EP1232271A2 - Techniques et dispositifs de regulation de l'expression genetique - Google Patents

Techniques et dispositifs de regulation de l'expression genetique

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Publication number
EP1232271A2
EP1232271A2 EP00979852A EP00979852A EP1232271A2 EP 1232271 A2 EP1232271 A2 EP 1232271A2 EP 00979852 A EP00979852 A EP 00979852A EP 00979852 A EP00979852 A EP 00979852A EP 1232271 A2 EP1232271 A2 EP 1232271A2
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EP
European Patent Office
Prior art keywords
transcriptional activator
polypeptide
nucleic acid
trar
dna
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EP00979852A
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German (de)
English (en)
Inventor
Raffaele De Francesco
Riccardo Cortese
Petra Neddermann
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Istituto di Ricerche di Biologia Molecolare P Angeletti SpA
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Istituto di Ricerche di Biologia Molecolare P Angeletti SpA
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Publication of EP1232271A2 publication Critical patent/EP1232271A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • the present invention relates to regulation of gene expression, in particular regulation of expression in eukaryotic cells and expression systems, relevant for gene therapy applications.
  • nucleic acid molecules and proteins useful for regulating the expression of genes in eukaryotic cells and organisms in a highly controlled manner are disclosed.
  • transcription of a nucleotide sequence is activated by a transcriptional activator fusion protein composed of at least two and optionally at least three polypeptide components: (i) a first polypeptide component which binds DNA with selectivity for a particular operator sequence; (ii) a second polypeptide component comprising a regulatory domain which binds AcylHSL (or an analogue thereof) whereby upon AcylHSL binding, the DNA binding function of the first polypeptide is activated; and (iii) an optional third polypeptide which directly or indirectly activates transcription in eukaryotic cells.
  • the third polypeptide component can be provided separately, whereby the fusion protein and polypeptide component interact in order to provide a transcription factor.
  • regulatory systems In general these systems comprise three elements: (1) a target gene i.e. the gene whose expression needs to be regulated; (2) a gene coding for a regulatory protein, i.e. a protein that regulates the activity of the target gene via complexing with the third element; (3) a regulatory molecule, preferably of small molecular weight, that can be added to the system from outside.
  • a regulatory molecule preferably of small molecular weight, that can be added to the system from outside.
  • the relevant regulatory molecule can be added to the culture media or introduced in the body of the animal .
  • the two systems most commonly used to regulate gene expression are the one based on the use of steroid hormone receptor and the drug RU486 (Mifepristone or Mifegyne, a progesterone antagonist) , and the Bujard system based on the use of the tet-operon and the administration of tetracyclines .
  • TetR Tet repressor
  • the Tet repressor which binds to tet operator sequences in the absence of tetracycline and represses gene transcription, has been expressed in plant cells at sufficiently high concentrations to repress transcription from a promoter containing tet operator sequences (Gatz, C. et al .
  • TetR has been fused to the activation domain of VP16 to create a tetracycline-controlled transcriptional activator (tTA) (Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551).
  • the tTA fusion protein is regulated by tetracycline in the same manner as TetR, i.e., tTA binds to tet operator sequences in the absence of tetracycline but not in the presence of tetracycline.
  • Tc tetracycline-controlled transcriptional activator
  • the present invention pertains to a regulatory system which utilizes components of the prokaryotic quorum sensing system in order to regulate gene expression in eukaryotic cells.
  • Many prokayotic organisms are endowed with a control mechanism called quorum sensing (Fuque, et al (1994), J. Bacteriol . ,
  • the quorum sensing mechanism consists primarily of (1) the product of the gene LuxR; (2) a small effector molecule called the autoinducer, chemically a N-acyl- homoserine-lactone (AcylHSL) , freely diffusible across bacterial membranes and that binds to the LuxR gene product; (3) a DNA operator sequence to which the LuxR/AcylHSL complex binds thus activating transcription, termed the lux box B a 20 nucleotide inverted repeat; (4) the Luxl gene product, which is the AcylHSL synthase and whose expression is under the control of a lux box.
  • Several bacteria genera contain regulatory system homologous to the quorum sensing system of V.
  • the AcylHSL inducer employed may be N (3-oxooctanoyl) -L homoserine lactone.
  • the system of the invention utilizes components of the prokaryotic quorum sensing/autoinducer pathway in order to regulate gene expression in eukaryotic cells.
  • this invention provides AcylHSL-regulated transcriptional activators which are useful for activating gene expression, in a controlled manner, of a gene linked to one or more selected operator sequences .
  • the present invention relates to nucleic acid molecules and proteins which can be used to regulate the expression of genes in eukaryotic cells or animals. Regulation of gene expression by the system of the invention involves at least two components: A gene which is operably or operatively linked to a regulatory sequence and a protein which, in presence of an inducible agent, binds to the regulatory sequence and activates transcription of the gene.
  • the system of the invention utilizes components of the prokaryotic quorum sensing pathway in order to regulate gene expression in eukaryotic cells. More specifically, the present invention is based on the prokaryotic LuxR-type transcription f ctors.
  • the term "LuxR-type transcription factor" is intended to include, homologues of the Vibrio fischeri LuxR protein. According to the general teaching of Henikoff, S., et al (Henikoff, S.
  • DNA-binding proteins that are a component of an N-acyl homoserine lactone based gene regulatory system
  • (2) comprise a first cluster of sequence similarity in a region that aligns with the putative AcylHSL-binding region of LuxR;
  • Michel-Servet 1211, Geneve, 4 Switzerland
  • Addition LuXR signature patterns may be defined with reference to the ABlocks® database at the Fred Hutchinson Cancer Research Center in Seattle, Washington, USA or at the Weizmann Institute of Science in Israel.
  • a list of LuxR-type transcription factors that may be employed in embodiments of the present invention is shown in Table I.
  • One preferred embodiment employs LuxR DNA binding-domain and/or regulatory domain.
  • Another preferred embodiment employs TraR DNA binding domain and/or regulatory domain.
  • the invention is widely applicable to a variety of situations where it is desirable to be able to turn gene expression on and off, or regulate the level of gene expression.
  • the invention may be employed for gene therapy purposes, e.g. in treatments for genetic or acquired diseases.
  • cells of a subject in need of gene therapy are modified to contain 1) nucleic acid encoding a transactivator of the invention in a form suitable for expression of the transactivator in the host cells and 2) a gene of interest (e.g., for therapeutic purposes) operatively linked to an AcylHSL-regulated transcription unit.
  • a gene of interest e.g., for therapeutic purposes
  • Expression of the gene of interest in the cells of the subject is then stimulated by administering AcylHSL or a AcylHSL analogue to the patient.
  • the level of gene expression can be modulated by adjusting the dose of the AcylHSL or analogue administered to the patient.
  • the regulatory system of the invention offers the advantage over constitutive regulatory systems of allowing for modulation of the level of gene expression depending upon the requirements of the therapeutic situation.
  • the system of the invention can be used to conditionally express a suicide gene in cells, thereby allowing for elimination of the cells after they have served an intended function.
  • cells used for vaccination can be eliminated in a subject after an immune response has been generated the subject by inducing expression of a suicide gene in the cells by administering AcylHSL or a AcylHSL analogue to the subject.
  • a protein of interest can be accomplished using cultured cells in vi tro which have been modified to contain a nucleic acid encoding a transactivator fusion protein of the invention in a form suitable for expression of the transactivator in the cells and a gene encoding the protein of interest operatively linked to an AcylHSL-regulated transcription unit.
  • the invention also provides for large scale production of a protein of interest in transgenic animals.
  • Transgenic livestock carrying in their genome the components of the inducible regulatory system of the invention can be constructed, wherein a gene encoding a protein of interest is operatively linked to an AcylHSL-regulated transcription unit. Gene expression, and thus protein production, is induced by administering AcylHSL or analogue thereof to the transgenic animal .
  • the transcriptional activator proteins of the invention can be used alone or in combination to stimulate or inhibit expression of specific genes in animals to mimic the pathophysiology of human disease to thereby create animal models of human disease.
  • Ability to regulate the expression of the specific gene may be advantageous over gene "knock out" by homologous recombination to create animal models of disease, since the AcylHSL-regulated system described herein allows for control over both the levels of expression of the gene of interest and the timing of when gene expression is regulated.
