EP0998560A1 - Proteines multifonctionnelles hybrides et recepteurs de lepidopterane modifies destines a etre utilises pour reguler l'expression transgenique - Google Patents

Proteines multifonctionnelles hybrides et recepteurs de lepidopterane modifies destines a etre utilises pour reguler l'expression transgenique

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Publication number
EP0998560A1
EP0998560A1 EP98934353A EP98934353A EP0998560A1 EP 0998560 A1 EP0998560 A1 EP 0998560A1 EP 98934353 A EP98934353 A EP 98934353A EP 98934353 A EP98934353 A EP 98934353A EP 0998560 A1 EP0998560 A1 EP 0998560A1
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European Patent Office
Prior art keywords
nucleic acid
binding domain
ligand
cells
host
Prior art date
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EP98934353A
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German (de)
English (en)
Inventor
Fred H. Gage
Steven T. Suhr
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Salk Institute for Biological Studies
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Salk Institute for Biological Studies
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Priority claimed from US08/891,298 external-priority patent/US6300488B1/en
Application filed by Salk Institute for Biological Studies filed Critical Salk Institute for Biological Studies
Publication of EP0998560A1 publication Critical patent/EP0998560A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

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 exogenous genes in mammalian systems, and products useful therefor.
  • an inducible tetracycline regulated system has been devised and utilized in transgenic mice, whereby gene activity is induced in the absence of the antibiotic and repressed in its presence (see, e.g, Gossen et al. (1992) PNAS 89, 5547-5551; Gossen et al.(1993) TIBS 18, 471-475; Furth et al . (1994) PNAS 91, 9302-9306; and Shockett et al . (1995) PNAS 92, 6522-6526) .
  • disadvantages of the inducible tetracycline system include the requirement for continuous administration of tetracycline to repress expression and the slow clearance of antibiotic from bone, which interferes with regulation of gene expression. While this system has been improved by the recent identification of a mutant tetracycline repressor which 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 . (1995) Science 268, 1766-1769) .
  • transgenes with potential pathogenic and cytotoxic properties has been difficult in the context of stable cell lines since cells chronically producing significant levels of the toxic transgene generally die from the cytopathic effects.
  • CAG repeats and concomitant polyglutamine (polyQ) expression has been linked to a variety of neurodegenerative conditions, including Hungtington' s disease (Koide et al . (1994) Nat Genet 6:9-13), dentatorubro-pallidoluysian atrophy (Ranum et al . (1994) Nat Genet 8:280-284), spinocerebellar ataxias (Kawaguch et al.
  • Neuroblastoma cells transiently transfected with an 82 -Q construct derived from an expanded huntingtin gene exon 1 were found to be 2-3 times more susceptible to staurosporine induced apoptosis than cells transfected with a LacZ control construct (18) , however, spontaneous cell death was not described. Variations in culture techniques, transfection efficiencies, and cell lines, however, make it difficult to rigorously characterize the progression of polyQ expression, intracellular aggregate (IA) formation, and cell death.
  • IA intracellular aggregate
  • a non- mammalian-based transcription regulating system would be desirable for general application to transgene regulation in in vi tro, ex vivo, and in vivo applications, as well as transgenic animals.
  • Such systems would be extremely desirable to conditionally express pathogenic or toxic compounds in order to produce stable cells .
  • a system that is simple, compact and dependent on ligands which are relatively inexpensive, readily available and of low toxicity in animals would prove useful for stimulation of regulated systems.
  • nuclear receptor proteins isolated from the silk moth bombyx mori are useful for the regulation of transgene expression.
  • bR is generally thought to be a strong transcriptional regulator within cells of the silk moth.
  • bR is also functional in mammalian cells without the addition of exogenous dimer partners therefor.
  • VbR open-reading frame
  • FIG. IA Further modifications to the bR ligand binding domain result in receptors with unique transactivation characteristics (see, e.g., Figure IB).
  • hybrid proteins produced by fusion of modified bRs with other ligand-regulated proteins have been found to be capable of high level, regulated transactivation of sequences controlled by both hormone response elements and tetracycline operators.
  • VbR variants and hybrid proteins in combination with the appropriate promoters and transgenes, can be introduced into target cells by common methods such as transfection of plasmids or by virus mediated gene transfer. The small size and simplicity of these proteins makes them particularly attractive for use in retroviral vectors.
  • Invention receptor peptides have proven capable of providing a high degree (>100 fold) of ligand-dependent transgene expression in a variety of infected and transfected cell types including primary cell types.
  • transgenes including luciferase, LacZ, human GH, GFP, tyrosine hydroxylase, and the low-affinity NGF receptor, have all been shown to function in a ligand-dependent fashion in cells infected with invention constructs .
  • Invention methods provide for regulated gene expression by exogenous non-mammalian inducers, and therefor can be advantageously employed in a variety of in vivo and in vi tro mammalian expression systems.
  • inducible expression of flp recombinase in transgenic mammals would enable those of skill in the art to accomplish temporally specific inducible gene targeting of the adult or the developing embryo (See, e.g., U.S. Patent Nos. 5,654,182 and 5,677,177).
  • Stable cell lines conditionally expressing desired genes provide a valuable tool for the study and treatment of diseases.
  • a retroviral construct in which expression of the transgene is controlled by an exogenous ligand.
  • a desired transgene such as polyglutamine expression cassette ( PQEC)
  • PQEC polyglutamine expression cassette
  • a retroviral construct in which expression of the transgene is controlled by an exogenous ligand.
  • CAG glutamine
  • Two aspects of polyQ-mediated cytotoxicity are well-suited to characterization in vi tro; the expression, distribution, and aggregation of proteins with expanded polyQ tracts within individual cells, and the progression of polyQ expression to death of the cell.
  • the huntingtin protein is expressed predominantly in the cytoplasm of both neuronal and non-neuronal cell types.
  • the expression of polyglutamine tracts separately from the huntingtin protein will allow one to determine what toxic properties, if any, polyglutamine tracts themselves may harbor.
  • a variable length polyglutamine fused to cytosolic reporter proteins such as green fluorescent protein (GFP) facilitates the selection and characterization of PQEC ligand-responsive cell populations and clonal lines.
  • GFP green fluorescent protein
  • the use of recombinant retroviruses as PQEC transfer vectors will allow the stable integration of the transgene into a wide variety of cultured cell types.
  • Figure IA provides a schematic drawing of the nuclear receptor protein isolated from the silk moth bombyx mori (bR) , as well as a construct containing a VP16 activation domain fused at an internal Mlul site near the N-terminus of the bombyx mori nuclear receptor, referred to as VbR.
  • Figure IB depicts the replacement of several amino acids of the ligand binding domain of the bombyx mori nuclear receptor with sequences from the retinoic acid receptor or the thyroid hormone receptor (VbRRA and VbRTR, respectively) .
  • FIG. 1C provides a schematic of a hybrid transactivating construct, designated TTMT, containing a tetracycline-responsive transactivator (Tet-responsive region) , a VP16 activation domain and a peptide derived from bombyx mori .
  • TTMT tetracycline-responsive transactivator
  • FIG. 2A shows the induction of fusion proteins (VP16 activation domain fused to either Drosophila ecdysone receptor (dVEcR) or bombyx mori nuclear receptor derived (VbR) ) with 5 ⁇ g/ml tebufenozide in the absence of exogenous dimer partners .
  • dVEcR Drosophila ecdysone receptor
  • VbR bombyx mori nuclear receptor derived
  • Figure 2B shows that a peptide derived from bombyx mori responds to tebufenozide (teb) with greater than 200-fold induction, while Drosophila EcR responds weakly, it at all.
  • FIG. 2C illustrates the action of tebufenozide (teb) on the expression of EcRE-Luc reporter when co- transfected with the following receptors: LINX: which encodes the tet-transactivator (TTA) , VbR: which encodes VbR,
  • TTMT which encodes the TTA-VbR fusion protein
  • TTMT-2V similar to TTMT but with two VP16 t-domains.
  • Figure 2D depicts the same experiment illustrated in Figure 2C, utilizing a TetO-luc reporter, which responds only to TTA or fusion proteins in the absence of doxycycline .
  • Figure 2E shows the results of an experiment employing CV-1 cells transfected with TTMT and two reporters: 6TetO-luc and 4-EcRE- ⁇ -galactosidase.
  • Figure 3 shows that lower levels of teb are as effective as higher levels for stimulating full activity.
  • Figure 4 is a schematic drawing of representative single-plasmid constructs involving VbR use.
  • Figure 5 illustrates the construction of the pBW plasmid.
  • Figure 6A provides a comparison of Bombyx mori ecdysone receptor (BE) and Drosophila melanogaster ecdysone receptor (DE) by transient transfection with both receptors and the dimer partners Usp or RXR in 293 cells.
  • BE Bombyx mori ecdysone receptor
  • DE Drosophila melanogaster ecdysone receptor
  • Figure 6B provides a similar comparison as in Figure 6A, however, in CV-1 cells.
  • Figure 7A is a schematic drawing of native and chimeric ecdysone receptors .
  • Figure 7B is a graph depicting the transient transfection of VEH into CV-1 cells.
  • Figure 8 is a schematic drawing comparing several different chimeric receptors .
  • Figure 9A provides a schematic of receptor variant viral vectors and an ecdysone response element responsive reporter virus (MS) .
  • Figure 9B illustrates induction levels of luciferase in 293, CV-1 and primary fibroblast cells infected with MS- luc virus .
  • Figure 10 illustrates the construction of the Boris I plasmid.
  • Figure 11 illustrates the construction of the Boris III plasmid.
  • Figure 12 illustrates the construction of the Boris IV plasmid.
  • Figure 13 illustrates the construction of the pBW-6E- GFP-CB-VBE plasmid.
  • Figure 14 illustrates the retroviral construct, BORIS III transcribes a transgene in the presence and absence of a ligand.
  • Figures 15A and B illustrate the construction of a PolyQ expression construct.
  • Figure 16 provides a comparison of various polyQ-GFP vectors constructed.
  • nucleic acid constructs encoding a receptor peptide comprising:
  • nucleic acid constructs encoding a receptor peptide comprising:
  • a DNA binding domain wherein the DNA binding domain is not obtained from the same member as the ligand binding domain and hinge region, wherein the receptor peptide activates a regulatory element in the absence of an exogenous dimer partner therefor and in the presence of a ligand for said ligand binding domain.
  • method (s) employing such constructs.
  • nucleic acid construct comprising a promoter and said exogenous nucleic acid(s) under the control of a regulatory element
  • a receptor peptide comprising a DNA binding domain, and the ligand binding domain and hinge region of a non-mammalian member of the nuclear receptor superfamily which is not normally present in the cells of said host, wherein said receptor peptide activates said regulatory element in the absence of an exogenous dimer partner therefor and in the presence of a ligand for said ligand binding domain,
  • said method comprising administering to said host an amount of ligand effective to modulate the transcription of said exogenous nucleic acid(s); wherein said ligand is not normally present in the cells of said host.
