WO2004108906A2

WO2004108906A2 - Bacterial carriers for nucleotide sequences coding for active substances

Info

Publication number: WO2004108906A2
Application number: PCT/DE2004/001180
Authority: WO
Inventors: Werner Goebel; Sabine Pilgrim; Jochen Strizker; Ivaylo Gentschev; Christoph Schoen
Original assignee: Zentaris Gmbh
Priority date: 2003-06-06
Filing date: 2004-06-07
Publication date: 2004-12-16
Also published as: DE112004001496D2; WO2004108906A3; DE10326189A1

Abstract

The invention relates to a bacterial microorganism containing a foreign nucleic acid sequence that codes for an active substance. A natural nucleic acid sequence of the bacterium, which codes for expression of a bacterial enzyme is deleted or mutated in the chromosomal DNA microorganism provided that a translation product obtained therefrom is not functional. The invention also relates to cells that are infected with such microorganisms, methods for producing such organisms, and the use thereof, especially for producing pharmaceutical compositions.

Description

Bacterial carriers for nucleotide sequences coding for

drugs

Field of the Invention.

The invention relates to a bacterial microorganism with foreign DNA, a cell which is infected with such a microorganism, methods for producing such organisms, pharmaceutical compositions with such organisms, and uses of such organisms.

Background of the Invention and Prior Art.

Bacteria such as Shigella fl exeri, Salmonella spp., Escherichia coli, Yersinia enterocoli tica and Listeria monocytogen.es can be used as ferries for the transfer of nucleic acid sequences, preferably in the form of plasmids, in mammalian cells. This bacterial DNA transfer is called bactofection (Shata et al., 2000; Grillot-Courvalin et al., 2002; Al-Mariri et al., 2002; Hense et al., 2001; Powell et al., 1999, USP 5877159 ).

Bacteria as ferries for DNA vaccines offer the additional advantage of co-stimulation through their bacterial components.

Depending on the bacterium, the genes are transferred in the infected mammalian cell in different ways: Shigella and listeria reach the cytosol of the host cell and release the gene here after lysis. Salmonella, Yersinia and E. coli remain in Phagoso en. It is practically unknown how the released DNA penetrates from there into the cell nucleus. The release of the DNA within the infected cell can take place spontaneously or can be facilitated by antibiotics. In addition, specific mutants of the bacteria can be used which are characterized by easier disintegration in the host cell (Sizemore et al., 1997; Grillot-Courvalin et al., 2002; Darj i et al., 1997; Grillot-Courvalin et al. ., 1998

The self-destructing L has a special shape. onocytogenes carrier system (sLM), which causes expression and secretion of heterogeneous endolysins derived from Listeria bacteriophages when the Listeria enters the cytosol of the host cell to dissolve the Listeria (Dietrich et al., 1998; Loessner et al., 1995 ).

The use of this sLM system as well as all bacterial systems other than gene ferries has so far been opposed to the problems of stability and biological safety, that is to say the virulence of the bacterial carriers.

For example, if sLM transfected with plasmid coding for a desired protein (vaccine plasmid) was administered to mice, a considerable number, generally more than 30%, of sLM lost their plasmids. To solve these problems, a new so-called "balanced lethal" system was established (Pilgrim, S., dissertation University of Würzburg, 2002). For this purpose, the essential gene trpS in the chromosome was deleted, while at the same time a functional trpS gene copy was transferred to the vaccine plasmid. This system ensures that only bacteria carrying vaccine plasmids survive. To reduce the virulence of listeria, Pilgrim (doctoral thesis University of Würzburg 2002) deleted the gene iap. This deletion caused an incorrect arrangement of the ActA protein on the surface of the listeria, a defect in the mobility of the listeria and at the same time a strong virulence attenuation. At the same time, however, it was also found that a defect in the mobility of the listeria, for example due to deletion of the genes iap and actA, also affects the ability of the listeria to release plasmid DNA in the cytosol from cells. It was therefore concluded that the ability of listeria to move within cells and to penetrate neighboring cells is an important prerequisite for an efficient transfer of plasmid DNA. Deletion of the iap gene

1 c,, to reduce the virulence of listeria has therefore proven unsuitable for bactofection. ^•

With Salmonella, E.coli or Pasteurella has been around since

It has long been known that the deletion or negative mutation o n of at least one gene aroA coding for an enzyme for

Synthesis of an aromatic amino acid leads to a reduction in virulence (Holseth et al., Nature 291: 238-239

(1981); Stocker et al. , Dev Biol Stand 53: 47-54 (1983);

Jones et al., Vaccine 9: 29-34 (1991); Scott et al. , Αvian

9π

Dis. 43: 83-88 (1999)). Such germs have been successfully used for immunization against the respective germ as well as against an antigen expressed by a transgene introduced in, for example, Salmonella (Turner et al., Infect Immun 61: 5374-5380 (1993). On the other hand, it is known that a negative mutation of the aroA gene in Yersinia pestis leads to a reduction in the virulence of these germs in guinea pigs, whereas the virulence of the mutated germ is largely preserved in mice (Oyston et al., Microbiology 142: 1847-1853 (1996). However, it is not known whether such a mutation in Listeria maintains its functionality for bactofection.

