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WO1997020055A1 - Utilisation de sequences anti-sens pour augmenter la sensibilite par rapport a l'amplification genique - Google Patents

Utilisation de sequences anti-sens pour augmenter la sensibilite par rapport a l'amplification genique Download PDF

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Publication number
WO1997020055A1
WO1997020055A1 PCT/US1995/015098 US9515098W WO9720055A1 WO 1997020055 A1 WO1997020055 A1 WO 1997020055A1 US 9515098 W US9515098 W US 9515098W WO 9720055 A1 WO9720055 A1 WO 9720055A1
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Prior art keywords
dhfr
gene
sense
cell
sequence
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Yves Mory
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WEINWURZEL HENRY
Interpharm Laboratories Ltd
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WEINWURZEL HENRY
Interpharm Laboratories Ltd
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Priority to ZA959429A priority Critical patent/ZA959429B/xx
Application filed by WEINWURZEL HENRY, Interpharm Laboratories Ltd filed Critical WEINWURZEL HENRY
Priority to AU42408/96A priority patent/AU718896B2/en
Priority to PCT/US1995/015098 priority patent/WO1997020055A1/fr
Priority to AT95940765T priority patent/ATE222957T1/de
Priority to DE69527994T priority patent/DE69527994D1/de
Priority to US09/077,253 priority patent/US6399377B1/en
Priority to EP95940765A priority patent/EP0863990B1/fr
Priority to CA002235962A priority patent/CA2235962A1/fr
Publication of WO1997020055A1 publication Critical patent/WO1997020055A1/fr
Anticipated expiration legal-status Critical
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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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/01—Oxidoreductases acting on the CH-NH group of donors (1.5) with NAD+ or NADP+ as acceptor (1.5.1)
    • C12Y105/01003—Dihydrofolate reductase (1.5.1.3)
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • the present invention is generally in the field of mammalian gene expression systems used for the cell culture production of proteins.
  • the present invention concerns new gene expression systems for increasing the efficiency and the amount of protein production in eukaryotic cell lines and, in particular, the well-known Chinese hamster ovary (CHO) cell culture systems which utilize the dihydrofolate reductase
  • DHFR DHFR -methotrexate
  • MTX metalhotrexate
  • CHO cell lines are then transformed or co-transformed with a plasmid/vector carrying a gene sequence encoding the protein of choice to be expressed together with the DHFR gene carried either on the same vector or on a different one.
  • the enzyme DHFR serves as a selectable marker for these transformed cells.
  • the vector is usually of the type which can undergo stable recombination and hence subsequent stable incorporation into the genome of CHO DHFR" cells thereby rendering such cells DHFR + in a stable and constitutive manner.
  • the positively transformed DHFR + cells are selected by growing the cells in a standard culture medium, in which DHFR' cells cannot grow, and subjecting the cell cultures to a number of culture cycles or passages.
  • a standard culture medium usually contains sufficient methotrexate to kill DHFR ' cells but which is generally not lethal to the DHFR + cells.
  • stably transformed DHFR + cells i.e., in which the entire transforming vector or co- ransforming vectors or the essential portions thereof such as the DHFR + sequence and sequences adjacent thereto which include the gene encoding the protein of choice are stably integrated into the host chromosome.
  • stably transformed DHFR + cells are further cultured and cloned, i.e., individual colonies of cells are taken and cultured separately to provide a number of cloned, transformed DHFR + cell lines.
  • DHFR + cell lines are then examined for their ability to express the desired protein and those cell lines showing good expression (i.e., expressing the protein of choice in its expected form either as an intact protein or as an intact fusion product, depending on how the gene encoding the protein of choice was originally constructed on the transforming vector) are selected.
  • Axel et al. discloses a system for co-transforming eukaryotic cells with a foreign DNA encoding a desired proteinaceous material and with an unlinked DNA encoding a selectable phenotype such as DHFR conferring methotrexate resistance.
  • Axel et al. discloses amplifying a gene encoding a desired protein linked to the DNA encoding a selectable phenotype by challenging with successive- sively higher amounts of the selecting agent.
  • MTX during cell culture causes gene amplification of gene sequences at and around the DHFR sequence, such as large stretches of flanking DNA that include the gene sequence encoding the desired protein (when unlinked DNAs are cotransfected into a cell, they tend to form a cointegrate that link the DNAs prior to integration into the host genome by non-homologous recombination) .
  • the degree of amplification is regulated by the MTX inhibitory effect of DHFR.
  • the above CHO DHFR/MTX system has a number of drawbacks, the major one being that the necessary constitutive expression of the DHFR gene during cell culture results in increased levels of DHFR which act to inhibit the effect of MTX.
  • the cell culture progresses through successive stages of amplifying stable transfectants from the previous stage, more and more MTX is required for gene amplification until a limit is reached whereby the elevated MTX levels become toxic to the cells; in other words, the upper concentration limit of MTX to which the cells are still MTX-resistant is reached.
  • the current CHO protein production systems have an upper limit as to the amount of desired protein that can be produced.
  • the level of constitutive heterologous (desired) protein expression is relatively limited; for example, only production levels of as high as 10-30 mg/l of culture can be obtained after MTX treatment. Consequently, in order to further increase the amounts of protein produced in these systems, either additional cultures are required or larger cultures need to be grown, which adds considerably to the production costs.
  • current CHO protein production systems are employed for the production of medically and veterinarily important proteins on a commercial scale. There has therefore been a long-felt need to improve these systems to increase the amount of desired protein produced, on the one hand, and on the other hand, to reduce the costs for producing this increased amount of protein.
  • Anti-sense R ⁇ A is transcribed from an upstream promoter of a coding sequence oriented in the anti-sense direction, i.e., opposite the normal or sense direction of the D ⁇ A and its transcribed sense R ⁇ A.
  • the expression of anti ⁇ sense R ⁇ A complementary to the sense R ⁇ A is a powerful way of regulating the biological function of R ⁇ A molecules. Through the formation of a stable duplex between the sense R ⁇ A and anti-sense R ⁇ A, the normal or sense R ⁇ A transcript can be rendered inactive and untranslatable.