  • the terms “modulate” and “modulating” refer to the ability of a given ligand/receptor peptide complex to activate/deactivate and/or up-regulate/down-regulate transcription of exogenous nucleic acids, relative to the transactivation activity of said receptor peptide in the absence of ligand.
  • the actual effect of complex formation on the transactivation activity of a receptor peptide will vary depending on the specific ligand and DNA binding domains employed in the receptor peptide and on the regulatory element with which the ligand/receptor peptide complex interacts.
  • hosts refers to the cell, tissue, organ or organism in need of transcriptional regulation of exogenous or endogenous nucleic acids.
  • hosts are mammalian or mammalian derived cells or tissue.
  • Exemplary mammals include: humans; domesticated animals, e.g., rat, mouse, rabbit, canine, and feline; farm animals, e.g., chicken, bovine, porcine and ovine; and animals of zoological interest, e.g., monkey and baboon; and the like.
  • a "dimer partner” refers to members of the nuclear receptor superfamily to which other members preferentially bind to form heterodimeric species.
  • members of the nuclear receptor superfamily preferentially form heterodimers with a common partner, the retinoid X (or 9-cis retinoic acid) receptor (RXR, see, for example, Yu et al . (1991) Cell 67:1251-1266; Bugge et al . (1992) EMBO J. 11:1409-1418; Kliewer et al . (1992) Nature 355:446-449; Leid et al, (1992) Cell 68:377-395; Marks et al. (1992) EMBO J.
  • Receptor peptides utilized in the present invention are characterized as being fully functional in mammalian cells without the addition of any exogenous dimer partners therefor.
  • the presence of native level or concentration of endogenous dimer partner is sufficient to promote transcription.
  • the phrase "exogenous dimer partner” refers to a dimer partner for the receptor peptide that is introduced into the host . Exogenous dimer partner may be employed because the dimer partner is not present in the host cell (i.e., Usp) or because the dimer partner is required at elevated levels in the host (i.e., higher levels of RXR) .
  • endogenous dimer partner refers to dimer partners which are native to the unmodified mammalian host cell.
  • Invention receptors are advantageous because dimer partner is not necessary at elevated levels in order for formation of heterodimers to occur, thereby promoting transcription.
  • the invention receptor peptide comprises the hinge region and ligand binding domain derived from insect receptors of 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 . Biol .
  • presently preferred insect receptors from which the hinge region and/or ligand binding domain is derived substantially lack the C-terminal "F" domain.
  • presently preferred insect receptors share less than 50% sequence similarity with Drosophila ecdysone receptors, comparisons performed by Higgins and Sharp sequence alignment.
  • the term "hinge region” or "D domain” refers to the domain located between the DNA binding domain and the ligand binding domain of intact members of the nuclear receptor superfamily.
  • the hinge region facilitates high affinity interaction with dimer partners endogenous to mammalian cells, also referred herein as "endogenous dimer partners.”
  • endogenous dimer partners Preferably, the hinge region of a non-mammalian member of the nuclear receptor superfamily can be characterized as any sequence having substantial sequence identity with amino acid residues 273-362 set forth in SEQ ID NO: 3, or substantial portions thereof which confer sufficiently high affinity for endogenous dimer partner.
  • the hinge region can be functionally located in either orientation and at various positions within the receptor peptide.
  • the hinge region can be positioned at either the amino or carboxy terminus of the receptor peptide, or therebetween.
  • the hinge region is positioned internally between the ligand binding and DNA binding domains of the receptor peptide (see Figure IA) .
  • ligand binding domain of a non-mammalian member of the nuclear receptor superfamily refers to ligand binding domains derived from receptors which are not endogenous to a mammalian host.
  • Ligand binding domains which are not endogenous to a mammalian host include ligand binding domains which are modified to be non-responsive to ligands endogenous or native to the host .
  • Ligand binding domains contemplated for use according to the present invention can be derived from non-mammalian member (s) of the nuclear receptor superfamily which members are not normally present in the cells of a host.
  • Ligand binding domains are preferably derived from the carboxy-terminal portion of non-mammalian receptors which are capable of activating transcription of regulatory elements in the absence of exogenous dimer partner therefore.
  • Exemplary receptors which are not normally present in mammalian cells include insect receptors, plant receptors, and the like.
  • Ligand binding domains can be functionally located in either orientation and at various positions within the receptor peptide.
  • the ligand binding domain can be positioned at either the amino or carboxy terminus of the receptor peptide, or therebetween.
  • the ligand binding domain is positioned at the carboxy terminus of the receptor peptide (see Figure IA) .
  • Exemplary ligand binding domains can alternatively be characterized as comprising substantial sequence identity with amino acid residues 393-508 ("E 2 "), preferably amino acid residues 393-586 (E 2 and E 3 , wherein "E 3 " is amino acid residues 509-586), as set forth in SEQ ID NO : 3 , or substantial portions thereof (i.e., typically at least 46 or more contiguous nucleotides thereof) . It has been determined that the specific ligand response determinant of bombyx lies within the E 2 region of the native bR, whereas both E 2 and E 3 regions facilitate heterodimer formation with endogenous dimer partners.
  • Modifications of this sequence contemplated for use in the practice of the present invention include replacing several amino acids of the ligand binding domain with sequences from ligand binding domains of other members of the nuclear receptor superfamily, such as retinoic acid receptor and/or thyroid hormone receptor ( Figure IB) . These modifications provide unique transactivating characteristics and/or eliminate restriction sites, which facilitate the construction of useful peptide-based viral constructs.
  • DNA-binding domains contemplated for use in the preparation of invention receptor peptides are typically obtained from DNA-binding proteins (e.g., transcription factors) .
  • DNA-binding domain is understood in the art to refer to an amino acid sequence that is able to bind to DNA.
  • 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 regulatory element.
  • DNA-binding domains are known to function heterologously in combination with other functional domains by maintaining the ability to bind DNA recognition sequence (see, e.g., Brent and Ptashne, (1985) Cell , 43:729-736). For example, with respect to hormone receptors, DNA-binding domains are interchangeable, thereby providing numerous chimeric receptor proteins (see, e.g., U.S. Patent 4,981,784; and Evans, R. , (1988) .Science, 240:889-895). Similar to the ligand binding domain, the DNA-binding domain can be positioned at either the carboxy terminus or the amino terminus, or the DNA-binding domain can be positioned between the ligand binding domain and the activation domain. In preferred embodiments of the present invention, the DNA-binding domain is positioned internally between the ligand binding domain and the activation domain .
  • 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.
  • Exemplary DNA-binding proteins contemplated for use herein include transcription control proteins (e.g., transcription factors and the like; Conaway and Conaway, 1994, “Transcription Mechanisms and Regulation", Raven Press Series on Molecular and Cellular Biology, Vol. 3, Raven Press, Ltd., New York, NY) .
  • 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, Harrison, S., "A Structural Taxonomy of DNA-binding Domains," Nature, 353:715-719.
  • Homeobox D ⁇ A- binding proteins suitable for use herein include, for example, HOX, STF-1 (Leonard et al . , 1993, Mol . Endo . , 7:1275-1283), Antp, Mat ⁇ -2, I ⁇ V, and the like. See, also,
  • Suitable zinc finger D ⁇ A-binding proteins for use herein include Zif268, GLI, XFin, and the like. See also, Klug and Rhodes (1987) Trends Biochem . Sci . , 12:464; Jacobs and Michaels (1990) New Biol . , 2:583; and Jacobs (1992), EMBO J. , 11:4507-4517.
  • the D ⁇ A 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, with the 147 amino terminal amino acid residues of yeast GAL4 being presently most preferred.
  • D ⁇ A-binding domain(s) used herein is (are) obtained from a member of the nuclear receptor superfamily.
  • the phrase "member (s) of the nuclear receptor superfamily” also known as “intracellular receptors” or “steroid/thyroid hormone superfamily of receptors” refers to hormone binding proteins that operate as ligand-dependent transcription factors, including identified members of the nuclear receptor superfamily for which specific ligands have not yet been identified (referred to hereinafter as "orphan receptors" .
  • Exemplary members of the steroid/thyroid hormone superfamily of receptors include steroid receptors such as glucocorticoid receptor (GR) , mineralocorticoid receptor (MR) , estrogen receptor (ER) , progesterone receptor (PR) , androgen receptor (AR) , vitamin D 3 receptor (VDR) , various isoforms of peroxisome proliferator-activated receptors (PPARs) , and the like; plus retinoid receptors, such as the various isoforms of retinoic acid receptor (e.g., RAR , RAR ⁇ , or RARy) , the various isoforms of retinoid X receptor (e.g., RXR ⁇ , RXR ⁇ , or RXRy) , and the like (see, e.g., U.S.
  • GR glucocorticoid receptor
  • MR mineralocorticoid receptor
  • ER estrogen receptor
  • PR progesterone receptor
  • TR thyroid receptor
  • TR ⁇ such as TR ⁇ , TR ⁇ , and the like
  • insect derived receptors such as the ecdysone receptor, and the like
  • other gene products which, by their structure and properties, are considered to be members of the superfamily, as defined hereinabove, including the various isoforms thereof.
  • orphan receptors contemplated for use herein as a source of DNA binding domain include HNF4 (see, for example, Sladek et al . , in Genes & Development 4: 2353-2365 (1990)), the COUP family of receptors (see, for example
  • COUP-like receptors and COUP homologs such as those described by Mlodzik et al . , in Cell 60: 211-224 (1990) and Ladias et al . , in Science 251: 561-565 (1991) , the insect derived knirps and knirps- related receptors, and the like.
  • the DNA-binding domains of all members of the 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:
  • X designates non-conserved amino acids within the DNA-binding domain; an asterisk denotes the amino acid residues which are almost universally conserved, but for which variations have been found in some identified hormone receptors; and the residues enclosed in parenthesis are optional residues (thus, the DNA-binding domain is a minimum of 66 amino acids in length, but can contain several additional residues) .
  • DNA-binding domains which are engineered with novel DNA-recognition specificity (see, e.g., Pomerantz et al . Science 267:93-96, 1995, ZFHD1, an engineered transcription factor with a composite DNA- binding domain) are also contemplated.
  • Two polynucleotides or polypeptides are said to be "identical” if the sequence of nucleotides or amino acid residues in the two sequences is the same when aligned for maximum correspondence.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl . Math . , 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J “ . Mol . Biol . , 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc . Natl . Acad. Sci .