Technical problem of the invention.

The invention is therefore based on the technical problem of creating a bacterial carrier for plasmid DNA with the following combination of properties: i) the virulence of the bacteria is drastically reduced, a strong bactofection is ensured, and iii) stable expression of the plasmid DNA is made possible ,

Basics of the invention and embodiments.

The invention is based on the knowledge that the virulence of bacteria, for example the virulence of sLM, can be drastically reduced if genes coding for enzymes for the synthesis of essential metabolic products are deleted in their chromosomes. Surprisingly, such virulence enables attenuated bacteria, despite attenuation as carriers of expression plasmids and, in comparison to non-attenuated bacteria, to achieve at least an equally strong bactofection, namely if these attenuated carrier bacteria are capable of spreading, that is to say have retained the ability to move within the infected cell move and penetrate into neighboring cells.

The invention thus relates to bacteria as carriers of at least one nucleotide sequence coding for at least one active substance, characterized in that in at least one chromosome of these bacteria has deleted or mutated at least one gene for a metabolic enzyme so that the metabolic enzyme is defective.

The invention furthermore relates to bacteria according to the invention, with the deletion or mutation of at least one gene for at least one metabolic enzyme, which are competent for dissemination and which, after infection in a mammalian cell, express lytic proteins in the cytoplasm of the bacteria, these lytic proteins dissolving the bacteria and which release at least one nucleotide sequence coding for at least one active substance within the cell, preferably in the cytosol of the cell

A preferred example of the bacteria according to the invention are listeria, in which in particular a gene for an enzyme for the synthesis of aromatic amino acids in the chromosome has been deleted, for example the aroA gene which codes for the first enzyme in the biosynthesis of aromatic amino acids, so that these bacteria are dependent on their growth, are determined by the presence of aromatic amino acids those proteins ^■ enabling the motility of the bacteria expressing unaffected, iap example, the function of the genes and the gene trpS obtained actA encoding tryptophanyl-tRNA synthetase deleted in the chromosome has been

and that plasmids have been introduced into these bacteria, whose replication was stabilized by a suitable "replication origin", for example by ori pAMßl (Simon and Chopin, 1988), which contain the trpS gene coding for tryptophanyl-tRNA synthetase and which contain a gene for an endolysin, for example the lysis gene of phage A118 { ply 118; Loessner et al., 1995) under the control of a promoter that can be activated in the cytosol of mammalian cells, for example the actA promoter (PactA, Dietrich et al., 1998), which encodes at least one nucleotide sequence for at least one active ingredient under the control of a bacterium or contained in promoters which can be activated in mammalian cells, the activation of the promoter being cell-specific, cell-specific, cell cycle-specific, cell function-specific or dependent on metabolites, drugs or on the oxygen concentration.

Bacteria of the genus Listeria are gram-positive, mostly coccoid and catalase-positive rod bacteria. An example of this is Listeria monocytogenes.

Bacteria such invention have by deri-loss of at least one gene for an essential metabolic selprote ^'in a drastic reduction in their virulence, for example in relation to their in vivo viability and nevertheless show a considerably increased Bactofection, a lysis of the bacteria in the cytosol, a Fre - Setting the plasmids contained in the bacteria and stable expression of the active substance encoded by the plasmid. Active substances in the sense of the invention are proteins, for example, selected from a group comprising:

Antigens from infectious agents such as viruses, bacteria, mycoplasmas, parasites - antigens specific for tumors, in particular proteins encoded by oncogenes

Antibodies, epitope-binding fragments of antibodies and fusion proteins containing at least one epitope-binding fragment of an antibody, for example directed against an antigen on a tumor cell, a lymphocyte such as a T-lymphocyte or an endothelial cell such as a tumor endothelial cell

Enzymes, in particular enzymes for activating inactive precursors of a medicament, such as, for example, a β-glucuronidase, a phosphatase, a hydrolase, a lipase,

Immunosuppressive cytokines such as IL-10

Immune stimulating cytokines such as IL-1, IL-2, 11-3 or IL-6 - chemokines

interferons

Growth factors such as G-CSF, GM-CSF, M-CSF, FGF; VEGF or EGF or

Inhibitory proteins for cytokines, chemokines, interferons or growth factors

The invention furthermore relates to the use of a bacterium according to the invention for the prevention or treatment of a disease.

The bacteria according to the invention are preferably used to treat a tumor disease or an immune disease to treat. For this purpose, the gene inserted into the bacteria encodes a protein which is tumor cytolytic, has a pro-inflammatory effect, - negative regulating immune cells are inhibited, for example by inhibiting CTLA-4, B7-H1 or CD25, or TGFß, which has an immunosuppressive effect or an inactive precursor can convert a cytotoxic, immunomodulating or immunosuppressive substance into an active ingredient. angiogenesis inhibits angiogenesis promotes coagulation - the coagulation inhibits clot formation dissolves a hormone effect has a circulatory effect has the growth of connective or supporting tissue promotes - promotes wound healing

100 to 10 10 bacteria are administered to prevent or treat a disease. The bacteria are preferably administered as a suspension in a pharmaceutically prepared aqueous solution for injection or in a pharmaceutically prepared ointment or lotion for local administration. Such pharmaceutical preparations are known to the person skilled in the art.