  • anti-sense R ⁇ A is believed to control plasmid C0LE1 replication (Tomizawa et al., Proc. ⁇ at'l. Acad. Sci. USA 78:1421. 1981; Lacatena et al.. Nature 294:623.1981) and regulation of outer membrane protein production (Mizuno et al., Proc. Nat'l Acad. Sci. USA 11:1966,1984) as well as many others. Izant et al., Cell 3_£:107 (1984), showed that anti-sense RNA also inhibits gene expression in eukaryotes. They constructed a plasmid with a promoter directing the transcription of an anti-sense RNA complementary to the normal thymidine kinase (tk) transcript which substantially reduced expression of the normal thymidine kinase gene.
  • tk normal thymidine kinase
  • anti-sense DNA sequences have been used to express anti-sense RNA complementary to normal or sense RNA transcripts of numerous genes.
  • Kaufman et al., US 4,912,040 discloses a system for expressing an anti-sense GRP78 DNA sequence capable of hybridizing to part or all of the endogenous GRP78 (similar to immunoglobulin heavy chain binding protein) -encoding mRNA transcript and thereby preventing its translation into GRP78 protein.
  • Maher's oligonucleotides contained either anionic diester or neutral methyl phosphonate internucleoside linkages prepared by automated synthesis. Shotkoski et al., J. Trop. Med. Hyg. 5_0_:433-439
  • anti-sense DHFR has not previously been used to increase the responsiveness of DHFR + cells to methotrexate.
  • the present invention is based on the surprising and unexpected discovery that upon transfection of a eukaryotic cell line that is DHFR" 1" , MTX-resistant and capable of producing a desired protein, with a vector encoding an anti- sense DHFR sequence, the cell line became more responsive (sensitive) to MTX, thus allowing higher levels of gene amplification and hence higher production levels of the desired protein at lower MTX levels. Even higher levels of protein production can be achieved by increasing MTX levels to the upper limit where the cells are still MTX-resistant. In some cases, the increased level of protein production was 500% (5-fold) higher than that achieved for control cultures not transformed with anti-sense DHFR-encoding plasmid.
  • the present invention also provides an expression enhancing system for regulating the amount of protein production in eukaryotic cells having protein production levels regulated by a DHFR/MTX regulatory system.
  • the expression enhancing system comprises an expression enhancing vector according to the present invention, that is capable of transfecting eukaryotic cells which are DHFR + and MTX- resistant, and which produce, under suitable conditions, a desired protein product and, when expressed in the eukaryotic cell line, is capable of producing anti-sense DHFR RNA which is complementary to the normal DHFR mRNA produced in the same cells.
  • the anti-sense DHFR RNA can specifically hybridize to normal DHFR in the eukaryotic cell line and consequently, can increase both the MTX sensitivity and the desired protein production at lower MTX levels in these eukaryotic cells.
  • the above expression system of the invention may be used to control the expression of a desired protein selected from the group of desired proteins consisting of the known medically and veterinarily important proteins produced in CHO cells under DHFR/MTX regulation, for example, interleukins (ILs) , interferons (IFNs) , receptors and others.
  • ILs interleukins
  • IFNs interferons
  • the present invention provides an expression enhancing vector for regulating the expression of the normal DHFR gene carried by an eukaryotic cell line, the expression enhancing vector comprising: a) a double stranded sequence encoding the DHFR gene or a portion thereof in the anti-sense or reverse orientation instead of the sense or normal orientation, the anti-sense DHFR sequence being transcribable by RNA polymerase under the control of a promoter sequence to yield an anti-sense RNA product that is complementary to the normal DHFR mRNA sequence or a portion thereof, and capable of specifically hybridizing to the normal DHFR mRNA sequence, sufficiently to inhibit translation of the normal DHFR mRNA; b) a promoter sequence situated adjacent to the anti-sense DHFR sequence and controlling the expression of the anti ⁇ sense DHFR sequence.
  • the promoter sequence is located upstream of the 5' end of the anti-sense DHFR sequence so that the 5' ⁇ >3' directionality of the promoter sequence is in phase with the 5' ⁇ 3' directionality of the anti-sense sequence enabling transcription to proceed from the promoter in the 5'-*3' direction to yield an anti-sense DHFR RNA product; and, optionally, c) a genetic marker gene sequence encoding a product, the expression of which is readily screenable or selectable in cells transfected with and expressing the expression enhancing plasmid.
  • the present invention also provides a method for regulating the level of desired protein production in eukaryotic cells which comprises genetically manipulating an eukaryotic cell line so that it expresses a protein of interest, together with both DHFR (so that it is MTX- resistant) and an anti-sense DHFR RNA which hybridizes with normal DHFR mRNA to inhibit the translation thereof, thus leading to a decrease in DHFR production and in the level of inhibition of MTX.
  • This decreased level of MTX-inhibition results in an increase in the amplification of the desired gene and its subsequent expression.
  • this genetic manipulation involves: a) constructing an eukaryotic cell line that is DHFR + , MTX-resistant and capable of producing a desired protein, the level of production of which is at least partly regulated by the MTX-amplifiable copy number of the gene encoding the desired protein.
  • the level of MTX-induced amplification is regulated by the level of normal DHFR expression in these cells) ; and b) transfecting the eukaryotic cell line with an expression enhancing vector according to the present invention.
  • the genetic elements can be introduced in any order, and on the same or different vectors, provided that the DHFR gene and the gene of interest are or become associated such that amplification of the DHFR gene results in amplification of the gene of interest.
  • the new expression system provides for both an increase in the amount of protein produced and a reduction in the cost of producing medically and veterinarily important proteins in CHO cell cultures.
  • the method of the present invention results in an increase in expression, which is to a level preferably at least about 200%, more preferably at least about 300%, most preferably at least about 500%, that achieved in control DHFR + cultures not transferred with the anti-sense sequence.
  • This method is generalizable to other gene amplification systems in which a directly amplifiable gene (the counterpart of the DHFR gene in our model system) whose expression product is required to protect the cell from a toxic agent (the counterpart of the methotrexate in our model system) is cotransfected with a gene of interest, which thereby becomes indirectly amplifiable by exposing the cell to the toxic agent.
  • a directly amplifiable gene the counterpart of the DHFR gene in our model system
  • a toxic agent the counterpart of the methotrexate in our model system
  • This exposure causes the cell to elevate expression of the protective expression product in such a manner as to result in the elevated expression of the gene of interest.
