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WO1997014810A1 - Utilisation de cellules hotes procaryotes pour l'expression de genes dans des cellules eucaryotes - Google Patents

Utilisation de cellules hotes procaryotes pour l'expression de genes dans des cellules eucaryotes Download PDF

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Publication number
WO1997014810A1
WO1997014810A1 PCT/US1996/016129 US9616129W WO9714810A1 WO 1997014810 A1 WO1997014810 A1 WO 1997014810A1 US 9616129 W US9616129 W US 9616129W WO 9714810 A1 WO9714810 A1 WO 9714810A1
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WIPO (PCT)
Prior art keywords
donor
pathogenic bacterium
gene
eucaryotic cell
genome
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PCT/US1996/016129
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English (en)
Inventor
Jerry M. Buysse
Paul K. Tomich
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Pharmacia & Upjohn Company
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Publication date
Application filed by Pharmacia & Upjohn Company filed Critical Pharmacia & Upjohn Company
Priority to AU72614/96A priority Critical patent/AU7261496A/en
Publication of WO1997014810A1 publication Critical patent/WO1997014810A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • procaryotic hosts for expression of gene i n eucaryotic cel l s
  • the present invention relates to the field of molecular biology and a method of altering the genetic material of a cell or organism.
  • the invention relates to the use of pathogenic bacteria as vectors suitable for the expression, at various levels, of heterologous genetic material.
  • the present invention has a wide variety of applications, for example, in the understanding and treatment of various genetic and viral diseases and normal cellular processes.
  • Attenuated bacterial pathogens such as Salmonella typhimirium (8,8a) and BCG (9) have been used to induce immunity by expression of heterologous DNA.
  • Salmonella typhimirium (8,8a) and BCG (9) have been used to induce immunity by expression of heterologous DNA.
  • the pathogen invades macrophage, dies and leaves the heterologous DNA in the macrophage.
  • the macrophage present a proteolytically processed expressed protein(s) on their cell surface for the induction of an immune response.
  • the functionality of the processed protein is only to induce an immune response and not its original encoded purpose.
  • Intracellular antibodies that are synthesized by a bacterial cell and targeted to specific host cellular compartments represent a new addition to the molecular techniques used in the analysis and manipulation of eucaryotic cellular pathways.
  • Intracelluar antibodies have been used to inactivate proteins in the endoplasmic reticulum (ER), cytoplasm and nucleus which has allowed the study of particular proteins to determine their role(s) in different physiological and pathological contexts.
  • ER endoplasmic reticulum
  • nucleus As reported recently by Marusco, W.A., Immunotechnologv. (1995) 1, 1-19 (36), the recent advances in antibody engineering have allowed antibody genes to be manipulated and antibody molecules to be reshaped (30, 31).
  • the present invention relates to using recombinant DNA technology to introduce at least one gene encoding a protein molecule into a donor pathogenic bacterium which can attach itself and invade a given eucaryotic cell. Following invasion of the eucaryotic cell, the donor pathogenic bacterium then expresses the gene within itself and then exports the protein into the recipient eucaryotic cell resulting in the transfer of the expressed protein from the donor pathogenic bacterium to the eucaryotic cell. The effect, if any, the presence of this protein has on the recipient eucaryotic cell is then determined.
  • the donor pathogenic bacterium is attenuated or avirulent.
  • the gene expressed in the donor pathogenic bacterium is heterologous to the genome of the recipient eucaryotic cell and the genome of the donor pathogenic bacterium.
  • the gene expressed in the donor pathogenic bacterium is heterologous to the genome of the recipient eucaryotic cell and is homologous to the genome of the donor pathogenic bacterium.
  • the gene expressed in the donor pathogenic bacterium is homologous to the genome of the recipient eucaryotic cell and is heterologous to the genome of the donor pathogenic bacterium.
  • Figure 1 illustrates a map of plasmid pCVD422 which is a decendent of plasmid pCVD421 disclosed in M. E. Donnenberg and J. B. Kaper (1991) Infection & Immunity 59:4310-4317 (37).
  • pCVD422 is s ⁇ B-containing suicide vector for general use composed of the mob, ori, and bla regions from pGO704 and the sacB gene. Five unique endonuclease sites, including a blunt-end Smal site, are available on this vector for cloning.
  • the present invention involves the use of virulent or avirulent forms of pathogenic microorganisms for the expression of cloned genes within eucaryotic cells which are targets of the microorganism.
