MXPA98005835A - Cells genetically modified and their utilization in prophylaxy or therapy of the enfermeda - Google Patents

Abstract

The invention relates to cells for application in gene therapy which are obtainable by a) isolation of cells from blood or bodily fluids containing cells b) cultivation of cells obtained in step a) in a medium of culture containing gangliosides, phospholipids, glycolipids and / or growth factors, c) optionally, immortalization of the cells obtained in steps a) or b) by transformation with an oncogene, activation of an oncogene or inactivation of a suppressor gene; transfection of the cells obtained in steps a) and b) or in step c) with an artificial nucleic acid structure for gene therapy, containing an effector gene, which can be activated by appropriate promoter systems in a specific manner for target cells, specifically for cell cycles, specifically for viruses and / or hypoxia, and the use of these cells for the preparation of a medicament or intended for the treatment of a disease that is selected from the group consisting of tumors, leukemias, autoimmune diseases, allergies, arthritis, inflammations, rejection of organs, transplant reactions against host, blood coagulation diseases, diseases of the circulation , anemia, infections, hormonal diseases and nervous system injuries centr

Description

Genetically modified cells and their use in the prophylaxis or therapy of diseases The present invention relates to cells for application in gene therapy, obtainable by a) isolation of mononuclear cells from blood or body fluids containing cells, -b) cultivation of the cells obtained in step a) in a cell culture medium, containing gangliosides, phospholipids, glycolipids and / or growth factors for endothelial cells, including factors that influence the characteristics of differentiation, survival, migration and / or vascularization; (c) optionally, immortalization of the cells obtained in step a) or b) by transformation with an oncogene, activation of an oncogene or inactivation of a suppressor gene; and d) optionally, transfecting the cells obtained in steps a) and b) or in step c) with an artificial nucleic acid structure for gene therapy, containing an effector gene, which can be activated by appropriate promoter systems of specific mode for target cells, specifically for cell cycles, specifically for viruses and / or hypoxia. 1. Introduction The administration of somatic cells transfected in vi tro, transduced for the expression of an active substance, is a method of gene therapy, which is currently broadly propagated at both preclinical and clinical scales. In this case, cells of various types are used, including fibroblasts, lymphocytes, keratinocytes and tumor cells. The endothelial cells began to be applied for the first time in 1989 for this purpose. To do this, the endothelial cells were transfected in vitro with the aid of a retroviral vector (Zwiebel et al., Science 243: 220 (1989)) for the expression of an active substance. "'The endothelial cells transduced in such a way, which had grown in vitro together with prostheses of blood vessels made of a synthetic material, were in a position, after an in vivo transplantation of these prostheses, to express the transgene (Zwiebel et al. Science 243: 220 (1989), Wilson et al., Science 244: 1, 344 (1989)) Accordingly, it was proposed by Zwiebel and Wilson to administer to patients transduced endothelial cells, adhered to a synthetic material or a collagen support. This proposal was carried out on an experimental scale by Nathan and colleagues (PNAS USA 92: 8.130 (1995).) As an extension of this proposal, it was described by Nabel et al. (Science 244: 1.342). (1989)) for the first time the possibility of administering a cell suspension of transduced endothelial cells into the bloodstream as a pathway of gene therapy. The authors could demonstrate that endothelial cells, which had been obtained by curettage from vessels of a living mammal and had been transduced in vitro for the expression of a reporter gene after local administration, p. ex. inside blood vessels with a lesion in endothelial cells, they grow there and express the reporter gene. By virtue of these results, the authors describe the possibility of administering genetically modified endothelial cells in order to effect gene therapy, active substances being delivered by these endothelial cells directly to the blood circulation in order to effect the therapy of systemic diseases or hereditary This idea was perfected by Bernstein et al. (FASEB J. 4: 2, 665 (1990)). Pulmonary endothelial cells were transfected in vitro for the expression of an active substance with plasmids and then injected to mice devoid of immunity intraperitoneally, intravenously, subcutaneously or under the renal capsules. In animals treated in this way, the active substance produced by transplants with endothelial cells was detected locally (in cysts of the kidneys) or in the blood (after application intraperitoneally [ip], intravenously [iv] or subcutaneously [sc]) which demonstrated the utility of in vivo administration of endothelial cells transduced in vi tro, for gene therapy. Next, Zwiebel et al., 1992 (PCT international patent application with publication number WO) 93/13807) and Ojeifo et al. (Cancer. Res. 55: 2.240 (1995)) demonstrated in several examples the possibility of employing the method of administering endothelial cells transduced in vi tro into the bloodstream in order to effect gene therapy. Zwiebel et al. (1992) transduced human endothelial cells from the umbilical cord and endothelial cells from the fatty tissue of rats with u? retroviral vector for the expression of an active substance and injected these endothelial cells intravenously to animals, in which by injection of irradiated cells, which secreted FGF, a local vascular lesion and angiogenesis had previously been generated. These authors were able to demonstrate that the injected endothelial cells are located at the site of vascular injury and angiogenesis and express the active substance there. In view of this background, the authors claimed in their patent application the use of transduced endothelial cells in vitro for the expression of adenosine deaminase, blood coagulation factors, hematopoietic growth factors, cytokines, antithrombotic agents as well as inhibitors of enzymes and hormones. Parallel to these works, the technique for the isolation of endothelial cells was improved by other authors and the migration behavior of transduced endothelial cells in vi tro was also studied in more detail. Thus, Messina et al. (PNAS USA 89: 12. 