  • the method would then involve expression of anti ⁇ sense RNA which inhibits translation of the mRNA encoded by the directly amplifiable gene so as to render the cell more sensitive to the elicitor.
  • Figure 1 is a schematic outline of the construction of the anti-sense DHFR-encoding plasmids showing the parent plasmids from which various regions were excised and subsequently ligated to provide the anti-sense DHFR-encoding plasmids, as described in Example 1.
  • FIG. 1(a) pSVE3DHl and pSVE3DH2 are constructed, using pSVE3 as a starting material. Also, the DHFR gene is inserted into pSVE3DHl in an antisense orientation to yield pSVEaDHFR. In Fig. Kb), the plasmid pSVEaDHFR is used as a starting material in the production of pSVEadHFRl and pSVEadHRF2. Finally, in fig.
  • Plasmids pSVEadHFRl, pMAMneo and pSVEadHFR2 are used in the construction of a DHFRl/NeoR, a DHFRi/Neo, a DHFRl/NeoR, and a DHFR2/Neo.
  • Figures 2-5 are schematic representations of the anti-sense DHFR-encoding plasmids in which the various arrowheads denote the orientation of various coding regions, each different coding region being denoted by a different pattern next to which appears the abbreviated name of the region, as described in Example 1.
  • the present invention in a preferred embodiment, concerns (1) an expression vector encoding an anti-sense DHFR sequence, (2) an expression enhancing system using this vector to regulate production of a desired protein in eukaryotic cell line, and (3) a method for regulating the production level of a desired protein in an eukaryotic cell line by transfecting these cells with the vector of the invention.
  • Cell lines transformed with the so-called "an i- sense DHFR" vector are, however, more sensitive to MTX than the original DHFR + cell lines because, upon expression of the anti-sense DHFR sequence, the translation of endogenous DHFR + mRNA into the DHFR enzyme is blocked and the transformed cells become almost DHFR".
  • the selection of transformed cell lines selects those which still retain net DHFR + expression, i.e., where the expression of normal DHFR + mNRA is greater than the newly-acquired anti-sense DHFR RNA expression.
  • the cells are also rendered more sensitive to
  • these anti-sense DHFR-expressing cells produce elevated levels of the desired protein as exemplified by some clones achieving a 500% higher expression level of the desired protein. This increase may be explained by the fact that addition of a small amount of MTX preferentially amplifies the DHFR + gene sequence and its flanking sequences (including the desired protein) in contrast to the anti-sense DHFR sequence which is either amplified to a lower extent or not at all.
  • the vectors of the invention were constructed using standard methods of molecular genetics (Sambrook et al. Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)), and used to transfect DHFR" 1" , MTX-resistant CHO cell lines which also produce a desired protein. Some of the resulting clones (see Examples 1 and 2) which were rendered more MTX-sensitive were capable of producing significantly elevated amounts of the desired protein, i.e. specific productivity was increased (in some cases up to 5-fold or 500% more) .
  • the present method for increasing the responsiveness of an amplifiable gene to a toxic agent such as methotrexate is generalizable to other gene amplification systems such as those listed in Table 1 of Kaufman, op. cit., herein incorporated by reference.
  • ornithine decarboxylase ODC
  • ODC ornithine decarboxylase
  • Example 3 hypothetically shows how anti-sense ODC DNA can be expressed in transformed cells to increase the responsiveness of the cell to added DFMO in regulating the level of DFMO-amplified copy number of the gene encoding the desired protein.
  • Anti-sense ODC RNA expressed from transformed cells will also selectively hybridize to ODC mRNA, reducing the level of ODC enzyme produced and thereby increasing the responsiveness of the transformed cells to DFMO.
  • vectors of the invention may be any suitable eukaryotic or prokaryotic vector normally used for transfecting eukaryotic cells such as CHO cells (see Sambrook et al., chapters 1, 2, 3 and 16, op. cit. for some examples of known vectors used for transfecting eukaryotic cells)
  • plasmids are the most preferred vectors for carrying the anti ⁇ sense DNA sequence that acts to control expression of DHFR and the desired protein of interest.
  • the plasmid vectors of the invention were constructed to contain the following key elements: an anti ⁇ sense DHFR sequence; an efficient promoter adjacent to the anti-sense DHFR sequence and positioned with respect to the anti-sense DHFR sequence in such a way that only anti-sense DHFR mRNA can be transcribed by a RNA polymerase initiating transcription from that promoter; and a selectable genetic marker other than the normal DHFR gene, such as an antibiotic resistance-encoding gene sequence, which allows for the rapid selection of those CHO cells transfected by the plasmid vector of the invention. This selection is simply achieved by growing the cells in the presence of the antibiotic where successfully transfected cells are being protected by the antibiotic resistance-encoding sequence.
  • the "normal" DHFR gene may be on the same or a different vector from the antisense DHFR gene, or it may be a native element of a chromosome of the host cell. Normally, the cell will be already DHFR+ and then transformed with an anti-DHFR vector.
  • the "normal" DHFR gene will be expressed, in a suitable host cell, to produce a DHFR polypeptide.
  • Expression comprises transcription of the gene, resulting in production of a messenger RNA; followed by translation of the messenger RNA transcript, resulting in production of the encoded DHFR polypeptide.
  • the DHFR polypeptide may also feature post ⁇ translational modifications.
  • the anti-sense DHFR can inhibit expression by hybridizing to the gene, thereby inhibiting transcription, and/or to the messenger RNA, thereby inhibiting translation.
  • normal DHFR gene is intended to include any gene which encodes a DHFR polypeptide, as later defined.
  • the term “normal” is used for contrast with “anti-sense”, and not to indicate identity with a natural DHFR gene.
  • the DHFR coding sequence may be that of a DHFR gene which occurs in nature. It may also be advantageous to modify the DHFR DNA sequences, e.g., to "silently" mutate degenerate codons to improve stability, reduce RNA secondary structure formation or raise the melting temperature (T M ) of the annealed DHFR mRNA and anti-sense DHFR RNA. By mutating degenerate codons, the nucleotide sequence of the DHFR gene and its complementary anti-sense DHFR sequence can be modified without modifying the amino acid sequence of DHFR.
