DE10326189A1 - Bacterial carriers for nucleotide sequences encoding drugs - Google Patents

Abstract

The The invention relates to a bacterial microorganism containing a foreign nucleic acid sequence, which for an active ingredient, wherein in the chromosomal DNA of the microorganism a natural one and for the expression of a bacterial enzyme encoding nucleic acid sequence the bacterium either delektiert or mutated with the proviso, that a resulting translation product is non-functional is. The invention further relates to cells containing such Microorganisms are infected, methods of producing such Organisms and their use, in particular for the production of pharmaceutical compositions.

Description

Field of the invention.

The The invention relates to a bacterial microorganism with foreign DNA, a cell that infects with such a microorganism is, process for producing such organisms, pharmaceutical Compositions containing such organisms, as well as uses of such Organisms.

Background of the invention and state of the art.

Bacteria, such as Shigella flexeri, Salmonella spp., Escherichia coli, Yersinia enterocolitica and Listeria monocytogenes 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 will Bactofection (Shata et al., 2000, Grillot-Courvalin et al. 2002; Al-Mariri et al., 2002; Hense et al., 2001; Powell et al. 1999, US Pat. No. 5,877,159).

bacteria as ferries for DNA vaccines offer the extra Advantage of co-stimulation by their bacterial components.

ever After bacterium, the transfer of genes takes place in the infected Mammalian cell over different Pathways: Shigella and Listeria reach the cytosol of the host cell and release the gene here after lysis. Salmonella, Yersinia and E. coli remain in phagosomes. It is practically unknown, as from there The released DNA penetrates into the cell nucleus.

The Release of the DNA within the infected cell can be spontaneous be made or relieved by antibiotics. In addition, specific Mutants of the bacteria are used, which are characterized by a lighter Characterize disintegration in the host cell (Sizemore et al., 1997; Grillot-Courvalin et al., 2002; Darji et al., 1997; Grillot-Courvalin et al., 1998.

A special form represents the self-destructive L. monocytogenes carrier system (sLM), which is expressed by expression and secretion of heterogeneous, of Listeria bacteriophages derived endolysins upon entry of Listeria into the cytosol the host cell a resolution listeria (Dietrich et al., 1998; Loessner et al., 1995).

one Use of this sLM system as well as all other bacterial Systems as gene shuttles So far, the problems of stability and biosafety have this means the virulence of bacterial carriers opposite.

were For example, sLM transfected with plasmid encoding a desired Protein (vaccine plasmid) mice administered, so lost a considerable number, as a rule more than 30% of sLM their plasmids. To solve these problems was established a new, so-called "Balancedlethal" system (Pilgrim, S., Dissertation University of Würzburg, 2002). This was the deleted essential trpS gene in the chromosome, while at the same time a functional trpS gene copy transferred to the vaccine plasmid has been. This system ensures that only bacteria bearing vaccine plasmids continue to survive.

to Reduction of the virulence of Listeria was reported by Pilgrim (Dissertation university Würzburg 2002) deleted the gene iap. This deletion caused a faulty one Arrangement of the protein ActA on the surface of the Listeria, a defect in the mobility of 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 Listeria, for example, by deletion of genes iap and actA, also the ability the Listeria is impaired, To release plasmid DNA in the cytosol from cells. Thus, it was concluded that ability from Listeria to move within cells and into neighboring ones Invading cells is an important prerequisite for one efficient transfer of plasmid DNA. The deletion of the iap gene thus reducing the virulence of Listeria proved to be unsuitable for the Bactofection.

at Salmonella, E. coli or Pasteurella has been known for some time, that the deletion or negative mutation encoding at least one gene aroA for a Enzyme for the synthesis of an aromatic amino acid to a reduction of Virulence leads (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., Avian Dis. 43: 83-88 (1999)). Such germs could successful for the immunization against the respective germ as well as against an antigen, expressed by a transgene introduced into, 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 gene aroA in Yersinia pestis to reduce the virulence of these germs in the guinea pig, against it in mice the virulence of the mutated germ is largely preserved (Oyston et al., Microbiology 142: 1847-1853 (1996). However, not known is whether such a mutation in Listeria their operability for the Bactofection receives.

