EP2205747A1

EP2205747A1 - Attenuated invasive e. coli strains and applications thereof as intracellular vector for therapeutic molecule

Info

Publication number: EP2205747A1
Application number: EP08836217A
Authority: EP
Inventors: Catherine Liliane Andrée Grillot-Courvalin; Sylvie Dominique Goussard; Patrice Marie Denis Paul Courvalin
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut Pasteur de Lille
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut Pasteur de Lille
Priority date: 2007-10-02
Filing date: 2008-10-02
Publication date: 2010-07-14
Also published as: CA2701542A1; EP2045331A1; US20100216233A1; WO2009043921A9; WO2009043921A1

Abstract

The invention relates to a vectorial system capable of delivering a nucleic acid of interest into eukaryotic target cells, said vectorial system including a recombinant non-pathogenic and non-replicative Escherichia coli bacterium (E. coli) having integrated into its chromosome, in a targeted manner and without any antibiotic marker, one or more genes imparting to said E. coli bacteria the capacity to penetrate into the cytoplasm of said eukaryotic target cells and to lyse the penetration vacuole. The present invention also relates to the use of such E. coli bacteria for the production of therapeutic compositions and their delivery without any purification step for preventing or treating diseases by vaccination or gene therapy.

Description

Invasive attenuated E. coli strains and their applications as an intracellular vector of a therapeutic molecule

The present invention relates to a vector system capable of delivering, in eukaryotic target cells, a nucleic acid of interest, or which encodes a protein, this vector system comprising a non-pathogenic recombinant Escherichia coli (E. coli) bacterium which has integrated in a targeted manner and without antibiotic marker in its chromosome one or more genes conferring this bacterium E. coli the ability to enter the cytoplasm of these eukaryotic target cells. The present invention also includes the use of such E. coli bacteria to deliver therapeutic compositions for the prevention or treatment of disease by vaccination or gene therapy.

The transfer of genetic material into mammalian cells using non-pathogenic invasive E. coli bacteria has already been published (French patent application FR 95 15556 published under No. 2,743,086; Grillot-Courvalin C, Goussard S., Huetz F., Ojcius DM, and Courvalin P. (1998), Nature Biotechnol., 16: 862-866).

The techniques described in these documents relate to a genetically engineered E. coli bacterium for the purpose of penetrating and reaching the cytoplasm of specific target eukaryotic cells, preventing its multiplication and survival in target cells upon entry into the cytoplasm. of these target eukaryotic cells. This bacterium is further transformed by a plasmid coding for a gene, which it is desired to transfer into the target cell, which is a prokaryotic-eukaryotic shuttle vector that can be replicative in said E. coli bacterium, or else integrative in said cells. eukaryotic target hosts.

More specifically, these documents describe such E. coli bacteria in which the genes conferring on E. coli bacteria its ability to penetrate and reach the cytoplasm of target eukaryotic cells, such as the inv invasion gene of Yersinia pseudotuberculosis (Y pseudotuberculosis), and the hly gene coding for the hemolysin of Listeria monocytogenes (L. monocytogenes) are provided by plasmids (type τpGB2Ω.inv-hly). Among recombinant, non-pathogenic and invasive E. coli bacteria thus transformed, E. coli strain BM2710 / pGB2Ωmv- / z / y is particularly described (see also Courvalin et al., CR Acad ScL, 1995, 318, pages 1207-1212). This strain BM2710 was deposited at the CNCM (National Collection of Cultures of Microorganisms, Pasteur Institute, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France), October 27, 1995 under the number 1-1635.

This initial work has shown that the E. coli BM2710 auxotrophic strain for diaminopimelic acid (which lyses without dividing by lack of synthesis of its wall in a dap deficient environment as is the case in cells eukaryotic) and made invasive was able to deliver plasmid DNA to different cell types in which transgene expression could be detected (Courvalin et al., 1995). For efficient transfer, two steps appeared necessary: the entry of the bacterium into the cell and its exit (or that of the plasmid DNA) from the vacuole of induced phagocytosis. The internalization of the bacterial vector in many cell types has been made possible by the introduction into E. coli BM2710 dap of the inv gene of Y. pseudotuberculosis. This gene directs the synthesis of invasin that binds with high affinity to cell membrane receptors, β1-integrins. The release of the phagocytosis vacuole is controlled by the acquisition by E. coli BM2710 of the hly gene of L. monocytogenes which directs the synthesis of listeria O. Listeria lysine O allows E. coli, or its plasmid content in case of lysis of the bacterium, to leave the vacuole of phagocytosis thanks to the formation of membrane pores. In this first generation of the bacterial vector, the inv and hly genes were cloned into the low copy number plasmid pGB2 which confers resistance to spectinomycin and streptomycin. It has been shown that this bacterial vector makes it possible, by abortive invasion, to transfer and to express by mammalian cells integrative or replicative vectors with good efficacy (5 to 20% of the cells depending on the cell type transfected in vitro: HeLa cells, CHO or COS-I). The transfer efficiency of the plasmid of interest is evaluated using a reporter gene coding for the "green fluorescent protein" (GFP) placed under the control of a eukaryotic promoter. The transfer is also observed, but with a lower efficiency, in the absence of cell division or when the invasion is carried out at cell confluence (Grillot-Courvalin et al, 1998). The advantages of gene transfer by abortive bacterial invasion lie in the low toxicity to the recipient cells, the rarity of the rearrangements in the delivered DNA, the good efficiency of transfection and also in the fact that the donor bacterium, non-pathogenic and well-defined genetically, is unable to multiply and even survive in the receptor cell and in the environment due to auxotrophy for diaminopimelic acid.

