WO1997014810A1

WO1997014810A1 - Use of procaryotic hosts for expression of gene in eucaryotic cells

Info

Publication number: WO1997014810A1
Application number: PCT/US1996/016129
Authority: WO
Inventors: Jerry M. Buysse; Paul K. Tomich
Original assignee: Pharmacia & Upjohn Company
Priority date: 1995-10-18
Filing date: 1996-10-15
Publication date: 1997-04-24
Also published as: AU7261496A

Abstract

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. In an alternate embodiment of the invention, the donor pathogenic bacterium is attenuated or avirulent. In a further embodiment, 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. In yet a further embodiment, 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. In yet a further embodiment, 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.

Description

Use of procaryotic hosts for expression of gene i n eucaryotic cel l s

Technical Field The present invention relates to the field of molecular biology and a method of altering the genetic material of a cell or organism. In particular, 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.

Background A variety of methods have been utilized to introduce recombinant DNA molecules into eucaryotic cells for expression of encoded genes of interest. Genes have been transferred by incubating cells with DNA, possibly in the presence of chemicals such as polyions or calcium phosphate (1), or DEAE/Dextran sulfate precipitation (2), micro-injection (3), liposomes (4), retroviral infection (5), or electroporation (6). The efficiencies of transfection, however, vary not only on the method chosen but also upon the cell type with some cell lines being transformed very poorly or not at all (7).

Attenuated bacterial pathogens such as Salmonella typhimirium (8,8a) and BCG (9) have been used to induce immunity by expression of heterologous DNA. In this process 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. However, the functionality of the processed protein is only to induce an immune response and not its original encoded purpose. More recently plasmids encoding cloned DNA for a desired gene have been introduced into an asd^' Shigella strain which was subsequently used to infect live guinea pigs; release of intracellular content including the cloned plasmid into the eucaryotic host permitted expression of the desired gene product in this host (23).

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. 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). By harvesting the genetic information of the immune system in the form of rearranged immunoglobin genes, intracellular antibodies of high affinity and fine specificity can be created. These technological advances, combined with the wealth of information that has been obtained on classical intracellular protein trafficking signals (32), has created a new and powerful research tool to analyze and manipulate microbial and cellular proteins. Until the efficiencies of transfection are improved, however, there will be a delay in the advances which this marriage of technologies should realize. Accordingly, there remains a need for an improved method for introducing recombinant DNA molecules into eucaryotic cells for expression of encoded genes of interest.

Summary of The Invention 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.

In an alternate embodiment of the invention, the donor pathogenic bacterium is attenuated or avirulent. In a further embodiment, 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. In yet a further embodiment, 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. In yet a further embodiment, 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.

Brief Description of the Drawings 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 sααB-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. Detailed Description

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. Although any pathogen that can attach and invade a given eucaryotic cell(s) type could be used, 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.

In order to prevent the cell death typically caused by Shigella flexneri invasion of epithelial and macrophage cells, 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)).

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. 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.

Several means exist to secrete cloned gene(s) of interest from S. flexneri into the eucaryotic cytosol. The two discussed here are its virG gene and the E. coli M13 phage/plasmid system. The virG gene product in wild-type S. flexneri secretes with high efficiency into the eucaryotic host cytosol (26). Genes expressed utilizing the necessary signals encoded within virG are used to export proteins directly into the eucaryotic cytosol.

For those gene products which can function as fusion proteins, 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

-A- will release its contents into the eucaryotic cell cytosol.

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.

EXAMPLES A. Introduction of single chain antibodies (scR,) into eucarvotic cells either as fusion proteins or as soluble antibodies.

To test the expression of a cloned gene introduced into eucaryotic cells via Shigella flexneri, two anti-phosphokinase C (anti-PKC) monoclonal antibodies (mAbs) are selected: 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 pCANTABδ using standard, commercially available procedures. On infection with wild-type M13 bacteriophage, 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. (In sup^' strains 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.

These strains are then used to infect either an epithelial (e.g., CHO cells) or macrophage (J774) cell lines. For example, 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). Both soluble and fusion scF_v blocking antibodies will inhibit IL-2 production after cell activation, but the nonblocking antibodies will not. However, 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 presence of plasmid and/or production of antibody levels are monitored by PCR and/or Western analysis. The latter technique takes advantage of a peptide tag which is part of the pCANTABδ plasmid. The same experiments are tested with asd^' and thyA^' strains to confirm that similar results are obtainable. ThyA Shigella spp. were selected as described ("Experiments in Molecular Genetics", (1972) Jeffrey H. Miller, ed., Cold Spring Harbor Laboratory, pp. 218-220), (38). Succinctly, Shigella spp. were plated on minimal plates that contain appropriate nutritional requirements for the strain of interest plus trimethoprim at 10 μg mL and thymidine at 50 μg mL. Plates were incubated for 3-7 days at 37 C Those colonies which grew were single colony purified twice on the same type of plates either with or without thymidine. Those colonies which grew with thymidine but did not grow upon its omission were kept.