  • the percentage of sequence identity between two sequences is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • a preferred method for comparing sequences uses the GAP program based on the algorithm of Needleman, et at., supra. Typically, the default values for all parameters are selected. These are gap weight: 5.0, length weight: 0.30, average match: 1.0, and average mismatch: 0.0.
  • the phrase "substantial sequence identity" refers to nucleotide or amino acid sequences which share at least 80% sequence identity, preferably 90%, more preferably 95 % or more, regardless of the algorithm used to determine sequence identity, compared to a reference sequence over a comparison window of about 20 bp to about 2000 bp, typically about 50 to about 1500 bp, usually about 350 bp to about 1200.
  • the values of percent identity are preferably determined using the GAP program, referred to above. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. It is recognized, however, that proteins (and DNA or mRNA encoding such proteins) containing less than the above- described level of homology produced as splice variants or as a result of conservative amino acid substitutions (or substitution of degenerate codons) are contemplated to be within the scope of the present invention.
  • sequences that have slight and non-consequential sequence variations from the actual sequences disclosed herein refers to sequences that have slight and non-consequential sequence variations from the actual sequences disclosed herein. Species that are substantially the same are considered to be equivalent to the disclosed sequences.
  • sequences that are substantially the same as the DNA, RNA, or proteins disclosed and claimed herein are functionally equivalent to the sequences disclosed and claimed herein. Functionally equivalent sequences will function in substantially the same manner to produce substantially the same effect as the sequences disclosed.
  • Receptor peptides employed in the present invention can be modified by the introduction of activation domains.
  • 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.
  • Activation domains contemplated for use herein 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 a suitable ligand binding domain.
  • An activation domain can be positioned at any convenient site within the receptor peptide, i.e., at the carboxy terminus, the amino terminus or between the ligand binding domain and the DNA binding domain. In preferred embodiments of the present invention, the activation domain is positioned at the amino terminus of the receptor peptide.
  • Suitable activation domains can be obtained from a variety of sources, e.g., from the N-terminal region of a member of the steroid/thyroid hormone superfamily of receptors, from a transcription factor activation domain, such as, for example, VP16, GAL4 , NF- ⁇ B or BP64 activation domains, and the like.
  • a transcription factor activation domain such as, for example, VP16, GAL4 , NF- ⁇ B or BP64 activation domains, and the like.
  • the presently most preferred activation domain contemplated for use in the practice of the present invention is obtained from the N-terminal region of the VP16 protein.
  • the invention nucleic acid construct further comprises promoters and regulatory elements operatively associated with exogenous nucleic acids.
  • receptor peptide in the presence of a ligand therefor, binds the regulatory element and activates transcription of the exogenous nucleic acids.
  • Regulatory elements contemplated for use in the practice of the present invention include elements responsive to the invention receptor peptide.
  • such elements are exogenous regulatory elements not normally present in the cells of the host.
  • One class of exogenous regulatory elements contemplated for use herein includes hormone response elements which modulate transcription of exogenous nucleic acid when bound to the DNA binding domain of an invention receptor peptide.
  • Additional regulatory elements that may be utilized in the practice of the present invention include exogenous regulatory elements responsive to a non-mammalian transactivator .
  • One such transactivator-responsive regulatory element is an operator which confers responsiveness to antibiotics.
  • Exemplary operators contemplated for use in this aspect of the invention include the tetracycline-analog regulated operator, the TET operator, the Lac operator, and the like.
  • Exogenous response elements contemplated for use herein are short cis-acting sequences (i.e., having about 12-20 bp) that are required for activation of transcription in response to association with a suitable ligand, such as diacyl hydrazines, and an invention receptor peptide.
  • Response element sequences contemplated for use herein function in a position- and orientation-independent fashion.
  • Exemplary response elements include hormone response elements, GAL4 response elements and the like.
  • Hormone response elements contemplated for use in the practice of the present invention are response elements which are responsive to members of the nuclear receptor superfamily. These response elements comprise at least two half-sites (in either direct repeat or inverted repeat orientation to one another) , separated by a spacer of 0-5 nucleotides. As used herein, the term "half-site" refers to a contiguous 6 nucleotide sequence that is bound by a particular member of the nuclear receptor superfamily. Typically, two half-sites with a corresponding spacer make up a hormone response element. Hormone response elements can be incorporated in multiple copies into various transcription regulatory regions.
  • Preferred hormone response elements employed in the practice of the present invention comprise a first half- site and a second half-site, separated by a spacer of 0-5 nucleotides;
  • each half-site has the sequence:
  • Exemplary half-sites having the -RGBNNM- motif for use in preparing response elements useful in the practice of the present invention include, for example, half-sites selected from -AGGGCA- , -AGTTCA- , -AGGTAA- , -AGGTCA- , -GGTTCA-, -GGGTTA-, -GGGTGA- , -AGGTGA- , -GGGTCA- , and the like.
  • a particularly preferred first half-site is -AGTGCA- .
  • GAL4 response element Additional response elements included the GAL4 response element.
  • Exemplary GAL4 response elements are those containing the palindromic 17-mer: 5' -CGGAGGACTGTCCTCCG-3' (SEQ ID NO: 4),
  • Regulatory elements employed in the practice of the present invention are operably linked to a suitable promoter for transcription of exogenous nucleic acid(s) product(s) .
  • exogenous nucleic acid(s) operatively linked to a suitable promoter, is (are) introduced into the cells of a suitable host, expression of the exogenous nucleic acid(s) is (are) controlled by the presence of ligands, which are not normally present in the host cells.
  • exogenous to said mammalian host refers to nucleic acids not naturally found at levels sufficient to provide a function in the particular cell where transcription is desired.
  • exogenous 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 vi tro applications) , or the nucleic acids of interest can be introduced directly or indirectly into a host 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.
  • Exogenous 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, respectively.
  • a synthetic or natural gene coding for human insulin would be exogenous 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 which 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 which 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.
  • Exogenous 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 said 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 said 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 or nucleotides which characterize specific conditions or diseases (i.e., CAG repeat expansion and concomitant polyglutamine (polyQ) expression has been linked to a variety of neurodegenerative conditions including Huntington' s disease, dentatorubro-pallid
  • Additional nucleic acids contemplated for use in accordance with the present invention include genes which 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 exogenous nucleic acid(s) contemplated for use employed 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 either obtain the corresponding nucleic acid molecule directly from a public depository or 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
  • Selectable markers contemplated for use in the practice of the present invention include radiolabeled molecules, fluorescent molecules, ligands, enzymes, and the like.
  • Preferable selectable markers are enzymes such as antibiotic resistance genes, genes which enable cells to process metabolic intermediaries, and the like.
  • Exemplary antibiotic resistance genes include genes which impart tetracycline resistance, genes which impart ampicillin resistance, zeomycin resistance, neomycin resistance, hygromycin resistance, puromycin resistance, and the like.
  • Genes which 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 invention nucleic acid construct further comprises, in addition to the receptor peptide, a non-mammalian transactivator, not a member of the nuclear receptor superfamily and not normally present in the cells of said host, and a compatible transactivator responsive regulatory element not normally present in cells of said host.
  • the transactivator responsive regulatory element controls transcription of the exogenous nucleic acid(s) or a second nucleic acid construct comprising a second exogenous nucleic acid(s).
  • Examples of transactivator responsive regulatory elements include operators which are responsive to non-mammalian transactivators which confer responsiveness to antibiotics.
  • Exemplary operators contemplated for use in this aspect of the invention include the tetracycline-analog regulated operator, the TET operator, the Lac operator, and the like.
  • the transactivator responsive regulatory elements employed in the practice of the present invention are operably linked to a suitable promoter for transcription of exogenous nucleic acid(s) proteins.
  • Non-mammalian transactivators other than members of the nuclear receptor superfamily, contemplated for use in the practice of the present invention function in the absence of exogenous dimer partner.
  • Examples of transactivators that typically function in the absence of exogenous dimer partner are tetracycline-controlled transactivators, Vpl6-Lac fusion transactivators, and the like.
  • the transactivator can be positioned at any convenient site within the construct, i.e., at the carboxy terminus or the amino terminus of the transactivating construct. In preferred embodiments of present invention, the transactivator is positioned at the amino terminus of the transactivating construct ( Figure 1C) .
  • the non-mammalian transactivator confers responsiveness to antibiotics.
  • An example of a non- mammalian transactivator which confers responsiveness to antibiotics, as contemplated for use in the practice of the present invention is the tetracycline-controlled transactivator.
  • the tetracycline inducible system is well- known in the art (see, e.g, Gossen et al . (1992) PNAS 89, 5547-5551; Gossen et al . (1993) TIBS 18, 471-475; Furth et al. (1994) PNAS 91, 9302-9306; Shockett et al .
  • Non-mammalian transactivators well known in the art include the IPTG inducible system based on a VP16-Lac repressor fusion which functions through lac operator sequences inserted into heterologous promoters (see, e.g., Baim et al . (1991) PNAS 88:5072- 5076) .
  • transactivators can be used herein, 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, Harrison, S., "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 . , (1993) Mol . Endo .
  • Suitable zinc finger DNA-binding proteins for use herein include Zif268, GLI , XFin, and the like. See also, Klug and Rhodes (1987) Trends Biochem. Sci . , 12:464; Jacobs and Michaels (1990) New Biol . , 2:583; and Jacobs (1992) EMBO J. , 11:4507-4517.
  • expression cassettes are prepared by operably linking invention nucleic acid constructs to a suitable promoter for expression of the encoded receptor peptide.
  • 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 use in the practice of the present invention include inducible (e.g., minimal CMV promoter, minimal TK promoter, modified MMLV LTR) , constitutive (e.g., chicken b-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 b-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; andU.S. #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 l (EFl ) 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, phophoglycerokinase (PGK) promoter, Stfl promoter, Low Density Lipoprotein (LDL) promoter, chicken b-actin promoter (used in conjunction with ecdysone response element) and the like.
  • MMLV Moloney murine leukemia virus
  • tissue specific refers to the substantially exclusive initiation of transcription in the tissue from which a particular promoter, which drives expression of a given gene, 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
  • constructs comprising a promoter, a tetracycline-controlled transactivator, a VP16 activation domain, a DNA binding domain and bombyx mori-derived hinge region and ligand binding domain encoding sequence, wherein the components of the construct are operatively associated with the other components of the construct.
  • 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 linkage of DNA to 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.
  • constructs comprising nucleic acid(s) encoding a a VP16 activation domain operatively associated with nucleic acid encoding a DNA binding domain and bombyx mori-derived hinge region and ligand binding domain.
  • gene transfer vectors useful for the introduction of invention constructs into suitable host cells preferably comprise a first reporter under the control of a regulatory element, a second reporter under the control of an operator which is responsive to a ligand-mediated receptor which confers responsiveness to antibiotics, and a construct comprising a promoter and nucleic acid encoding a VP16 activation domain, a DNA binding domain and bombyx mori-derived hinge region and ligand binding domain, optionally, a tetracycline-controlled transactivator.