018 (1992)) were able to demonstrate that endothelial cells transfected in vi tro, after injection into the circulation, can both adhere to the intact endothelial cell layer and also integrate within this. An exclusive localization of endothelial cells administered intramuscularly to areas with vascular lesions and angiogenesis could therefore be excluded with these results. On the other hand, it could be demonstrated that endothelial cells transfected in vitro and injected in a mixture with tumor cells participate in the angiogenesis of the bed of tumor vessels (Lal et al., PNAS USA 91: 9.695 (1994), Nam et al., Brain Ree 731: 161 (1996)). Conditioned therefor, the tumor cells, transplanted in admixture with endothelial cells, present in vivo a clear growth advantage (Stqpeck et al., Proc. Am.Aseoc.Cancer Ree. 38: 265 (1997)). On the other hand, transduced endothelial cells can be administered locally p. ex. to the brain or to a brain tumor, they can be pharmacologically active or antitumor effective by expression of the active substance encoded by the transgene (Nam et al Brain Res. 731: 161 (1996), Quiñonero et al., Gene Ther. 4: Ill (1997 )). For example, Robertson et al. (Proc. Am. Assoc. Cancer Res. 38: 382 (1997)) applied human endothelial cells (HUVEC), transduced them in vitro with an AV vector for the expression of HSV-TK, in a mixture. with human cells of ovarian carcinoma. After the administration of Ganciclovir, which is activated in the tumor by HSV-TK to form a cytostatic agent, these authors could observe a manifest regression of the tumor in mice devoid of immunity. The use of endothelial cells as cellular carriers of transgenes in gene therapy has been considerably limited up to now however by two essential groups of problems: Obtaining appropriate endothelial cells in sufficient numbers has been shown up to the present time as extremely difficult. Allogeneic endothelial cells can certainly be obtained relatively easily from the umbilical cord or from cell cultures, but because of their immunogenicity in the receptor they are usable only to a limited extent, and on the other hand their reproduction in the cell culture is possible only to a limited extent. Certainly, autologous endothelial cells can be obtained for example by mechanical means by "curettage" of varicose veins or fatty tissue. This method of obtaining, however, is not possible in all patients and also means considerable harm to the patient. Therefore, angioblasts or endothelial cell precursor cells were alternatively obtained from peripheral blood (Asahara and collaborator, Science 75: 964 (1997)). The extraction of blood, which is needed for this, is certainly not very burdensome for patients, but it is very expensive to isolate hypothetical angioblasts from mononuclear blood cells and differentiate these angioblasts to form endothelial cells. Thus, from blood leukocytes (isolated with the help of density gradient centrifugation) the mononuclear blood cells CD34 + or Flk-1 +, which are present in the blood in only small concentration (= 0.1%), they are enriched by immunoadsorption in monoclonal antibodies bound to supports (specifically for CD34 or Flk-1). Next, these cells, coated in tissue trays with collagen type 1 or fibronectin, are incubated for approximately 4 weeks in a culture medium containing bovine brain for differentiation into the form of endothelial cells as well as for their reproduction. However, the reproduction of these cells is possible only in a limited way. In addition to this, the incubation of endothelial cells with brain substance, p. ex. of bovine brain, poses considerable safety problems. The migration of the endothelial cells and the selective expression of the transgene in the desired target region can not be controlled in a sufficient way. After intravascular administration of the endothelial cells, these are localized (as already described above) both in regions of angiogenesis as well as to and within the resting layer of endothelial cells. Furthermore, it is not clear whether the endothelial cells, which have been formed in the cell culture from precursor cells, can be retrodifferentiated in vivo after an injection again to form precursor cells and can be distributed throughout the organism. With the present invention both essential groups of problems have finally been solved. 2) General description of the invention A) The invention consists of: • r i) In an improved, ie simple and safe, method for isolating and culturing mononuclear cells, especially endothelial cell precursor cells, from blood and other body fluids containing cells, and for using these cells in order to effect the prophylaxis or therapy of a disease. 2) Optionally, in a transformation of these cells, specific for cells, especially for endothelial cells, possibly pharmacologically controllable, so that with the help of cell culture can easily obtain high amounts of such cells. 3) In the production of cells, especially endothelial cells, as vectors for effector genes, such that in these cells prepared according to 1), or with 1) and 2), at least one effector gene is inserted, which by the choice of appropriate promoter systems it is expressed specifically for cells, especially for endothelial cells and possibly for hypoxia, specifically for a cell cycle and / or specifically for viruses. . • 4) In the administration of these cells, especially endothelial cells, genetically modified, thus obtained, for the prophylaxis or therapy of a disease.