Among the improvements to be made include the possibility of introducing any plasmid expression vector without having to respect the compatibility rules of the origins of replication and also the possibility of reducing or even eliminating the genes for resistance to antibiotics present in the vector bacterium.

The inventors have demonstrated that it is possible to obtain such improvements by integrating in the bacterial chromosome the genes conferring on the bacterium E. coli its ability to penetrate and reach the cytoplasm of the target eukaryotic cells, such as the genes inv and hly.

The inventors have thus generated an E. coli strain which hosts these two genes in the chromosome.

Apart from the aforementioned advantages, this chromosomal integration makes it possible to stabilize the genes inv and hly in progeny.

The subject of the present invention is therefore a vector system capable of delivering, in eukaryotic target cells, a nucleic acid of interest or encoding a protein of interest, a vector system comprising a non-pathogenic recombinant E. coli bacterium, said E. coli bacterium. non-pathogenic being furthermore:

modified by introduction (by allelic exchange) of one or more genes conferring on this non-pathogenic recombinant E. coli bacterium the ability to enter the cytoplasm of said eukaryotic target cells;

unable to survive in the cytoplasm of said target cell; and - modified by foreign DNA fragments, characterized in that the gene (s) conferring on said non-pathogenic recombinant E. coli bacterium the ability to enter the cytoplasm of said eukaryotic target cells are integrated into the chromosome of said non-pathogenic E. coli.

The terms "vector system" and "target cell" reflect that the E. coli strain is used here as a means of transferring the nucleic acid of interest into said eukaryotic cells and not as a host bacterium for the expression of said fragment of E. coli. DNA, which can take place where appropriate in said target cells after penetration of the bacterium into the cytoplasm. This vector system also covers recombinant E. coli strains as defined as a vector system and defined in the present invention.

By "nucleic acid of interest" is meant here any nucleotide sequence, DNA or RNA, with or without enzymatic restriction sites. Since most gene transfer systems do not allow the transfer of large DNA fragments, viral vectors can not accommodate fragments larger than 150 kb; by lipofection it is first necessary to produce large quantities of these large fragments from cultures of bacteria that replicate these artificial bacterial chromosomes, then purify the DNA and introduce it into the cells by lipofection, all these steps often result in denaturation genetic material and result in low transfection efficiency. Preferably, the bacterial vector makes it possible to propagate and transfer directly into the cytoplasm of the cells these artificial bacterial chromosomes of all sizes without prior purification and thus maintaining their physical integrity.

Thus, in a also preferred embodiment, the nucleic acid of interest may be of any size up to 100 kb, or 100 to 150 kb but also greater than 150 kb the upper limit not being defined.

In a particularly preferred embodiment, the nucleic acid comprises at least 150 kb.

By "nucleic acid of interest" is meant here any nucleotide sequence hetero logue to the target cell or any nucleotide sequence present in the target cell but whose expression is desired to modify or regulate. Preferably, it may be an exogenous nucleic acid. In a preferred embodiment, the vector system according to the invention is characterized in that said non-pathogenic E. coli bacterium is transformed by chromosomal integration of two genes, originating from one or two other bacteria, conferring on it the ability to enter the cytoplasm of said target cells. In a preferred embodiment, the vector system according to the invention is characterized in that the gene or genes integrated into the chromosome of the non-pathogenic E. coli bacterium and conferring on it its ability to penetrate the cytoplasm of said target cells, allow to lyse the membrane of the vacuoles of said target cells. When the specificity of piloting towards said particular target cells is conferred by a gene originating from another bacterium coding for the ability to specifically penetrate said target cells, it is the choice of the gene responsible for the recognition and penetration of the cell membrane which confers the specificity of the piloting. Said eukaryotic target cells may be animal cells, in particular mammalian cells, in particular human cells, or yeast cells or even plant cells.

The nature of the bacterium of origin of the gene responsible for the recognition and specific penetration into the target cells determined, used according to the present invention, confers on the vector system of the invention a specificity of control towards a type of eukaryotic target cells, in particular mammalian cells, especially human cells, corresponding to the cellular host spectrum of said bacterium.

Among the genes conferring on the bacteria the ability to penetrate certain target cells, mention may be made of:

a gene or a set of genes of the bacterium Salmonella typhimurium for transferring DNA into lymphoid tissue cells and liver cells;

a gene or a set of genes of the bacterium Shigella flexneri for transferring DNA into mammalian epithelial cells;

a gene of the bacterium of the genus Legionella, in particular Legionella pneumophila, in particular for transferring DNA more specifically into lung epithelial cells;

a L. monocytogenes gene for transferring DNA into epithelial cells of the central nervous system; a gene of the bacterium of the genus Yersinia, in particular Y. pseudotuberculosis or Y. enterocolitica for transferring DNA into epithelial cells; and

a bacterium gene of the genus Mycobacterium, in particular Mycobacterium tuberculosis, Avilum complex, scrofulaceum for transferring DNA into macrophages.