  • a pathogenic procaryotic cell secretes proteins that affect its target eucaryotic cell (e. g., 10,11). More importantly, some pathogenic bacteria, notably Shigella species, attach to and invade host target cells (12,13,13a). Combining these two functions, an expressed protein(s) can be secreted by a given pathogen into the cytosol of its target eucaryotic cell.
  • the proteins can be expressed either as fusion proteins or as soluble molecules with, if needed, appropriate known localization signals engineered into the DNA construct to direct the expressed protein(s) to appropriate eucaryotic subcellular sites.
  • appropriate known localization signals engineered into the DNA construct to direct the expressed protein(s) to appropriate eucaryotic subcellular sites.
  • this invention employs Shigella flexneri as the prime example.
  • S. flexneri has extensive homology with Escherichia coli (14,14a). Plasmids, phage and vectors that function in the latter work in the former. Thus, anything cloned and expressed in E. coli will behave similarly in S. flexneri (e. g., 15,15a,15b). More importantly, any gene(s) cloned in E. coli can be transferred (16) or transformed (17,17a) at high frequency to S. flexneri, thus obviating the need for subcloning into eucaryotic expression vectors. Furthermore, with the ability to introduce S. flexneri into eucaryotic cells via an infective process, the need to subclone into eucaryotic expression vectors and perform subsequent transfection protocols can be eliminated.
  • Shigella flexneri invades epithelial and macrophage cells (11,18). It resides in the cytosol of these cell types where it then replicates (18). For epithelial cells, death occurs by general necrotic mechanisms from replicating and/or dying bacteria (19). Macrophage undergo apoptosis (20). The attachment and invasive process, however, are similar for all invasive cell types.
  • modified strains of S. flexneri are used. Some are made avirulent by deletion of appropriate gene(s) (21). Similarly others are programmed for their own eventual demise after infection of the host eucaryotic cell by making the pathogen deficient for its survival, e.g., thyA ' (a mutated thymidylate synthetase gene which renders the cell thymine deficient preventing DNA synthesis (22)); and or asd ' (a mutated aspartic semialdehyde dehydrogenase gene which renders the cell diaminopimelic acid deficient preventing cell wall biosynthesis (23)).
  • thyA ' a mutated thymidylate synthetase gene which renders the cell thymine deficient preventing DNA synthesis
  • asd ' a mutated aspartic semialdehyde dehydrogenase gene which renders the cell diaminopimelic acid deficient preventing cell wall biosynthesis (23)
  • Eucaryotic cells that Shigella flexneri does not invade are rendered susceptible to this pathogen by cloning and expressing invasive gene(s) from other microbial pathogens (bacterial, viral, parasitic) that do invade the eucaryotic cell type(s) of interest.
  • Genes such as the Staphylococcus aureus fibronectin receptor (24) should be expressed properly on the cell surface of Shigella flexneri. Since (almost) all eucaryotic cells express fibronectin (25), transient growth of eucaryotic cell line(s) in defined, serum-free, culture medium is required for S. flexneri to attach and infect. After infection, the cell line is transferred to normal culture medium.
  • modified Shigella flexneri One particularly advantageous aspect of using modified (e.g., attenuated or avirulent) Shigella flexneri is that the organism can have a commensalistic relationship with the infected eucaryotic cell.
  • the modified Shigella flexneri organism can live within the eucaryotic cell and continue to produce the heterologous protein without causing the cell death typically associated with cellular invasion by Shigella flexneri.
  • the M13 phage system appears ideal. Infected E. coli extrude this phage into culture medium. Introduction of an E. coli F plasmid with a selective marker (e.g., F ' ::T_nlO which encodes tetracycline resistance) into S. flexneri should allow this phage to infect this host. Thus all constructs made in E. coli and/or its M13 phage will be expressed and processed accordingly. Rather than being expressed as a fusion protein, the M13 system also has the capability, with appropriate stop codons and in a suppressor deficient S. flexneri host, to extrude the expressed protein(s) into the bacterial periplasmic space. With appropriate lesions (e. g. asd ' or thyA ), S. flexneri
  • Shigella flexneri A variety of mutations are introduced into Shigella flexneri using standard mutagenesis and/or selection techniques. For example, standard E. coli expression vectors, plasmids, and phage can be used to generate asd ' and thyA ' mutations, among others. Some avirulent forms of Shigella flexneri having either mutated or deleted virulence genes or cured of the invasion plasmid are already available for use.
  • anti-PKC anti-phosphokinase C
  • mAbs two anti-phosphokinase C monoclonal antibodies
  • one is a blocking, or inhibitory, mAb against PKC enzymatic activity; the other is nonblocking or non-inhibitory.