  • Any modification to the DHFR DNA coding sequence would normally mean a corresponding modification of the anti-sense DHFR DNA coding sequence in order to maintain complementarity between the sense (normal) and anti-sense DHFR sequences.
  • the greater the number of complementary base pairs the greater the number of non- complementary bases that can be tolerated, especially if the non-complementary bases are scattered.
  • the GC content of the DHFR and anti- DHFR genes can be increased in a complementary manner to raise the T M of the annealed DHFR mRNA/anti-sense DHFR RNA, thereby providing further stability to the annealing of anti-sense
  • DHFR RNA to DHFR mRNA in the inhibition of DHFR translation RNA to DHFR mRNA in the inhibition of DHFR translation.
  • Sequence modification can also be used to reduce secondary structure formation by self-annealing of DHFR mRNA.
  • degenerate codons can be used to replace specific codons of the DHFR sequence which disrupt RNA secondary structure by preventing self-annealing through selective elimination of complementary bases that give rise to secondary structure.
  • the choice of specific nucleotide(s) to modify in the DHFR sequence can be determined from an estimation of the DHFR MRNA secondary structure using RNA estimation techniques reported in, for example, Tinoco et al., Nature New Biology 246:40-41 (1973), Tinoco et al.. Nature 23_0_:362-367 (1971), and Bachra J. Mol. Evol.51:155-173 (1976).
  • the DHFR gene may also be non-silently modified, i.e., so that a mutant DHFR polypeptide, of greater or lesser enzymatic activity, is produced, provided that the polypeptide is still capable of imparting, at its level of expression, sufficient resistance to methotrexate so that the gene of interest is amplified.
  • DHFR polypeptide is any DHFR form which occurs in nature, or a mutant (including a fragment or chimera) which is substantially identical to a natural DHFR (preferably a mammalian DHFR) , and retains at least 10% of the native DHFR activity of the most homologous native DHFR.
  • the DHFR may be a DHFR polypeptide which occurs in nature, or a mutant thereof, especially one obtained by conservative substitution of amino acids. Many DHFRs have been sequenced, and the relative importance of residues may be ascertained by aligning the sequences, with residues important to activity being more strongly conserved. Substitution of amino acids with others of similar size, hydrophobicity and charge, or amino acids found at that position in other DHFR, is less likely to perturb activity.
  • the DHFR is preferably a mammalian DHFR, especially a mouse DHFR. The sequence of the mouse DHFR is cited in the examples.
  • the anti-sense DHFR sequence must not only be expressible in the host/target cells, but the expressed anti ⁇ sense RNA must be stable (i.e., does not undergo rapid degradation) . Moreover, the anti-sense DHFR RNA, will essentially specifically only hybridize to the sense DHFR mRNA expressed in host cells, and form a stable double-stranded RNA molecule that is essentially non-translatable. In other words, the anti-sense DHFR RNA expressed in transfected host cells prevents the expressed sense DHFR mRNA from being translated into active DHFR enzyme.
  • the vector-borne anti- sense DHFR sequence may carry either the entire DHFR gene sequence or merely a portion thereof as long as the anti-sense DHFR sequence is capable of hybridizing to "sense" DHFR mRNA and preventing its translation into DHFR enzyme.
  • an "anti-sense" sequence of the invention can be defined as a sequence which is capable of being expressed in transformed/transfected cells and which is also capable of specifically hybridizing to "sense" DHFR mRNA to form a non- translatable double-stranded RNA molecule.
  • the anti-sense DHFR sequence need not hybridize to the entire length of the DHFR mRNA.
  • the anti-sense DHFR sequence is preferably at least 17, more preferably at least 30, base pairs in length.
  • shorter sequences may still be useful, i.e., they either fortuitously do not hybridize to other mammalian sequences, or such "cross-hybridization" does not interfere with the metabolism of the cell in a manner and to a degree which prevents the accomplishment of the objects of this invention.
  • Both the preferred hybridization target and the preferred anti-sense sequence length are readily determined by systematic experiment. Standard methods such as described in Sambrook et al. , 1989 can be used to systematically remove an increasingly larger portion of the anti-sense DHFR sequence from the plasmid vector. Besides the full length anti-sense DHFR sequence, a series of staggered deletions may be generated, preferably at the 5' -end of the anti-sense DHFR sequence.
  • the series of plasmid vectors generated by staggered deletions of anti-sense DHFR sequence, each having a different modified anti-sense sequence, can then subsequently be tested for their effectivity in DHFR + host cells.
  • plasmid vectors containing a range of truncated anti-sense sequences can be obtained and used individually or in combination as expression control vectors for amplifying the protein of choice as well as the DHFR + marker.
  • promoters used to control transcription of the anti-sense gene to yield the anti-sense RNA may be any of those which are functional in the host cells.
  • promoters functional in mammalian cells include the SV40 early promoter, adenovirus major late promoter, herpes simplex (HSV) thymidine kinase promoter, rous sarcoma (RSV) LTR promoter, human cytomegalovirus (CMV) immediate early promoter, mouse mammary tumor virus (MMTV) LTR promoter, interferon ⁇ promoter, heat shock protein 70 (hsp70) promoter, as well as many others well known in the art.
  • HSV herpes simplex
  • RSV thymidine kinase promoter
  • RSV rous sarcoma
  • CMV human cytomegalovirus
  • MMTV mouse mammary tumor virus
  • hsp70 heat shock protein 70
  • promoters may be either constitutive or regulatable. All else being equal, constitutive promoters are preferred because an extra treatment step such as temperature shift, addition of chemical agents or inducers, etc., is not required for expression from constitutive promoters. Nonethe ⁇ less, regulatable promoters may be desirable to modulate the level of anti-sense DHFR expression to maximize the degree of amplification. If the expression level is too high, the cells may not grow or even survive. If it is too low, the cells may not respond adequately to the methotrexate. When regulatable promoters are to be used for expressing the anti-sense sequence, it may be desirable to suitably modify the host cells to increase their tolerance to the inducing conditions used, e.g., temperature shifts.
  • the "marker" gene used to verify transformation of the cells with the anti-sense vector is preferably an anti ⁇ biotic resistance gene.
  • the types of antibiotic resistance sequences which may be carried by the vector may be any of the well known antibiotic resistance sequences, for example neomycin-resistance (or G418-resistance) and hygromycin resistance. Other screenable or selectable markers may be used instead of antibiotic resistance genes.