In one embodiment illustrating this aspect of the invention, it is preferred to transform the E. coli bacterium according to the invention by two exogenous genes from different bacteria. In particular, the invading gene of Y. pseudotuberculosis bacteria can be used to confer the ability to enter the cells. The L. monocytogenes hemolysin gene, an approximately 2kb gene that confers intracellular release capacity by lysing the vacuole membranes, can also be used.

In a preferred embodiment, the vector system according to the invention is characterized in that said non-pathogenic E. coli bacterium is transformed by chromosomal integration by the bacterium invasion gene or Y. pseudotuberculosis which confers the property of penetrating in the cytoplasm of epithelial cells.

In a preferred embodiment, the vector system according to the invention is characterized in that said non-pathogenic E. coli bacterium is transformed by chromosomal integration by the gene coding for hemolysin of L. monocytogenes or of another E. pathogenic E. coli which confers the property of lysing the membranes of the vacuoles of said target cells. The function of recognition and penetration of the cell membrane and the function of lysis of the membranes of vacuoles or phagosomes, the latter making it possible to reach the cytoplasm, may be conferred by one or more distinct genes, in particular two distinct genes.

In a preferred embodiment, the vector system according to the invention is characterized in that said non-pathogenic E. coli bacterium is transformed by chromosomal integration at the same time by the invasion gene of the bacterium Y. pseudotuberculosis which confers the property of penetrating the cytoplasm of the epithelial cells. And by the gene encoding L. monocytogenes hemolysin which confers the property of lysing the membranes of the vacuoles of said target cells.

In a preferred embodiment, the vector system according to the invention is characterized in that said non-pathogenic E. coli bacterium is transformed by chromosomal integration both by:

1. by the inv invasion gene of Yersinia pseudotuberculosis, preferably of sequence SEQ ID NO: 10 or sequence identical to at least 80% and capable of conferring the property of penetrating the cytoplasm of the epithelial cells; 2. the hly gene encoding Listeria monocytogenes hemolysin, preferably of sequence SEQ ID NO: 4 or sequence identical to at least 80% and capable of conferring the property of lysing the membranes of vacuoles.

In a preferred embodiment, the vector system according to the invention is characterized in that:

1. the inv invasion gene of Yersinia pseudotuberculosis is under the control of the Ptrc promoter; and or

2. The hly gene coding for Listeria monocytogenes hemolysin is under the control of the Ftet promoter. In a preferred embodiment, the vector system according to the invention is characterized in that said E. coli strain has been rendered incapable of surviving in said cells as soon as it enters the cytoplasm of eukaryotic cells.

The E. coli strain can be rendered incapable of multiplying and surviving in eukaryotic cells in multiple ways, particularly by rendering it auxotrophic for a factor necessary for its survival and absent from eukaryotic cells.

In the preferred embodiment according to the invention, the E. coli bacterium is rendered incapable of multiplying and thus of surviving as soon as it enters the cytoplasm of eukaryotic cells. For this purpose, in a preferred embodiment according to the invention, said E. coli bacterium has been modified so as to be made auxotrophic for diaminopimelic acid which is an essential compound for the synthesis of the bacterial wall and whose pathway biosynthesis is well known. In a further preferred embodiment, the E. coli bacterium is rendered dap ^" following a double homologous recombination event in two diaminopimelic acid synthesis genes that is prokaryotic specific and absent in the cyptoplasm of the cells. The first two steps in the synthesis of diaminopimelate, which is not present in mammalian cells, are catalyzed by the enzymes encoded by the dap A and dapB genes.

The gene can be doubly inactivated by deletion and insertion-inactivation in the dap A and dapB genes. Inactivation of the metabolic chain in the first step avoids the accumulation in cells of a metabolic intermediate that can be metabolized by another enzyme in the cell.

In a preferred embodiment, the vector system according to the invention is characterized in that one of the genes conferring on said non-pathogenic recombinant E. coli bacterium the ability to enter the cytoplasm of said eukaryotic target cells is integrated into the chromosome. of said E. coli by homologous recombination at the level of the dapA or dapB gene of said non-pathogenic E. coli bacterium, resulting in at least a partial deletion of this gene and its inactivation.

In a preferred embodiment, the vector system according to the invention is characterized in that one of the genes or the two genes conferring on said non-pathogenic recombinant E. coli bacterium the ability to enter the cytoplasm of said eukaryotic target cells are integrated into the chromosome of said E. coli by:

insertion of the penetration gene and deletion of the dap A gene between its 5 'fragment of sequence SEQ ID NO: 3 and its 3' fragment of sequence SEQ ID NO: 5, or between a 5 'and 3' fragment of the resulting dapA gene deleting a fragment of the dapA gene which is sufficient to inactivate this gene and / or to render auxotrophic said E. coli bacterium non-pathogenic for diaminopimelic acid;

insertion of the lysis gene and deletion of the dapB gene between its 5 'fragment of sequence SEQ ID NO: 9 and its 3' fragment of sequence SEQ ID NO: 11, or between a 5 'and 3' fragment of the dapB gene resulting in deletion of a fragment of the dapB gene sufficient to inactivate this gene and / or to render auxotrophic said E. coli bacterium non-pathogenic for diaminopimelic acid. In a preferred embodiment, the vector system according to the invention is characterized in that the selection of non-pathogenic recombinant E. coli bacteria having integrated the penetration gene is carried out by means of a tetracycline resistance cassette flanked by FRT sites permitting its excision of the bacterial chromosome by the yeast FIp recombinase, preferably by the introduction of a plasmid into said E. coli bacterium capable of producing the transient form FIp recombinase.

