirety. These documents provide a description of methods for efficiently introducing nucleic acids into human cells using such retroviral vectors.
- retroviral vectors include, for example, mouse mammary tumor virus vectors (e.g., Shackleford et al. , (1988) PNAS, USA, 85 :9655-9659), human immunodeficiency virus (e.g., Naldini et al. (1996) Science 272:165-320), and the like.
- helper cells which produce retroviral vector particles that are essentially free of replicating virus. See, for example, U.S. Patent 4,650,764; Miller, Human Gene Therapy, 1 :5-14, 1990; Markowitz, et al. , Journal of Virology, 61(4) : 1120- 1124, 1988 ; Watanabe, et al. ,
- Retroviral virions suitable for carrying out the invention methods are produced employing well-known methods for producing retroviral virions. See, for example, U.S. Patent 4,650,764; Miller, supra 1990; Markowitz, et al, supra 1988; Watanabe, et al, supra 1983; Danos, et al, PNAS, 85:6460-6464, 1988; and Bosselman, et al, Molecular and Cellular Biology, 7(5): 1797-1806, 1987.
- a modular assembly retroviral vector can be utilized to express both the first and second invention chimeric proteins and an antibiotic resistance gene.
- a "covector” (referred to herein as MARSHA) can be utilized to provide a nucleic acid construct comprising the promoter, the regulatory element and non-endogenous nucleic acid, and a second antibiotic resistance gene.
- the MARSHA vector carrying non-endogenous nucleic acid also has LTRs modified to promote high- level expression only in the presence of the invention chimeric protein encoded by MARV and non-endogenous ligand therefor.
- Co-infected primary mammalian cells can then be selected using both antibiotics, resulting in a cell population that is dependent on ligand for high-level expression of the non-endogenous nucleic acid.
- the simultaneous preparation of the invention first and second chimeric proteins chains by co-expression is presently preferred.
- the association of different subunits of complex proteins takes place after protein synthesis.
- Other components of the cellular apparatus frequently participate in this association as catalysts or controlling elements, with folding of the original structures taking place on occasion.
- Disturbances of the association e.g. by an equal synthesis of the individual components, can have negative consequences both for the proteins which are to be formed and for the host cell.
- this system is subject to sophisticated regulation, which is for the most part cell-specific. Since this regulation is in general not adjustable in genetically manipulated cells, the alternatives explained below have been developed and used for the simultaneous preparation of several foreign proteins:
- the genes separately encoding the first and second chimeric proteins can be integrated separately into expression vectors and then cotransfe ⁇ ed in an appropriate ratio into the cells. This presupposes that several plasmid copies are taken up at the same time in a stable manner and continue to be harbored during division.
- the ratio of the expression of the different genes to each other depends both on the copy number and on the site of integration in the genome of the host cell. It is possible, by elaborate screening processes, to isolate cell clones which express the individual gene products in the desired ratio.
- the different genes can be placed in independent transcription units on one vector. While this, to a large extent, ensures stoichiometric representation of the genes, this process is also subject to problems. Thus, even if expression units having promoters of equal strength are used, it is in no way guaranteed that the mRNAs, which encode the different proteins, have the same stability and translation efficiency. Nor does the transcriptional efficiency of the two genes necessarily need to be identical. In this case, the stoichiometry of expression is produced step- wise using recombinant DNA stratagems (positioning of the transcription units with respect to each other and modulation of the strength of the individual promoters by removing or adding individual elements).
- Bicistronic or multicistronic vectors have been developed in order to avoid the problems connected with the stability of the mRNA of different transcripts.
- the individual reading frames of the gene segments— cistrons—encoding the chimeric proteins lie on one transcription unit (expression unit).
- Expression of the multicistronic gene is effected using a single promoter. While the first cistron in such vectors is normally translated very efficiently, translation of the subsequent cistrons depends on the intercistronic sequences.
- LRES nucleic acid sequences
- poliovirus and encephalomyocarditis virus are known in the art and include the untranslated regions of individual picorna viruses, e.g. poliovirus and encephalomyocarditis virus as well as some cellular proteins, e.g. BiP.
- a short segment of the 5' untranslated region the so-called the internal ribosomal entry site (IRES)
- IRS internal ribosomal entry site
- VHB functionally couples the VP16 domain with the BEcR high-affinity hormone responsive domains (see Figure 3). This DNA was introduced into a number of expression constructs for expression in transduced cells.
- the DNA-binding dimer partner protein fused the 95 amino acid Gal4 DNA binding domain via a BspE I site at the 3' end to a region of either the RXR or Usp protein with high affinity ligand dependent interaction with BEcR .
- These variants used amino acids 200 - 462 of the human RXR alpha protein or amino acids 169-507 of the Drosophila Usp protein (see Figure 3).
- These proteins are termed GR (for the RXR variant) or GU (for the Usp variant).
- GRU fusion chimeras were produced by similar methods and coupled individual regions of both proteins as shown in Figure 5.
- GRU1.2D-3 was performed by PCR-mediated site directed mutagenesis of the GRUl .2 HBD to convert cysteine 329 (numbering in the native Drosophila Usp protein) to a glycine residue ( Figure 6).
- TR tetracycline-repressor
- Transient transfection assays were performed on common cultured cell types using calcium-phosphate precipitation and standard methods as described by Suhr et al., supra. Transfected cells were treated uniformly with 1 micromolar ligand in ethanol vehicle for 48 firs, after transfection before harvest and assay for activity, as described by Suhr et al., supra.
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EP01912956A EP1259537A4 (en) | 2000-02-24 | 2001-02-23 | Gene expression system based on chimeric receptors |
US10/181,325 US20040102367A1 (en) | 2001-02-23 | 2001-02-23 | Gene expression system based on chimeric receptors |
AU2001241682A AU2001241682A1 (en) | 2000-02-24 | 2001-02-23 | Gene expression system based on chimeric receptors |
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- 2001-02-23 EP EP01912956A patent/EP1259537A4/en not_active Withdrawn
- 2001-02-23 WO PCT/US2001/005750 patent/WO2001062780A1/en not_active Application Discontinuation
- 2001-02-23 AU AU2001241682A patent/AU2001241682A1/en not_active Abandoned
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