The bacteria are preferably in a suitable

Preparation locally on the skin, in the circulation, in a body cavity, in a tissue, in an organ or orally, rectally or bronchially administered at least once. The invention further relates to the infection of a cell, for example a macrophage, lymphocyte, granulocyte, a connective tissue cell, a muscle cell, a tumor cell or leukemia cell ex vivo with a bacterium according to the invention and the injection of the cell thus infected into an organism for prophylaxis or therapy Illness.

Diseases in which the bacteria according to the invention are used represent, for example, tumor diseases, autoimmune diseases, chronic inflammation, organ transplants, circulatory diseases, diseases of blood clotting, metabolism, the stomach and intestines, the kidneys, the lungs and wound healing.

The invention is explained in more detail below on the basis of exemplary embodiments.

Example 1: Materials and methods.

The bacterial strains and DNA sequences used are shown in Table 1. The infection of various Zeil lines with listeria, flow cytometry (FASC) of EGFP-expressing cells and the mouse infection are described in detail by Pilgrim (dissertation University of Würzburg 2002). The microinjection protocol is described in Götz et al., 2000.

Example 2: Production of virulence-attenuated suicide listeria (aroA) with stably replicating plasmids Because L. monocytogenes is an optional animal and human pathogenic species, for the development of these listeria as carriers for plasmid DNA coding for pharmaceutical active substances according to this invention, attenuations regarding their virulence were introduced. For this purpose, the aroÄ gene was additionally deleted in the genome of the trpS deletion utante (WL-140, Pilgrim, dissertation University of Würzburg 2002). This gene codes for the first enzyme in the biosynthesis of aromatic amino acids, so that the growth of these bacteria depends on the presence of aromatic amino acids.

The aroA / trpS (WL-141) double mutant was produced using the deletion mutagenesis technique based on the WL-140 strain and the oligonucleotides aroA-Al, 2 and aroA-Bl, 2 (Tab. 1).

To construct an aroÄ mutagenesis plasmid, a deletion region 5 'flanking fragment A (365 bp) was amplified with the oligonucleotides aroA-Al and aroA-A2, while using the oligonucleotides aroA-Bl and aroA-B2 a 3' - flanking fragment B (438 bp) was formed. The sequence of both fragments was derived from strain L sequenced in the "Listeria monocytogenes genome sequencing project". monocytogenes EGD-e taken (Glaser et al., 2001). Both

Fragments became fragment via a PspAI interface

AB ligated, this was again amplified in a PCR reaction with the aid of the oligonucleotides aroA-Al and aroA-B2 and inserted into the plasmid pLSVl via .EcoRI and Ba π interfaces. After transformation of the aroÄ mutagenesis plasmid pLSVl-aroA-AB into the L. monocytogenic strain WL-140, intermediate cultivation at 30 ° C and subsequent Cultivation on BHI agar plates with erythromycin at 43 ° C made it possible to prepare a few clones which had inserted the plasmid chromosomally. A clone was selected from these which had performed a homologous recombination with the plasmid via the aroA-A fragment.

After 20 passages at 37 ° C the aroA, trpS deletion mutant (trpS, aroA / pTRPS) could be isolated. This newly constructed strain was called WL-141.

Example 3: Bactofections experiments "in vitro".

The Listeria strains WL-140 and WL-1.41 with the plasmids pSPO-EGFP or pSP118-EGFP (Tab. 2), which the gene for the enhanced green fluorescent protein (EGFP) under the control of the "Major immediate-early promoter / enhancer "region of the human cytomegalovirus (Pcmv) were tested for their bactofection efficiency in several cell cultures (Table 3). While most EGFP-expressing cells were found in the CaCo-2 and COS-1 cell cultures after infection with WL-141 / pSP118-EGFP, the best results were found in HeLa and HepG2 cell cultures

WL-140 / pSP118-EGFP can be achieved. That means the

Strain WL-141, similar to WL-140, very good for in vi tro

Gene transfer experiments are suitable.

Example 4: in vivo virulence tests.

The WL-140 and WL141 strains were tested in the mouse infection model (BALB / c) for their infection efficiency and virulence. In these experiments, a similar number of bacteria was found in the spleen and liver after infection with WT and WL140 / pSP0 (Table 4). In contrast, the strain WL141 / pSP0 is spread in the same organs dramatically attenuated (Table 4) and thus only leads to a very mild infection of the host.

Example 5: The bactofection efficiency is particularly dependent on the spread of the bacteria

(Spreading attempts)

Surprisingly, it was found in the context of the invention that the actΛ-negative strain WL-142 (incompetent for distribution) has a low bactofection rate after a microinjection into Caco-2 cells (Table 5). In contrast, bactofection with actΛ-positive strains WL-140 / pSPO-EGFP or pSP118-EGFP (widespread) is much more efficient, especially at a later point in time (48-72h; Tab. 6). The explanation for this is that by spreading into several cells, the bacteria capable of spreading enable a much more efficient bactofection (Tab. 7). It was therefore concluded that the spreading property of the bacteria is an important factor for an efficient plasmid DNA release.

Literature:

Al-Mariri, A. et al. , Infect. Immune. 70, 1915-1923 (2002).