In a preferred embodiment, the vector system according to the invention is characterized in that the chromosomal integration of the gene (s) conferring on said non-pathogenic recombinant E. coli bacterium the ability to enter the cytoplasm of said eukaryotic target cells, is performed by homologous recombination in the presence of red α (exo) and red β (bet) proteins of bacteriophage λ expressed by a plasmid.

In a preferred embodiment, the vector system according to the invention is characterized in that it is a non-pathogenic recombinant E. coli bacterium of genotype:

- [thi-1, Endal, hsdR17 (rκ mκ ^~ ^+), supE44, Δ (lac) X74, ΔdapAΩinv, ΔdapBΩhly, ReCAL]; or

- [BM2710, ΔdapAΩinv, ΔdapBΩhly] or [BM2710, ΔdapAΩPtrc-inv, ΔdapBΩPtet-hly], the strain BM2710 having been deposited on October 27, 1995 under the number 1-1635 to the CNCM; or

the E. coli strain BM4570 deposited on July 18, 2007 under number 1-3788 at the CNCM (National Collection of Microorganism Cultures), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France; or - strain E. coli BM4569 deposited on December 13, 2007 under number 1-3877 at the CNCM (National Collection of Cultures of Microorganisms), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France; or

the E. coli strain BM4658 filed on January 17, 2008 under number 1-3894 at the CNCM (National Collection of Microorganism Cultures), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.

One of the problems with in vivo use of E. coli is its potential toxicity related to LPS. It has recently been shown that the potential for Inflammatory LPS by attenuating the immunogenicity of lipid A by genetic modification (Somerville et al., 1996, d'Hauteville et al., 2002).

Thus, in a particular aspect, the subject of the present invention is a vector system according to the invention, characterized in that the msbB structural gene of said non-pathogenic recombinant E. coli bacterium has been mutated in order to generate a strain whose lipid A LPS is devoid of myristoylated fatty acids.

In a particularly preferred embodiment, the vector system according to the invention is characterized in that it is a non-pathogenic recombinant E. coli bacterium of genotype: [thi-1, endAl, hsdR17 (rκ ^~ , mκ ⁺ ), supE44, Δ (lac) X74, ΔmsbB, ΔdapAΩinv, ΔdapBΩhly, recAl]; or

- [BM2710, ΔmsbB, ΔdapAΩinv, ΔdapBΩhly] or [BM2710, ΔmsbB, ΔdapAΩPtrc-inv, ΔdapBΩPtet-hly], the strain BM2710 having been deposited on October 27, 1995 under the number 1-1635 to the CNCM; or - strain E. coli BM4573 filed on July 18, 2007 under number 1-3790 at the CNCM (National Collection of Cultures of Microorganisms), Pasteur Institute, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France; or

the E. coli strain BM4571 deposited on December 13, 2007 under number 1-3878 at the CNCM (National Collection of Microorganism Cultures), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France; or

the E. coli strain BM4572 deposited on December 13, 2007 under number 1-3879 at the CNCM (National Collection of Microorganism Cultures), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France; or

E. coli strain BM4657 deposited on January 17, 2008 under number 1-3893 at the CNCM (National Collection of Microorganism Cultures), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.

The present invention also allows the generation of an invasive E coli strain allowing the production in large amounts by the vector of hetero log proteins and short hairpin RNA (sh RNA, interfering RNA molecules synthesized by the bacterium) under control. of the T7 promoter, the gene coding for T7

RNA polymerase is integrated into the bacterial chromosome. Thus, the present invention comprises an in vitro, ex vivo or in vivo method of producing heterologous proteins or nucleic acids, in particular RNAs, in particular short hairpin RNA (for short hairpin RNA) RNAs. , in a eukaryotic cell, characterized in that it implements a vector system according to the invention wherein said nucleic acid is under the control of the T7 promoter and the gene coding for the T7 RNA polymerase is integrated into the bacterial chromosome of said recombinant E. coli bacterium.

In a preferred embodiment, the vector system according to the invention is characterized in that the T7 RNA polymerase gene has been integrated, preferably under the control of the lacUV5 promoter, into the chromosome said non-pathogenic recombinant E. coli bacterium. .

In a particularly preferred embodiment, the vector system according to the invention is characterized in that it is a non-pathogenic recombinant E. coli bacterium of genotype:

- [thi-1, Endal, hsdR17 (rκ mκ ^~ ^+), supE44, Δ (lac) X74, ΔmsbB, ΔdapAΩinv, ΔdapBΩhly, ReCAL (DE3)]; or

- [BM2710, ΔdapAΩinv, ΔdapBΩhly, (DE3)] or [BM2710, ΔdapAΩPtrc-inv, ΔdapBΩPtet-hly, (DE3)], the strain BM2710 having been deposited on October 27, 1995 under the number 1-1635 at the CNCM; or

the strain E. coli BM4570 (DE3) deposited on July 18, 2007 under number I-3789 at the CNCM (National Collection of Microorganism Cultures), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.