B) Cells are therefore an object of the invention for their application in gene therapy, obtainable by a) isolation of mononuclear cells from blood or body fluids containing cells, b) cultivation of the cells obtained in step a) in a cell culture medium containing gangliosides, phospholipids, glycolipids and / or growth factors, growth factors for endothelial cells including factors influencing differentiation, survival , migration and / or vascularization; c) optionally, immortalization of the cells obtained in step a) or b) by transformation with an oncogene, activation of an oncogene or inactivation of a suppressor gene; d) transfection of the cells obtained in steps a) and b), or in step c), with an artificial structure (construction) of nucleic acid for gene therapy, containing an effector gene, which can be activated by appropriate promoter systems specifically for target cells, specifically for cell cycles, specifically for viruses and / or hypoxia .

Other objects of the invention, embodiments of the invention, as well as corresponding Examples, are described below. 3) Production of endothelial cells 3. 1) Isolation and cultivation of endothelial cell precursor cells The method according to the invention for the isolation of endothelial cell precursor cells has to be broken down into the following sections: Body fluids containing cells are extracted from the respective organs with invasive procedures known to a person skilled in the art. These body fluids containing cells belong, for example, blood obtained from veins, capillaries, arteries or umbilical cord and respectively placenta - suspensions of bone marrow cells suspensions of spleen cells (splenic) suspensions of ganglion cells lymphatic suspensions of peritoneal cells suspensions of pleural cells - liquid lymph of conjunctive tissue (which e.g. exits on the surface of a superficially injured epidermis, eg mechanically).

The erythrocytes, granulocytes and other cellular components are separated from these body fluids by density gradient centrifugation, and the thrombocytes are separated by differential centrifugation correspondingly to methods known to a person skilled in the art. The mononuclear cells (which contain nuclei thus isolated, are suspended in a cell culture medium containing serum.) The related cell culture medium contains the gangliosides, phospholipids and / or growth factors that are discussed later in this specification. In a special embodiment of this invention, isolated mononuclear cells (containing nuclei) are cultured in this cell culture medium and are differentiated to form endothelial cell-like cells, in another special embodiment of this invention, the mononuclear cells containing isolated nuclei are incubated with an antibody against surface markers typical for monocytes / macrophages (CDll, CDllb, CD13, CD14, CD34, CD64 and CD68) which is coupled to iron particles or iron oxides, coated with a polysaccharide, are incubated and washed, and then the cells thus loaded are obtained with the help of a The cells are added to a cell culture medium, which contains the gangliosides, phospholipids and / or growth factors which are discussed below, and are further reproduced in vitro and differentiated to form endothelial cells. Immortalization and / or transfection are carried out in vitro after reproduction and / or differentiation of the cells.