  • These mAbs exist as stable hybridoma cell lines (27). They are cloned as scF v antibodies into the vector pCANTAB ⁇ using standard, commercially available procedures.
  • virus particles are obtained with the scF v antibodies expressed as fusion proteins of the phage surface protein pill in suppressor strains (sup*) ofE. coli.
  • suppressor strains sup*
  • the scF v antibodies are as expressed free, soluble proteins.
  • M13 phage expressing the antibody fusions are isolated and used to infect both sup* and sup ' Shigella flexneri strains which harbor an E. coli F plasmid.
  • strains are then used to infect either an epithelial (e.g., CHO cells) or macrophage (J774) cell lines.
  • epithelial e.g., CHO cells
  • macrophage J774 cell lines.
  • the ability of the scF v antibody to block PKC activity in macrophage is determined by measuring inhibition of IL-2 production following activation of the eucaryotic cells with appropriate mitogens (28).
  • mitogens 28
  • Both soluble and fusion scF v blocking antibodies will inhibit IL-2 production after cell activation, but the nonblocking antibodies will not.
  • the soluble form will not inhibit as effectively because the amount of antibody available in soluble form in the eucaryotic cytosol depends on how many scF v molecules leak out of the periplasmic space of Shigella spp.
  • the aspartic semialdehyde dehydrogenase gene ⁇ C Haziza, P. Stragier, and J.-C. Patte (1982) EMBO J. 1:379-384 (39) ⁇ has been mutated in other species to generate strains that require diaminopimelic acid (DAP) acid for cell growth ⁇ K. Nakayama, S. M. Kelly, and R. Curtiss III, (1988) Bio/Technology 6:693-697 (40) ⁇ .
  • DAP is required for cell wall biosynthesis and DAP mutants absolutely require this supplement. DAP cells will spontaneously lyse upon removal of this nutrient (ibid).
  • Oligonuclecotide primers (Primer 1: GCGTATGCATGCATGTTGGTTTTATCGGCTGGCGCG [SEQ ID NO:l] and Primer 2: CGCACCGAGCTCTTACGCCAGTTGACGAAGCATCCG [SEQ ID NO:2] have been designed to subclone the E.
  • E. coli cells are resuspended in 50 ⁇ L of H 2 O (ca. IO 4 to 10° cells) and a fraction used in a PCR reaction with these primers.
  • the primers generate SphI and Sad restriction sites at the 5 and 3 of the amplified DNA fragment.
  • the purified DNA is digested with these enzymes and cloned into the plasmid pCVD422 (M. E. Donnenberg and J. B. Kaper (1991) Infection & Immunity 59:4310-4317 (37); Fig.
  • the constructed plasmid is reisolated and digested with EcoRV, gel purified to remove an internal 395 base pair asd gene fragment and religated. This generates an internal deletion in the asd gene.
  • This derived plasmid is then transformed into SmlO ⁇ pir (ibid) and the constructed strain mated with the Shigella flexneri harboring an E. coli F'::7 ⁇ l0 selecting for ampicillin and tetracycline resistant exconjugants.
  • the pCVD422/ ⁇ sd ⁇ plasmid will integrate into the S. flexneri chromosome via homology with the asd gene. Under appropriate selection and screening as described by Donnenberg and Kaper (vide supra), the plasmid is cured and the asdA resides in the S. flexneri chromosome.
  • the scF v blocking antibody is fused in a variety of constructs with the Shigella flexniri virG gene to determine the optimal construct for excretion of this antibody by Shigella spp. into culture medium as monitored by Westerns. This same construction is then performed with the nonblocking scF v to ascertain whether this procedure could be extended to other proteins. The strains harboring these constructs will then be infected into eucaryotic cells and the functional activity of the antibodies are then determined in the same manner as previously described (vide supra). If this concept proves viable, an entire recombinant library of scF v antibodies can be cloned into this same construct.
  • a lysozyme gene is cloned onto a vector with either a weak or regulated promoter such that sufficient enzyme is expressed to generate viable protoplasts but not lyse
  • Shigella spp. This expression unit is subcloned onto the anti-PKC scF v vector and tested in this system. This approach ensures that all materials expressed by Shigella spp. that would have been in the periplasmic volume will be exported into the eucaryotic cytosol.