  • the number of copies of regulatory elements can readily be varied by those of skill in the art.
  • transcription regulatory regions can contain from 1 up to about 50 copies of a particular regulatory element, preferably 2 up to about 25 copies, more preferably 3 up to about 10-15 copies, with about 4-6 copies being especially preferred.
  • Gene transfer vectors contemplated for use herein are recombinant nucleic acid molecules that are used to transport invention nucleic acid constructs 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 include a 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.
  • a recombinant DNA or RNA construct 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.
  • 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 ligand/receptor peptide complex.
  • a minimal promoter when combined with a regulatory element functions to initiate mRNA transcription in response to a ligand/receptor peptide complex.
  • transcription typicallywill not occur unless the required inducer (ligand therefor) is present.
  • the transcription regulatory region further comprises a binding site for ubiquitous transcription factor (s) .
  • s ubiquitous transcription factor
  • 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.
  • Expression vectors suitable for use in the practice of the present invention are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells as well as those that remain episomal and those that integrate into the host cell genome. Expression vectors typically further contain other functionally important nucleic acid sequences encoding antibiotic resistance proteins, and the like.
  • Exemplary eukaryotic expression vectors include eukaryotic constructs, such as the pSV-2 gpt system
  • a gene transfer vector contemplated for use herein is a viral vector, such as Adenovirus, adeno-associated virus, or herpes-simplex virus based vectors, and synthetic vectors for gene therapy, and the like (see, e.g., Suhr et al . (1993) Arch , of Neurol . 50:1252-1268).
  • Adenovirus and adeno-associated virus are extremely suitable as gene transfer vectors to produce receptor peptides.
  • a gene transfer vector employed herein is a retroviral vector. Retroviral vectors are gene transfer plasmids that have an expression construct containing an exogenous nucleic acid residing between two retroviral LTRs .
  • Retroviral vectors typically contain appropriate packaging signals (e.g., a retroviral psi ( ⁇ ) packaging signal) , elements which enable invention construct integration into a host cell (e.g., a 5' and/or a 3 ' long terminal repeat (LTR) ) , and/or genes encoding proteins required for retroviral packaging (e.g., the pol gene, the gag gene and the env gene) , that enable the retroviral vector, 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) .
  • appropriate packaging signals e.g., a retroviral psi ( ⁇ ) packaging signal
  • elements which enable invention construct integration into a host cell e.g., a 5' and/or a 3 ' long terminal repeat (LTR)
  • LTR long terminal repeat
  • genes encoding proteins required for retroviral packaging
  • retroviral vectors for use herein are described, for example, in U.S. Patents 5,399,346 and 5,252,479; and in WIPO 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 their 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., ⁇ aldini et al . (1996) Science 272:165-320), and the like.
  • helper cells which produce retroviral vector particles which 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 . , Molecular and Cellular Biology, 3 (12) : 2241-2249 (1983); Danos, 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, Human Gene Therapy, 1:5-14 (1990); Markowitz, et al . , Journal of Virology, 61(4) :1120- 1124 (1988); Watanabe, et al . , Molecular and Cellular Biology, 3 (12) :2241-2249 (1983); Danos, et al . , PNAS, 85:6460-6464 (1988); and Bosselman, et al . , Molecular and Cellular Biology, 7 (5) : 1797-1806 (1987).
  • a recombinant retroviral vector can be utilized to express receptor peptide.
  • the retroviral vector will further comprise a regulatory element and exogenous nucleic acid under the control of the regulatory element.
  • the retroviral vector can express an antibiotic resistance gene
  • a "covector” can also be utilized to provide a nucleic acid construct comprising the promoter, the regulatory element and exogenous nucleic acid and a second antibiotic resistance gene.
  • the co-vector carrying exogenous nucleic acid also has LTRs modified to promote high-level expression of exogenous nucleic acid (s) only in the presence of the receptor peptide encoded by the recombinant retrovirus and exogenous 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 exogenous nucleic acid.
  • invention nucleic acid constructs allows the stable integration and expression of an exogenous nucleic acid(s) or transgene into a wide variety of cultured cell types.
  • recombinant cells containing invention nucleic acid constructs encoding receptor peptides as described herein, optionally further containing regulatory elements operatively associated with exogenous nucleic acid(s).
  • recombinant cells constructs comprising a regulatory element, such as the bombyx mori receptor response element, operatively associated with exogenous nucleic acids, optionally further containing invention receptor peptides.
  • the amount of exogenous nucleic acid introduced into a host can be varied by those of skill in the art.
  • the amount of nucleic acid introduced can be increased by increasing the amount of plaque forming units (PFU) of the viral vector.
  • PFU plaque forming units
  • transducing Suitable means for introducing (transducing) expression vectors containing invention nucleic acid constructs into host cells to produce transduced recombinant cells (i.e., cells containing recombinant heterologous nucleic acid) are well-known in the art (see, for review, Friedmann, (1989) Science, 244:1275-1281; Mulligan, (1993) Science, 260:926-932, each of which are incorporated herein by reference in their entirety) .
  • Exemplary methods of transduction 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.
  • 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.
  • Exemplary eukaryotic cells suitable for introducing invention expression vectors include, e.g., CV-1 cells, P19 cells and NT2/D1 cells (which are derived from human embryo carcinomas) , ES cells (embryonic stem cells) , COS cells, mouse L cells, Chinese hamster ovary (CHO) cells, primary human fibroblast cells, human embryonic kidney cells, African green monkey cells, HEK 293 (ATCC accession #CRL 1573; U.S. Patent No. 5,024,939), Ltk " cells (ATCC accession #CCL1.3), COS-7 cells (ATCC under accession #CRL 1651), DG44 cells (dhfr- CHO cells; see, e.g., Urlaub et al .
  • cultured primary tissues such as hcn/v-myc (a tetracycline sensitive retroviral vector selected by G418 resistance)
  • neuronal progenitor or precursor cells such as hcn/v-myc (a tetracycline sensitive retroviral vector selected by G418 resistance)
  • neuronal cells lines such as cerebellum derived neuronal precursors and PC12 cells, neurons, primary astrocytes, oligodendrocytes, and the like.
  • Presently preferred cells include CV-1 and 293 cells.
  • Invention nucleic acid constructs may be stably incorporated into cells or may be transiently expressed using methods known in the art.
  • 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 reporter gene such as the E. coli ⁇ -galactosidase gene
  • nucleic acid construct comprising a promoter and said exogenous nucleic acid(s) under the control of a regulatory element
  • nucleic acid encoding a receptor peptide comprising a DNA binding domain, and the ligand binding domain and hinge region of a non- mammalian member of the nuclear receptor superfamily which is not normally present in the cells of said host, wherein said receptor peptide activates said regulatory element in the absence of an exogenous dimer partner therefor and in the presence of a ligand for said ligand binding domain .
  • transgenic animal refers to an animal that contains one or more inheritable expression constructs containing one or more exogenous nucleic acid(s) under the transcription control of an operator and/or hormone 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, a receptor peptide as described above (e.g., VbR). Tissue specificity is conferred by the selection of a tissue-specific promoter
  • transgenic animals are produced by the transfection/infection of embryonic stem cells which are employed to produce invention transgenic animals employing methods known to those of skill in art.
  • an invention transgenic animal contains one or more expression constructs containing nucleic acid encoding receptor peptide and exogenous nucleic acid under the transcription control of a regulatory element.
  • nucleic acid construct comprising a promoter and said exogenous nucleic acid(s) under the control of a regulatory element
  • nucleic acid encoding a receptor peptide comprising a DNA binding domain, and the ligand binding domain and hinge region of a non- mammalian member of the nuclear receptor superfamily which is not normally present in the cells of said host, wherein expression of said receptor peptide is under the control of an inducible promoter, wherein said receptor peptide activates said regulatory element in the absence of an exogenous dimer partner therefor and in the presence of a ligand for said ligand binding domain;
  • said method comprising subjecting said host to conditions suitable to induce expression of said receptor peptide .
  • nucleic acid construct comprising a promoter and exogenous nucleic acid(s) which express said recombinant product under the control of a regulatory element; wherein said regulatory element is not normally present in the cells of said host, and
  • nucleic acid encoding a receptor peptide comprising a DNA binding domain, and the ligand binding domain and hinge region of a non- mammalian member of the nuclear receptor superfamily which is not normally present in the cells of said host, wherein said receptor peptide activates said regulatory element in the absence of an exogenous dimer partner therefor and in the presence of a ligand for said ligand binding domain, and
  • 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.
  • inducible expression of a toxin such as the diptheroid toxin would allow for specific ablation of tissue (Ross et al . Genes and Development 7:1318-1324 (1993)).
  • the numerous gene products that are 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) .
  • high level expression of 79-amino acid polyglutamine tracts in cells results in apoptosis and rapid cell death.
  • nucleic acid constructs and cell lines which express a polyglutamine expression cassette (PQEC) under the control of an exogenous ligand to maximize obtaining stable transfected/infected cultured cells which do not undergo undesired apoptosis.
  • PQEC polyglutamine expression cassette
  • Expression of polyglutamine tracts separately from the huntingtin protein facilitates identification of the toxic properties associated with the polyglutamine tracts themselves.
  • methods for employing these cell lines to identify novel proteins that could target developing intracellular aggregates (IAs) and either block subsequent growth or facilitate or mark the IAs for degradation are provided.
  • methods for modulating the transcription of nucleic acid(s) in an in vi tro system comprising administering to said system an amount of a ligand effective to modulate the transcription of said nucleic acid(s); wherein said ligand is not normally present in said cellular system; wherein said system comprises:
  • nucleic acid construct comprising a promoter and said nucleic acid(s) under the control of a regulatory element
  • nucleic acid encoding a receptor peptide comprising a DNA binding domain, and the ligand binding domain and hinge region of a non- mammalian member of the nuclear receptor superfamily which is not normally present in the cells of said host, wherein said receptor peptide activates said regulatory element in the absence of an exogenous dimer partner therefor and in the presence of a ligand for said ligand binding domain,
  • nucleic acid construct comprising a promoter and said exogenous nucleic acid(s) under the control of a regulatory element ;
  • nucleic acid encoding a receptor peptide comprising a DNA binding domain, and the ligand binding domain and hinge region of a non- mammalian member of the nuclear receptor superfamily which is not normally present in the cells of said host, wherein expression of said receptor peptide is under the control of an inducible promoter, wherein said receptor peptide activates said regulatory element in the absence of an exogenous dimer partner therefor and in the presence of a ligand for said ligand binding domain . administering, to said host, ligand for said ligand binding domain.