In another embodiment of this invention, isolated nuclei-containing cells are pre-incubated in said cell culture medium for more than 1 hour for further differentiation and reproduction. Under these conditions, cells recognized as endothelial cell precursor cells increasingly develop surface markers typical of monocytes / macrophages (CDll, CDllb, CD13, CD14, CD34, CD64, CD68). These cells are then isolated, for example they are obtained with the aid of a magnet with an antibody which is directed against these markers for monocytes (eg CDll, CD14) and is coupled to iron particles coated with dextran. The cells are reproduced in vitro additionally and differentiated to form endothelial cells. Alternatively to it, from non-adherent mononuclear cells, as described for example by Asahara et al., Science 275, 964 (1997), CD34-positive cells are isolated (hematopoietic cells) and subsequently reproduced in vi tro and differentiated to form endothelial cells. In a special embodiment of the invention, the cells containing isolated nuclei are suspended in a cell culture medium and the remaining phagocytic cells (eg monocytes, macrophages and granulocytes) are removed by adhesion to the surface or by phagocytosis of iron particles coated with dextran and loaded with proteins, with the aid of a magnet and / or by countercurrent centrifugation, in a manner corresponding to the procedures known to a person skilled in the art, and the remaining mononuclear cells, which contain CD34-positive cells, are cultured in the cell culture medium according to the invention and differentiated to form endothelial cell-like cells. The cell culture vessels can be coated with an extracellular matrix component (see Attachment and Matrix Factors, eg from the Sigma entity) such as for example fibronectin. To the cell culture medium are added either gangliosides, phospholipids and / or glycolipids and / or, however, preferably according to this invention, growth factors for endothelial cells are added, for example a vascular endothelial growth factor (VEGF) and / or other KDR or FLt ligands such as a fibroblast growth factor (FGFar, FGFJ) and / or an epidermal growth factor (EGF) and / or a similar growth factor to insulin (IGF-1, IGF-2) and / or a β-endothelial cell growth factor (EGCF) and / or an endothelial cell binding factor (ECAF) and /? interleukin-3 (IL-3) and / or GM-CSF and / or G-CSF and / or -interleukin-4 (11-4) and / or interleukin-1 (11-1) and / or a factor of colony stimulation (CSF-1) and / or interleukin-8 (11-8) and / or a platelet-derived growth factor (PDGF) and / or IFN? and / or oncostatin M and / or LIF and / or B61 and / or - a platelet-derived endothelial cell growth factor (PDEGF) and / or a stem cell factor (SCF) and / or transforming growth factor- jS (TGF-ß) and / or. angiogenin and / or - pleiotropin and / or Flt-3 (FL) ligand and / or Fie-2 ligands, such as p. ex. angiopoietin-1 and / or a stromal-derived factor (SDF-1) and / or midquins.