  • neomycin-resistance or G418-resistance
  • hygromycin resistance Other screenable or selectable markers may be used instead of antibiotic resistance genes.
  • Table 1 of the Kaufman reference op.
  • adenosine deaminase (ADA) , one of many other selectable markers, can be selectable at cytotoxic concentrations of adenosine or 9-0-D-xylofuranosyl adenine.
  • the target cell may be any cell which, either naturally or, as a result of genetic manipulation, is DHFR + .
  • CHO cells are preferred as the target cells of the invention because well-characterized DHFR" CHO cell lines which are easily and readily transformed to DHFR + are available.
  • CHO cell lines are widely used for the industrial production of a variety of mammalian proteins and culture requirements are well established.
  • Other well-known eukaryotic cell lines such as mammalian, yeast and insect cell lines may be used in the expression system of the invention if suitably modified in a similar manner as the CHO cell lines.
  • mammalian cell lines, and especially CHO cells remain preferred.
  • a DHFR' cell line is established first, and then transformed/transfected with a vector carrying both the DHFR + gene sequence and a gene encoding the protein of choice. Positive transformants are selected by growing the transformed/transfected cells in a basic minimal culture medium such that only those cells expressing the essential DHFR enzyme (DHFR + cells) will survive. Before amplifying with MTX, the DHFR "1" transformed cell lines would need to be tested for the level of MTX-resistance, whether MTX-induced amplification is effective, and whether anti-sense DHFR regulation is necessary for further increases in expression of the desired protein.
  • the DHFR gene sequence be incorporated into the genome upon transformation/transfection to ensure that the transformants stably and constitutively express the essential DHFR enzyme.
  • the gene encoding the protein of choice is stably co-integrated into the genome with the DHFR gene sequence with the protein of choice being expressed in either a constitutive or regulatable manner.
  • the anti-sense DHFR sequence carried on a separate selectable vector, may be introduced into cells by any of well-knowntechniques fortransforming/ ransfeetingeukaryotic cells, such as electroporation, calcium phosphate treatment, and liposome-mediated transformation, etc. (see Ausubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Assoc, New York (1987-1994) chapter 9) and maintained in cells either via stable integration into the cell genome or on a non-integrated vector. If integration is desired, then it must be ensured that not only does integration of the anti ⁇ sense sequence not lead to loss of the sense DHFR + sequence, but that once integrated, the anti-sense DHFR sequence is still expressed.
  • DHFR + cells which have both DHFR and the gene of interest integrated into the genome
  • anti-sense DHFR DNA a different order of introducing the various sequences is also contemplated.
  • the anti ⁇ sense DHFR DNA along with a selectable marker can be introduced first into the genome followed later by the introduction and integration of the DHFR group together with the gene of interest into the genome. Again, it must be ensured that integration of the DHFR gene and the gene of interest does not lead to loss of the anti-sense DHFR sequence.
  • the anti-sense DNA is preferably expressed from a regulatable promoter so that prior to a first series of DHFR/gene of interest amplification with methotrexate, there is no expression of anti-sense DNA. This would allow the copy number of DHFR/gene of interest to be first amplified prior to increasing the responsiveness to further amplification with methotrexate.
  • the two different order of introducing sequences described above are non-limiting examples; other possibilities can be readily devised based on the disclosure presented herein.
  • both the DHFR + gene and the co-integrated protein of choice gene, which flanks the DHFR + gene, are amplified together.
  • the protein of choice can be any protein having pharmaceutical, veterinary or industrial importance that is currently being produced or can be produced.
  • proteins which can be categorized as follows: a) Pharmaceutically important proteins/hormones: (i) cytokines and cytokine receptors such as interleukins (IL-1, IL-2, IL-6, etc.) and their receptors; interferons (IFN- ⁇ , - ⁇ , -y , etc.) and their receptors (RBIF, etc.); tumor necrosis factors (TNF- ⁇ , - ⁇ , etc.) and their receptors (TBP-l, TBP-2); (ii) hormones for growth regulation of the reproductive system, regulation of the circulatory (blood) system and regulation of the nervous system, e.g., all the various growth factors, GH, aFGF, bFGF, KGF and their receptors; FSH, LH, CG and their receptors; erythropoietin (EP) , plasminogen activator (tPA) , the various platelet factors (blood clotting agents)
  • cytokines and cytokine receptors such
  • the promoter used for expressing the protein of choice can be the native promoter for the gene encoding the desired protein or it can be any suitable promoter capable of expressing the desired protein, preferably at high levels.
  • the promoter can be constitutive or regulatable and can be any one of those currently used in mammalian expression systems as described above for promoters expressing the anti-sense sequence. It should be noted that the promoter used for expressing the desired protein may be different from the one used to express the anti-sense DHFR or it may be the same.
  • the promoter for the gene encoding the desired protein is preferably different from the promoter for expres ⁇ sing anti-sense DHFR.
  • the culture conditions used for culturing the CHO cells were the standard culture conditions generally used for CHO DHFR + cells that are treated with MTX (see Kaufman, R.J., Methods in Enzymol., 185. 537-566 (1990)) . If other cells are used, the culture conditions may be whatever is suitable fcr growth of said cells and compatible with gene amplification as disclosed herein.
  • Plasmids containing the murine DHFR gene (see Chang, et al., Nature, 275:617-24 (1978) and Genbank sequences J00382, J00383, J00384, J00385, J00386, J00387, J00388; L26316; NCBI sequence 387160) in the anti-sense orientation were constructed with an antibiotic resistance gene as an additional selectable genetic marker. This additional selectable marker is necessary since the plasmids encoding the anti-sense DHFR sequence are intended for transfection of CHO cells already transfected with the normal DHFR gene.
  • antibiotic-resistance genes for this additional selectable marker are those encoding hygromycin B or neomycin (G418) resistance
  • other antibiotic resistance genes may be used under the condition that their expression in CHO host cells do not cause cell death or interfere with the gene amplification mechanism.
  • the antibiotic resistance marker is preferably G418, with this preferred marker being inserted into the various anti-sense DHFR-encoding plasmids.