Darji, A. et al. , CeU 91,765-775 (1997).

Dietrich, G. et al. , Nat. Biotechnol. 16.181-185 (1998)

Glaser, P. et al. , Sci ence 294, 849-852 (2001).

Goetz, M. et al. , Proc. Natl. Acad. Be . U S A 9.12221-12226 (2001).

Grillot-Courvalin, C, Goussard, S., & Courvalin, P., Cell. Mi crobi ol. 4, 177-186 (2002). Grillot-Courvalin, C. et al. , Nat. Biotechnol. 16.862-866 (1998).

Hense, M. et al. , Cell. Microbiol. 3,599-609 (2001).

Loessner, M.J., Wendlinger, G., & Scherer, S., Mol. Microbiol. 16.1231-1241 (1995).

Pilgrim, S., PhD thesis, University of Würzburg, Development of a "DNA Delivery" system based on virulence-attenuated Listeria (2002)

Powell, R.J., Lewis, G.K., & Hone, D.M., U.S. Patent No. 5,877,159.

Shata, M.T., Steveeva, L., Agwale, S., Lewis, G.K., & Hone, D.M., Mol. Med. Today 6, 66-71 (2000). • Simon, D., & Chopin, A., Biochimi e 70, 599-66 (1988)

Sizemore, D.L., Branstrom, A.A., & Sadoff, J.C. Vacine 15, 804-807 (1997).

Strizker, J. H. "Studies on the improvement of DNA transfer in animal cells by Listeria monocytogenes", diploma thesis, University of Wuerzburg, 2001.

Wuenscher, MD, Kohler, S., Goebel, W., & Chakraborty T, Mol. Gene. Genet. 228, 177-82 (1991)

Table 1. Bacterial strains and DNAs:

Table 2. DNA sequence of the base plasmid pSP118

1 gaattcgcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 61 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 121 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 181 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 241 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 301 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 361 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 421 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 481 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 541 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 601 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 661 ctacggggtc tgacgctcag tgaattcccc tcggtaccat gcgaaaaaag cgcttcaaca 721 agcagcgaaa gatttatatg gtgaagatgc ttctaaaaaa gttgctgaag cttgggaagc 781 agttggggtt aactgattaa caaatgttag agaaaaatta attctccaag tgatattctt 841 aaaataattc atgaatat tt tttcttatat tagctaatta agaagataac taactgctaa 901 tccaattttt aacggaacaa attagtgaaa atgaaggccg aattttcctt gttctaaaaa 961 ggttgtatta gcgtatcacg aggagggagg atccatgaca agttattatt atagtagaag 1021 tttagcgaat gtaaataagt tagcagacaa tacgaaagcg gcagctagaa aattgctaga 1081 ttggtctgaa agcaacggga ttgaagtatt aatctacgaa acaattagaa cgaaagaaca 1141 acaagccgca aatgttaaca gtggagcgtc tcaaacaatg cgctcttatc acttggtagg 1201 acaagcgcta gatttcgtca tggcgaaagg taaaacggtc gattggggtg cttatcgttc 1261 agacaaaggc aagaaatttg tggcaaaggc aaaatcttta ggttttgagt ggggtggtga 1321 ttggtctgga tttgtagaca atccgcacct tcaatttaat tataaaggct atgggactga 1381 tacttttgga aaaggagcta gtactagtaa ttcatctaaa ccgagcgcag acacaaacac 1441 aaacagtcta ggattagtag attatatgaa tttaaataaa ctagattcaa gctttgcgaa 1501 tcgcaaaaaa ctagcgacaa gttacggaat taaaaattac agtggaacag caacgcagaa 1561 cacaacatta ttagcgaagt taaaagcagg aaaaccacac acaccagcaa gcaaaaacac 1621 atactacaca gaaaatccgc gaaaagttaa aacactagta caatgtgatc tatacaaatc 1681 agtagacttt acaacaaaaa accaa acagg tggaacattt ccgccaggca cagtcttcac 1741 gatttcaggg atggggaaaa cgaaaggcgg aacacctcgc ttgaagacga agagcggtta 