The adult cells, after a period of time selected between 6 hours and 8 weeks, are further treated according to the invention. The endothelial cells thus isolated can also be used directly for the promotion of the endothelialization of injured vessels and for the promotion of angiogenesis. 3. 2) Endothelial cell isolation Alternatively to the method according to the invention, set forth in paragraph 3.1.), Endothelial cells can be obtained, however, also by methods known to a person skilled in the art, for example from fatty tissue, by curettage of veins or by detachment from the umbilical cord endothelium. Its cultivation is carried out as already described in paragraph 3.1.) 4) Immortalization of endothelial cells Correspondingly to this invention, in one or several non-adherent mononuclear endothelial cells or cells a nucleotide sequence (component a) can be introduced for a protein, which results in these cells progressively traversing the cell division cycle and consequently become in a cell line that is "permanently" divided and does not age. Such nucleotide sequences or genes that are immortalized, are already known. The oncogenes belong, for example, to these nucleotide sequences. Correspondingly to this invention, the oncogenes may be of cellular or viral origin. Examples of cellular oncogenes have already been described summarily by Wynford-Thomas, J. Pathol. 165: 187 (1991); Harrington et al., Curr. Opin. Genet Developm. 4: 120 (1994); Gonos et al., Anticancer Res. 13: 1117 (1993) and Baserga et al., Cancer Surveye 16: 201 (1993). Such oncogenes can be introduced into the cell with methods known to a person skilled in the art. However, the cells also carry proto-oncogenes in their genome, which according to this invention can be activated in the cell by methods known to a person skilled in the art, ie they can be transformed into oncogenes. In a special embodiment of this invention, component a) constitutes a nucleotide sequence, which encodes a protein, which inactivates the protein of a suppressor gene. Examples of suppressor genes have already been described comprehensively by Carp and Broder, Nature Med. 4: 309 (1995); Skuee and Ludlow, The Lancet 345: 902 (1995); Duan et al., Science 269: 1 .402 (1995); Hugh et al., Cancer Ree. 55: 2225 (1995); Knudeon, PNAS USA 90: 10. 914 (1993)). Example of a gene (component a), which encodes a protein, which inactivates the product of expression of a suppressor gene: Table 1 Protein of the suppressor gene according to the invention (component a) which encodes: Retinoblastoma protein Adenovirus E1A protein (Rb protein) and proteins (Wythe et al., Nature 334: 124 (1988)) related, such as the large T antigen of the SV40 virus p107 and p130 (De Caprio et al., Cell 54: 275 (1988)) the E7 protein of the papillomavirus (eg HPv-16 and HPV-18) (Dyson et al. Science 243: 934, (1989)) a protein containing the amino acid sequence LXDXLLXXL-II-LXCXEXXXXXSDDE (SEQ ID NO: 1), in which X represents a variable amino acid and -II- an arbitrary chain of amino acids with -80 amino acids (selected) - among the 20 natural amino acids that are presented in translation products; Münger et al; Cancer Surveys 12: 197 (1992)) p53 The E1 B protein of the adenovirus (Sarnow et al., Cell 28: 287 (1982)) The large T antigen of the SV-40 virus (Lane et al., Nature 278: 261 (1979)) ) An E6 protein from papillomavirus r - (eg, HPV-16, HPV-18) (Werpess et al., Science 248: 76 (1990) Scheffner et al., Cell 63: 1129 (1990)) MDM protein (Momand et al., Cell 69: 1237 (1992), Oliner et al., Nature 362: 857 (1993) Kussie et al., Science 274: 948 (1996)) In another special embodiment of this invention, component a) it constitutes a mutated nucleotide sequence for a cell cycle regulation protein, which has been modified by the mutation in such a way that, certainly, it can still fully activate the cell cycle, but in this function it can no longer be inhibited by inhibitors cell phones. In the sense of this invention, mutated sequences of nucleotides encoding cyclin-dependent kinases belong to it, which, despite the mutation, retain their activity as kinases, but have lost the ability to bind to cellular kappa inhibitors. Examples of component a) are: cdk-4, mutated in such a way that they can no longer inhibit pl6, pl5 and / or p2l cdk-6, mutated in such a way that they can no longer inhibit pl5 and / or pl8 cdk-2 , mutated in such a way that they can no longer inhibit p21 and / or p27 and / or WAF-1. For example, the mutation of cdk4 can constitute the exchange of an arginine with a cysteine at position 24, such that this mutated cdk has kinase activity, but can already be inhibited by pl5 and pl6 (Wolfel et al., Science 269: 1281 (1995)). In another embodiment of this invention, component a) constitutes a transforming gene, the expression of which is regulated by a self-reinforcing promoter element, optionally in combination with a pharmacologically controllable promoter (see, for example, paragraph 6.4). In another special embodiment of this invention, they are introduced into endothelial cells, but especially also into endothelial cell precursor cells or cells of a mixture of cells, which contains a certain proportion of endothelial cells or endothelial cell precursor cells. or a certain proportion, increased in comparison to blood, of cells positive for CD34, CDll, CDllb, CD14, CD13, CD64 and CD68, whose sequence consists of a promoter or enhancer sequence, which is specific for endothelial cells (component b). ), and of component a), the transcription of component a) being activated by binding of transcription factors of the endothelial cells to component b). For the best permanence of the expression product of component a) in the nucleus of the cells, component a) can be added with a nuclear localization signal (co by c). The arrangement of the compone which results from them, is represented by way of example in Figure 1. By introduction of an artificial nucleic acid structure, which contains the componeshown in Figure 1, only endothelial cells are transformed and endothelial cell precursor cells contained in a heterogeneous cell mixture in an immortalized phase, that is to say in permanently dividing cells, so that after a few days these endothelial cells dominate proportionally in the cell culture and after a dependent period of time of cultivation conditions, but estimable, are present exclusively in cell culture. With these artificial nucleic acid structures and processes according to the invention, in a relatively short period of time and with little expenditure, also from few endothelial cells, or from a few endothelial cell precursor cells, also when these are present in a heterogeneous mixture of cells, large quantities of uniform endothelial cells can be produced for prophylaxis or therapy.