  • a high multiplicity of infection or ratio of Shigella spp. to eucaryotic host has to be utilized. This means that although every eucaryotic cell is guaranteed to become infected with at least one Shigella spp., many will receive multiple copies based on Gaussian distribution. Consequently, if Shigella spp. harbors a library of different cloned genes in its expression vector, (e.
  • the recipient eucaryotic cell upon infection may carry two or more copies of vectors that express different proteins.
  • mixed infections are performed. Two strains of Shigella spp. are used: one carries a vector that expresses the anti-PKC scF v antibody; the other does not.
  • Various ratios of the two strains are used to infect epithelial cells (e.g., CHO). That ratio which demonstrates a single copy of the plasmid expressing the anti-PKC scF v antibody should be used for all subsequent infections.
  • the experiment is also done in which the strain containing the anti-PKC scF v antibody is asd ' and/or thyA* and the other strain with no plasmid is correspondingly asd* and/or thyA ' .
  • Shigella spp. can infect epithelial and macrophage cells. This range can be extended by cloning adherence/invasion genes from other pathogenic organisms that invade other cell types (29,29a,29b).
  • the fibronectin receptor from Staphylococcus aureus could also be used since fibronectin exists on the vast majority of eucaryotic cell types. After expressing in Shigella spp. a clone of this receptor, the strain is tested for its ability to infect a variety of cell types, Eucaryotic cell types having fibronectin should be capable of being infected with this engineered construct.
  • this system can now be used to introduce and express any cloned gene or library of genes into the eucaryotic cell of choice.
  • ipaB gene Upon invasion of macrophage S. flexneri resides in the cytoplasm (33) and secretes several proteins, one of which is encoded by the ipaB gene (34). This gene product triggers apoptosis in this cell type (35).
  • the purified gene product is sent to a commercial company for injection into mice from which spleens are obtained. They are frozen in liquid nitrogen and shipped on dry ice. The spleens are the source from which mRNA for murine antibody heavy and light chains are isolated. After isolation of poly(A) + mRNA, a library of recombinant anti-ip ⁇ B scF v antibodies is generated in a procaryotic expression plasmid pCANTAB ⁇ E.
  • the scF v molecules are expressed either as soluble antibodies or as fusion molecules of the M13 geneS protein.
  • the Sfil and Notl restriction sites are converted into appropriate cloning sites for eucaryotic expression vectors with designed oligonucleotides. These vectors include but are not limited to pCDM8 having SV40 and EBV origin of replications. For the former vector the Sfil site is converted to a Hindlll site using ohgonucleotide 1; the Notl site remained unchanged.
  • the mouse anti-ip ⁇ JB scF v library cloned into a suitable vector is transfected into an established macrophage cell line.
  • the transfected library is then infected with Shigella flexneri. Those cells that survived the apoptotic event are subcloned and cultured in larger volume.
  • Total DNA is obtained from the transfected cell line(s) and, using appropriate ohgonucleotide primers the plasmid DNA(s), which encodes the scF v antibody(ies), is amplified by PCR and transformed into E. coli.
  • Plasmid DNA(s) are re-isolated and transfected into macrophage which subsequently are infected with Shigella flexneri. In this case, macrophage surviving Shigella flexneri- induced apoptosis were used to recover antibody constructs.
  • the recovered mAbs are expressed in E. coli and used to purify the target protein (ipa
  • Shigella flexneri genetics of entry and intercellular dissemination in epithelial cells. Curr.Top. Microbiol. Immunol. 192: 217-41 34. Menard, R., Sansonetti, P. and Parsot, C. (1994). The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by ipaB and ipaD. EMBO. J.13:6293-5302. 35. Zychlinsky, A., Kenny, B., Menard, R.
  • IpaB mediates macrophage apoptosis induced by Shigella flexneri. Mol. Microbiol. 11: 619-627.
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention porte sur un moyen d'introduction par une technique de recombinaison d'ADN d'au moins un gène codant pour une molécule de protéine dans une bactérie donneuse pathogène pouvant se fixer à une cellule eucaryote donnée et l'envahir. Après l'invasion de la cellule eucaryote, la bactérie donneuse exprime en elle même le gène puis exporte la protéine dans la cellule eucaryote réceptrice, ce qui se traduit par le transfert de la protéine exprimée de la bactérie pathogène donneuse vers la cellule eucaryote. On peut alors déterminer l'effet éventuel de la présence de ladite protéine sur la cellule eucaryote réceptrice. Dans une variante, la bactérie donneuse pathogène peut être atténuée ou non virulente. Dans une deuxième variante, le gène exprimé dans la bactérie donneuse pathogène est hétérologue du génome de la cellule eucaryote réceptrice et du génome de la bactérie donneuse pathogène. Dans une troisième variante, le gène exprimé dans la bactérie donneuse pathogène est hétérologue du génome de la cellule eucaryote réceptrice et homologue du génome de la bactérie donneuse pathogène. Dans une quatrième variante, le gène exprimé dans la bactérie donneuse pathogène est homologue du génome de la cellule eucaryote réceptrice et hétérologue du génome de la bactérie donneuse pathogène.