In a preferred embodiment, the vector system according to the invention is characterized in that said non-pathogenic recombinant E coli strain is transformed by a replicative or non-replicative vector into E coli carrying said nucleic acid of interest or coding for said protein. interest, and, where appropriate, placed under the control of regulatory elements in said eukaryotic target cells. Advantageously, said nucleic acid of interest comprises a DNA fragment coding for a protein of interest, this latter fragment being placed under the control of regulatory elements in said eukaryotic target cells. The term "regulatory elements" means regulatory sequences suitable for transcription then translation such as promoter, including codons "start" and "stop", "enhancer" and "operator". The means and methods for identifying and selecting these elements are well known to those skilled in the art. In an advantageous embodiment, said strain is transformed by a replicative or non-replicative vector in E. coli carrying said nucleic acid of interest and optionally said regulatory elements.

In a preferred embodiment, the vector system according to the invention is characterized in that said vector carrying said nucleic acid of interest further comprises elements allowing integration into the genome of the target eukaryotic cells.

In a preferred embodiment, the vector system according to the invention is characterized in that said vector carrying said nucleic acid of interest further comprises an origin of replication allowing the vector to replicate extra chromosomally in said target eukaryotic cells. .

According to the present invention, said nucleic acid of interest is carried by a replicative and non-integrative plasmid in the bacterium. In addition, the foreign DNA fragment is placed under the control of functional expression elements in the target eukaryotic cells. Said carrier vector may be replicative or integrative in the target eukaryotic cells, that is to say that it may comprise elements allowing integration into the genome of the target eukaryotic cells, or may include an origin of replication allowing the vector to to replicate extra chromosomally in the target eukaryotic cells. In a preferred embodiment, the vector system according to the invention is characterized in that said eukaryotic cells are mammalian cells, yeast or plant cells, preferably mammalian cells.

In another aspect, the present invention relates to a vector system according to the invention for the preparation of a therapeutic composition. In another aspect, the present invention provides a method for in vivo or in vitro transfer of DNA into eukaryotic cells other than animal cells. or human, characterized in that it implements a vector system according to the invention.

In another aspect, the present invention relates to an in vitro or ex vivo method for transferring DNA into human or animal eukaryotic cells from a biological sample of human or animal origin, characterized in that it implements a vector system according to the invention.

In another aspect, the present invention relates to the use of a vector system according to the invention, for the preparation of a vaccine composition, characterized in that the nucleic acid of interest encodes an antigen of an infectious agent whose infection can be prevented by means of an antibody directed against this antigen.

In another aspect, the subject of the present invention is the use of a vector system according to the invention, the preparation of an anti-tumor vaccine composition, characterized in that the nucleic acid of interest codes for an antigen tumor whose tumor or cancer can be prevented by means of an antibody directed against this antigen.

In a final aspect, the present invention relates to the use of a vector system according to the invention, for the preparation of a therapeutic composition for the treatment or prevention of diseases by gene therapy.

Other features and advantages of the present invention will become apparent from the examples and figures below.

Legends of the figures

Figure 1: Figure 1 shows a construction scheme of the bacterial vectors. Figure 2: Figure 2 shows a diagram showing the different building steps to lead to different vector systems BM4570, BM4573 and BM4570 (DE3). FIGS. 3A-3C: FIGS. 3A to 3C represent the comparative results obtained by FACS analysis for the various vector systems derived from the BM2710 strain, expression of the inv gene on the surface of the E. coli bacterium. Figure 4: Figure 4 shows the quantification of the hemolityc activity in the three vectors.

Figure 5: Figure 5 shows the ability of pEGFP-C1 gene transfer by BM4570 compared with that of BM2710pGB2Ωmv- / z / y.

EXAMPLE 1 Construction of E. coli BM4570 by Integration of Inv and My Genes in the Chromosome of E. coli BM2710

It has been shown that the expression of the bacteriophage λ Red α (exo) and Red β (bet) proteins, as well as the Red γ (gam) protein, can efficiently promote homologous recombination in a strain of E. coli recA lacking homologous recombination. Using this system it is therefore possible to perform the allelic exchange of genes located in the chromosome following a double electroporating recombination event in the strain to modify a DNA fragment containing terminal sequences homologous to the target (JPP Muyrers, 2001). The Red αβγ functions of the phage λ are carried by the plasmid pKOBEGA which has a heat-sensitive origin of replication and are expressed under the control of the promoter inducible by arabinose pBAD (Chaveroche et al, 2000).

The E. coli strain harboring pKOBEGA was electrotransformed by fragments which include the inv gene or the hly gene flanked, respectively, by the 5 'and 3' portions of the dapA gene or the dapB gene.

To select recombinant strains, we used a tetracycline resistance cassette that could be excised secondarily from the chromosome. The Tet cassette ^R (D. Mazel, unpublished) is flanked by short direct repetitions (FRT sites) which allow its excision by the yeast FIp recombinase (MM Cox, 1983) thus generating a stable bacterial vector and devoid of resistance. Tetracycline resistance was deleted from the chromosome by introducing into the strain the plasmid pCP20 (Cherepanov and Wackernagel, 1995) which allows transient production of the FIp recombinase in trans (Figure 1). We integrated with this system: - in the E. coli BM2710 dapA gene, the inv gene under the control of the strong Ptrc promoter. Ptrc is a hybrid promoter composed of the sequence -35 of the trp promoter and the -10 sequence of / αcUV5; in the E. coli BM2710 dapB gene, the hly gene lacks a signal sequence under the control of the Vtet promoter of pACYC184 (Higgins et al, 1999).