Technical problem of Invention.

Of the Invention is thus based on the technical problem of a bacterial carrier for plasmid To create DNA with the following property combination: i) the Virulence of the bacteria is drastically reduced, a strong bactofection is guaranteed and iii) stable expression of the plasmid DNA is made possible.

Broad features of the invention and embodiments.

Of the The invention is based on the finding that the virulence of Bacteria, such as the virulence of sLM, drastically reduced can be, if in their chromosomes genes coding for enzymes be deleted for the synthesis of essential metabolites. Surprisingly, allow such virulence attenuates attenuated bacteria despite attenuation as a carrier of expression plasmids and compared to non-attenuated bacteria an at least equally strong bactofection, when they attenuated Carrier bacteria distribution competent are, that is, maintain the ability have to move within the infected cell and in neighboring cells penetrate.

object The invention thus bacteria are encoding as a carrier at least one nucleotide sequence for at least an active substance, characterized in that in at least one Chromosome of these bacteria deleted at least one gene for a metabolic enzyme or mutated so that the metabolic enzyme is defective.

• – welche verbreitungskompetent sind
• – welche nach Infektion in eine Säugerzelle lytische Proteine in das Cytoplasma der Bakterien exprimieren, wobei diese lytischen Proteine die Bakterien auflösen
• – und welche mindestens eine Nukleotidsequenz kodierend für mindestens einen Wirkstoff innerhalb der Zelle, vorzugsweise im Cytosol der Zelle freigeben

The invention further 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 distribution competent
• - Which after infection into a mammalian cell express lytic proteins into the cytoplasm of the bacteria, whereby these lytic proteins dissolve the bacteria
• - And which at least one nucleotide sequence coding for at least one active ingredient within the cell, preferably in the cytosol of the cell release

• – ein Gen für ein Enzym zur Synthese von aromatischen Aminosäuren im Chromosom deletiert wurde, – beispielsweise das aroA Gen, welches für das erste Enzym in der Biosynthese von aromatischen Aminosäuren kodiert –, sodass diese Bakterien in ihrem Wachstum abhängig sind von der Anwesenheit aromatischer Aminosäuren
• – diejenigen Proteine, welche die Motilität der Bakterien ermöglichen, unbeeinträchtigt exprimiert werden, beispielsweise die Funktionsfähigkeit der Gene iap und actA erhalten ist
• – das Gen trpS kodierend für tryptophanyl-tRNR-synthetase im Chromosom deletiert wurde und dass in diese Bakterien Plasmide eingeführt worden sind,
• – deren Replikation stabilisiert wurde durch einen geeigneten "Replication origin", beispielsweise durch ori pAMß1 (Simon and Chopin, 1988)
• – die das trpS Gen kodierend für tryptophanyl- tRNA-synthetase enthalten
• – die ein Gen für ein Endolysin, beispielsweise das Lysis Gen des Phagen A118 (ply 118; Loessner et al., 1995) unter der Kontrolle eines im Cytosol von Säugerzellen aktivierbaren Promoters, beispielsweise des actA Promoters (PactA, Dietrich et al., 1998) enthalten,
• – die mindestens eine Nukleotidsequenz kodierend für mindestens einen Wirkstoff unter der Kontrolle eines in Bakterien oder in Säugerzellen aktivierbaren Promotors, enthalten, wobei die Aktivierung des Promoters zellunspezifisch, zellspezifisch, zellzyklusspezifisch, zellfunktionsspezifisch oder abhängig von Metaboliten, Arzneimitteln oder von der Sauerstoffkonzentration erfolgen kann.