  • in vivo delivery refers to 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.
  • ligand refers to a non-steroidal substance or compound which, in its native state (or after conversion to its "active" form), binds to the receptor peptide, thereby creating a ligand/receptor peptide 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 . In accordance with one aspect of the present invention, unless and until a suitable ligand is administered to the host, substantially no transcription of the desired exogenous nucleic acids occurs .
  • Preferred ligands contemplated for use in the practice of the present invention are ligands characterized as not normally present in the cells of the host to be treated. Such ligands are referred to as being exogenous to the host.
  • An example of a class of ligands not naturally present in mammalian systems are compounds referred to as hydrazines, preferably diacyl hydrazines.
  • Hydrazines contemplated for use in the present invention include compounds which are readily available and are/or relatively inexpensive to manufacture.
  • One such compound, tebufenozide is a non-steroidal ecdysone agonist which is used commercially as an insecticide. 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 mimics of the naturally occurring ecdysones, i.e., synthetic organic compounds which have transactivation activity characteristic of the naturally occurring ecdysones.
  • Examples of such compounds, referred to herein as ecdysone mimics include 1,2 -diacyl hydrazines (e.g., tebufenozide and others described in U.S. Patent Nos.
  • N' -substituted-N,N' -di-substituted hydrazines e.g., those described in U.S. Patent No. 5,117,057, the entire contents of which are hereby incorporated by reference herein
  • dibenzoylalkyl cyanohydrazines e.g., those described in European Application No. 461,809, the entire contents of which are hereby incorporated by reference herein
  • N-substituted-N-alkyl-N,N' -diaroyl hydrazines e.g., those described in U.S.
  • diacyl hydrazines are neither toxic, teratogenic, nor known to affect mammalian physiology, they are ideal candidates for use as inducers in cultured cells and transgenic mammals according to invention methods.
  • Ligands are administered in a manner compatible with the 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 micrograms 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 (e.g., diacyl hydrazine (s) ) required to achieve the desired level of transcription and/or translation of exogenous nucleic acid.
  • a therapeutically effective amount is typically an amount of a ligand or ligand precursor that, when administered in a physiologically acceptable composition, is sufficient to achieve a plasma concentration of the transcribed or expressed nucleic acid product from about 0.1 ⁇ g/ml to about 100 ⁇ g/ml, preferably from about 1.0 ⁇ g/ml to about 50 ⁇ g/ml, more preferably at least about 2 ⁇ g/ml and usually 5 to 10 ⁇ g/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 receptor peptide.
  • such ligands can be administered topically, orally, intravenously, mtraperitoneally, mtravascularly, 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.
  • compositions 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 as discussed hereinafter.
  • 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.
  • nucleic acid construct comprising a promoter and exogenous nucleic acid(s) under the control of a regulatory element
  • nucleic acid encoding a receptor peptide comprising a DNA binding domain, and the ligand binding domain and hinge region of a non- mammalian member of the nuclear receptor superfamily which is not normally present in the cells of said host, wherein said receptor peptide activates said regulatory element in the absence of an exogenous dimer partner therefor and in the presence of a ligand for said ligand binding domain;
  • the exogenous nucleic acid is introduced directly into cells obtained from a donor (host or separate donor) . Such cells are then implanted within the host.
  • 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 .
  • 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 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.
  • the VP16 t-domain (activation domain) is fused in frame onto bombyx mori-derived nuclear receptor (bR) at an internal Mlul site near the N-terminus (VbR, Figure IA) .
  • An Ascl site immediately downstream of the ATG start codon allows multimerization of VP16 domains for VbRs with multiple VP16 activation domains.
  • bR has virtually no C-terminal domain downstream of the LBD.
  • Several amino acids of the hormone binding domain are replaced with sequences from the retinoic acid receptor or the thyroid hormone receptor (VbRRA and VbRTR, respectively) (Figure IB) .
  • These mutant variations have properties slightly different from VbR, and in addition have silent mutations that eliminate Bell and BstBl sites in VbR. These are useful in the construction of many VbR-based retroviral constructs .
  • VbR Tebufenozide/Tetracycline Modulated Transactivator
  • TTMT Tebufenozide/Tetracycline Modulated Transactivator
  • This protein functions jointly as a ligand-modulated regulator of gene expression from both tetO and EcRE (ecdysone response element) -containing promoters either separately or simultaneously.
  • This compact protein confers constitutive activation of promoters containing tetracycline operators (tetOs) in the absence of tetracycline/analogs .
  • tetOs tetracycline operators
  • the VbR half of the chimeric protein is constitutively inactive on EcRE-containing promoters, but may be stimulated to transactivate responsive promoters to a high level by the addition of muristerone A (murA) or tebufenozide (teb) .
  • two separate responsive promoters may be simultaneously regulated by two separate ligands through a single protein. Additional variants using the reverse-Tet-transactivator, other VbR variants, novel response elements, and other regulatory proteins, will presumably result in further customized variations on this same theme.
  • a single transgenic animal generated with the TTMT protein would be simultaneously responsive to transgene regulation of two separate transgenes by two separate ligands. Further, individual promoters can be produced that respond to both halves of the protein, allowing the transgene to be regulated by both ligands, at the discretion of the investigator.
  • Transient transfection experiments are carried out in CV-1 cells or 293 cells using modified drosophila ecdysone receptor (VdEcR) or modified bombyx hormone receptor (VbR) with or without dimer partners.
  • Luc activity is examined 40 hours after transfection and stimulation with ligand (Figure 2A) .
  • Both receptors are assayed in CV-1 cells with 1 ⁇ M MurA and no exogenous dimer partner.
  • bR responds, while dEcR does not.
  • Both receptors are also assayed in the presence of 5 ⁇ g/ml tebufenozide ( Figure 2B) .
  • VbR responds with greater than 200-fold induction while drosophila EcR does not respond at all, and in fact, decreases slightly. Both receptors are also assayed with and without dimer partners. VbR is stimulated with 5 ⁇ g/ml tebufenozide and VdEcR is stimulated with 1 ⁇ M MurA. Note that RXR and Usp are necessary for VdEcR while VbR is constitutively activated by the presence of exogenous dimer partner and is inhibited by ligand addition. VbR, with no exogenous dimer partner, has an extremely low base-line which is stimulated 200-300 fold by ligand. Note also from this experiment that VdEcR tends to have a much higher baseline than VbR without dimer partner.
  • Tebufenozide is administered to cells co-transfected with nucleic acids encoding receptors : LINX (which encodes the tet-transactivator (TTA) ) , VbR, TTMT (which encodes the TTA-VbR fusion protein, and TTMT-2V which is like TTMT, but with two VP16 t-domains) to assay for EcRE-Luc activity (Figure 2C) . Note that teb stimulates VbR, and the TTMT fusions, but has no action on TTA (LINX) .
  • TTA tet-transactivator
  • Transgene regulation by teb or MurA in stably infected rat fibroblasts is assayed utilizing recombinant retroviral constructs as the reporter, and VbR encoding retroviral vectors to provide the receptor.
  • Bulk infected selected populations stimulated with 2.5 ⁇ g/ml teb or 1 ⁇ M MurA indicate b-gal or tyrosine hydroxylase infected cells.
  • Quantitative analysis of an individual VbR-luc clonal fibroblast line, and a VbR-GH clonal line show that 0.25 ⁇ g/ml teb is as effective at stimulating full activity as higher levels (Figure 3) .
  • the plasmid, pBO utilizes a responsive internal promoter to regulate transgene expression and also contains a VbR-responsive 3 ' LTR to autoregulate the VbR receptor itself ( Figure 4) .
  • the plasmid, pNA is constructing using a 3 ' -responsive LTR to regulate transgene expression.
  • pBO utilizes an EcR responsive internal promoter and a TTA responsive 3' LTR. Both are host to regulation by the TTMT protein, simultaneously. The purpose of this design is to provide chronic stimulation of the duplicated 5' LTR along with regulated expression of the internal EcR responsive promoter. By removing all of the native promoting sequences from the 6To LTR with the exception of the TetO's, variants of this construct are also inactivatable .
  • VbR and TTMT systems including constitutive and inducible promoters are integrated into a modular plasmid vector known as "pBW" ( Figure 5) .
  • the purpose of pBW is to integrate all of the elements of bombyx receptor- or tetracycline-analog responsiveness into a single plasmid vector destined for use in producing transgenic animals by methods such as pronuclear injection.
  • pBW is to facilitate construction of transgenic animals or stably transfected cells using VbR variants.
  • tissue-specific expression cassette to produce the receptor and/or antibiotic resistance genes.
  • the second is an expression cassette with a responsive promoter and a transgene.
  • Polyadenylation signals flank all of the expression cassettes to provide efficient polyadenylation (p (A) ) of transcribed RNAs .
  • p (A) polyadenylation
  • An adjunct shuttle vector derived from pBSK (Stratagene) called SKSP contains rare sites flanking the polylinker and compatible with pBW.
  • pBW also shares a number of sites with pBO to further simplify construction.
  • pBW is constructed within the plasmid pcDNA3 (Invitrogen) .
  • Recombinant retrovirus starts as a polylinker composed of 5' Notl -Mlul - NruI - EcoRI -AscI - Pml l -Bs tB I -BamHI -Hindl l l -ffpal - CIal -Nsil - Kpnl 3' sites.
  • the retroviral ⁇ gin (see Adam and Miller in J. Virol . 62:3802 (1988) and Barklis et al . , in Cell 47:391 (1986)) is inserted into sites of the recombinant retroviral polylinker.
  • the internal CMV promoter is inserted BamHI-Clal into the BamHI-Clal sites of the evolving recombinant retroviral vector.
  • All LTRs destined for insertion into the 5' location are produced by low- cycle/high-fidelity PCR production and end primers with compatible Notl - lul sites for insertion into the Notl - lul sites.
  • 3' LTRs are generated with Clal -Nsil compatible ends for insertion into these sites.
  • the R region of the 3' LTR is included in the downstream primer sequence.
  • Transgenes, receptor cD ⁇ As, or selectable marker genes are inserted into the remaining polylinker from the EcoRI site through the BamHI site, or the remaining polylinker after the internal promoters (Hindlll -Clal) .
  • the resulting vectors are characterized by restriction digests and mapping, amplified by large-scale plasmid preparation, and prepared for transfection into packaging cells by standard methods.
  • Transient retroviral production in 293 and 293T cells has been previously described (see Pear et al . , in PNAS 90:8392 (1993)).
  • the conditioned media is removed, filtered through 0.45 mm filters, and frozen at -70°C until use. 10-cm dishes of primary-cultured Fisher rat abdominal fibroblasts (Rosenberg et al .