  • a schematic outline of how anti-sense DHFR-encoding plasmids carrying the G418 resistance marker were constructed from pre-existing (parent) plasmids is set forth in Figs. Ka)-(c). The methods used for constructing these plasmids are the well-establishedmethods of molecular genetics/genetic engineering (see Sambrook et al., op cit.).
  • pBRM is a 2 kb fragment of pBR322, containing the jS-lactamase gene conferring resistance to ampicillin, and the plasmid origin of replication.
  • the incorporation of the pBRM fragment into a mammalian expression vector makes possible the propagation of the plasmid in E. coli.
  • SV40 Tag is the coding region for SV40 T Antigen, located in the early region of SV40, between SV40 early promoter and polyadenylation sequences. See Tooze, J. (ed.) (1980) DNA Tumor Viruses: Molecular Biology of Tumor Viruses. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) Part 2.
  • pSVE3 is an expression vector contains pBRM and the whole early region of SV40 (Tag) . In the paper which summarizes the construction of pSVE3, a description of its components is given. See Hartman, J.R. et al. (1982) Proc.
  • PR22103 and PR22104 are both oligonucleotides of 22 bases long each, which combine to give an adaptor fragment, used in the first step of construction of the plasmid containing DHFR sequence in pSVE3.
  • pMAMneo is a mammalian expressionvector (Clontech) .
  • Neomycin resistance gene was removed from pMAMneo and introduced into antisense-DHFR plasmids, in order to enable selection of cells carrying this plasmid. See Lee, F., et al.
  • the structure of the plasmids was characterized by restriction map analysis.
  • Figs. 2-5 show the restriction and sequence orientation maps of these plasmids.
  • the four plasmids were designated: adHFRl/Neo (Fig. 2); adHFRl/NeoR (Fig. 3); adHFR2/Neo (Fig. 4), and adHFR2/NeoR (Fig. 5) .
  • the difference between the four plasmids resides in the position of the neomycin resistance gene, 5' (upstream) or 3' (downstream) in relation to the anti-sense DHFR gene and in the orientation of the neomycin resistance gene with respect to the orientation of the anti-sense DHFR gene.
  • the neomycin resistance gene confers resistance to G418 and converts host cells to G418-resistant cell lines.
  • DHFR sequence is under the control of the SV40 early promoter) and T-antigen sequences, i.e. the anti-sense DHFR sequence was inserted into the SV40 EP/Tag region of the parent plasmid
  • the expressed anti-sense DHFR RNA sequence will be comple ⁇ mentary to the normally expressed (in the CHO cells) DHFR mRNA (DHFR sense mRNA sequence) , with the result that when expres- sed in the same cell these sequences will hybridize to each other, thereby effectively inhibiting the translation of the normal DHFR mRNA.
  • the above four anti-sense DHFR plasmids were tested for their ability to transfect, as secondary transfections, CHO clones that were transfected previously and presently expressing normal murine DHFR (DHFR + clones) and a desired protein, TBP-I (a soluble TNF receptor molecule) . These tests were directed toward determining whether the transfected CHO clones are rendered sensitive to MTX since the expression of normal DHFR in these cells should be significantly inhibited following expression of the anti-sense DHFR sequence, thus causing the cells to be more responsive, or sensitive to MTX.
  • the anti-sense DHFR plasmid successfully transfected the CHO cells and was expressed in these cells.
  • the results show that a higher amount of anti-sense DHFR plasmid per cell (per dish) used for transfection resulted in increased inhibition of DHFR + expression and hence, increased MTX sensitivity, i.e., the 9 ⁇ M concentration of MTX to which the cells of CHO clone SBP13-30 are normally resistant, becomes a toxic concentration in cel-s in which DHFR + expression is inhibited.
  • the greater the DHFR " * ' inhibition the greater the MTX toxicity.
  • CHO Clones SBV14-24 and SK-108-1-22-12, producing TBP-I were transfected with the anti-sense DHFR clone, adHFR2/Neo (Fig. 4) .
  • CHO clone SBV14-24 a stable TBP- I producer, was isolated by one approach where, after several rounds of MTX selection, clone SBV14-24 was found to be highly resistant to MTX, at least up to the relatively high level of lO ⁇ M MTX, a level which is usually toxic to CHO cells.
  • CHO clone SK-108-1-22-12 was isolated using a second approach where selection was carried out at lower MTX levels (1.2 ⁇ M), resulting in clone SK-108-1-22-12 apparently having fewer copies of the normal DHFR gene, thus making those fewer copies more readily neutralized by the expression of the anti-sense DHFR plasmid.
  • Clone SBV14-24 was transfected with 30, 100 and 300 ⁇ g, respectively, of the above anti-sense DHFR plasmid, and the resulting neomycin-resistant clones were isolated and subjected to a MTX-sensitivity test.
  • the isolated neomycin- resistant clones were then grown under standard CHO cell culture conditions, in which the cells of each clone were grown in two of the wells of a six-well Costar culture plate in standard CHO- cell-culture medium. 10 ⁇ M MTX was added to one of the two wells with the second well serving as a control (cells did not receive any MTX) .
  • Each Costar plate also had two additional control wells in which cells of the parental clones, i.e., non-transfected, non-neomycin resistant cells of the SBV14-24 clone, were grown. To one of these wells 10 ⁇ M MTX was added, while the other received no MTX. In much the same manner as described in Example l (C) above, when the cells in the wells without MTX grew to approach confluence, the experiment was terminated (this usually being after the cells were grown for a duration between several days and more than two weeks) and the surviving cells were stained with crystal violet for visualization (results not shown) .
  • neomycin resistant clones Forty seven such neomycin resistant clones were tested, some of which showed no sensitivity to lO ⁇ M MTX, i.e., behaving similarly to the parental clones which are resistant to 10 ⁇ M MTX.
  • Other clones for example, the ones designated 30dl3 and 30dl6 (the "30" denoting the amount of plasmid DNA, 30 ⁇ M, used for transfection) showed intermediate sensitivity to 10 ⁇ M MTX.
  • a third group of clones for example, the ones designated 30dl5, 30dl7, 30dl8, 30dl9 and 30d20, showed a high level of sensitivity to 10 ⁇ M MTX; i.e., much reduced numbers of cells surviving after incubation in the presence of 10 ⁇ M MTX.
  • These highly sensitive MTX clones thus represent those having the highest anti-sense DHFR plasmid transfection efficiency and anti-sense DHFR gene expression.