1801 ctatctcact gctaacacga agtttgttaa aaagatttag gtcgactagt attttatgta 1861 tgtttttgtg attgacaagc cgttttgtaa actgatagaa tgaaagtaat ataactgatg 1921 cgttaatagg aagattagta cattttgttg ctttataata gagagtctgt ggttggtgga 1981 aacagataaa gcgcaaaatc gaatggactt ctaaggactg tttgtgaaag cgagtagcga 2041 acagcggcta aagaccgtta cttctttaat agaaactttc tattgctaag agccttttta 2101 ggtacttagg gtggcaccgc ggataaccgt tcgtccctaa catttatatg ttgggatgga 2161 cggtttttta ttttcgcaat cagtgaaaaa attggaggaa taatcatgaa aaaagtaata 2221 ttttccggaa ttcaacctag tggacaatta actttaggaa attatattgg ggctttaaaa 2281 cagtttgggc agtttcaaga tgaatatgaa tgtttttatt gtatcgttga cgaacatgct 2341 attacggttc cacaagatag attaaaactt cgcgagcaaa caagaagttt agcagctctt 2401 tatttagcag ttggactgga tcctgaaaaa tccactctat ttattcaatc tgaagtggcg 2461 gcacatgcgc aagcggcatg gattttacaa tgtaacgtct atattggtga attggagcgt 2521 atgacacaat ttaaagacaa atcagctggt aaagcaggag tgagcgccgg actattaacc 2581 tatccaccat taatggctgc tgatatttta ctttatcaaa cagatttagt tccagttggt 2641 gaggatcaaa aacaacatat cgaattaact cgcgacttag ctgaacgatt taataaaaag 2701 catgcagata ttttcacgat gccagaagta ttcattccta aacaaggcgc acgagtaatg 2761 tccttacaag atccaacgaa aaaaatgagt aaatctgatg ccaatttgaa aaatgctatc 2821 tttttacttg atccaccagc aactattaga aaaaaaatca aaagcgctgt gaccgattct 2881 agtggtatta ttgaatataa taaagaagaa aagcccggcg tatctaattt gctaactatc 2941 tattcggtta ttaccggcga aacaattgct agtatagaag aaaaatatgt tggaaaaggc 3001 tatggtgatt tcaaaacaga tttagctgaa ttagttgttt ctgaacttga gccaattcaa 3061 gaaagatact atgcttatct caaatctgag gagctggaca ctatattaga tgcaggagca 3121 gaaaaagcag ctcgcgtggc taataaaaca ttgaagaaaa tggaaaatgg cgttgggctt 3181 ggccgtaaac gcagaaaata atataaaaaa agccaatgac ctagttaatc taagtcatcg 3241 gcttttttta tttaatcatt aacctacttt aatttccacg tcgactctag aggatccgaa 3301 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta 3361 acttgtttat tgcagcttat aatggttaca AATAAA GCAA tagcatcaca aatttcacaa 3421 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 3481 atcatgtctg gatccccgcg gccgcgggta caattccgca gcttttagag cagaagtaac 3541 acttccgtac aggcctagaa gtaaaggcaa catccactga ggagcagttc tttgatttgc 3601 accaccaccg gatccgggac ctgaaataaa agacaaaaag actaaactta ccagttaact 3661 ttctggtttt tcagttcctc gagtaccgga tcctctagag tccggaggct ggatcggtcc 3721 cggtgtcttc tatggaggtc aaaacagcgt ggatggcgtc tccaggcgat ctgacggttc 3781 actaaacgag ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc 3841 aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa 3901 ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca 3961 cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag 4021 atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc 4081 catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact 4141 gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt 4201 ggcgttacta tgggaacata cgtcattatt gacgtcaatg g gcgggggtc gttgggcggt 4261 cagccaggcg ggccatttac cgtaagttat gtaacgacct gcagccaagc ttatcgattc 4321 tagactcgag agctctcgag tctagaatcg atacgatttt gaagtggcaa cagataaaactat ataaaataca 4441 agatagcgac agagaaggcg aaaacattgc ctggtcgatc attcataaag caaatgcctt 4501 ttctaaagat aaaacgtata aaagactatg gatcaatagt ttagaaaaag atgtgatccg 4561 tagcggtttt caaaatttgc aaccaggaat gaattactat cccttttatc aagaagcgca 4621 aaagaaaaac gaaatgatac accaatcagt gcaaaaaaag atataatggg agataagacg