  • the short (24 hour) course is performed in 24 -well Costar dishes with cells at approximately 80% confluency. 5 ⁇ l of ligand (final concentration of 1 ⁇ M) or vehicle (20% EtOH) is added to the 1 ml of media in each well. Eight hours after first ligand administration, the cells are all washed in PBS and the media is replaced. ⁇ - galactosidase histochemical reaction is performed on 1.5% glutaraldehyde-fixed cells essentially as described by Shimohama et al . in Brain Res Mol Brain Res 5:271 (1989) in a 37°C environment for 2 hours.
  • the 4-day time course is performed in an identical fashion, except that the cell population is plated at only 20% confluency at the start of the experiments (and grow to near-total confluency by the end of the 4 day period) and neither ligand nor media is changed or replaced.
  • MMBG fibroblasts are plated in triplicate at high density in 24 -well plates.
  • 1 ⁇ M ligand or vehicle is added to one group of plates, while others are left completely untreated.
  • the wells are then placed in a 37°C- 10% C0 2 environment for the next 25 days without any media changes.
  • the plates that had received neither vehicle nor ligand on day 1 are removed and half of the wells are given vehicle and the other half are given 1 ⁇ M ligand for 40 hours.
  • all of the plates are processed for ⁇ gal histochemical staining as above.
  • MMGH fibroblasts are produced by infecting a fibroblast line with the recombinant retroviral vectors containing desired elements. Cells are selected under both antibiotics as described previously. After preliminary examination of ligand-induced hGH production in the bulk population, the MMGH population is plated at high density in 6-well Costar plates. Forty-eight hours after the initial plating and when the cells are essentially completely growth inhibited by contact, the medium of all plates is replaced with DME containing 2% FBS . Under these conditions, primary rat fibroblasts stop dividing even if they are not confluent, and settle out into a distended morphology with prominent nuclei characteristic of severely growth-arrested fibroblasts.
  • Control wells are TH (tyrosine hydroxylase) producing fibroblasts, but display all of the characteristics of the recombinant retrovirus except for the hGH transgene and secreted protein.
  • VBR (or CVBR) when referring to the retrovirus) was produced by insertion of VP16 (14) sequences with a synthetic ATG start codon into the amino-terminal region of BE up to, and in-frame with, the lul site (corresponding to amino acid 26).
  • VP16 primers (5') 5 ' -GAGAGAAGCTTATGGCGCGCCCGACCGATG (SEQ ID NO: 5) and (3') 5 ' -CACACACGCGTGTACTCGTCAATTCCAAG (SEQ ID NO: 6) .
  • VDE (or CVDE) was produced in a similar fashion by fusing VP16 sequences in-frame at a novel Ncol site corresponding to amino acid 68.
  • the downstream primer was made Ncol compatible. Plasmid template (100 ng) , each primer (500 ng) , and reaction conditions outlined by the manufacturer for Pwo (Boehringer Mannheim) high fidelity polymerase, were used with a program of 1 min at 94 °C, 1 min at 45°C, and 1 min at 72°C for 20 cycles for production of all PCR products used herein.
  • LNCX-Usp and RXR were produced from BcoRI fragments encoding this complete cDNAs, filling with the Klenow fragment of DNA polymerase, and inserting the fragments into the Hpal site of LNCX. Orientation was determined by restriction endonuclease digestion. Transient transfection in CV-1 and 293 cell was performed by using standard methods (Sambrook et al (1989) Molecular Cloning: A Laboratory Manual 2 nd edition) in triplicate in 24-well plates by calcium phosphate coprecipitation of 100-ng receptor (s), reporter plasmid E4-luc, and pCHHO as an internal control.
  • E4-luc is four tandem EcREs inserted upstream of a thymidine kinase gene minimal promoter directing luciferase expression
  • EcRE oligonucleotides were as described (Thomas et al . (1993) Nature 362:471-475) with BamHI -Bg/l I compatible ends.
  • Ligand (1 ⁇ M) was added at the time of transfection, and 40 h later cells were lysed and extracts were used for ⁇ -galactosidase assay and measurement of luciferase activity in an analystical bioluminescence photometer.
  • VDEiR and MS vectors used only as retroviruses in production of stable cell lines were produced as follows:
  • the VDEiR expression cassette was constructed by modifying the ATG start codon of human RXR to contain an overlapping BstXI site for fusion into the ATG start codon of the 0.8-kb EMCV IRES (Jang et al (1989) J. Virol 63:1651-1660 and Hoshimaru et al (1996) PNAS 93:1518-1523) sequence.
  • the IRES-RXR cassette was then subcloned downstream of VDE in LNCX-ofVDE (CofVDE) , a VDE variant vector with lower basal transactivation levels in superphysiological RXR environments, to produce CVDEiR.
  • the MS retroviral reporter was produced by removing the Moloney murine leukemia virus enhancer core from the 3 ' long terminal repeat (LTR) with Nhel -Xbal digestion and replacing this sequence with four tandem EcREs, resulting in a reporter analogous to the E4-luc reporter plasmid.
  • LTR long terminal repeat
  • Double-stranded EcRE probes corresponding to response elements described above were labeled by filling of the Klenow fragment of DNA polymerase with [ 32 P] dCTP and unlabeled dGAT by standard methods.
  • Reaction conditions for protein-probe interaction and gel electrophoresis were essentially as described by Yao et al . ((1992) Cell 71:63- 72) except, to facilitate comparison between samples, reaction mixtures (including dimer partners) and probe) were prepared as a mixture and distributed equally to individual tubes with receptor proteins. The reactions were allowed to proceed at 23°C for 5 min. at which time ligand or vehicle was added and the reaction allowed to continue for an additional 20 min.
  • Chimeric receptors were produced by taking advantage of conserved DNA sequences and sites within the receptors and by introducing compatible sites by low cycle, high fidelity PCR (described above) of bombyx mori receptor (BE) and drosophila melanogaster ecdysone receptor (DE) templates to produce fusion proteins.
  • DEBE-A/B was produced by PCR and replaced the BE /AB domain (amino acids 1-197, as set forth in SEQ ID NO:3) with amino acids 1-255 of DE introducing a novel Apal restriction site.
  • DEBE-C replaced the A/B and C domains (amino acids 1-273, as set forth in SEQ ID NO: 3) of BE with amino acids 1-331 of the DE protein by introduction of a compatible Eagl site into BE at the fusion junction.
  • DEBE-D replaced BE from amino acids 1-363, as set forth in SEQ ID NO: 3, with amino acids 1-430 of DE by Kpnl digest.
  • BEDE was the reverse of this construct and replaced amino acids 1-430 of DE with amino acids 1-363 (as set forth in SEQ ID NO: 3) of BE by using Kpnl partial digest.
  • DEBH was produced from DEBE-C by removing all BE sequences except the D domain by Kpnl digestion and inserting sequences encoding the intact E and F domains (amino acids 430-878) of the original DE after partial Kpnl digestion.
  • VEH was produced by excision of the central region of DEBH, including the heterologous hinge domain, and inserted into VDE, replacing the analogous region.
  • the chimera, BEDB was produced by insertion of the 654 -bp Kpnl -Bg/l ⁇ fragment encoding precisely the DE hormone-binding domain into the identical analogous sites in BE. Two unique sites within the hormone-binding domain of BEDB were used in the production of the chimeras. A unique Aatll site lies approximately 120 bp downstream of the 5' .Kpnl site, and a unique Eagl sites lies approximately 440 bp downstream.
  • PCR was used as described above, and corresponding fragments of BE with appropriate compatible ends were generated and subcloned into BEDB digested with Kpnl + Aatll (BKE) , Aatl l + Eagl (BAE), Aatll + Bg/11 (BAB), and Eagl -Bg/l ⁇ for BEB .
  • BKE Kpnl + Aatll
  • BAE Aatl l + Eagl
  • BAB Aatll + Bg/11
  • Eagl -Bg/l ⁇ for BEB .
  • the resulting constructs were translated, examined by SDS/PAGE, quantified, and normalized as described above. Conditions for gel mobility shift assay were also as described above.
  • Transient restroviral production in 293 cell types has been described (Pear et al . ((1993) PNAS 90:8392-8396). Forty-eight hours after transient retroviral production, the conditioned medium was removed, filtered through 0.45- ⁇ m filters, and frozen at - 70°C until use. Ten-centimeter dishes of primary-cultured Fischer rat abdominal fibroblasts (Schinstine et al . (1992) Neurochem 58:2019-2029) and 293 and CV-1 cells at approximately 50% density were infected at a multiplicity of infection of approximately 0.05 with virus-containing media and 8 mg/ml Polybrene for 48 h.
  • Infected cells were then selected with 4-6 mM L-histidinol until noninfected cells were cleared from the population: approximately 10 d for FF12s, 21 dafor 293s, and >30 d for CV-ls (CV-1 cell populations were resistant to rigorous selection with l . -histidinol) . Resistant colonies from multiple plates were trypsinized, pooled, and passaged. The resulting bulk population was then split and infected with receptor-bearing virus and reselected again in medium containing both L-histidinol and 400-800 ⁇ g/ml G418. The infection and selection process for the second round of viral infection proceeded as described for the first round.
  • Both receptors were tested to determine the effect that differential endogenous dimer partner availability had on transactivation of responsive promoters.
  • Relative luciferase activity employing the reporter plasmid, E4-luc were performed from VDE and VBR transiently cotransfected into 293 or CV-1 cells with either Usp, RXR, or no added heterodimer partner. Luciferase activity was assayed in the presence of either vehicle, 1 ⁇ M murA, or 1 ⁇ M tebufenozide .
  • VDE and VBR transactivation were similar in both cell types.
  • both proteins were induced less than 2 -fold by ligand and displayed a high level of basal transactivation.
  • RXR VDE displayed an average relative 5.35-fold induction across both cell types, whereas VBR was induced only 2.35-fold, even though the absolute level of induction matched or exceeded the expression with VDE.
  • the decreased relative induction of VBR + RXR resulted from approximately doubled basal activity levels compared with VDE + RXR.
  • both receptors in both cell types exhibited dramatic 15- to 80-fold decreases in basal transactivation.
  • VDE failed to respond to tebufenozide
  • VBR continued to respond well to both murA and tebufenozide.
  • the addition of RXR to VDE-transfected 293 cells increased the maximum murA-treated expression level by only 20%, indicating that the high level of endogenous RXR in 293 cells is near saturation for heterodimerization with DE .
  • CV-1 cells supported murA stimulation of VDE at only 13% of the expression level of VDE with added RXR (Fig. IB) .
  • VBR was active at levels similar to VDE in the high RXR background of the 293 cells; however, in the CV-1 cells, tebufenozide-stimulated VBR exhibited both the highest absolute level of transactivation and the greatest relative induction (160.2-fold) of any of the other combinations of receptors and ligands tested. In the CV-1 cells, tebufenozide-stimulated VBR displayed 21-fold greater relative induction and an absolute expression level 9.25 times the level of VDE treated with mutA.