  • These clones were selected for tests to ascertain the effect of the acquired MTX-sensitivity on TBP-I production in these clones.
  • clone SK-108-1-22-12 was transfected with 30 or 100 ⁇ g of the above-noted anti-sense DHFR plasmid.
  • the resulting neomycin-resistant clones were isolated and subjected to a MTX-sensitivity test in 6-well Costar plates, in which the test-wells received 1.2 ⁇ M MTX and the other control wells being equivalent to those noted above, with the exception that the control parental line cells were of clone SK-108-1-22-12.
  • the specific productivity of parental clone SBV14-24 was measured by a standard ELISA procedure, specific for detection of quantification of TBP-I production. This ELISA procedure was carried out over a period of 5-16 months to ascertain the stability of this clone for TBP-I production. Over this period, the average specific productivity of this clone was calculated to be 7.25 ⁇ 1.82 ⁇ g/l0 6 cells/24 hours.
  • the culture procedures used were as follows: The clone was grown in Petri dishes in standard CHO culture medium under standard CHO culture conditions with or without the addition of MTX. One day before the ELISA the medium was changed to fresh medium and the cells were incubated for a further 24 hours.
  • TBP-I production was measured in aliquots of the cell supernatants. Cell numbers in each culture were determined following trypsinization. TBP-I production was calculated per IO 6 cells, as well as per 24 hour culture, with the calculated value representing the specific productivity.
  • the above noted neomycin-resistant anti-sense DHFR-transfected SBV14-24 clones having high MTX sensitivity were subjected to the same ELISA procedure over a two-month period.
  • the clones having the highest TBP-I specific productivities are listed in Table 1 below. It should be noted that clones designated with names beginning with "30d”, "100d” and "300d” are those in which the transfections were with 30 ⁇ g, lOO ⁇ g, and 300 ⁇ g, respectively, of plasmid DNA.
  • these eight clones were selected for further gene amplification studies using MTX.
  • the cells of each clone were grown in each well of a six-well Costar plate in standard CHO culture medium and to each well was added a different concentration of MTX
  • clones lOOdO ⁇ and 100dl6 were treated with 5 ⁇ M MTX
  • clone 100dl2, lOOdOl, , 10dl8, 10d22 were treated with 4 ⁇ M MTX
  • clone 30dl7 was treated with 3 ⁇ M
  • clone 100dl3 was treated with 2 ⁇ M MTX.
  • clones were derived from each of the above MTX-treated clones and their specific productivities of TBP-I were measured by ELISA as above. In Tables 2-9 below, the results of the ELISA are shown; the values of specific productivity being average values obtained from a number of ELISA determinations over about a 2-month period in which the clones were grown.
  • Clones 100d08 produced about 7 ⁇ g/10* cells/24 hours at the time the MTX treatment was started.
  • Selection with 5 ⁇ M MTX enabled the isolation of clones producing an average of 10 or 12 ⁇ g/10 6 cells/24 hours. This represents an increase of about 50-70%.
  • Clones I00dl6 produced about 4.6 ⁇ g/l0 6 cells/24 hours at the time the MTX treatment was started.
  • Selection with 5 ⁇ M MTX enabled the isolation of clones producing averages of about 12 or I3 ⁇ g/10* cells/24 hours. This represents an increase of about 260- 280%.
  • Clones 100dl3 produced relatively low levels (1.5 ⁇ g/10 6 cells/24 hours) of TBP-I at the time the MTX treatment was started. Selection with ⁇ M MTX enabled the isolation of clones producing averages of 8 ⁇ g/l0 6 cells/24 hours. This represents an increase of about 500%. Although the starting productivity was low, this increase in productivity is very impressive, especially considering that it has been achieved with only 2 ⁇ M MTX. Clones 300d01 and 30dl7 increased their productivity by about 100% upon MTX treatment. Most of the clones isolated by treatment of clone 30dl8 with 4 ⁇ M MTX showed no enhancement in productivity. A few showed a two ⁇ fold increase. In view of the above, it may therefore be concluded that:
  • the parental clone was shown to have an average specific productivity of 3.01 ⁇ 1.28 ⁇ g TBP-1/10 6 cells/24 hours.
  • neomycin resistant, anti-sense DHFR-carrying clones derived from the parental clone were shown to have TBP- I specific productivities of average values ranging from 1.25 to 7.70 ⁇ g/l0 6 cells/24 hours, most however having values comparable to that of the parental clone (results not shown) . Nevertheless, these neomycin-resistant SK-108-l-22-l2-derived clones, some of which are capable of increased TBP-I specific productivity, illustrate that transfection with the anti-sense DHFR carrying plasmid was likewise successful and that the anti-sense DHFR sequence was expressed in these clones.
  • a second transfection can be performed with an anti- sense expression vector in clones that have already been prepared by a first transfection, to become desired protein (TBP-I) producing clones.
  • TBP-I desired protein
  • a second selectable genetic marker eg. neomycin-resistance
  • DHFR + used for the initial transfection
  • DHFR normal gene
  • a promoter eg. SV40 early promoter
  • the level of translation inhibition of DHFR mRNA depends on the relative levels of expression of the two DHFR sequences, partial inhibition being achieved when the normal gene sequence is expressed at higher levels than the anti-sense sequence.
  • the TBP-I producing clones are established MTX- resistant clones, i.e., selected after extensive growth in the presence of MTX. This selection leads to an amplification of the integrated DHFR sequence as well as its surrounding sequences (eg. that encoding TBP-I) in these clones thus giving rise to relatively high DHFR expression levels as a result of MTX-induced amplification of DHFR gene copy number.
  • the anti-sense DHFR sequence newly integrated into the clones is in lower copy numbers relative to the normal DHFR sequence, and consequently, normal DHFR was usually observed to be only partially inhibited. Indeed, DHFR inhibition must be only partial in all surviving clones grown in the presence of MTX, otherwise, complete inhibition would lead to MTX-induced cell death. This partial inhibition of DHFR in the surviving selected clones therefore provides clones which have been rendered more responsive/sensitive to MTX and the DHFR-induced inhibition of MTX is consequently reduced in these clones.