A preferred example of the bacteria according to the invention are listeria, in particular in these bacteria

• - a gene for an enzyme for the synthesis of aromatic amino acids in the chromosome was 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 the growth of the presence of aromatic amino acids
• - Those proteins which allow the motility of the bacteria are expressed without interference, for example, the functionality of the genes iap and actA is obtained
• The gene trpS coding for tryptophanyl-tRNR synthetase has been deleted in the chromosome and that plasmids have been introduced into these bacteria,
• The replication of which has been stabilized by a suitable "replication origin", for example by ori pAMβ1 (Simon and Chopin, 1988)
• - Containing the trpS gene encoding tryptophanyl tRNA synthetase
• A gene for an endolysin, for example the lysis gene of the phage A118 (ply 118, Loessner et al., 1995) under the control of a mammalian cell-activatable promoter, for example the actA promoter (PactA, Dietrich et al., 1998 ) contain,
• Which contain 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-specific, cell-specific, cell cycle-specific, cell function-specific or dependent on metabolites, drugs or oxygen concentration.

bacteria The genus Listeria are Gram-positive, mostly cocoa and catalase-positive Rod-shaped bacteria. An example of this is Listeria monocytogenes.

Such inventive bacteria by the loss of at least one gene for an essential metabolite protein For example, a drastic reduction in their virulence as measured their in vivo reproducibility and still show a significantly increased bactofection, one Lysis of the bacteria in the cytosol, a release of the bacteria contained plasmids and stable expression of the plasmid encoded drug.

• – Antigene von Infektionserregern wie Viren, Bakterien, Mycoplasmen, Parasiten
• – Antigene spezifisch für Tumore, im Besonderen Proteine kodiert von Oncogenen
• – Antikörper, Epitop-bindende Fragmente von Antikörpern und Fusionsproteine enthaltend mindestens ein Epitop-bindendes Fragment eines Antikörpers, gerichtet beispielsweise gegen ein Antigen auf einer Tumorzelle, einem Lymphozyten wie beispielsweise einem T-Lymphozyten oder einer Endothelzelle wie beispielsweise einer Tumorendothelzelle
• – Enzyme, im besonderen Enzyme zur Aktivierung von inaktiven Vorstufen eines Arzneimittels wie beispielsweise eine β-Glucuronidase, eine Phosphatase, eine Hydrolase, eine Lipase,
• – Immunsuppressive Cytokine wie beispielsweise IL-10
• – Immunstimulierende Cytokine wie beispielsweise IL-1, IL-2, Il-3 oder IL-6
• – Chemokine
• – Interferone
• – Wachstumsfaktoren wie beispielsweise G-CSF, GM-CSF, M-CSF, FGF; VEGF oder EGF oder
• – Inhibitorische Proteine für Cytokine, Chemokine, Interferone oder Wachstumsfaktoren

Active substances according to the invention are proteins, for example selected from a group comprising:

• - Infections of infectious agents such as viruses, bacteria, mycoplasma, 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 egg an endothelial cell, such as a tumor endothelial cell
• Enzymes, in particular enzymes for activating inactive precursors of a medicament, such as a β-glucuronidase, a phosphatase, a hydrolase, a lipase,
• Immunosuppressive cytokines such as IL-10
• - Immunostimulatory cytokines such as IL-1, IL-2, IL-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

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

• – tumorzytolytisch ist,
• – proinflammatorisch wirkt,
• – negativ regulierende Immunzellen inhibiert wie beispielsweise durch Inhibition von CTLR-4, von B7-H1 oder von CD25 oder von TGFß,
• – immunsuppressiv wirkt oder
• – eine inaktive Vorstufe einer zytotoxischen, immunmodulierenden oder immunsuppressiven Substanz in einen aktiven Wirkstoff verwandeln kann.
• – die Angiogenese hemmt
• – die Angiogenese fördert
• – die Gerinnung fördert
• – die Gerinnung hemmt
• – eine Gerinnselbildung auflöst
• – eine Hormonwirkung aufweist
• – eine Kreislaufwirkung aufweist
• – das Wachstum von Binde- oder Stützgewebe fördert
• – die Wundheilung fördert

Preferably, the bacteria of the invention are used to treat a tumor disease or an immune disease. For this purpose, the inserted into the bacteria gene encodes a protein which

• Is tumor cytolytic,
• - acts proinflammatory,
• Inhibits negatively regulating immune cells, for example by inhibiting CTLR-4, B7-H1 or CD25 or TGFβ,
• - immunosuppressive or
• - Can transform an inactive precursor of a cytotoxic, immunomodulatory or immunosuppressive substance into an active ingredient.
• - inhibits angiogenesis
• - promotes angiogenesis
• - promotes coagulation
• - inhibits coagulation
• - Clot formation dissolves
• - has a hormone effect
• - Has a circulatory effect
• - promotes the growth of connective or supporting tissue
• - promotes wound healing

to Prevention or treatment of a disease will be 100 to 10 ^ 10 bacteria administered. The administration of the bacteria is preferably carried out as a suspension in a pharmaceutically prepared aqueous solution for one Injection or in a pharmaceutically prepared ointment or lotion for one local administration. Such pharmaceutical preparations are known to the skilled person.