  • Figure 1 is a schematic diagram of the construction of LuxR/VP16 fusion contructs by in- frame fusion of nucleic acid encoding LuxR and the activation domain of the VP16 protein.
  • Figure 2 is a schematic diagram of the construction of HNF- l/LuxR/VP16 fusion contructs by in-frame fusion of nucleic acid encoding the DNA binding domain, of HNF-1, the regulatory domain of LuxR and the activation domain of the VP16 protein.
  • Figure 3 is a schematic diagram of a promoter construct for regulation of a genes of interest operatively linked to the lux-box containing promoter for regulation by a AcylHSL- regulated transcriptional activator.
  • Figure 4 is a schematic diagram of a promoter construct for regulation of a genes of interest operatively linked to an hHNFl-box containing promoter for regulation by a AcylHSL- regulated transcriptional activator.
  • Figure 5 illustrates individual embodiments that are preferred in accordance with different aspects of the present invention. Shown are constructs providing a eukaryotic chimeric transactivator of transcription that is regulated by N-(3-oxo- octanoyl) -L-homoserine lactone.
  • Figure 5A illustrates a transcriptional activator according to individual embodiments of the present invention in which a eukaryotic trans-activation domain (e.g. VP16, F3 , p65, LFB 1/HNF-1 or other) is fused in frame with the TraR protein or another member of the LuxR protein family.
  • a eukaryotic trans-activation domain e.g. VP16, F3 , p65, LFB 1/HNF-1 or other
  • the activation domain is placed in the fusion protein N-terminal to TraR (or other DNA binding/regulatory protein) .
  • the activation is placed C-terminal.
  • TraR (or other) may be separated from the activation domain by a linker amino acid sequence (linker) .
  • a nuclear localization sequence (NLS) is an optional component of embodiments of the invention.
  • An NLS where used may, as illustrated, be provided at the N-terminal or at the C-terminal of the fusion protein.
  • the NLS is introduced between TraR and the activation domain or as part of the linker sequence.
  • Figure 5B illustrates how in other embodiments of the present invention TraR or a portion of it can be used to replace the dimerization domain in a eukaryotic dimeric transcription factor.
  • TraR* indicates full-length TraR protein or a portion of it containing the TraR dimerization or the N-(3-oxo- octanoyl) -L-homoserine lactone binding domain or both.
  • DNA binding domain 1 ' indicates the DNA binding domain of a eukaryotic transcription factor whereby the original dimerization domain has been deleted or inactivated.
  • the activation domain module may be placed at either the N- terminal of the fusion protein, or at the C-terminus. If required, a nuclear localization sequence maybe added to the fusion protein.
  • a transcriptional activator which comprises a fusion protein comprising at least two polypeptide components: (i) a first polypeptide component which binds in a sequence specific manner to an operator sequence in DNA; and (ii) a second polypeptide component, comprising a Lux-R transcription factor regulatory domain which binds cognate AcylHSL or an analogue thereof whereby upon binding of the AcylHSL or analogue the DNA binding function of the first polypeptide component is activated.
  • the transcriptional activator additionally comprises a third polypeptide which activates transcription in eukaryotic cells, and this may be a third polypeptide component of the fusion protein. Where it is not a component of the fusion protein the third polypeptide is a transcriptional activator that interacts with the fusion protein comprising the first and second polypeptide components in order to activate transcription when the fusion protein binds to a cognate operator sequence.
  • a transcriptional activator according to the invention activates transcription when bound to an operator sequence, and binds to the operator sequence in the presence of AcylHSL, or an analogue thereof.
  • transcription of a gene operatively linked to an operator may be controlled by altering the concentration of AcylHSL (or analogue) in contact with the host cell (e.g. adding AcylHSL from a culture medium, or administering AcylHSL to a host organism, etc.) .
  • the invention further pertains to transcription units for regulation by the transcriptional activators of the invention. Methods for stimulating transcription of a gene using AcylHSL (or analogues thereof) are also encompassed by the invention.
  • AcylHSL analogue encompasses compounds which need not be structurally related to AcylHSL but which bind to at least one of the LuxR-type transcription factors and induce the requisite change to induce DNA binding in an operably linked DNA binding component.
  • an AcylHSL analogue binds a LuxR transcription factor with a K a of at least about 10 6 M "1 , and more preferably K a of about 10 9 M "1 or greater.
  • Transcription may be induced in a cell in vi tro by culturing the cell in a medium containing AcylHSL or analogue thereof.
  • concentration of AcylHSL or analogue thereof is preferably between about 10 and about 1000 ng/ml .
  • To induce transcription in vivo AcylHSL or analogue thereof may be administered to the body, or a tissue of interested (e.g. by injection) .
  • the body to be treated may be that of an animal, particularly a mammal, which may be human or non- human, such as rabbit, guinea pig, rat, mouse or other rodent, cat, dog, pig, sheep, goat, cattle or horse, or which is a bird, such as a chicken, or a plant.
  • the dosage may preferably be designed to achieve a serum concentration of between about 0.05 and 1.0 ⁇ g/ml.
  • Suitable routes of administration include oral (e.g. in drinking water) and, in the case of a plant, water administered to the plant.
  • the first, second, and third polypeptide components of a fusion protein may be arranged in any order or sequence in a fusion transactivator protein of the invention.
  • the first and second polypeptide components may be derived from a bacterial LuxR transcription factor.
  • the first and/or second polypeptide components may be derived from TraR of Agrobacterium tumefaciens (see below, including
  • both the first (the DNA binding) and the second (the regulatory) polypeptides of the trans-activator fusion protein are derived from the DNA binding domain and the regulatory domain, respectively, of LuxR or a LuxR-type transcription factor.
  • the DNA binding and the regulatory polypeptide components are both derived from the Vibrio fischeri LuxR protein.
  • the DNA binding domain may consist of or comprise residues 160-250 of Vibrio fischeri LuxR (containing a 20-amino acid region (amino acids 190-210) believed to fold in a helix-turn- helix structure that is predicted to be involved in DNA recognition) , and/or the regulatory polypeptide component may consist of or comprise residues 20-159 of that protein.
  • This part of the LuxR protein encompasses the AcylHSL-binding region (amino acids 79-120) .
  • the first polypeptide component (the DNA binding domain) of the trans-activator fusion protein is derived from the DNA binding domain of a protein other than a LuxR transcription factor.
  • the first polypeptide may be derived from the DNA-binding domain of a eukaryotic transcription factor.
  • a HNF-1 DNA-binding domain may be used, which may for example consist of or comprise residues 1-281 of hHNF-1 (Bach, et al (1990) , Genomics , 8 (1) : 155-164 (Sequence accession number P20823)).
  • DNA-binding domain of hHNF-1 may be fused to the regulatory domain of a LuxR transcription factor in order to achieve AcylHSL- inducibility of DNA-binding.
  • the third polypeptide component transcriptional activator domain may be any available to those skilled in the art.
  • Polypeptides which activate transcription in eukaryotic cells are well known in the art.
  • transcriptional activation domains of many DNA binding proteins have been described and have been shown to retain their activation function when the domain is transferred to a heterologous protein.
  • a preferred polypeptide for use in the fusion protein of the invention is the herpes simplex virus virion protein 16 (referred to herein as VP16, the amino acid sequence of which is disclosed in Triezenberg, S. J. et al . (1988) Genes Dev. 2:718-729) .
  • VP16 herpes simplex virus virion protein 16
  • about 127 of the C-terminal amino acids of VP16 are used.
  • a polypeptide having the amino acid sequence of the 127 C-terminal amino acids of VP 16 may be used as the second polypeptide component in a fusion protein according to the present invention.
  • at least one copy of about 11 amino acids from the C-terminal region of VP16 (amino acids 437-447) which retain transcriptional activation ability is used as the second polypeptide component.
  • multimers (two to four monomers) of this region are used.
  • a dimer of this region i.e., about 22 amino acids
  • Suitable C-terminal peptide portions of VP16 are described in Seipel, K. et al. (EMBO J. (1992) 13:4961-4968).