  • EXAMPLE 3 Increasing responsiveness to gene amplification for the ornithine decarboxylase (ODC) in wild-type CHO cells (ODC+ ⁇
  • An ODC expression vector is to be constructed similar to the plasmid pdhODl as described in Chiang et al. Afol. Cell . Biol . 8.:764-769 (1988) where the mouse ODC gene is flanked by SV40 early promoter and SV40 polyadenylation signals.
  • the DHFR expression unit shown on pdhODl can be replaced with the gene encoding the protein of choice and a promoter for expressing such a gene. The type of promoter used for expressing the protein of choice is described above.
  • ODC + can be maintained on Dulbecco modified Eagle medium containing 10% fetal calf serum.
  • ODC-deficient can be maintained on Dulbecco modified Eagle medium containing 10% fetal calf serum.
  • ODC ODC CHO cells
  • C55.7 ODC CHO cells
  • ODC CHO cells are to be grown as described in Steglich et al., Somatic Cell
  • CHO host cells transfected with the ODC expression vector is to be selected for resistance to serially increasing levels of difluoromethyl ornithine (DFMO) , a suicide-substrate inhibitor of ODC, such as sequential selection for resistance to 160 ⁇ M, 600 ⁇ M, 1 mM, 3mM, 9mM and 15 mM DFMO.
  • DFMO difluoromethyl ornithine
  • a suicide-substrate inhibitor of ODC such as sequential selection for resistance to 160 ⁇ M, 600 ⁇ M, 1 mM, 3mM, 9mM and 15 mM DFMO.
  • approximately IO 5 cells are to be plated per 100 mm dish into medium containing DFMO, and the plates are to be refed with fresh medium every 5 days until a resistant population emerges.
  • the level of expression of ODC and the protein of choice can be readily determined in DFMO-resistant CHO cells.
  • DFMO-resistant CHO cells showing high level expression of the protein of choice can be used as host cells to be transfected/transformed with an anti-sense expression plasmid vector constructed to carry an anti-sense ODC gene in a manner similar to that described in Example l.
  • Expression of anti-sense ODC RNA in DFMO-resistant CHO cells carrying amplified copies of both the ODC gene and the desired gene (encoding the protein of choice) will increase cell sensitivity to lower amounts of added DFMO, thereby increasing cell responsiveness to further amplification at lower levels of DFMO.
  • ODC/DFMO will permit further amplification of the desired gene as well as a concomitant increase in the level of expression of the protein of choice.

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Abstract

L'invention concerne un procédé pour réguler le niveau de production d'une protéine souhaitée dans une cellule mammalienne transformée, faisant appel à: une séquence d'un gène directement amplifiable, sous une forme permettant son expression, qui, lors de l'expression, rend la cellule plus résistante à un agent toxique; un gène utile, sous une forme permettant son expression, codant pour une protéine souhaitée étrangère à la cellule; et une séquence d'un gène anti-sens, sous une forme se prêtant à une transcription, codant pour un ARN anti-sens capable de former sélectivement un hybride avec au moins une portion de l'ARNm obtenu par la transcription de ladite séquence de gène directement amplifiable, ce qui inhibe la traduction dudit ARNm, de sorte qu'un niveau d'amplification plus élevé dudit gène directement amplifiable et dudit gène utile peut être obtenu, lorque la cellule est exposée à un niveau suffisant de l'agent toxique, qu'en l'absence dudit ARN anti-sens.
PCT/US1995/015098 1995-11-07 1995-11-27 Utilisation de sequences anti-sens pour augmenter la sensibilite par rapport a l'amplification genique Ceased WO1997020055A1 (fr)

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ZA959429A ZA959429B (en) 1995-11-07 1995-11-07 Use of anti-sense sequences to increase responsiveness to gene amplification
AU42408/96A AU718896B2 (en) 1995-11-27 1995-11-27 Use of anti-sense sequences to increase responsiveness to gene amplification
PCT/US1995/015098 WO1997020055A1 (fr) 1995-11-07 1995-11-27 Utilisation de sequences anti-sens pour augmenter la sensibilite par rapport a l'amplification genique
AT95940765T ATE222957T1 (de) 1995-11-27 1995-11-27 Verwendung von antisense-sequenzen zur erhöhung der antwort auf gen-amplifizierung
DE69527994T DE69527994D1 (de) 1995-11-27 1995-11-27 Verwendung von antisense-sequenzen zur erhöhung der antwort auf gen-amplifizierung
US09/077,253 US6399377B1 (en) 1995-11-27 1995-11-27 Use of anti-sense sequences to increase responsiveness to gene amplification
EP95940765A EP0863990B1 (fr) 1995-11-27 1995-11-27 Utilisation de sequences anti-sens pour augmenter la sensibilite par rapport a l'amplification genique
CA002235962A CA2235962A1 (fr) 1995-11-27 1995-11-27 Utilisation de sequences anti-sens pour augmenter la sensibilite par rapport a l'amplification genique

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988003558A1 (fr) * 1986-11-14 1988-05-19 Genetics Institute, Inc. Systeme d'expression d'eucaryotes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988003558A1 (fr) * 1986-11-14 1988-05-19 Genetics Institute, Inc. Systeme d'expression d'eucaryotes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LI, L. ET AL.: "Establishment of a Chinese hamster ovary cell line that expresses grp78 antisense transcripts and suppresses A23187 induction of both GRP78 and GRP94.", JOURNAL OF CELLULAR PHYSIOLOGY, (1992 DEC) 153 (3) 575-82., XP000575845 *
MC IVOR, R. ET AL.: "ISOLATION AND CHARACTERIZATION OF A VARIANT DIHYDROFOLATE REDUCTASE cDNA FROM METHOTREXATE-RESISTANT MURINE L5178Y CELLS", NUCLEIC ACIDS RESEARCH, vol. 18, no. 23, 11 December 1990 (1990-12-11), pages 7025 - 7032, XP000257045 *
SHOTKOSKI, F. ET AL.: "Expression of an antisense dihydrofolate reductase transcript in transfected mosquito cells: effects on growth and plating efficiency.", AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, (1994 APR) 50 (4) 433-9., XP000575913 *
WANG, S. ET AL.: "Quantitative evaluation of intracellular sense: antisense RNA hybrid duplexes.", NUCLEIC ACIDS RESEARCH, (1993 SEP 11) 21 (18) 4383-91., XP000394395 *

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