EXAMPLE 2 Construction of E. coli BM4573 Derived from E. coli BM2710 Invasive with Modified LPS

Mutations in the msbB structure gene of E. coli generating strains whose LPS lipid A lacks myristoylated fatty acids thereby decreasing their proinflammatory activity (Somerville et al., 1996).

Plasmid pCVD442 msbB1 :: km, a suicide vector that only replicates in strains producing the Pir protein, was used (d'Hauteville et al., 2002). Insertion into this plasmid comprises the 5 'portion and the upstream region of the msbB gene, the kafamycin resistance determinant aphA of pUC4K and the 3' portion and the region downstream of the msbB gene. The aphA gene of this construct was replaced by the tet ^R cassette flanked by the FRT sites. E. coli strain BM4573 (AmsbB) was constructed using the same approach as previously described.

EXAMPLE 3 Construction of E. coli BM4570 (DE3) by Integration of the T7 RNA Polymerase Gene into the Chromosome of E. coli BM4570

The λDE3 lysogenization kit (Novagen) was used to integrate the T7 RNA polymerase gene under the control of the laclIV5 promoter.

The main steps in the construction of the strains are shown in Figure 2.

For each of the strains constructed, the expression of invasin on the surface of the bacterium and the production of listeria listerine were studied and compared with those of the original strain that hosts the plasmid pGB2Ω.inv-hly (Figure 3).

Similarly, the ability of these strains to transfer the EGFP gene has been studied in a comparative manner (Figure 5). BIBLIOGRAPHIC REFERENCES

- Chaveroche MK, Ghigo JM, and Enfert C (2000) A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res., 28 (22) e97.

- Courvalin P, Goussard S and Grillot-Courvalin C (1995) Gene transfer from bacteria to mammalian cells. CR. Acad. Sci. Ser.I.I., 318: 1207-1212.

- Cox MM. (1983) The FLP protein of the yeast 2-μm plasmid: Expression of a eukaryotic genetic recombination system in Escherichia coli. Proc. Natl. Acad. Sci. USA, 80: 4223-4227.

- Hauteville H, Khan S, Maskell DJ, Kussak A, Weintraub A, Mathison J, Ulevitch RJ, Wuscher N, Parsot C, and Sansonetti PJ (2002) Two msbB genes encoding maximal acylation of lipid A are required for invasive Shigella flexneri to mediate inflammatory rupture and destruction of the intestinal epithelium. J. Immunol., 168: 5240-5251. - Grillot-Courvalin C, Goussard S, Huetz F, Ojcius DM, and Courvalin P (1998) Functional gene transfer from intracellular bacteria to mammalian cells. Nature Biotechnol, 16: 862-866.

- Robbens J, Raeymaekers A, Steidler L, Proud W, and Remaut E (1995) Production of soluble and active murine recombinant interleukin-2 in Escherichia coli: high level expression, Kil-induced release, and purification. Protein Expr. Purif, 6: 481-486.

- Somerville Jr. JE, Cassiano L, Bainbridge B, Cunningham MD, and Darveau RP (1996) A novel Escherichia coli lipid A mutant that produces an antiinflammatory lipopolysaccharide. J. Clin. Invest., 97 (2): 359-365.

Claims

1. Vector system capable of delivering into eukaryotic target cells a nucleic acid of interest or coding for a protein of interest, this vector system comprising a non-pathogenic recombinant E. coli bacterium, said non-pathogenic E. coli bacterium also being :

- modified by introduction (by allelic exchange) of one or more genes conferring on this non-pathogenic recombinant E. coli bacterium the ability to penetrate the cytoplasm of said eukaryotic target cells; - incapable of surviving in the cytoplasm of said target cell; And

- modified by foreign DNA fragments, characterized in that the gene(s) conferring to said non-pathogenic recombinant E. coli bacterium the ability to penetrate the cytoplasm of said eukaryotic target cells are integrated into the chromosome of said non-pathogenic E. coli pathogenic.

2. Vector system according to claim 1, characterized in that said non-pathogenic E. coli bacteria is transformed by chromosomal integration of two genes, coming from one or two other bacteria, giving it the capacity to penetrate into the cytoplasm of said cells targets.

3. Vector system according to claim 1 or 2, characterized in that the gene(s) integrated into the chromosome of the non-pathogenic E. coli bacterium and giving it its ability to penetrate the cytoplasm of said target cells, allow it to lyse the membrane of the vacuoles of said target cells to reach the cytoplasm.

4. Vector system according to one of claims 1 to 3, characterized in that said non-pathogenic E. coli bacterium is transformed by chromosomal integration by the invasion gene of the Y. pseudotuberculosis bacteria which confers the property of penetrating into the cytoplasm of epithelial cells.

5. Vector system according to one of claims 1 to 3, characterized in that said non-pathogenic E. coli bacterium is transformed by chromosomal integration by the gene coding for the hemolysin of L. monocytogenes which confers the property of lysing membranes vacuoles of said target cells.