4681 gttcgtgttc gtgctgactt gcaccatatc ataaaaatcg aaacagcaaa gaatggcgga

4741 aacgtaaaag aagttatgga aataagactt agaagcaaac ttaagagtgt gttgatagtg

4801 cagtatctta aaattttgta taataggaat tgaagttaaa ttagatgcta aaaatttgta

4861 attaagaagg agtgattaca tgaacaaaaa tataaaatat tctcaaaact ttttaacgag

4921 tgaaaaagta ctcaaccaaa taataaaaca attgaattta aaagaaaccg ataccgttta

4981 cgaaattgga acaggtaaag ggcatttaac gacgaaactg gctaaaataa gtaaacaggt

5041 aacgtctatt gaattagaca gtcatctatt caacttatcg tcagaaaaat taaaactgaa

5101 tactcgtgtc actttaattc accaagatat tctacagttt caattcccta acaaacagag

5161 gtataaaatt gttgggagta ttccttacca tttaagcaca caaattatta aaaaagtggt

5221 ttttgaaagc catgcgtctg acatctatct gattgttgaa gaaggattct acaagcgtac

5281 cttggatatt caccgaacac tagggttgct cttgcacact caagtctcga ttcagcaatt

5341 gcttaagctg ccagcggaat gctttcatcc taaaccaaaa gtaaacagtg tcttaataaa

5401 acttacccgc cataccacag atgttccaga taaatattgg aagctatata cgtactttgt

5461 ttcaaaatgg gtcaatcgag aatatcgtca actgtttact aaaaatcagt ttcatcaagc

5521 aatgaaacac gccaaagtaa acaatttaag taccgttact tatgagcaag tattgtctat

5581 ttttaatagt tatctattat ttaacgggag gaaataattc tatgagtcgc ttttgtaaat

5641 ttggaaagtt acacgttact aaagggaatg tagataaatt attaggtata ctactgacag

5701 cttccaagga gctaaagagg tccctagcgc tcttatcatg gggaagctcg gatcatatgc

5761 aagacaaaat aaactcgcaa cagcacttgg agaaatggga cgaatcgaga aaaccctctt

5821 tacgctggat tacatatcta ataaagccgt aaggagacgg gttcaaaaag gtttaaataa

5881 aggagaagca atcaatgcat tagctagaac tatatttttt ggacaacgtg gagaatttag

5941 agaacgtgct ctccaagacc agttacaaag agctagtgca ctaaacataa ttattaacgc

6001 tataagtgtg tggaacactg tatatatgga aaaagccgta gaagaattaa aagcaagagg

6061 agaatttaga gaagatttaa tgccatatgc gtggccgtta ggatgggaac atatcaattt

6121 tcttggagaa tacaaatttg aaggattaca tgacactggg caaatgaatt tacgtccttt

6181 acgtataaaa gagccgtttt attcttaata taacggctct ttttatagaa aaaätcctta

6241 gcgtggtttt tttccgaaat gctggcggta ccccaagaat tagaaatgag tagatcaaat

6301 tattcacgaa tagaatcagg aaaatcagat ccaaccataa aaacactaga acaaattgca

6361 aagttaacta actcaacgct agtagtggat ttaatcccaa atgagccaac agaaccagag

6421 ccagaaacag aatcagaaca agtaacattg gatttagaaa tggaagaaga aaaaagcaat

6481 gacttcgtgt gaataatgca cgaaatcgtt gcttattttt ttttaaaagc ggtatactag

6541 atataacgaa acaacgaact gaatagaaac gaaaaaagag ccatgacaca tttataaaat

6601 gtttgacgac attttataaa tgcatagccc gataagattg ccaaaccaac gcttatcagt

6661 tagtagagg aactcttccc tcgtaagaag ttatttaatt aactttgttt gaagacggta

6721 tataaccgta ctatcattat atagggaaat cagagagttt tcaagtatct aagctactga

6781 atttaagaat tgttaagcaa tcaatcggaa atcgtttgat tgcttttttt gtattcattt

6841 atagaaggtg gagtttgtat gaatcatgat gaatgtaaaa cttatataaa aaatagttta

6901 ttggagataa gaaaattagc aaatatctat acactagaaa cgtttaagaa agagttagaa

6961 aagagaaata tctacttaga aacaaaatca gataagtatt tttcttcgga gggggaagat

7021 tatatatata agttaataga aaataacaaa ataatttatt cgattagtgg aaaaaaattg

7081 acttataaag gaaaaaaatc tttttcaaaa catgcaatat tgaaacagtt gaatgaaaaa

7141 gcaaaccaag ttaattaaac aacctatttt ataggattta taggaaagga gaacagctga

7201 atgaatatcc cttttgttgt agaaactgtg cttcatgacg gcttgttaaa gtacaaattt 7261 aaaaatagta aaattcgctc aatcactacc aagccaggta aaagcaaagg ggctattttt 7321 gcgtatcgct caaaatcaag catgattggc ggtcgtggtg ttgttctgac ttccgaggaa 7381 gcgattcaag aaaatcaaga tacatttaca cattggacac ccaacgttta tcgttatgga 7441 acgtatgcag acgaaaaccg ttcatacacg aaaggacatt ctgaaaacaa tttaagacaa

7501 atcaatacct tctttattga ttttgatatt cacacggcaa aagaaactat ttcagcaagc 7561 gatattttaa caaccgctat tgatttaggt tttatgccta ctatgattat caaatctgat 7621 aaaggttatc aagcatattt tgttttagaa acgccagtct atgtgacttc aaaatcagaa 7681 tttaaatctg tcaaagcagc caaaataatt tcgcaaaata tccgagaata ttttggaaag 7741 tctttgccag ttgatctaac gtgtaatcat tttggtattg ctcgcatacc aagaacggac 7801 aatgtagaat tttttgatcc taattaccgt tattctttca aagaatggca agattggtct 7861 ttcaaacaaa cagataataa gggctttact cgttcaagtc taacggtttt aagcggtaca 7921 gaaggcaaaa aacaagtaga tgaaccctgg tttaatctct tattgcacga aacgaaattt 7981 tcaggagaaa agggtttaat agggcgtaat aacgtcatgt ttaccctctc tttagcctac 8041 tttagttcag gctattcaat cgaaacgtgc gaatataata tgtttgagtt taataatcga 8101 ttagatcaac ccttagaaga aaaagaagta atcaaaattg ttagaagtgc ctattcagaa 8161 aactatcaag gggctaatag ggaatacatt accattcttt gcaaagcttg ggtatcaagt