The Bacteria are preferably localized in a suitable preparation on the skin, in the circulation, in a body cavity, in a tissue, in an organ orally, rectally or bronchially at least once.

object Furthermore, the invention is the infection of a cell, for example a macrophage, lymphocytes, granulocytes, a connective tissue cell, a muscle cell, a tumor cell or leukemia cell ex vivo with a the bacterium of the invention and injecting the thus infected cell into an organism Prophylaxis or therapy of a disease.

diseases in which the bacteria of the invention can be used, for example, tumors, autoimmune diseases, chronic inflammation, Organ transplants, circulatory diseases, blood clotting disorders, of the metabolism, the stomach and intestines, the kidney, the lungs and the wound healing.

in the The invention will be explained in more detail below with reference to exemplary embodiments.

Example 1: Materials and methods.

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

Example 2: Preparation of virulence-attenuated suicide-listeria (aroA) with stably replicating plasmids

There L. monocytogenes a facultative animal and human pathogenic species has been used to develop these listeria as a carrier for plasmid DNA coding for Pharmaceutical agents according to this Invention related to introduced to their virulent. In addition, the trpS deletion mutant was identified in the genome (WL-140, Pilgrim, Dissertation University of Würzburg 2002) additionally the aroA gene deleted. This gene encodes the first enzyme in biosynthesis of aromatic amino acids, so that these bacteria depend on the presence of aromatic in their growth Amino acids are.

The aroA / trpS (WL-141) double mutant was obtained using the deletion mutagenesis technique based on the WL-140 strain and the oligonucleotides aroA-A1,2 and aroA-B1,2 (Table 1).

to Construction of an aroA mutagenesis plasmid became the deletion region 5'-flanking Fragment A (365 bp) with the oligonucleotides aroA-A1 and aroA-A2 amplified while using the oligonucleotides aroA-B1 and aroA-B2, a 3'-flanking fragment B (438 bp) was created. The sequence of both fragments was from the in the "Listeria monocytogenes genome sequencing project "sequenced Strain L. monocytogenes EGD-e (Glaser et al., 2001). Both Fragments were over ligated a PspAI site to Fragment AB, this with help the oligonucleotides aroA-A1 and aroA-B2 again in a PCR reaction amplified and over EcoRI and BamHI sites inserted into the plasmid pLSVl. After transformation of the aroA mutagenesis plasmid pLSV1-aroA-AB into the L. monocytogenes Strain WL-140, intermediate breeding at 30 ° C and subsequent cultivation BHI agar plates with erythromycin at 43 ° C were able to prepare some clones which had inserted the plasmid chromosomally. Of these a clone was selected which is a homologous recombination with the plasmid via the Fragment aroA-A had completed. Subsequently, after 20 passages at 37 ° C the aroA, trpS deletion mutant (trpS, aroA / pTRPS) can be isolated. This newly-constructed strain was named WL-141.

Example 3: Bactofection experiments "in vitro".

The Listeria strains WL-140 and WL-141 with the plasmids pSP0-EGFP or pSP118-EGFP (Tab. 2), which is the gene for the reinforced 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 tested (Table 3). While most EGFP-expressing Cells detected in CaCo-2 and COS-1 cell cultures after infection with WL-141 / pSP118-EGFP were able to get the best results in HeLa and HepG2 cell cultures achieved with WL-140 / pSP118-EGFP. That means the trunk WL-141, similar like WL-140, very good for In vitro gene transfer experiments is suitable.

Example 4: In vivo virulence experiments.