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). The E. coli asd gene has been cloned and used to generated internal deletion in a variety of procaryotic cells, including Shigella flexneri {D. R. Sizemore, A. A. Branstrom, and J. C. Sadoff (1995) Science 270:299-302 (41)}. Oligonuclecotide primers (Primer 1: GCGTATGCATGCATGTTGGTTTTATCGGCTGGCGCG [SEQ ID NO:l] and Primer 2: CGCACCGAGCTCTTACGCCAGTTGACGAAGCATCCG [SEQ ID NO:2] have been designed to subclone the E. coli asd gene having first eight nucleotides deleted, missing the first two a ino acids, and consequently generating an out of frame open reading frame. E. coli cells are resuspended in 50 μL of H₂O (ca. IO⁴ to 10° cells) and a fraction used in a PCR reaction with these primers. In addition to obtaining the almost full length clone, 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. 1). 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 SmlOλpir (ibid) and the constructed strain mated with the Shigella flexneri harboring an E. coli F'::7 ιl0 selecting for ampicillin and tetracycline resistant exconjugants. The pCVD422/αsdΔ 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.

Since the amount of soluble scF_v antibody expressed in a sup^" host that leaks out of the periplasmic space could be insufficient at times, depending on the affinity of the expressed antibody, further modifications to the system are devised. 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. B. Optimization of Infection

To obtain optimal infection of a eucaryotic host, i.e., to guarantee that every cell is infected, 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. g., a shotgun library to complement a defect in the eucaryotic cell or an scF_v antibody library), the recipient eucaryotic cell upon infection may carry two or more copies of vectors that express different proteins. To minimize this problem, 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^'.

C. Extension of the host range for Shigella spp.

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). In addition, 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. Thus, this system can now be used to introduce and express any cloned gene or library of genes into the eucaryotic cell of choice.

D. Shigella flexneri Induced Apoptosis in Macrophage.

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-ipαB scF_v antibodies is generated in a procaryotic expression plasmid pCANTABδE. Depending on the Escherichia coli host type and whether co-infection with a wild-type M13 phage is used, 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. For the latter plasmid the Sfil and Notl sites are converted to BamHI and Hindlll sites using oligonucleotides 2 and 3 respectively. Detailed protocols for the generation of this library are delineated in written material supplied from Pharmacia Biotech .

The mouse anti-ipαJB 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 (ipaB).

While specific embodiments of the invention have been described, it should be apparent to those skilled in the art that various modifications thereof can be made without departing from the true spirit and scope of the invention. Accordingly, it is intended that the following claims cover all such modifications within the full inventive scope. References:

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SEQUENCE LISTING

( 1 ) GENERAL INFORMATION :

(i) APPLICANT: Buysse, Jerry M. To ich, Paul K.

(ii) TITLE OF INVENTION: ENHANCED TRANSFECTION OF EUCARYOTIC CELLS

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(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : GCGTATGCAT GCATGTTGGT TTTATCGGCT GGCGCG 36 (2) INFORMATION FOR SEQ ID NO: 2 :

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CGCACCGAGC TCTTACGCCA GTTGACGAAG CATCCG 36

Claims

WE CLAIM:

-1- A process for the transfer of at least one expressed gene into a eucaryotic cell comprising the steps of: (a) cloning a gene into a vector for expression;

(b) introducing said vector into a donor pathogenic bacterium;

(c) infecting a recipient eucaryotic cell with said donor pathogenic bacterium;

(d) expressing said gene as protein in said donor pathogenic bacterium; (e) exporting said protein from said donor pathogenic bacterium into said recipient eucaryotic cell; and

(f) screening or selecting for a phenotypic change of said recipient eucaryotic cell.

-2- The process of claim 1 in which the donor pathogenic bacterium is Shigella flexneri.

-3- The process of claim 1 in which the gene encodes a peptide, antibody, antigen, hormone, or drug not normally expressed in the cell, at biologically significant levels.

-4- The process of claim 1 in which the donor pathogenic bacterium is attenuated or avirulent.

-5- The process of claim 1 in which 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.

-6- The process of claim 1 in which 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.

-7- The process of claim 1 in which 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.