8221 g_atttaacca gtaaagattt atttgtccgt caagggtggt ttaaattcaa gaaaaaaaga 8281 agcgaacgtc aacgtgttca tttgtcagaa tggaaagaag atttaatggc ttatattagc 8341 gaaaaaagcg atgtatacaa gccttattta gtgacgacca aaaaagagat tagagaagtg 8401 ctaggcattc ctgaacggac attagataaa ttgctgaagg tactgaaggc gaatcaggaa 8461 attttcttta agattaaacc aggaagaaat ggtggcattc aacttgctag tgttaaatca 8521 ttgttgctat cgatcattaa agtaaaaaaa gaagaaaaag aaagctatat aaaggcgctg 8581 acäaattctt ttgacttaga gcatacattc attcaagaga ctttaaacaa gctagcagaa 8641 cgccctaaaa cggacacaca actcgatttg tttagctatg atacaggctg aaaataaaac 8701 ccgcactatg ccattacatt tatatctatg atacgtgttt gttttttctt tgctgtttag 8761 cgaatgatta gcagaaatat acagagtaag attttaatta attattaggg ggagaaggag 8821 agagtagccc gaaaactttt agttggcttg gactgaacga agtgagggaa aggctactaa 8881 aacgtcgagg ggcagtgaga gcgaagcgaa cacttgattt tttaattttc tatcttttat 8941 aggtcattag agtatactta tttgtcctat aaactattta gcagcataat agatttattg

9001 aataggtcat ttaagttgag catattagag gaggaaaatc ttggagaaat atttgaagaa 9061 cccgattaca tggattggat tagttcttgt ggttacgtgg tttttaacta aaagtagtga 9121 atttttgatt tttggtgtgt gtgta tatta gtta ga Table 3. Gene transfer efficiency of the / sfer / a strains WL-140 and WL141, which contain plasmids pSP118-EGFP or pSOP-EGFP. Three days after infection, EGFP expression was determined quantitatively in different Zeil lines using flow cytometry (Epics XL flow cytometer, Beckman Coulter).

* The percentage of live EGFP-expressing cells (propidium iodide (Pl) negative)

# The ratio between Pl positive (dead) to Pl positive cells after infection with WL140 / pSP0-EGFP

Lisürta straiπ Caco-2 COS-1 FieLa HeoG2

WL-HÜ / pSPÜ-EC 5 ^ 7A0 ϊ7 * 3.93 * β * G2 1.04 * {>, <$ 1.78 * 0.37

(1.00 * 0.11 (f.0O. * OH) (1.00 * 0.31) (1.00 * 0.01) L-HO / ^ SPl KS-EGFP 15.50 * 1.56 5, S9 * + 7 , «Ö * _, 24 5. ϊ 9 * 1.26 Ütάö.Ol) <0, <J9fcO.23) (1.27 * 0.33) fθ.52 * 0.28}

W 41 SPw-EöfP 6.73 * ^' 2 6.38 - ** »0.31 * 0.10 1.87 * 0.0

(0.11 * 0.03.) Ψ 2 * ^{) M) «1.42 * 0.04) (0.33 * 0.01)

WL-14i / pS? Πg-EGfP ϊ 9ά 3.1.5 28.31 -U), 47 .1.13 * 0.55 2.71 * 0.44

(0.05: i: 0.00} (ö, öϊ:! • ^■ 0,! 5) (0.26 k 0.3!) (0.25: i: 0.01}

Table 4. Orogastric infection of BALB / c mice (Charles River Laboratories, Germany; groups of 5 animals each) with wild type L monocytogens, WL-140 / pSPO or WL-141 / pSPO. Bacteria numbers in spleen and liver (4 days p.i.)

Table 5

(A) EGFP expressing Caco-2 cells (in%) after microinjection with the incompetent strains WL-142 / pSPO-EGFP or WL142 / pSP118-EGFP, 24, 48 and 72h post infection (p.i.).

Table 6

EGFP expressing Caco-2 cells (in%) after microinjection with the distribution-competent strains WL-140 / pSPO-EGFP or WL140 / pSP118-EGFP, 24, 48 and 72h p.i ..

Table 7

EGFP exposure in Caco-2 cells after infection with different numbers of WL-140 / pSPO-EGFP, WL-140 / pSP118-EGFP, WL-142 / pSP0-EGFP or WL-142 / pSP118-EGFP ("multiplicity of infection ", MOI = 5 or 100). (A) Number of intracellular bacteria in 10 ⁶ Caco-2 cells 2h ip (B) EGFP-expressing cells (in%), 48h ip

Claims

claims

1) Bacteria, in particular of the genus Listeria, as carriers of at least one nucleotide sequence coding for at least one active substance, characterized in that at least one gene for a metabolic enzyme is deleted in at least one chromosome of these bacteria or is mutated in such a way that the metabolic enzyme is defective.

2) Bacteria according to claim 1), characterized in that a gene for an enzyme for the synthesis of aromatic amino acids has been deleted in them.

3) Bacteria according to claim 2), characterized in that the aroA gene has been deleted.

4) Bacteria according to one of claims 1) to 3), characterized in that they are competent to spread.

5) Bacteria according to one of claims 1) to 4), characterized in that after infection in a mammalian cell they express lytic proteins in the cytoplasm of the bacteria, these lytic proteins dissolving the bacteria and thereby the bacteria coding for at least one nucleotide sequence for at least one Release active substance within the cell, preferably in the cytosol of the cell.