The WL-140 and WL141 strains were in the mouse infection model (BALB / c) on their infection efficiency and virulence tested. In these experiments became a similar one Number of bacteria after infection with WT and WL140 / pSP0 in spleen and Liver (Table 4). In contrast, the strain WL141 / pSP0, however in its dissemination in the same organs dramatically attenuated (Tab. 4) and leads thus only a very mild infection of the host.

Example 5: The Bactofection Efficiency is particularly dependent from the spreading of the bacteria (spreading attempts)

Surprisingly was found in the invention that the actA negative Strain WL-142 (spreading incompetent) a low bactofection rate after microinjection into Caco-2 cells (Table 5). On the other hand is the bactofection with actA-positive strains WL-140 / pSP0-EGFP or pSP118-EGFP (distribution competent) much more efficient, especially for a later Time (48-72h, Table 6). The explanation for this is that through the spread in multiple cells, the spread-competent bacteria a much more efficient Enable bactofection (Tab. 7). Thus it was concluded that the dissemination property of Bacteria an important factor for represents an efficient plasmid DNA release.

Literature:

• Al-Mariri, A. et al., Infect. Immune. 70, 1915-1923 (2002).
• Darji, A. et al., Cell 91, 765-775 (1997).
• Dietrich, G. et al., Nat. Biotechnol. 16, 181-185 (1998).
• Glaser, P. et al., Science 294, 849-852 (2001).
• Goetz, M. et al., Proc. Natl. Acad. Sci. USA 9, 12221-12226 (2001).
• Grillot-Courvalin, C., Goussard, S., & Courvalin, P., Cell. Microbiol. 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., Doctoral 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., US Pat. 5,877,159.
• Shata, M.T., Stevceva, L., Agwale, S., Lewis, G.K., & Hone, D.M., Mol. Med. Today 6, 66-71 (2000).
• Simon, D., & Chopin, A., Biochimie 70, 559-66 (1988) Sizemore, D.L., Branstrom, A.A., & Sadoff, J.C., Vaccine 15, 804-807 (1997).
• Strizker, J. H. "Investigations to improve DNA transmission in animal cells by Listeria monocytogenes ", diploma thesis, University Wuerzburg, 2001
• Wuenscher, M.D., Kohler, S., Goebel, W., & Chakraborty T, Mol. Genet. 228, 177-82 (1991)

It follows a sequence listing according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA become.

Claims (20)