6. Vector system according to one of claims 1 to 5, characterized in that said non-pathogenic E. coli bacteria is transformed by chromosomal integration at both by the invasion gene of the Y. pseudotuberculosis bacteria which confers the property of penetrating the cytoplasm of epithelial cells and by the gene coding for hemolysin of L. monocytogenes which confers the property of lysing the membranes of the vacuoles of said target cells and by the gene.

7. Vector system according to one of claims 1 to 6, characterized in that said non-pathogenic E. coli bacteria is transformed by chromosomal integration by both:

1. by the inv invasion gene of Y. pseudotuberculosis, preferably of sequence SEQ ID NO: 10 or of sequence identical to at least 80% and capable of conferring the property of penetrating the cytoplasm of epithelial cells; And

2. the hly gene encoding the hemolysin of L. monocytogenes, preferably of sequence SEQ ID NO: 4 or of sequence identical to at least 80% and capable of conferring the property of lysing the membranes of vacuoles.

8. Vector system according to claim 7, characterized in that: 1. the inv invasion gene of Yersinia pseudotuberculosis is under the control of the Ptet promoter;

2. the hly gene encoding L. monocytogenes hemolysin is under the control of the Ptrc promoter.

9. Vector system according to one of claims 1 to 8, characterized in that said E. coli strain has been rendered incapable of survival in said cells as soon as it enters the cytoplasm of eukaryotic cells.

10. Vector system according to claim 9, characterized in that said non-pathogenic recombinant E. coli bacteria has been modified so as to be made auxotrophic for diaminopimelic acid.

11. Vector system according to claim 10, characterized in that the gene conferring on said non-pathogenic recombinant E. coli bacterium the ability to penetrate the cytoplasm of said eukaryotic target cells is integrated into the chromosome of said E. coli bacterium by homologous recombination at the level of the dapA gene of said non-pathogenic E. coli bacterium, resulting in at least partial deletion of this gene and its inactivation, preferably that the dapA gene coding for the enzyme dihydrodipicolinate synthase is inactivated.

12. Vector system according to claim 10 or 11, characterized in that the gene conferring on said non-pathogenic recombinant E. coli bacterium the ability to penetrate the cytoplasm of said eukaryotic target cells is integrated into the chromosome of said E. coli bacterium by homologous recombination at the level of the dapB gene of said non-pathogenic E. coli bacterium, resulting in at least partial deletion of this gene and its inactivation.

13. Vector system according to one of claims 9 to 12, characterized in that the gene conferring on said non-pathogenic recombinant E. coli bacterium the ability to penetrate the cytoplasm of said eukaryotic target cells and to lyse the penetration vacuole is integrated in the chromosome of said E. coli bacteria by:

- insertion of the penetration gene and deletion of the dapA gene between its 5' fragment of sequence SEQ ID NO: 3 and its 3' fragment of sequence SEQ ID NO: 5, or between a 5' and 3' fragment of the dapA gene resulting in the deletion of a fragment of the dapA gene sufficient to inactivate this gene and/or render said non-pathogenic E. coli bacteria auxotrophic for diaminopimelic acid;

- insertion of the lysis gene and deletion of the dapB gene between its 5' fragment of sequence SEQ ID NO: 9 and its 3' fragment of sequence SEQ ID NO: 11, or between a 5' and 3' fragment of the dapB gene resulting in the deletion of a fragment of the dapB gene sufficient to inactivate this gene and/or render said non-pathogenic E. coli bacteria auxotrophic for diaminopimelic acid.

14. Vector system according to one of claims 9 to 13, characterized in that the selection of non-pathogenic recombinant E. coli bacteria having integrated the penetration gene and the lysis gene is carried out by means of a resistance cassette to tetracycline flanked by FRT sites allowing its excision from the bacterial chromosome by the yeast FIp recombinase, preferably by the introduction of a plasmid into said E. coli bacteria capable of producing the FIp recombinase in transient form.

15. Vector system according to one of claims 1 to 14, characterized in that the chromosomal integration of the gene(s) conferring on said non-pathogenic recombinant E. coli bacterium the ability to penetrate the cytoplasm and lyse the penetration vacuole of said eukaryotic target cells, is carried out by homologous recombination in the presence of Red α(exo) and Red β(bet) proteins of bacteriophage λ expressed by a plasmid.

16. Vector system according to one of claims 1 to 15, characterized in that it is a non-pathogenic recombinant E. coli bacterium of genotype:

- [thi-1, endAl, hsdR17 (rκ ^~ mκ ⁺ ), supE44, Δ(lac)X74, ΔdapAΩinv, ΔdapBΩhly, recAl]; or - [BM2710, ΔdapAΩinv, ΔdapBΩhly] or [BM2710, ΔdapAΩPtrc-inv, ΔdapBΩPtet-hly], the strain BM2710 having been deposited on October 27, 1995 under number 1-1635 at the CNCM with genotype [thi-1, endAl, hsdR17 (rκ ^~ mκ ⁺ ), supE44, Δ(lac)X74, ΔdapAΩ, recAl]; Or

- the E. coli strain BM4570 deposited on July 18, 2007 under number 1-3788 at the CNCM (National Collection of Cultures of Microorganisms), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France; Or

- the E. coli strain BM4569 deposited on December 13, 2007 under number 1-3877 at the CNCM (National Collection of Cultures of Microorganisms), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France; or - the E. coli strain BM4658 deposited on January 17, 2008 under number 1-3894 at the CNCM (National Collection of Cultures of Microorganisms), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.