6) Bacteria according to one of claims 1) to 5), characterized in that they represent listeria. ) Bacteria according to one of claims 1) to 6), characterized in that in them

- the aroA gene, which codes for the first enzyme in the biosynthesis of aromatic amino acids, has been deleted, so that these bacteria in their

Growth depends on the presence of aromatic amino acids

- those proteins which enable the motility of the bacteria to be expressed unimpaired, for example the functionality of the genes iap and actA is preserved

- The gene coding for tryptophanyl-tRNA synthetase was deleted in the chromosome

and that plasmids have been introduced into these bacteria,

- whose replication was stabilized by a suitable "replication origin", for example by ori pAMßl (Simon and Chopin, 1988) - which contain the trpS gene coding for tryptophanyl-tRNA synthetase

- which is a gene for an endolysin, for example the lysis gene of phage A118 {ply 118; Loessner et al., 1995) under the control of a promoter which can be activated in the cytosol of mammalian cells, for example the actA promoter (PactA, Dietrich et al., 1998),

- Contain the at least one nucleotide sequence coding for at least one active ingredient under the control of a promoter which can be activated in bacteria or in mammalian cells, the activation of the promoter being cell-unspecific, cell-specific, cell cycle-specific, cell function-specific or dependent on Metabolites, drugs or from the ■ oxygen concentration can occur.

8) Bacteria according to one of claims 1) to 7), characterized in that the active ingredient was selected from the group consisting of:

- Antigens of infectious agents such as viruses, bacteria, ycoplasmas, parasites

- Antigens specific for tumors, in particular proteins encoded by oncogenes

- Antibodies, epitope-binding fragments of antibodies and fusion proteins containing at least one epitope-binding fragment of an antibody, directed for example against an antigen on a tumor cell, a lymphocyte such as a T

Lymphocytes or an endothelial cell such as a tumor endothelial cell

- Immunosuppressive cytokines such as IL-10

- Immunostimulating cytokines such as IL-1, IL-2, 11-3 or IL-β - chemokines

- interferons

- growth factors such as G-CSF, GM-CSF, M-CSF, FGF; VEGF or EGF

- Inhibitory proteins for cytokines, chemokines, interferons or growth factors

- Coagulation-active factors

- Fibrinolytically active factors

- hormones and - hormone antagonists

9) Bacterium according to claim 8), characterized in that the active ingredient

- is tumor cytolytic,

- has a pro-inflammatory effect,

inhibits negative regulating immune cells such as by inhibiting CTLA-4, B7-H1 or CD25 or TGFß,

- has an immunosuppressive effect or

- can convert an inactive precursor of a cytotoxic, immunomodulating or immunosuppressive substance into an active ingredient, - inhibits angiogenesis

- promotes angiogenesis

- promotes coagulation

- inhibits coagulation

- dissolves clot formation - has a hormonal effect

- has a circulatory effect

- promotes the growth of connective or support tissue and / or

- promotes wound healing

10) Pharmaceutical composition containing a bacterium according to any one of claims 1) to 9), wherein the bacteria are suspended in an aqueous solution and galenically prepared for injection, or in an ointment or lotion for local administration. 11) Pharmaceutical composition according to claim 10) for administration locally to the skin, into the circulation, into a body cavity, into a tissue, into an organ or orally, rectally or bronchially.

12) Pharmaceutical composition containing bacteria according to one of claims 1) to 9), wherein the bacteria have been introduced into a cell by infection and the cell is galenically prepared for administration to an organism.

13) Cells containing bacteria according to one of claims 1) to 9), selected from a group consisting of macrophages, lymphocytes, granulocytes, connective tissue cells, muscle cells, parenchyma cells, tumor cells and leukemia cells.

14) Use of a bacterium, a pharmaceutical composition or a cell according to one of claims 1) to 13) for the prophylaxis or therapy of a disease, an organism which is exposed to a disease or at risk of a physiologically effective dose of bacteria , pharmaceutical composition or cells.

15) Use according to claim 14) for the prophylaxis or therapy of a tumor disease, an autoimmune disease, a chronic inflammation, an organ transplant, a circulatory disease, a blood clotting disorder, a metabolic disorder, the stomach and intestine, the kidney, the lungs or the wound healing. 16) Bacterial microorganism, in particular according to one of claims 1) to 9), containing a foreign nucleic acid sequence which codes for an active ingredient and is optionally under the control of a regulatory 5 nucleic acid sequence, wherein in the chromosomal DNA of the microorganism is natural and for expression of a bacterial enzyme coding nucleic acid sequence of the bacterium is either deleted or mutated with the proviso that a translation product resulting therefrom is non-functional, and wherein the microorganism does not contain any foreign nucleic acid sequence which codes for the enzyme.

15 17) cell which is infected with a microorganism according to claim 16).

18) Process for the preparation of a microorganism according to

Claim 16), wherein the microorganism by means of a

20 a nucleic acid sequence coding for an active ingredient is transfected, and wherein in the chromosomal DNA of the microorganism a natural nucleic acid sequence coding for the expression of a bacterial enzyme is either deleted or its

25 suppresses transcription, or suppresses the translation of the transcription product, or mutates with the proviso that the translation product is non-functional.

30 19) Method for producing a cell according to claim

17), whereby the cell is infected with the microorganism and cultivated. 0) Use of a microorganism according to claim 16) or a cell according to claim 17) for the production of a pharmaceutical composition for the prophylaxis or treatment of a disease which can be treated by means of the active ingredient.