Bacteria, in particular of the genus Listeria, as carrier at least one nucleotide sequence coding for at least one active substance, characterized in that in at least one chromosome of this Bacteria at least one gene for a metabolic enzyme is deleted or mutated so that the metabolic enzyme is defective. Bacteria according to claim 1), characterized that in them a gene for an enzyme for the synthesis of aromatic amino acids has been deleted. Bacteria according to claim 2), characterized that the aroA gene was deleted. Bacteria according to one of Claims 1) to 3), characterized that they are distribution competent. Bacteria according to one of Claims 1) to 4), characterized that they are lytic proteins after infection in a mammalian cell into the cytoplasm of the bacteria expressing these lytic Proteins that dissolve bacteria and thereby encoding the bacteria at least one nucleotide sequence for at least an active substance within the cell, preferably in the cytosol of Release cell. Bacteria according to any one of claims 1) to 5), characterized that they represent Listeria. Bacteria according to any one of claims 1) to 6), characterized that in them - the aroA gene, which is for the first enzyme in the biosynthesis of aromatic amino acids encodes, deleted so that these bacteria are dependent on their growth from the presence of aromatic amino acids - those proteins which the motility allow the bacteria unimpaired For example, the operability of the genes iap and actA is obtained - the Gene trpS coding for tryptophanyl-tRNA synthetase was deleted in the chromosome and that in these bacteria plasmids introduced have been, - whose Replication was stabilized by a suitable "Replication origin", for example by ori pAMß1 (Simon and Chopin, 1988) - the encoding the trpS gene for tryptophanyl-tRNA synthetase contain - the a gene for an endolysin, for example the lysis gene of phage A118 (ply 118; Loessner et al., 1995) under the control of one in the cytosol of mammalian cells activatable promoter, for example the actA promoter (PactA, Dietrich et al., 1998), - The at least one nucleotide sequence coding for at least one active ingredient under the control of one in bacteria or in mammalian cells activatable promoter, containing the activation of the promoter cell-unspecific, cell-specific, cell-cycle-specific, cell function-specific or dependent of metabolites, medicines or oxygen concentration can be done. Bacteria according to one of claims 1) to 7), characterized that the active ingredient is selected was made up of the group: - Antigens of infectious agents like viruses, bacteria, mycoplasma, parasites - Antigens specific for Tumors, in particular proteins encoded by oncogenes - Antibody, epitope-binding Fragments of antibodies and fusion proteins containing at least one epitope-binding fragment of an antibody for example, 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 the activation of inactive precursors of a Medicinal product such as a β-glucuronidase, a phosphatase, a hydrolase, a lipase, - Immunosuppressive cytokines such as IL-10 - Immune stimulating Cytokines such as IL-1, IL-2, IL-3 or IL-6 - 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 - Clotting active factors - Fibrinolytic active factors - Hormones and - Hormone antagonists Bacterium according to claim 8), characterized in that the active ingredient is - tumor cytolytic, - acts proinflammatory, - inhibits negatively regulating immune cells such as by inhibition of CTLA-4, B7-H1 or CD25 or TGFß, - acts immunosuppressive or - can transform an inactive precursor of a cytotoxic, immunomodulatory or immunosuppressive substance into an active agent, - inhibit angiogenesis - promote angiogenesis - promote coagulation - inhibit coagulation - dissolves clot formation - has a hormonal effect - has a cardiovascular effect - promotes the growth of connective or supporting tissue and / or - promotes wound healing Pharmaceutical composition containing a bacterium according to one of the claims 1) to 9), wherein the bacteria suspended in an aqueous solution and galenic for injection, or in an ointment or lotion for local administration galenically prepared. Pharmaceutical composition according to claim 10) for the Local administration on the skin, in the circulation, in a body cavity, in a tissue, in an organ or peroral, rectal or bronchial. Pharmaceutical composition containing bacteria according to one of the claims 1) to 9), whereby the bacteria have been introduced into a cell by infection and the cell are galenic for administration to an organism is prepared. Cells containing bacteria according to any one of claims 1) to 9) from a group consisting of macrophages, lymphocytes, granulocytes, Connective tissue cells, muscle cells, parenchymal cells, tumor cells and Leukemia cells. Use of a bacterium, a pharmaceutical A composition or a cell according to any one of claims 1) to 13) for the prophylaxis or therapy of a disease, wherein an organism, who is at risk or sick of the disease, a physiologically effective dose of bacteria, pharmaceutical Composition or cells is presented. Use according to claim 14) for prophylaxis or therapy a tumor disease, an autoimmune disease, a chronic Inflammation, an organ transplant, a circulatory disease, a disease blood clotting, a disease of the metabolism, the stomach and bowel, kidney, lung or wound healing. Bacterial microorganism, especially after one of the claims 1) to 9) containing a foreign nucleic acid sequence which is for a Active substance coded and optionally under the control of a regulatory nucleic acid sequence wherein in the chromosomal DNA of the microorganism is a natural and for the Expression of a bacterial enzyme-encoding nucleic acid sequence the bacterium either deleted or mutated with the proviso is that a resulting translation product is non-functional and wherein the microorganism is not a foreign nucleic acid sequence contains which for encodes the enzyme. Cell, which with a microorganism according to claim 16) is infected. A method for producing a microorganism after Claim 16), wherein the microorganism by means of a for a Active substance-encoding nucleic acid sequence is transfected, and wherein in the chromosomal DNA of the microorganism a natural one and for the expression of a bacterial enzyme encoding nucleic acid sequence either deleted, or their transcription suppressed, or the Suppressed translation of the transcription product, or mutated with the proviso is that the translation product is non-functional. A method of manufacturing a cell according to claim 17), whereby the cell is infected with the microorganism and cultured becomes. Use of a microorganism according to claim 16) or a cell according to claim 17) for the preparation of a pharmaceutical A composition for the prophylaxis or treatment of a disease, which is treatable by means of the active ingredient.