  • a dimer of a peptide having an amino acid sequence DALDDFDLDML can be used as the second polypeptide in the fusion protein.
  • Other polypeptides with transcriptional activation ability in eukaryotic cells can be used in a transcriptional activator in accordance with the invention.
  • Transcriptional activation domains found within various proteins have been grouped into categories based upon similar structural features. Types of transcriptional activation domains include acidic transcription activation domains, proline-rich transcription activation domains, serine/threonine-rich transcription activation domains and glutamine-rich transcription activation domains. Examples of acidic transcriptional activation domains include the VP16 regions already described and amino acid residues 753-881 of GAL4.
  • proline-rich activation domains include amino acid residues 399-499 of CTF/NF1 and amino acid residues 31-76 of AP2.
  • serine/threonine-rich transcription activation domains include amino acid residues 1-427 of ITF1 and amino acid residues 2- 451 of ITF2.
  • glutamine-rich activation domains include amino acid residues 175-269 of Octl and amino acid residues 132-243 of Spl .
  • the amino acid sequences of each of the above described regions, and of other useful transcriptional activation domains, are disclosed in Seipel, K. et al. (EMBO J. (1992) 12:4961-4968).
  • the transcriptional activation ability of a polypeptide can be assayed by linking the polypeptide to another polypeptide having DNA binding activity and determining the amount of transcription of a target sequence that is stimulated by the fusion protein.
  • a standard assay used in the art utilizes a fusion protein of a putative transcriptional activation domain and a GAL4 DNA binding domain (e.g., amino acid residues 1-93). This fusion protein is then used to stimulate expression of a reporter gene linked to GAL4 binding sites (see e.g., Seipel, K et al . (1992) EMBO J. 11:4961-4968 and references cited therein) .
  • the fusion protein of the transactivator itself possesses transcriptional activation activity (i.e., the third polypeptide component directly activates transcription) .
  • the transcription is activated by an indirect mechanism, through recruitment of a transcriptional activation protein to interact with the fusion protein. This may, for example, be via a polypeptide domain (e.g., a dimerization domain) which mediates a protein-protein interaction with a transcriptional activator protein, such as an endogenous activator present in a host cell.
  • suitable interaction (or dimerization) domains include leucine zippers (Landschulz et al. (1989) Science 243:1681-1688), helix-loop-helix domains (Murre, C. et al . (1989) Cell 58:537-544) and zinc finger domains (Frankel, A. D. et al . (1988) Science 240:70-73).
  • a fusion protein of the invention may further comprise one or more additional polypeptide components, such as a fourth polypeptide component which promotes transport of the fusion protein into a cell nucleus, a nuclear localization signal (NLS) .
  • Nuclear localization signals typically are composed of a stretch of basic amino acids.
  • a heterologous protein e.g., a fusion protein of the invention
  • the nuclear localization signal promotes transport of the protein to a cell nucleus.
  • the nuclear localization signal is attached to a heterologous protein such that it is exposed on the protein surface and does not interfere with the function of the protein.
  • the NLS is attached to one end of the protein, e.g. the N-terminus.
  • amino acid sequence of a non- limiting example of an NLS that can be included in a fusion protein of the invention is Met-Pro-Lys- Arg-Pro-Arg-Pro .
  • a nucleic acid encoding the nuclear localization signal is spliced by standard recombinant DNA techniques in- frame to the nucleic acid encoding the fusion protein (e.g., at the 5' end) .
  • Preferred embodiments include transcriptional activators comprising a luxR+VP16 fusion, a TraR+VP16, TraR+LFBl/HNFl , TraR+ NF-KB p65.
  • the transcriptional activator component may be comprised in a fusion protein upstream or N-terminal to the regulatory and/or DNA binding domain.
  • the transcriptional activator component may be comprised in a fusion protein downstream or C-terminal to the regulatory and/or DNA binding domain.
  • One or more spacers may be used, e.g.
  • a transcriptional activator including a fusion protein according to the present invention may comprise of be composed of portions of naturally occurring proteins, of which examples have been mentioned.
  • one or more of the polypeptide components may comprise an amino acid sequence which differs by one or more amino acid residues from a natural amino acid sequence, whether a mutant, allele, isoform, variant or derivative of a specific sequence.
  • a transcriptional activator according to the present invention may include an amino acid sequence which differs by one or more amino acid residues from the wild-type amino acid sequence, by one or more of addition, insertion, deletion and substitution of one or more amino acids.
  • the amino acid sequence shares homology with a fragment of the relevant protein, preferably at least about 30%, or 40%, or 50%, or 60%, or 70%, or 75%, or 80%, or 85%, 90% or 95% homology.
  • a protein component may include 1, 2, 3, 4, 5, greater than 5, or greater than 10 amino acid alterations such as substitutions with respect to the wild- type sequence.
  • homology at the amino acid level is generally in terms of amino acid similarity or identity. Similarity allows for "conservative variation”, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine .
  • Similarity may be as defined and determined by the TBLASTN or other BLAST program, of Altschul et al . , (1990) J. Mol . Biol .
  • a further way of defining similarity or identity between sequences is to consider ability of nucleic acid to hybridize under stringent conditions.
  • a fusion protein and polypeptide components thereof according to the present invention are generally provide by expression from encoding nucleic acid. Such encoding nucleic acid may be employed in hybridisation experiments.
  • Preliminary experiments may be performed by hybridising under low stringency conditions.
  • Preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisations identified as positive which can be investigated further.
  • filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes - 1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes.
  • T m 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex.
  • suitable conditions include hybridisation overnight at 65°C in 0.25M Na 2 HP0 4 , pH 7.2 , 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0. IX SSC, 0.1% SDS.
  • One convenient way of producing a polypeptide or fusion protein according to the present invention is to express nucleic acid encoding it, by use of nucleic acid in an expression system.
  • the present invention also provides in various aspects nucleic acid encoding the transcriptional activator of the invention, which may be used for production of the encoded protein .
  • nucleic acid is provided as an isolate, in isolated and/or purified form, or free or substantially free of material with which it is naturally associated, such as free or substantially free of nucleic acid flanking the gene in the human genome, except possibly one or more regulatory sequence (s) for expression.
  • Nucleic acid may be wholly or partially synthetic and may include genomic DNA, cDNA or RNA. Where nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as encompassing reference to the RNA equivalent, with U substituted for T.
  • Nucleic acid sequences encoding a polypeptide or fusion protein in accordance with the present invention can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Sambrook, Fritsch and Maniatis, A Molecular Cloning, A Laboratory Manual, Cold Spring Harbour
  • PCR polymerase chain reaction
  • a DNA binding domain, or regulatory domain as the case may be may be generated and used in any suitable way known to those of skill in the art, including by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Modifications to the relevant sequence may be made, e.g. using site directed mutagenesis, to lead to the expression of modified peptide or to take account of codon preference in the host cells used to express the nucleic acid.
  • the sequences may be incorporated in a vector having one or more control sequences operably linked to the nucleic acid to control its expression.
  • the vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the polypeptide or peptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell.
  • Polypeptide can then be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the polypeptide is produced and recovering the polypeptide from the host cells or the surrounding medium.
  • Prokaryotic and eukaryotic cells are used for this purpose in the art, including strains of E . coli , yeast, and eukaryotic cells such as COS or CHO cells.
  • the present invention also encompasses a method of making a polypeptide or fusion protein as disclosed, the method including expression from nucleic acid encoding the product (generally nucleic acid according to the invention) .
  • This may conveniently be achieved by growing a host cell in culture, containing such a vector, under appropriate conditions which cause or allow expression of the polypeptide.
  • Polypeptides may also be expressed in in vitro systems, such as reticulocyte lysate.
  • Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others.
  • a common, preferred bacterial host is E. coli .
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate.
  • plasmids viral e.g. 'phage, or phagemid, as appropriate.
  • a recombinant expression vector's control functions may be provided by viral genetic material.
  • exemplary promoters include those derived from polyoma, Adenovirus 2, cytomegalovirus and SV40.
  • an expression vector similar to that described in Example 1 can be used.
  • a regulatory sequences of a recombinant expression vector used in the present invention may direct expression of a polypeptide or fusion protein preferentially in a particular cell type, i.e., tissue-specific regulatory elements can be used.