PCT/US1996/016129 1995-10-18 1996-10-15 Utilisation de cellules hotes procaryotes pour l'expression de genes dans des cellules eucaryotes WO1997014810A1 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
US7183105B2 (en) 2001-05-24 2007-02-27 Vaxiion Therapeutics, Inc. Eubacterial minicells and their use as vectors for nucleic acid delivery and expression
US7396822B2 (en) 2001-05-24 2008-07-08 Vaxiion Therapeutics, Inc. Immunogenic minicells and methods of use
US10005820B2 (en) 2011-02-15 2018-06-26 Vaxiion Therapeutics, Llc Therapeutic compositions and methods for antibody and Fc-containing targeting molecule-based targeted delivery of bioactive molecules by bacterial minicells

Citations (3)

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FR2564482A1 (fr) * 1984-05-18 1985-11-22 Int Genetic Sciences Transformation de cellules eucaryotes inferieures par alimentation naturelle au moyen de bacteries contenant des plasmides induits par le chloramphenicol
WO1990012867A1 (fr) * 1989-04-19 1990-11-01 The Board Of Trustees Of The Leland Stanford Junior University Microorganismes d'invasion
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FR2564482A1 (fr) * 1984-05-18 1985-11-22 Int Genetic Sciences Transformation de cellules eucaryotes inferieures par alimentation naturelle au moyen de bacteries contenant des plasmides induits par le chloramphenicol
WO1990012867A1 (fr) * 1989-04-19 1990-11-01 The Board Of Trustees Of The Leland Stanford Junior University Microorganismes d'invasion
EP0441071A1 (fr) * 1990-02-06 1991-08-14 Institut Pasteur Shigella transformé

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Title
COURVALIN P ET AL: "GENE TRANSFER FROM BACTERIA TO MAMMALIAN CELLS", COMPTES RENDUS DES SEANCES DE L'ACADEMIE DES SCIENCES SERIE III: SCIENCES DE LA VIE, vol. 318, no. 12, 1 December 1995 (1995-12-01), pages 1207 - 1212, XP000579701 *
SANSONETTI P J ET AL: "CONSTRUCTION AND EVALUATION OF A DOUBLE MUTANT OF SHIGELLA FLEXNERI AS A CANDIDATE FOR ORAL VACCINATION AGAINST SHIGELLOSIS", VACCINE, vol. 7, no. 5, 1 October 1989 (1989-10-01), pages 443 - 450, XP000080372 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7183105B2 (en) 2001-05-24 2007-02-27 Vaxiion Therapeutics, Inc. Eubacterial minicells and their use as vectors for nucleic acid delivery and expression
US7396822B2 (en) 2001-05-24 2008-07-08 Vaxiion Therapeutics, Inc. Immunogenic minicells and methods of use
US8101396B2 (en) 2001-05-24 2012-01-24 Vaxiion Therapeutics, Inc. Minicells displaying antibodies or derivatives thereof and comprising biologically active compounds
US8129166B2 (en) 2001-05-24 2012-03-06 Vaxiion Therapeutics, Inc. Immunogenic minicells and methods of use
US8524484B2 (en) 2001-05-24 2013-09-03 Vaxiion Therapeutics, Inc. Immunogenic minicells and methods of use
US9017986B2 (en) 2001-05-24 2015-04-28 Vaxiion Therapeutics, Inc. Minicell based delivery of biologically active compounds
US9670270B2 (en) 2001-05-24 2017-06-06 Vaxiion Therapeutics, Llc Minicell based delivery of biologically active compounds
US10005820B2 (en) 2011-02-15 2018-06-26 Vaxiion Therapeutics, Llc Therapeutic compositions and methods for antibody and Fc-containing targeting molecule-based targeted delivery of bioactive molecules by bacterial minicells
US10919942B2 (en) 2011-02-15 2021-02-16 Vaxiion Therapeutics, Llc Therapeutic compositions and methods for antibody and Fc-containing targeting molecule-based targeted delivery of bioactive molecules by bacterial minicells

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