17. Vector system according to one of claims 1 to 16, characterized in that the structural gene msbB of said non-pathogenic recombinant E. coli bacterium has been mutated in order to generate a strain whose lipid A of LPS is devoid of myristoylated fatty acids.

18. Vector system according to claim 17, characterized in that it is a non-pathogenic recombinant E. coli bacterium of genotype:

- [thi-1, endAl, hsdR17 (rκ ^~ , mκ ⁺ ), supE44, Δ(lac)X74, ΔmsbB, ΔdapAΩinv, ΔdapBΩhly, recAl]; Or

- [BM2710, ΔmsbB, ΔdapAΩinv, ΔdapBΩhly] or [BM2710, ΔmsbB, ΔdapAΩPtrc-inv, ΔdapBΩPtet-hly], the strain BM2710 having been deposited on October 27, 1995 under number 1-1635 at the CNCM of genotype [ thi-1 , endAl, hsdR17 (pC mκ ⁺ ), supE44, Δ(lac)X74, ΔdapAΩ, recAl]; or - the E. coli strain BM4573 deposited on July 18, 2007 under number 1-3790 at the CNCM (National Collection of Cultures of Microorganisms), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France; Or - the E. coli strain BM4571 deposited on December 13, 2007 under number 1-3878 at the CNCM (National Collection of Cultures of Microorganisms), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France; Or

- the E. coli strain BM4572 deposited on December 13, 2007 under number 1-3879 at the CNCM (National Collection of Cultures of Microorganisms), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France; Or

- the E. coli strain BM4657 deposited on January 17, 2008 under number 1-3893 at the CNCM (National Collection of Cultures of Microorganisms), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.

19. Vector system according to one of claims 1 to 18, characterized in that said nucleic acid of interest is of all sizes up to 100 kb or from 100 to 150 kb or larger than 150 kb.

20. Vector system according to one of claims 1 to 18, characterized in that the nucleic acid of interest, preferably coding for a heterologous protein or an RNA of shRNA type, is under the control of the T7 promoter, the gene coding for T7 RNA polymerase being integrated into the bacterial chromosome.

21. Vector system according to one of claims 1 to 18, characterized in that the T7 RNA polymerase gene has been integrated, preferably under the control of the lacUV5 promoter, into the chromosome of said non-pathogenic recombinant E. coli bacterium .

22. Vector system according to claim 21, characterized in that it is a non-pathogenic recombinant E. coli bacterium of genotype:

- [thi-1, endAl, hsdR17 (rκ ^~ mκ ⁺ ), supE44, Δ(lac)X74, ΔmsbB, ΔdapAΩinv, ΔdapBΩhly, recAl, (DE3)]; or - [BM2710, ΔdapAΩinv, ΔdapBΩhly, (DE3)] or [BM2710, ΔdapAΩPtrc-inv, ΔdapBΩPtet-hly, (DE3)], the strain BM2710 having been deposited on October 27, 1995 under number 1-1635 at the CNCM of genotype [thi-1, endAl, hsdR17 (rκ ^~ mκ ⁺ ), supE44, Δ(lac)X74, ΔdapAΩ, recAl]; Or

- the E. coli strain BM4570 (DE3) deposited on July 18, 2007 under number 1-3789 at the CNCM (National Collection of Cultures of Microorganisms), Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.

23. Vector system according to one of claims 1 to 22, characterized in that said non-pathogenic recombinant E. coli strain is transformed by a replicative vector or not in E. coli carrying said nucleic acid of interest or coding for said protein of interest, and, where appropriate, placed under the control of regulatory elements in said eukaryotic target cells.

24. Vector system according to claim 22 or 23, characterized in that said vector carrying said nucleic acid of interest further comprises elements allowing integration into the genome of the target eukaryotic cells.

25. Vector system according to claim 22 or 23, characterized in that said vector carrying said nucleic acid of interest further comprises an origin of replication allowing the vector to replicate extrachromosomally in said target eukaryotic cells.

26. Vector system according to one of claims 1 to 25, characterized in that said eukaryotic cells are mammalian cells, yeast or plant cells, preferably mammalian cells.

27. Vector system according to one of claims 1 to 26, for the preparation of a therapeutic composition.

28. Method for in vivo or in vitro transfer of DNA into eukaryotic cells other than animal or human cells, characterized in that it uses a vector system according to one of claims 1 to 26.

29. In vitro or ex vivo method of transferring DNA and RNA into human or animal eukaryotic cells from a biological sample of human or animal origin, characterized in that it uses a vector system according to one of claims 1 to 26.

30. Use of a vector system according to one of claims 1 to 27, for the preparation of a vaccine composition, characterized in that the nucleic acid of interest codes for one or more antigens of an infectious agent or for antigenic fragments.

31. Use of a vector system according to one of claims 1 to 27, for the preparation of an anti-tumor vaccine composition, characterized in that the nucleic acid of interest codes for a tumor antigen.

32. Use of a vector system according to one of claims 1 to 27, for the preparation of a therapeutic composition intended for the treatment or prevention of diseases by gene therapy.