  • the recombinant expression vector of the invention is a plasmid, such as that described in Example 1.
  • a recombinant expression vector of the invention can be a virus, or portion thereof, which allows for expression of a nucleic acid introduced into the viral nucleic acid.
  • replication defective retroviruses, adenoviruses and adeno-associated viruses can be used.
  • Protocols for producing recombinant retroviruses and for infecting cells in vi tro or in vivo with such viruses can be found in Ausubel, et al . ⁇ supra) .
  • the genome of a virus such as adenovirus can be manipulated such that it encodes and expresses a transactivator protein but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle .
  • a further aspect of the present invention provides a host cell containing heterologous nucleic acid as disclosed herein.
  • the host cell can be, for example, a mammalian cell (e.g., a human cell), a yeast cell, a fungal cell or an insect cell.
  • the host cell can be a fertilized non-human oocyte, in which case the host cell can be used to create a transgenic organism having cells that express the transcriptional inhibitor fusion protein.
  • the recombinant expression vector can be designed to allow homologous recombination between the nucleic acid encoding the transactivator and a target gene in a host cell. Such homologous recombination vectors can be used to create homologous recombinant animals that express a fusion protein of the invention.
  • the nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.
  • the nucleic acid may be on an extra-chromosomal vector within the cell, or otherwise identifiably heterologous or foreign to the cell.
  • mammalian cell lines which may be used include CHO dhfr- cells (Urlaub and Chasin (1980) Proc. Natl. Acad Sci. USA 77:4216-4220), 293 cells (Graham et al . (1977) J. Gen. Virol. 36: pp 59) and myeloma cells like SP2 or NS0 (Galfre and Milstein (1981) Meth. Enzymol . 73 (B) : 3 -46) .
  • the invention is applicable to normal cells, such as cells to be modified for gene therapy purposes or embryonic cells modified to create a transgenic or homologous recombinant animal.
  • cell types of particular interest for gene therapy purposes include hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, muscle cells, neuronal cells and skin epithelium and airway epithelium. Additionally, for transgenic or homologous recombinant animals, embryonic stem cells and fertilized oocytes can be modified to contain nucleic acid encoding a transactivator fusion protein.
  • Nucleic acid a transactivator fusion protein can transferred into a fertilized oocyte of a non-human animal to create a transgenic animal which expresses the fusion protein of the invention in one or more cell types.
  • transgenic plants can be made by conventional techniques known in the art. Accordingly, aspects of the invention further provide non-human transgenic organisms, including animals and plants, that contains cells which express transcriptional activator protein of the invention (i.e., a nucleic acid encoding the transactivator is incorporated into one or more chromosomes in cells of the transgenic organism) .
  • transcriptional activator protein of the invention i.e., a nucleic acid encoding the transactivator is incorporated into one or more chromosomes in cells of the transgenic organism
  • a still further aspect provides a method which includes introducing the nucleic acid into a host cell.
  • the introduction which may (particularly for in vi tro introduction) be generally referred to without limitation as Atransformation®, may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage .
  • direct injection of the nucleic acid could be employed.
  • Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded product is produced. If the polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium. Following production by expression, a polypeptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e.g. see below) .
  • a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e.g. see below) .
  • nucleic acid encoding a polypeptide according to the present invention may take place in vivo by way of gene therapy.
  • One option is to introduce nucleic acid into cells ex vivo, which cells may then be implanted or otherwise administered to an individual. Such cells may have been taken from the individual and may be returned after treatment with nucleic acid of the invention.
  • a host cell containing nucleic acid according to the present invention may be comprised (e.g. in the soma) within an organism which is an animal, particularly a mammal, which may be human or non-human, such as rabbit, guinea pig, rat, mouse or other rodent, cat, dog, pig, sheep, goat, cattle or horse, or which is a bird, such as a chicken, or a plant. Genetically modified or transgenic animals, birds and plants comprising such a cell are also provided as further aspects of the present invention.
  • a host cell containing a transcriptional activator of the invention may additionally contain (e.g. as a result of transformation) one or more nucleic acids which serve as a target for the transcriptional activator.
  • a target nucleic acid comprises a nucleotide sequence to be transcribed operatively linked to at least one operator sequence.
  • a transcriptional activator in accordance with the present invention may be used to regulate transcription of a target nucleotide sequence which is operatively or operably linked to a regulatory sequence to which the transcriptional activator binds.
  • the nucleotide sequence to be transcribed typically includes a minimal promoter sequence which is not itself transcribed but which serves (at least in part) to position the transcriptional machinery for transcription.
  • the minimal promoter sequence is located upstream of the transcribed sequence to form a contiguous nucleotide sequence. The activity of such a minimal promoter is dependent upon the binding of a transcriptional activator to an operatively linked regulatory operator sequence.
  • the minimal promoter may be from the human cytomegalovirus (as described in Boshart et al. (1985) Cell 41:521-530), and other suitable minimal promoters are available to those skilled in the art.
  • the target nucleotide sequence is operatively linked to at least one oligonucleotide sequence to which a transcriptional activator of the invention binds, an operator sequence.
  • the operator is usually 5' to the sequence to be transcribed and, where appropriate, minimal promoter.
  • An operator sequence may be operatively linked downstream (i.e., 3') of the nucleotide sequence to be transcribed.
  • the operator sequence may correspond to that of a lux-box operator sequence.
  • lux-box operator sequence is intended to encompass all DNA sequences that are binding sites for LuxR-type transactivators (Fuque, et al (1994) , J " .
  • a nucleotide sequence to be transcribed can be operatively linked to a single lux box operator sequence, or to multiple lux-box operator sequences (e.g., two, three, four, five, six, seven, eight, nine, ten or more operator sequences) .
  • a transcription factor in accordance with the present invention binds to the appropriate DNA operator sequences in the presence but not the absence of a cognate acylhomoserine lactone (AcylHSL) or an analogue thereof.
  • a fusion protein of the invention binds to its cognate operator sequence. Where the operator sequence is operatively linked to a further sequence of interest, transcription of the further sequence of interest is thereby activated.
  • a LuxR DNA binding domain the operator sequence may be a Lux box (Fuque, et al (1994) ) .
  • Other DNA binding domains that may be employed include DNA binding domain of other prokaryotic or eukaryotic DNA-binding proteins with their cognate operator sequence.
  • a preferred embodiment of the invention utilises DNA-binding domains of eukaryotic origin, more preferably from mammals, more preferably from human .
  • DNA binding sites are GAL4 DNA, virus DNA binding sites and insect DNA binding sites.
  • a preferred embodiment of the invention utilises a DNA-binding domain of a human transcription factor.
  • the DNA binding domain of human transcription factor HNF-1 is utilised (amino acids 1-281, Sequence accession number P20823) .
  • the operator sequence may correspond to that of an HNF-1 cognate binding site.
  • a nucleotide sequence to be transcribed can be operatively linked to a single or multiple HNF-1 binding sites.
  • the further sequence operably linked to the promoter and operator sequences may be a coding sequence for a polypeptide or peptide, an antisense sequence or a ribozy e .
  • a polypeptide of which expression may be controlled using the present invention may be selected according to the desires and aims of the person performing the invention, and may be a therapeutic protein or a cytotoxic protein.
  • Polypeptide expression may be inhibited by using appropriate nucleic acid to influence expression by antisense regulation, and an antisense sequence may be placed under transcriptional control in accordance with the present invention.
  • an antisense sequence may be placed under transcriptional control in accordance with the present invention.
  • Double-stranded DNA is placed under the control of a promoter in a "reverse orientation" such that transcription of the "anti-sense" strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene.
  • the complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. Whether or not this is the actual mode of action is still uncertain. However, it is established fact that the technique works.
  • nucleic acid is used which on transcription produces a ribozyme, able to cut nucleic acid at a specific site - thus also useful in influencing gene expression.
  • Background references for ribozymes include Kashani-Sabet and Scanlon (1995) . Cancer Gene Therapy, 2, (3)
  • An AcylHSL-regulated transcription unit of the invention may be incorporated into a recombinant vector (e.g., a plasmid or viral vector) , and may be introduced into a host cell or animal, optionally along with a transcriptional activator as disclosed or encoding nucleic acid therefor.