  • DEBE-C with ligand was approximately 40% decreased relative to native BE; however, it still displayed 16 -fold greater binding than native DE .
  • MurA-stimulated BEDE + RXR produced a band shift 4 times the intensity of the native DE + RXR stimulated with murA, but BEDE was clearly significantly impaired to heterodimerization with RXR compared with native BE and other higher affinity chimeras like DEBE-A and DEBE-C.
  • the D region of DE was replaced with the BE D domain to produce DEBH.
  • Native DE and DEBH were compared side by side for binding to EcREs with both Usp and RXR dimer partners.
  • Gel mobility shift of in vi tro translated and normalized DE and hinge-substituted DEBH was performed, in the presence of either Usp or RXR, and further in the presence of vehicle or 1 ⁇ M murA.
  • DE + Usp DEBH + Usp averaged 5 -fold greater probe binding with murA and 9-fold greater binding without ligand.
  • DEBH + RXR exhibited both decreased probe shift in the absence of ligand and increased probe binding with murA (approximately 3 -fold over native DE) for a 10-fold relative induction by ligand compared with 2.5-fold for DE.
  • Transient transfection analysis of a VP16-DEBH construct (VEH) in CV-1 cells revealed that VEH shared characteristics of both DE and BE.
  • VEH displayed the high basal transactivation level and low relative induction (average of 2.5 fold) characteristic of BE with superphysiological RXR.
  • VEH displayed 5- to 8 -fold greater basal and murA-activated expression than native DE in the absence of any exogenous dimer partner.
  • MurA-stimulated VEH expression exceeded even the expression level for murA-stimulated BE by nearly 2 -fold.
  • DEBH did not to respond to tebufenozide.
  • Additional chimeric proteins with a subset for BED-region sequences did not function as well as DEBH with replacement of the entire D domain, suggesting that multiple discrete determinants presumably over much of the hinge region contribute to the high affinity phenotype.
  • BEDE E-domain chimeras
  • BEDB is identical with BE with the exception of complete replacement of the E domain with corresponding sequences from DE
  • BEDB was identical with BEDE with the exception of replacement of the large C-terminal DE F domain for the
  • BEDB 20-amino acid F domain of BE.
  • BKE, BAE, BAB, and BEB are derived from BEDB and are sequential, C-terminal moving replacements of three regions within the DE-derived E domain.
  • Gel mobility shift assays with BE, BEDE, and BEDB revealed that BEDE and BEDB are essentially identical in their properties of complex formation, suggesting that the F domain does not play a significant role in heterodimerization and DNA binding.
  • BEDB recapitulates the high affinity USP binding and the low affinity RXR binding of BEDE.
  • the BEDB-derived chimeric receptors reveal several functional subdomains over most of the BE E domain. By using unique internal sites, the E domain was subdivided into approximately thirds called E ⁇ E 2 and E 3 . Treatments were with vehicle (-) , 1 ⁇ M murA (M) , or 1 ⁇ M tebufenozide (T) . Chimera BKE with replacement of the DE E 1 and E 2 , regions and chimera BAE with replacement of only the E 2 region displayed very similar patterns of shift. Both chimeras were significantly impaired in complex formation with Usp and RXR relative to the original BEDB chimera, suggesting that there were fundamental incompatibilities between subdomains of the Bombyx and Drosophila E domains.
  • Parallel MS vectors encoding either the LacZ gene (MS-Z) or a luciferase transgene (MS-luc) were used in this comparative study, the former as an indication of the number of responding cells and the latter for quantitative purposes.
  • FF12 Three cell types, 293s, CV-Is, and rat primary fibroblasts (line FF12) , were selected for assay of reporter/receptor virus combinations based on their relative capacities to supports high level DE-mediated transactivation.
  • the human 293 cell line support essentially full DE activation with no requirement for added RXR, whereas CV-ls require the contransfection of exogenous dimer partner for DE function.
  • FF12s were selected as the third cell type because they are refractory to chemical methods of transduction and reliant on the use of biological vectors such as recombinant retroviruses for efficient gene transfer.
  • primary fibroblasts are frequently used as autologous donor cells for transplantation approaches to somatic gene therapy.
  • MS-LacZ cell types infected with an empty LNCX vector displayed no staining when stimulated with any ligand and further confirmed observations from vehicle-treated cells that the MS vector permitted only relatively low basal levels of transgene expression.
  • Approximately 30% of CVDE-, CV-DEiR- , and CVBR-infected MS-Z 293 cells responded to murA.
  • Tebufenozide stimulated ⁇ 1% of CVDE-infected cells and ⁇ 4% of CVDEiR-infected MS-Z 293s. More than 50% of CVBR-infected MS-Z 293s responded to tebufenozide, however, exceeding all DE-based vectors with murA. More dramatic differences were seen in CV-1 and FF12 MS-Z infected cells.
  • CVDE alone was not inducible in either cell type, indicating that the endogenous level of RXR in either CV-ls or primary fibroblasts is not sufficient to support VDE-mediated transactivation.
  • endogenous level of RXR is supplemented, as in CVDEiR, 3-10% of the infected population was observed to strongly respond to murA.
  • CVBR was clearly the most potent transactivator in the CV-1 and FF12 cell types with 20% of CV-1 and 55% of CVBR-infected FF12 cells histochemically positive.
  • Transient transfections are performed by calcium- phosphate coprecipitation employing standard methods (see Sambrook et al . , in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York, New York (1989)) . All tissue culture experiments are performed using DMEM 10% FBS in a 10% C0 2 incubator unless otherwise specified. All transfections are performed in triplicate in 24 -well Costar plates using CV-1 cells at an approximately 5X10 4 plating density. Immediately following transfection, 1 ⁇ m ligand in 20% EtOH/PBS is added to wells. After 40 additional hours of incubation, cells are harvested and luciferase activity measured in an analytical bioluminescence photometer.
  • TH tyrosine hydroxylase
  • invention constructs allows one to overcome the loss of transgene expression by providing stimulation of the retroviral LTR promoter through either ligand-activated transactivating complexes or through constitutive transactivating receptor variants. In this way, transgene expression may be maintained for longer periods of time, even indefinitely if desired.
  • Stem-type cells such as stem cells of the hematopoetic system, nervous system, or embryo, could be infected or transfected with VbR regulated transgenes and subsequently implanted into adult, fetus, or early embryonic animals for either therapeutic or research purposes.
  • Cells of the hematopoetic system could conditionally express proteins producing blood clotting factors such as factor IX, metabolic factors such as glucocerebrosidase, or protective factors including anti-HIV proteins.
  • VbRs have been introduced into both MLV and lentiviral-based retroviral systems. When these viruses are introduced directly into target cells of either a mature or developing organism, expression of the virally encoded transgenes may be regulated by systemic addition of ligands such as tebufenozide and derivatives.
  • ligands such as tebufenozide and derivatives.
  • Parkinson's disease An example of a disease that could be theoretically ameliorated by application of in vivo VbR encoding retroviruses is Parkinson's disease, described above.
  • VbR encoding retroviruses Another application would be to use VbR encoding retroviruses as an anti-viral agent.
  • Lentiviral vectors with regulated properties and harboring suicide genes or "protective" proteins could be used as a means of conditionally depleting or destroying HIV-positive cells.
  • L-dopa Treatment of Parkinson's disease with the chemical precursor of dopamine, L-dopa, has proven effective in ameliorating many of the deficits of Parkinsonism. With time, however, patients become refractory to L-dopa therapy, with the deleterious effects of chronic treatment outweighing even the serious symptoms of the disease itself. Eventually, patients are left with few therapeutic options. While the transplantation of TH expressing cells may be effective when constantly producing low-levels of L- dopa, a potentially far more beneficial approach would be to allow the physician some degree of control over L-dopa production in the patient. This would allow sufficient control to ensure that the transgenic factor is expressed at appropriate therapeutic levels.
  • the transgene may be allowed to become quiescent and transcriptionally inactive until needed again. Because the transcriptional induction of the invention retroviral constructs is dependent on an exogenous ligand, expression of an integrated therapeutic transgene can be placed under the control of the physician and patient.
  • PolyQ-GFP are constructed from an existing GFP clone, GFP-SKSP. PCR primers encoding a novel restriction site and ATG start codon at the 5' end, and an Age I site in-frame with the GFP ORF at the 3' end are obtained for PCR amplification of huntingtin polyQ regions. These PCR products are inserted in-frame into GFP-SKSP to produce the fusion constructs of pQ-GFP. The clones are analyzed for mutations and function by sequencing and in vitro translation to confirm the creation of a pQ-GFP ORF.
  • BORIS regulatable retroviral vectors are produced by removing the L-Histidinol gene from plasmid LSHL, by PCR, and inserting it in place on the neo r into a BORIS vector for production of BORIS-LHis.
  • the plasmid are large-scale prepped.
  • a preliminary test for function is transient transfection experiments into transfectable cell types such as 293 cells and examination for the expression of beta-galactosidase or GFP reporter genes.
  • Retrovirus are transiently produced and used for infection of target cells. Infected cells are selected in L-Histidinol containing media to ensure proper selection. polyQ-GFP is then inserted into regulated retroviral vectors.
  • pQ-GFPs are excised from SKSP and inserted into the regulatable BORIS and BORIS-LHis vectors.
  • the plasmids are large-scale prepped. Function is determined by examination for green fluorescence and altered phenotype. Additional plasmid are used for transient production of retroviruses.
  • rat fibroblasts and the hcnlv-myc are selected for initial testing of BPQGS . Both are infected with the appropriate vector (s) place under selection, and individual resulting colonies subcloned. The subclones are expanded and passaged for analysis . Infected cells are treated or not with their respective ligands and the resulting phenotypes analyzed. Preliminary analysis will examine GFP fluorescence and distribution within the cells. At timed intervals following ligand induced expression, the pattern of fluorescence are recorded to determine if the fusion protein changes in distribution. Morphological characteristics of stimulated cells their growth rates, and cell death will also be examined at intervals following induction. For the hcn/v-myc cells, the consequences of polyQ-GFP expression in the presence and absence of tetracycline are compared.
  • GFP fusion proteins with long (97Q) , short (13Q) , or no N-terminal polyQ tracts were constructed. Unique restriction endonuclease sites were designed into the regions flanking the polyQ tracts to allow insertion of an SV40 nuclear localization signal (NLS) or other sequences either C-terminal to the polyQ tract.
  • NLS nuclear localization signal
  • 97Q- and 97QN-GFP constructs initially manifested as uniform GFP positive fluorescence throughout the cytoplasm or nucleus, but within 24-72 hours after transfection condensed into bright granules indicative of intracellular aggregates (IAs) .