  • a recombinant vector e.g., a plasmid or viral vector
  • a further aspect of the present invention provides a composition
  • a composition comprising: (i) a transcriptional activator as disclosed, or a first nucleic acid encoding a transcriptional activator as disclosed (which nucleic acid may encode a fusion protein comprising first, second and third polypeptide components as disclosed or may comprise separate sequences encoding a fusion which comprises first and second polypeptide components and a third polypeptide to interact with the fusion to provide a transcription factor as discussed above) ; and
  • a second nucleic acid comprising a nucleotide sequence to be transcribed operatively linked to a AcylHSL- regulated transcription unit.
  • the first and second nucleic acids are separate molecules (e.g., two different vectors).
  • a host cell may be cotransfected with the two nucleic acid molecules or successively transfected first with one nucleic acid molecule and then the other nucleic acid molecule.
  • the components of a trancriptional activator comprising a fusion protein which comprises first and second components and another polypeptide providing transcriptional activation which interacts with the fusion to provide a transactivator may be provided as separate molecules.
  • the nucleic acids are linked (i.e., colinear) in the same molecule (e.g., a single vector) . In this case, a host cell may be transfected with the single nucleic acid molecule.
  • the invention further provides a method of treatment which includes administering to a patient an agent which comprises
  • a transcriptional activator according to the present invention may be used to regulate transcription of a sequence by means of an operator sequence operably linked to the sequence to be transcribed. As discussed, this operator/transcribed sequence construct may be introduced into host cells. In an alternative, a sequence to be transcribed may be endogenous to a host cell.
  • An endogenous sequence may be operatively linked to a AcylHSL-regulated transcription unit by means of homologous recombination.
  • a homologous recombination vector can be prepared which includes at least one Lux-box operator sequence and a miminal promoter sequence flanked at its 3 ' end by sequences representing the coding region of the endogenous gene and flanked at its 5' end by sequences from the upstream region of the endogenous gene by excluding the actual promoter region of the endogenous gene.
  • flanking sequences are of sufficient length for successful homologous recombination of the vector DNA with the endogenous gene.
  • several kilobases of flanking DNA are included in the homologous recombination vector.
  • a region of the endogenous promoter is replaced by the vector DNA containing one or more Lux-box operator sequences operably linked to a minimal promoter.
  • expression of the endogenous gene is no longer under the control of its endogenous promoter but rather is placed under the control of the AcylHSL-regulated transcription unit.
  • an operator sequence may be inserted elsewhere within an endogenous gene, preferably within a 5 ' or 3 ' regulatory region, via homologous recombination to create an endogenous gene whose expression can be regulated by a AcylHSL-regulated fusion protein described herein.
  • one or more Lux-box sequences can be inserted into a promoter or enhancer region of an endogenous gene such that promoter or enhancer function is maintained.
  • Application or provision of cognate Acyl-HSL e.g. by administration, may be used to regulate transcription from an AcylHSL-regulated transcription unit by means of a transcriptional activator according to the present invention.
  • a composition according to the present invention that is to be given to an individual, administration is preferably in a Aprophylactically effective amount or a " therapeutically effective amount" as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • compositions according to the present invention may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • Liposomes particularly cationic liposomes, may be used in carrier formulations.
  • the agent may be administered in a localised manner to a tumour site or other desired site or may be delivered in a manner in which it targets tumour or other cells.
  • Targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons, for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
  • these agents may be produced in the target cells by expression from an encoding gene introduced into the cells, e.g. in a viral vector.
  • the vector may targeted to the specific cells to be treated, or it may contain regulatory elements which are switched on more or less selectively by the target cells.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated, such as cancer, virus infection or any other condition in which an effect mediated by activity of the fusion protein is desirable.
  • Nucleic acid according to the present invention encoding a transcriptional activator may be used in methods of gene therapy, for instance in treatment of individuals, e.g. with the aim of preventing or curing (wholly or partially) a disorder or for another purpose as discussed elsewhere herein.
  • Vectors such as viral vectors have been used in the prior art to introduce nucleic acid into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transfection can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide.
  • the transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.
  • vectors both viral vectors and plasmid vectors
  • a number of viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpesviruses, including HSV and EBV, and retroviruses.
  • papovaviruses such as SV40
  • vaccinia virus vaccinia virus
  • herpesviruses including HSV and EBV
  • retroviruses retroviruses.
  • Many gene therapy protocols in the prior art have used disabled murine retroviruses.
  • nucleic acid into cells includes electroporation, calcium phosphate co-precipitation, mechanical techniques such as microinjection, transfer mediated by liposomes and direct DNA uptake and receptor- mediated DNA transfer.
  • Receptor-mediated gene transfer in which the nucleic acid is linked to a protein ligand via polylysine, with the ligand being specific for a receptor present on the surface of the target cells, is an example of a technique for specifically targeting nucleic acid to particular cells.
  • N- (3 -oxohexanoyl) -homoserine lactone- recfulated transcriptional activator fusion protein comprising the LuxR protein from Vibrio fischeri and VP16 activator domain from Herpes simplex virus
  • a nucleic acid fragment was amplified by the polymerase chain reaction (PCR) using the plasmid pHK724 (G. B. Kaplan and E. P. Greenberg. 1987. Proc. Natl. Acad. Sci. USA 84: 6639-6643) as template.
  • PCR polymerase chain reaction
  • the resulting DNA fragment was digested with BamHI and Xhol and ligated into the BamHI and Xhol sites of the pcDNA3 vector.
  • the resultant vector is named pcD-luxR.
  • a nucleic acid fragment encoding amino acids 363B490 of herpes simplex virus VP16 was amplified by PCR using the plasmid pUHD 15-1 (M. Grossen and H. Bujard. 1992. Proc. Natl. Acad. Sci. USA 89: 5547-5551) as template.
  • the amplified PCR fragment and pcD-luxR were digested with EcoRI and Xhol .
  • the amplified fragment was then ligated directionally into pcD-luxR to create the expression vector pcD-luxR/VP16.
  • the resultant expression vector contains nucleotide sequences encoding the fusion protein comprising amino acids 1B250 of wild type luxR protein linked in- frame to amino acids 363B490 of VP16.
  • the nucleotide and amino acid sequence across the junction of the fusion is as follows: TTT AAA AGT GAA TTC GCG TAC AGC and Phe-Lys-Ser-Glu-Phe-Ala-Tyr-Ser, respectively.
  • Phe-Lys-Ser corresponds to amino acids 248-250 of the luxR protein.
  • Glu-Phe are encoded by the EcoRI restriction site and Ala-Tyr-Ser correspond to the amino acids 363B365 of VP16.
  • Mouse-erythropoietin under the control of a minimal CMV promoter (Gossen and Bujard 1992) is used as reporter gene to measure N- (3 -oxohexanoyl) -homoserine lactone dependent transcription .
  • test operators present in the plasmid pOR/EPO (described in Rizzuto et al . (1999) Proc. Natl. Acad. Sci. USA 96, pp. 6417-6422) is removed by restriction digest using the restriction enzymes Ndel and Kpnl and protruding ends are removed by incubation with Klenow polymerase and T4 DNA polymerase .
  • Annealed oligonucleotides are ligated, digested with BamHI and Bglll in order to select only head-to-tail ligation products, filled-in with Klenow polymerase and cloned blunt into the reporter construct. Plasmids containing one to seven head-to-tail repeats of the luxR binding sites are isolated, verified by DNA sequencing. The resulting constructs are named pVIJ- nXlux-bdg mEpo, were n indicates the number of luxR-binding sites present. The plasmids are then examined for their ability to be activated by the LuxR/VP16 chimeric transcription factor utilizing transient transfection of DNA plasmids in cultured cells.
  • Hela cells grown in DMEM are transiently transfected by standard calcium phosphate method (F. L. Graham and A. J. van der Eb . 1973. Virology 52: 456-461) with 10 ug of the pcD- luxR/VP16 expression vector and 10 ug of the reporter plasmid pVIJ 6Xlux-bdg mEpo, in which hexameric luxR-binding sites are fused upstream of a minimal CMV-promoter and murine EPO reporter gene.
  • N- (3-oxo-octanoyl) -L-homoserine lactone- regulated transcriptional activator fusion protein comprising the TraR protein from Agrobacterium tumefaciens and either the full-length VP16 activator domain from Herpes simplex virus or a minimal activator domain derived from VP16 (F3)
  • the nucleic acid fragment encoding the TraR protein was cloned into the eukaryotic expression vector pcDNA3 (Invitrogen) digested with BamHI/ EcoRI . 5 ' -protruding ends of Ndel and
  • ATACGATCGCTCGAGTTAGAATTCGATGAGTTTCCGCCGGATGGCGAG-3 ' encodes the last eight C-terminal amino acids of TraR protein (amino acids 227-234) , a EcoRI restriction site, which can be used for C-terminal in-frame cloning of the desired activation domains, the stop codon UAA and a Xhol restriction site suitable for cloning into pcDNA3.
  • the resulting plasmid was called pcD-TraR/EcoRI .
  • LuxR/VP16 was digested with EcoRl/XhoI and the resulting DNA fragment coding for VP16 was cloned into pcD-TraR/EcoRI .
  • the resultant expression vector was named pcD-TraR/VP16.
  • the nucleotide and amino acid sequence across the junction of the fusion is as follows: AAA CTC ATC GAA TTC GCG TAC AGC and Lys-Leu-Ile-Glu-Phe-Ala-Tyr-Ser, respectively. Lys-Leu-Ile correspond to amino acids 232-234 of the TraR protein.
  • Glu-Phe are encoded by the EcoRI restriction site and Ala-Tyr-Ser correspond to the amino acids 363-365 of VP16.
  • VP16 activator domain in addition to the full-length VP16 activator domain in other embodiments we fused three minimal activation modules derived from VP16 (F3) either to the N-terminus or to the C-terminus of TraR. These minimal activation modules have been demonstrated to be equally active as the full-length VP16 activation domain (U. Baron, M. Gossen and H. Bujard. 1997. Nucl. Acids Res.: 2723-2729).
  • the amino acid sequence of one minimal activation module is Pro-Ala-Asp-Ala-Leu-Asp-Asp-Phe- Asp-Leu-Asp-Met-Leu and comprises the amino acids 435-447 of VP16.
  • F3 was fused in frame to the C-terminus of TraR using the EcoRI restriction site of pCD-TraR/EcoRI .
  • the nucleotide and amino acid sequence across the junction of the fusion is as follows: AAA CTC ATC GAA TTC CCG GCC GAC and Lys-Leu-Ile-Glu-Phe-Pro-Ala-Asp, respectively. Lys-Leu-Ile correspond to amino acids 232-234 of the TraR protein. Glu-Phe are encoded by the EcoRI restriction site and
  • Pro-Ala-Asp correspond to the last three amino acids of F3.
  • a BamHI site was introduced at the N-terminus of TraR (pcD-Bam/TraR) and at the C-terminus of F3 by PCR.
  • the resulting plasmid was called pcD-F3/TraR.
  • the nucleotide and amino acid sequence across the junction of the fusion is as follows: GAC ATG CTC GGA TCC ATG CAG CAC and Asp-Met-Leu-Gly-Ser-Met-Gln-His , respectively.
  • Asp-Met-Leu correspond to the last three amino acids of F3.
  • Gly-Ser are encoded by the BamHI restriction site and Met-Gln-
  • N- (3-oxo-octanoyl) -L-homoserine lactone- regulated transcriptional activator fusion protein comprising the TraR protein from Agrobacterium tumefaciens and the minimal activation domain F3 derived from VP16 separated from TraR in different embodiments by different peptide-linkers
  • Oligonucleotides were designed which introduce different peptides as linkers between the minimal activation domain F3 and the TraR protein either at the C-terminus or at the N- terminus of TraR.
  • a GS- linker is fused to the C-terminus of TraR by PCR using a 5 ' -oligonucleotide annealing to the internal coding region of TraR and the 3 ' -oligonucleotide (5'- GACTCTAGATTAACTCGAGCCTGAACCGCTCCCGGATCCGATGAGTTTCCGCCGGATGGC- 3') which adds the GS-linker and suitable restriction sites to TraR.
  • the PCR fragment coding for TraR fused to the GS linker is cloned into pcD-TraR by SacII/Xbal restriction digest.
  • the resulting plasmid is called pcD-TraR/GS.
  • the nucleotide and amino acid sequence at the C-terminus of TraR is as follows: AAA CTC ATC GGA TCC GGG AGC GGT TCA GGC TCG AGT TAA TCT AGA and Lys-Leu-Ile-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Ser-Ser-*, respectively. Lys-Leu-Ile correspond to the last three amino acids of TraR. All other amino acids are encoded by the GS- linker.
  • a second linker is cloned to the C-terminus of TraR by ligation of the two suitable, annealed oligonucleotides into pcD-TraR/GS digested with BamHI/XhoI restriction enzymes.
  • This plasmid is called pcD-TraR/Poly .
  • oligonucleotide sense 5 ' -GATCAGCTCTAGATGGCATGCTGCTAGCTGAGGATCCCCGGGGAGAATTC-3 '
  • oligonucleotide antisense 5 ' -TCGAGAATTCTCCCCGGGGATCCTCAGCTAGCAGCATGCCATCTAGAGCT-3 ' .
  • the amino acid sequence at the C-terminus of TraR is as follows : Lys-Leu-Ile-Gly-Ser-Ala-Leu-Asp-Gly-Met-Leu-Leu-Ala- Glu-Asp-Pro-Arg-Gly-Glu-Phe-Ser-Ser . Lys-Leu-Ile correspond to the last three amino acids of TraR. All other amino acids are encoded by the Poly- linker.
  • the C-terminal fusion protein of TraR and F3 , separated by the GS linker is obtained by restriction digest of pcD-Tra/F3 with EcoRl/Xbal and the DNA fragment coding for F3 is ligated into pcD-TraR/GS digested Xhol/Xbal . 5 ' -protruding ends of EcoRI and Xhol were filled with Klenow polymerase The resulting plasmid is called pcD-TraR/GS/F3.
  • the C-terminal fusion protein of TraR and F3 , separated by the Poly-linker is obtained by restriction digest of pcD-Tra/F3 with EcoRI/Xbal and the DNA fragment coding for F3 is ligated into pCD-TraR/Poly digested EcoRI/Xbal .
  • the resulting plasmid is called pcD-TraR/poly/F3.
  • oligonucleotide sense oligonucleotide sense:
  • oligonucleotide antisense 5 ' -AGCTTTCTAGAATGGGATCCGGGAGCGGTTCAGCTAGCA-3 ' ;
  • N-terminal amino acid sequence is as follows: Met-Gly-Ser- Gly-Ser-Gly-Ser-Ala-Ser-Arg-Ser-Met-Gln-His .
  • Met-Gly-Ser-Gly- Ser-Gly-Ser-Ala-Ser-Arg-Ser are encoded by the GS2 linker.
  • Met-Gln-His represent the first three amino acids of TraR.
  • two suitable oligonucleotides are designed such that they are annealed and directly cloned into the BamHI/Nhel restriction sites of pcD-GS2/TraR. The resulting plasmid is called pcD-Poly2/TraR.
  • the sequence of the oligonucleotides are: oligonucleotide sense:
  • N-terminal amino acid sequence is as follows: Met-Gly-Ser- Ala-Arg-Asp-Gly-Arg-Ile-Gln-Glu-Gly-Ile-Ser-Ala-Ala-Ser-Arg- Ser-Met-Gln-His .
  • Met-Gly-Ser-Ala-Arg-Asp-Gly-Arg-Ile-Gln-Glu- Gly-Ile-Ser-Ala-Ala-Ser-Arg-Ser are encoded by the Poly2 linker.
  • Met-Gln-His represent the first three amino acids of TraR.
  • the N-terminal fusion protein of TraR and F3 , separated by the GS2 linker is obtained by restriction digest of pcD-F3/TraR with Hindlll/BamHI and the DNA fragment coding for F3 is ligated into pCD-GS2/TraR digested Hindlll /BamHI .
  • the resulting plasmid is called pcD-F3/GS2/TraR.