  • IAs were localized within the cytoplasm, surrounding the nuclear envelope, and within the nucleus itself ( Figure IB) .
  • the presence of the SV40 nuclear localization signal (NLS) though observed to efficiently direct localization of either 13Q- or 97Q-GFP variants to the nucleus, resulted in a quantifiable difference in the rate of IA formation after transfection.
  • 13Q-GFP/97Q ⁇ doubly infected cells were distinctive from 97Q-GFP cells because they continued to display faint uniform cytoplasmic GFP positivity in addition to the formation of predominantly nuclear IAs, whereas IA formation within 97Q-GFP cells generally results in condensation of all GFP fluorescence into IAs leaving other areas of the cell nonfluorescent and dark.
  • Control cotransfection of NIT-97Q ⁇ with NIT-GFP constructs with no N-terminal polyglutamine tracts did not result in the formation of IAs under any conditions tested, indicating that the short polyQ tract of 13Q-GFP is necessary for participation in IA formation.
  • GFP positive cells contained GFP positive IAs. At a 1:1 ratio, nearly 40% of GFP positive cells also displayed IAs. At 10:1 ratio of NIT-97Q ⁇ to NIT-13Q-GFP, over 75% of positive cells displayed IAs 60 hours post-transfection.
  • Example 25 Characterization of IAs formed from a combination of long and short polvO-reporters
  • 97Q ⁇ was modified to contain an in-frame fusion of the LacZ gene encoding ⁇ -galactosidase (97QZ) .
  • Preliminary transient transfection studies of 97QZ revealed that like 97Q-GFP, 97QZ condensed within transfected cells to form both immunopositive and histochemically reactive IAs.
  • NIT-97QZ and NIT-13Q-GFP were cotransfected into cells and 60 hours later, performed immunohistochemistry and histochemical reactions on the transfected cells to simultaneously examine both reporter constructs.
  • ⁇ -galactosidase ( ⁇ -gal) histochemical reaction obscures GFP fluorescence, through slight modification of standard staining techniques and limiting the time of exposure to reagents, a low level of GFP fluorescence could be preserved and imaged along with the characteristic blue stain of the ⁇ -galactosidase reaction.
  • a population of IAs are observed that are both GFP and ⁇ -galactosidase positive. Numerous large ⁇ -gal only positive IAs are also present, presumably from the aggregation of 97QZ only (or predominantly) . Curiously, several IAs that appear only positive for GFP were also observed using the histochemical stain and native GFP fluorescence imaging.
  • VbR modified ecdysone receptor variant
  • TTA tetracycline transactivator
  • the TTA-based NIT vector tends not have the broad-based low level of "uninduced" expression characteristic of the BORIS vectors (identified in SEQ ID NO:6); however, because transactivator expression and transactivation is continuous in the absence of ligand, very high transgene expression levels may be achieved through the use of this vector.
  • 97Q-GFP was cloned via a custom shuttle vector into both retroviral vectors.
  • B3 -polyQ-GFP retroviruses B3-13Q or B3-97Q
  • NIT-13Q or NIT-97Q TTA-based NIT polyQ-GFP retroviruses
  • COS-7 cells were selected because several studies had examined IA formation and IA-induced apoptosis in this cell type.
  • 293 cells were utilized for the same reason and also because they are amenable to efficient transfection and would have significant utility in the development of gene-based factors that interact with or interrupt IA formation.
  • Primary rat fibroblasts were selected because they are non-transformed and readily enter a prolonged post-mitotic state following contact inhibition at high culture densities.
  • G418-resistant B3-97Q 293s displayed little to no visible GFP fluorescence during culture in the absence of ligand; however, introduction of 1 ⁇ M tebufenozide into the culture medium resulted in a rapid increase of cytoplasmic fluorescence within 24 hours post-induction. Nearly 100% of cells would display GFP-positive fluoresence within 24-72 hours of induction. After 24 hours, only widely scattered cells would contain small, detectable IAs. By 48 hours, >8% of the cell population would contain one or more IAs increasing to >15% of the population between 48 and 72 hours. Occasional IA containing cells would be observed floating in the culture medium; however, there was no evident "wave" of cell death at the 72 hour time point or later times.
  • G418-resistant B3-97Q C0S-7s displayed no visible GFP fluoresence during culture in the absence of ligand in a manner almost identical to the 293 B3-97Q population.
  • Addition of 1 ⁇ M tebufenozide into the culture medium resulted in a rapid increase of cytoplasmic fluorescence within 24-48 hours post-induction.
  • IA formation peaked at 4-5% of total cells (at any given moment) 72-96 hours post-induction.
  • a continuous population of G418 -resistant cells did not display even GFP fluoresence suggesting that one or more components of the regulatable system were lost from the COS population.
  • IA containing COS-7 cells were also observed floating in the culture medium; however a depletion of the expressing population was not observed presumably because the continuous formation of IAs in other cells.
  • B3-97Q infected primary fibroblasts became faintly GFP fluorescent within 72 hours of tebufenozide treatment but did not achieve a maximum level of 97Q-GFP expression sufficient to result in IA formation in a significant population of cells.
  • the NIT vectors were employed to generate regulated 97Q-GFP expression.
  • Infected NIT-97Q FF12s were infected and cultured continuously in the presence of doxycycline (dox) to prevent IA formation. Twelve individual colonies of NIT-97Q FF12s were selected in order to screen for individual cell populations with low dox inhibited expression and high 97Q expression in the absence of ligand.
  • dox doxycycline
  • FFN97Q-5 displayed GFP fluoresence within 90% of the confluent cell population within 5 days of ligand removal. IAs began forming on day 5 and continued to increase through day 8. Approximately 15% of the total cell population displayed IAs 10 days post ligand removal. IA formation in the confluent FFN97Q-5 population was distinctive from other cell types examined in that it was occasionally exclusively nuclear and at other times predominantly cytoplasmic in an unusual pattern within the cell body. IAs with a distinctive stellate or tri-partite appearance were often observed. Large cytoplasmic aggregates of multiple star-like IAs were also occasionally seen. Curiously, confluent FFN97Q-5 cells with multiple cytoplasmic or nuclear IAs appeared visually normal . Floating dead cells with or without IAs were rarely observed and in numbers no different from control wells. FF970Q-%s cultured in the presence of dox neither expressed visible 97Q-GFP nor formed IAs.
  • Transgenic animals are generally produced by either pronuclear injection of DNA or by transfection of embryonic stem (ES) cells followed by selection and injection of the stem cell into the inner cell mass of very early embryos.
  • Pronuclear injection results in approximately 5-10% stable gene transfer in the production of transgenic mice.
  • the use of ES cells in producing transgenics is likewise inefficient in generating mosaics with germ-line transmission of the transgene. It was proposed in the mid- 1980' s to use retroviruses to transfer transgenes with high efficiency into early embryos or ES cells to dramatically enhance the odds of producing transgenic animals. All attempts at this failed, not because the virus was incapable of stably integrating into the target cell genome, but because the integrated provirus did not express any of the genes encoded within the viral transcriptional cassette.
  • the present invention is capable of overcoming the transcriptional block to result in germ-line transgenic animals with full expression from the integrated transgene.
  • the level of transcription may still be regulated by controlling the supply of ligand to the transgenic animal.
  • the increased efficiency of producing transgenic animals by retroviral infection should open up the way to producing mutant animals of a variety of species previously impractical for genetic modification because of the potential cost of producing a large number of non-positive animals by classical methods.
  • transgenic mice To produce transgenic mice, the following nucleic acid constructs are prepared and subsequently injected into fertilized eggs: CD3-VbR and a ligand inducible ⁇ -gal reporter. Two separate lines of transgenic mice are generated harboring either a ligand inducible reporter, or a T-cell specific expression construct of VbR, respectively. The former are referred to as reporter mice, the latter are referred to as receptor mice, and double transgenic mice are referred to as receptor/reporter mice. Constructs CD3-VbR are injected, while the reporter is injected alone. Primary genotyping is performed by
  • Southern blot analysis and the transmission of transgenic mice is monitored by dot blot analysis.
  • Receptor mice are analyzed for VbR expression by Northern blot analysis of RNA collected from these mice.
  • Northern blot analysis 15 ⁇ g of total RNA obtained from the thymus, and various tissues as a control, is run on a denaturing gel and blotted onto a nitrocellulose membrane. The blot is probed with a radiolabeled ⁇ -gal-specific probe and exposed on film for 2 days.
  • the transgene can be transferred to the offspring as expected by Mendelian genetics .
  • invention constructs are a potent tool in the delivery of transgenes to somatic tissues of a developing embryo. With many diseases, considerable damage is done during embryonic development so that therapies applied after birth are essentially ineffective to ameliorate the disease phenotype .
  • the present invention provides methodology where one can infect cells of the embryo and can provide therapeutic factors to the developing fetus either constitutively, or under the regulation of exogenously produced ligand.
  • retroviruses as gene transfer agents.
  • titers of retroviruses from existing producer cell lines are only on the order of 1X10 4 or 1X10 5 .
  • expression of the retrovirus may be induced by greater than ten-fold, resulting in correspondingly higher titers of infectious virus .

Abstract

Selon la présente invention, il a été découvert que des protéines réceptrices nucléaires isolées et issues du ver à soie bombyx mori (bR) sont utilisées pour réguler l'expression transgénique. Le bR est généralement censé être un régulateur de transcription efficace à l'intérieur des cellules dudit ver à soie. Selon la présente invention, il a été découvert que le bR est également fonctionnel dans des cellules de mammifères, que l'ajout de domaines activateurs au cadre ouvert de lecture du bR stimule l'activité du régulateur modulé par des ligands et ce, dans le but d'obtenir une induction de transcription de haut niveau. Enfin, l'invention concerne d'autres modifications du domaine de liaison aux ligands du bR qui permettent d'obtenir des récepteurs présentant des propriétés de transactivation uniques.
EP98934353A 1997-07-10 1998-07-10 Proteines multifonctionnelles hybrides et recepteurs de lepidopterane modifies destines a etre utilises pour reguler l'expression transgenique Withdrawn EP0998560A1 (fr)

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US08/891,298 US6300488B1 (en) 1997-07-10 1997-07-10 Modified lepidopteran receptors and hybrid multifunctional proteins for use in transcription and regulation of transgene expression
US9187498P 1998-07-07 1998-07-07
US91874P 1998-07-07
PCT/US1998/014215 WO1999002683A1 (fr) 1997-07-10 1998-07-10 Proteines multifonctionnelles hybrides et recepteurs de lepidopterane modifies destines a etre utilises pour reguler l'expression transgenique

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