  • the same strategy is used to clone F3 in frame to the N-terminus of TraR separated by the Poly2 linker, except that the F3 -containing DNA fragment is cloned into pcD-Poly2/TraR, digested with Hindlll/ BamHI .
  • This plasmid is called pcD-F3/Poly2/TraR.
  • N- (3-oxo-octanoyl) -L-homoserine lactone- regulated transcriptional activator fusion protein comprising the TraR protein from Agrobacterium tumefaciens and the human
  • the human NF- ⁇ B p65 As an alternative to the Herpes simplex virus activation domain VP16 or its derivative F3 , the human NF- ⁇ B p65
  • activation domain is fused in frame either C-terminally or N-terminally to TraR.
  • the coding region of p65 is amplified by PCR using oligonucleotides which introduce restriction sites suitable for cloning and either an EcoRI restriction site at the N- terminus of p65 or a BamHI restriction site at the C-terminus of p65. These sites can be used for cloning p65 either to the C-terminus of TraR or to the N-terminus, respectively.
  • the plasmids pcD- TraR/GS and pcD-TraR/Poly are used, which introduce the two different linker-regions described above between TraR and p65.
  • the PCR fragment encoding p65 which contains an EcoRI restriction site at its N-terminus is digested with EcoRI cloned into pcD-TraR/GS digested Xhol . 5 ' -protruding ends of EcoRI and Xhol are filled in by Klenow polymerase.
  • the plasmid is called pcD-TraR/GS/p65.
  • the nucleotide and amino acid sequence across the junction of the fusion is as follows: AAA CTC ATC GGA TCC GGG AGC GGT TCA GGC TCG AAA TTC CAG TAC CTG and Lys-Leu-Ile-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Ser-Lys-Phe-Gln- Tyr-Leu. Lys-Leu-Ile correspond to the last three amino acids of TraR. Gly-Ser-Gly-Ser-Gly-Ser-Gly-Ser represent the GS linker region.
  • Lys-Phe correspond to the filled Xhol /EcoRI restriction sites and Gln-Tyr-Leu correspond to the first three amino acids of p65.
  • PCR fragment encoding p65 is digested with EcoRI and cloned into pcD-TraR/Poly, digested EcoRI/Xbal .
  • the plasmid is called pcD-TraR/Poly/p65. 5 ' -protruding ends of EcoRI and Xbal are filled in by Klenow polymerase.
  • the nucleotide and amino acid sequence across the junction of the fusion is as follows: AAA CTC ATC GGA TCA GCT CTA GAT GGC ATG CTG CTA GCT GAG GAT CCC CGG GGA GAA TTC CAG TAC CTG and Lys- Leu-lie-Gly-Ser-Ala-Leu-Asp-Gly-Met-Leu-Leu-Ala-Glu-Asp-Pro- Arg-Gly-Glu-Phe-Gln-Tyr-Leu. Lys-Leu-Ile correspond to the last three amino acids of TraR. Gly-Ser-Ala-Leu-Asp-Gly-Met- Leu-Leu-Ala-Glu-Asp-Pro-Arg-Gly represent the Poly linker region. Glu-Phe correspond to the EcoRI restriction site and
  • Gln-Tyr-Leu correspond to the first three amino acids of p65.
  • a similar stategy for cloning p65 to the N-terminus of TraR is used.
  • an ATG codon as the first amino acid of p65 is introduced by PCR.
  • the PCR fragment containing a BamHI restriction site at the C-terminus of p65 is digested with BamHI and inserted into the vectors pcD-GS2/TraR and pcD-
  • Poly2/TraR Poly2/TraR, respectively, which have been digested with Hindlll, /BamHI . 5 ' -protruding ends of Hindlll are filled in by Klenow polymerase.
  • the nucleotide and amino acid sequence across the junction of the fusion of pcD-p65/GS2/TraR is: ATC AGC TCC GGA TCC GGG AGC GGT TCA GCT AGC AGA TCC ATG CAG CAC and Ile-Ser-Ser-Gly-Ser-Gly-Ser-Gly-Ser-Ala-Ser-Arg-Ser-Met- Gln-His. Ile-Ser-Ser correspond to the last three amino acids of p65.
  • Gly-Ser correspond to the BamHI restriction site
  • Gly- Ser-Gly-Ser-Ala-Ser-Arg-Ser represent the GS2 linker region
  • Met-Gln-His are the first three amino acids of TraR.
  • the nucleotide and amino acid sequence across the junction of the fusion of pcD-p65/Poly2/TraR is: ATC AGC TCC GGA TCC GCT CGA
  • Ile-Ser-Ser are the last three amino acids of p65.
  • Gly-Ser correspond to the BamHI restriction site
  • Ala-Arg-Asp-Gly-Arg-Ile-Gln-Glu- Gly-Ile-Ser-Ala-Ala-Ser-Arg-Ser are encoded by the Poly2 linker and Met-Gln-His represent the first three amino acids of TraR.
  • N- (3-oxo-octanoyl) -L-homoserine lactone- regulated transcriptional activator fusion protein comprising the TraR protein from Agrobacterium tumefaciens and the rat LFB1/HNF1 activation domain
  • the activation domain of rat LFBl/HNFl (Accession No. J02170) (Frain et al . 1989. Cell 59: 145-157) is fused in frame to the C-terminus of TraR, leaving two different peptide linkers between the TraR protein and the LFBl/HNFl activation domain.
  • the plasmid contains the coding sequence of the full-length TraR protein from residue Ml to 1234 fused to the rat LFBl/HNFl activation domain (residues A282 to Q628) .
  • the cloning introduces four amino acids (Gly-Ser-Ala- Leu) between the C-terminus of TraR and the N-terminus of the activation domain of LFBl/HNFl.
  • a Mscl/Bglll fragment is excized from the plasmid pBl .2 (rat
  • TraR/Poly vector is filled-in in a reaction catalyzed by the Klenow enzyme .
  • PcD-TraR/GS/BlAD - The plasmid contains the coding sequence of the full-length TraR protein from residue Ml to 1234 fused to the N-terminus of rat LFBl/HNFl activation domain from residue M283 to Q628.
  • the cloning introduces nine amino acids (Gly- Ser-Gly-Ser-Gly-Ser-Gly-Ser-Thr) between the C-terminus of TraR and the N-terminus of LFBl/HNFl.
  • a Mscl/Bglll fragment is excized from the plasmid pBl .2 and cloned into the Xhol site of the plasmid pcD-
  • Xhol site of the receiving vector are filled-in a reaction catalyzed by the Klenow enzyme.
  • TraR functions as a N- (3-oxo-octanoyl) -L-homoserine lactone regulated dimerization domain in place of the LFBl/HNFl dimerizatin domain
  • LFBl/HNFl binds to DNA only as a dimer thereby activating transcription (Nicosia et al . 1990) .
  • the dimerization domain is contained within the N-terminal 28 amino acids.
  • PcD-TraRf1/BldelDD The plasmid contains the coding sequence of the full-length TraR protein from residue Ml to 1234 fused to the N-terminus of rat LFBl/HNFl from residue P33 to Q628, in place of the LFBl/HNFl dimerization domain.
  • an Sphl/Bglll fragment is excized from the plasmid
  • LFBl/HNFl ⁇ l-FL and cloned into the Sphl/BamHI sites of the plasmid pcD-TraR/Poly .
  • the plasmid LFBl/HNFl ⁇ l-FL derives from the construct LFBlmutant ⁇ l described by Nicosia et al . 1990 (Cell 61: 1225-1236), however, contains the sequence coding for the entire C-terminal activation domain of LFBl/HNFl protein followed by 220 nt of the 3 ' -non coding sequence of the LFBl/HNFl cDNA.
  • the cloning introduces a 7 aminoacids long spacer (Gly-Ser-Ala-Leu-Asp-Gly-Met ) between the TraR and LFB1 sequences .
  • PcD-TraR182/BldelDD - The plasmid contains the coding sequence of the TraR protein from residue Ml to L182 fused to the N- terminus of rat LFBl/HNFl from residue P33 to Q628, in place of the LFBl/HNFl dimerization domain.
  • An oligonucleotide comprising the sequence from nt 495 to nt 546 of the TraR open reading frame (oligo TRA166-182sense) is annealed to an oligonucleotide with the complementary sequence (oligo TRA166- 182rev) .
  • the resulting double-strand oligonucleotide is digested with the SacII restriction enzyme and cloned into the
  • TRA166-182sense and the oligo TRA166-182rev are as follows: oligo TRA166-182sense:
  • the plasmid contains the coding sequence of the TraR protein from residue Ml to T167 fused to the N- terminus of rat LFBl/HNFl from residue P33 to Q628, in place of the LFBl/HNFl dimerization domain.
  • an Sphl /Bglll fragment is excized from the plasmid LFBl/HNFl ⁇ l-FL and cloned into the SacIl/BamHI sites of the plasmid pcD-TraR. Both the Sphl and SacII sites are flushed in a reaction catalyzed by the Klenow enzyme.
  • NLS nuclear localization signal
  • the nuclear localization signal (NLS) Met-Pro-Lys-Arg-Pro-Arg- Pro is added to the TraR fusion protein in order to promote the transport of the protein to the cell nucleus.
  • Oligonucleotides encoding the above mentioned peptide sequence and restriction sites suitable for cloning into the expression vectors are designed.
  • oligonucleotide-sense 5 ' -AGCTTATGCCCAAAAGACCACGCCCTG-3 '
  • oligonucleotide-antisense 5 ' -GATCCAGGGCGTGGTCTTTTGGGCATA-3 '
  • the resulting plasmid is called pcD-NLS/TraR.
  • the DNA fragment coding for TraR containing the NLS at its N-terminus is cloned into any of the above mentioned vectors expressing the fusion proteins between TraR and C-terminal activation domains by Hindlll/SacII restriction digest.
  • oligonucleotide-sense 5 ' -AATTCCCCAAAAGACCACGCCCTTAAT-3 '
  • oligonucleotide-antisense 5 ' -CTAGATTAAGGGCGTGGTCTTTTGGGG-3 '
  • the resulting plasmid is called pcD- TraR/NLS .
  • the DNA fragment coding for TraR containing the NLS at its C-terminus is cloned into any of the above mentioned vectors expressing the fusion proteins between TraR and N-terminal activation domains by Xbal/SacII restriction digest.
  • the NLS sequence can also be inserted into the linker regions between TraR and the activation domain.
  • oligonucleotide-sense 5 ' -AATTCAGCCCAAAAGACCACGCCCTGG-3 '
  • oligonucleotide-antisense 5 ' -AATTCCAGGGCGTGGTCTTTTGGGCTG-3 '
  • the resulting plasmid is called pcD-p65/NLS/TraR.
  • the heptamerized tet-operators present in the plasmid pOr/EPO (described in Rizzuto et el. 1999. Proc. Natl. Acad. Sci. U.S.A. 96: pp6417-6422) are removed by restriction digest with Ndel/Kpnl .
  • tet-operators In place of the tet-operators we clone a DNA fragment containing the 18 bp inverted repeat sequence defined as DNA binding site of TraR (C. Fuqua and S.C. Winans . 1996. J. Bacteriol . 178: 435-440).
  • a DNA fragment containing the CMV minimal promotor and the trabox is excised from the plasmid p-trabox/EPO by restriction digest using the enzymes Aatll/EcoRI .
  • 3'- protruding ends produced by Aatll are removed by T4-DNA polymerase and the DNA fragment is cloned into the eukaryotic expression vector pSEAP2-Basic (Clontech) , digested with Nhel/EcoRI .
  • the 5 ' -protruding ends of the Nhel restriction site are filled in by Klenow polymerase.
  • the resulting plasmid is called p- trabox/SEAP2.
  • PcD-TraR linearized with the restriction enzyme Xbal , was taken as a DNA template for in vi tro transcription using T7
  • RNA polymerase The RNA transcript was subsequently used for in vi tro translation with nuclease-treated rabbit reticulocyte lysate in the presence of 35 S-labelled methionine . In vi tro translation was performed either in the presence or in the absence of 10 ⁇ M N- (3-oxo-octanoyl) -L-homoserine lactone.
  • TraR translated in vi tro was then tested for its DNA-binding activity on oligonucleotides containing the DNA sequence of the tra-box.
  • two complementary oligonucleotides (described in the section above) were annealed and labelled at the 5 '-end of the oligonucleotides with T4 -polynucleotide kinase using [ ⁇ - 32 P]ATP.
  • 40 fmoles of the resulting duplex were added to a reaction mixture containing 25 mM Hepes pH 7.5 , 1 mM DTT, 2 % glycerol, 5 mM EDTA, 100 mM NaCl, 100 ng poly [d (I .
  • the DNA-binding activity of TraR expressed in Hep3B cells using recombinant vaccinia virus vTF7-3 depends on the presence of N- (3 -oxo-octanoyl) -L-homoserine lactone
  • Hep3B cells (5.5 x 10 5 in 60-mm-diameter dishes) were infected with vaccinia virus vTF7-3 for 1 hour at 37 °C and then transfected with 20 ⁇ g of pcD-TraR or 20 ⁇ g of control vector (pcDNA3) by the calcium precipitation technique. Transfection was performed in the presence (+) or absence (-) of 10 ⁇ M N- (3-oxo-octanoyl) -L-homoserine lactone.
  • lysis buffer 25 mM Tris pH 8.0, 300 mM NaCl, 20 % glycerol, 2 mM DTT, 2 mM PMSF, 1 % Triton-XlOO
  • 80 ⁇ g of total protein were loaded onto a 12 % SDS-PAGE, proteins were transferred onto nitrocellulose and Western Blotting was performed using the SuperSignal West Pico system (Pierce) .
  • the specific antibody reacting with the TraR protein was prepared by us.
  • TraR protein was expressed at comparable amounts when transfected in the presence or absence of N- (3-oxo-octanoyl) - L-homoserine lactone. No signal was present in the control- transfection. Purified TraR was employed as a positive control. A non-specific cellular protein reacted with the ⁇ - TraR antibody.
  • Binding reaction was performed exactly as described above, except that 1 ⁇ g of poly [d (I . C) -d (I . C) and 10 ⁇ l of cellular extract were used. No NaCl was added to the reaction mix.
  • TraR expressed in Hep3B cells only bound to DNA, when N- (3-oxo- octanoyl) -L-homoserine lactone was present during transfection. No DNA-binding was detectable when TraR was expressed without ligand, even if N- (3-oxo-octanoyl) -L- homoserine lactone was added during the DNA-binding reaction.

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Abstract

L'invention concerne un activateur transcriptionnel comprenant une protéine de fusion, laquelle protéine comprend au moins deux constituants polypeptidiques, un premier constituant se liant à une séquence opérateur dans l'ADN de manière spécifique à la séquence, et un deuxième constituant polypeptidique comprenant un domaine de régulation du facteur de transcription Lux-R liant le AcylHLS parent ou un analogue. Lors de la liaison avec AcylHLS ou avec son analogue, la fonction de liaison à l'ADN du premier constituant polypeptidique est activée. L'activateur transcriptionnel comprend également un troisième constituant polypeptidique activant la transcription dans les cellules eucaryotes.
EP00979852A 1999-11-17 2000-11-17 Techniques et dispositifs de regulation de l'expression genetique Withdrawn EP1232271A2 (fr)

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EP3247378B8 (fr) 2015-01-09 2023-08-23 Revolution Medicines, Inc. Composés macrocycliques participant à une liaison coopérative et leurs utilisations médicales
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CA3042231A1 (fr) 2016-10-28 2018-05-03 Ginkgo Bioworks, Inc. Compositions et procedes pour la production de composes
CA3150224A1 (fr) 2019-09-10 2021-03-18 Obsidian Therapeutics, Inc. Compositions d'anhydrase carbonique 2 et methodes de regulation ajustable
KR20220109407A (ko) 2019-11-04 2022-08-04 레볼루션 메디슨즈, 인크. Ras 억제제
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AU2020379734A1 (en) 2019-11-04 2022-05-05 Revolution Medicines, Inc. Ras inhibitors
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GB9817704D0 (en) * 1998-08-13 1998-10-07 Zeneca Ltd Gene switch
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