) Production of endothelial cells modified by genes for prophylaxis and / or therapy Corresponding to the invention, in the endothelial cells obtained according to one of the methods according to the invention, an artificial nucleic acid structure containing at least the following components must be introduced: a promoter (component d) a structural gene coding for an active substance or an enzyme (component e). 6) Selection of promoter sequences In the sense of the invention, nucleotide sequences must be used as promoter sequences, which after the binding of transcription factors activate the transcription of a transgene located contiguously to the 3 'end, such as, for example, a structural gene. In the sense of the invention, at least one promoter sequence that is specific for endothelial cells (component b) and / or component d) is introduced into the endothelial cell. This promoter sequence that is specific for endothelial cells can be combined with at least one other promoter sequence. The choice of the promoter sequence (s) that has to be combined with the endothelial cell-specific promoter is targeted to the disease to be treated. Thus, the additional promoter sequence can be induced unlimitedly, specifically for endothelial cells, under certain metabolic conditions, such as for example by hypoxia or it can be induced or disconnected by a drug, and can be activated in a specific manner for viruses and / or in a specific way for cell cycles. Such promoters have already been mentioned in the European patent applications EP-95931204.2; EP95930524.4; EP95931205.9; EP95931933.6; EP96110962.2; German DE19704301.1; European EP97101507.8; EP97102547.3; German DE19710643.9; and European EP 97110995.8. These patent applications are referred to herein. The sequences of promoters that can be selected belong, for example, 6. 1) Promoters and promoter sequences unlimitedly activatable, such as, for example, the RNA polymerase III promoter, the polymerase II AR promoter? the CMV promoter and enhancer - the SV40 promoter. 6. 2) Sequences of metabolically activatable promoters and enhancers such as, for example, the hypoxia-inducible enhancer (Semenza et al., PNAS 88: 5, 680 (1991), McBurney and collaborator, Nuci, Acide, Ree. 19: 5, 755 (1991)). 6. 3) Promoters that can be activated specifically for cell cycles.

These are for example the promoter of the cdc25B gene, the cdc25C gene, the cyclin A gene, the cdc2 gene, the B-myb gene, the DHFR gene, the E2F-1 gene, or binding for transcription factors that appear or activate during cell proliferation. These binding sequences include, for example, binding sequences for c-myc proteins. Among these binding sequences, monomers or multimers of the nucleotide sequence designated as Myc E box (5'-GGAAGCAGACCACGTGGTCTGCTTCC-3 '(SEQ ID? O .: 2); Blackwood and Eisenmann, Science 251: 1.211 (1991 )). 6. 4) Self-reinforcing and / or pharmacologically controllable promoters In the simplest case, when combining the same or different promoters a promoter can be inducible, for example in the form of a promoter activable or disconnectable by tetracycline in the form of the tetracycline operator, in combination with a corresponding repressor.

Corresponding to the invention, the promoter can also, however, also be self-reinforcing with, or also without, a pharmacologically controllable promoter unit. Such self-reinforcing and / or pharmacologically controllable promoters have already been described in patent application DE19651443.6, to which reference is expressly made. 6. 5) Promoters specifically activated for endothelial cells These include promoters or activator sequences from promoters or enhancers of such genes, which encode proteins formed preferably on endothelial cells. In the meaning of the invention, for example promoters of the genes for the following proteins can be used: the endothelial glucose-1 transporter, specific for the endoglin brain, the VEGF receptor 1 (flt-1), the VEGF receptor 2 (flk). -1, KDR) tie-1 or tie-2 - the receptor of B61 (Eck receptor) B61 endothelin, especially endothelin B or endothelin-1 endothelin receptors, especially the endothelin receptor B - mannose-6 receptors phosphate von Willebrand factor IL-la, IL-l / S ,. the IL-1 receptor the vascular cell adhesion molecule (VCAM-l) - the interstitial cell adhesion molecule (ICAM-3) synthetic sequences of activators As an alternative to natural promoters specific for endothelial cells, sequences of synthetic activators that consist of oligomerized binding sites for transcription factors, which are preferably or selectively active in endothelial cells. An example of this is the transcription factor GATA-2, whose binding site in the endothelin-1 gene is 5'-TTATCT-3 '(Lee et al., Biol. Chem. 16. 188 (1991), Dormann et al. colaboradoree, J. Biol. Chem. 1279 (1992)) and Wileon and collaboraree, Mol. Cell. Biol. 4.854 (1990)). 7) Combination of the same or different promoters The combination of equal promoters is effected, for example, by sequential chaining of several promoters in the reading direction from 5 'to 3' of the nucleotide sequence. However, for the combination of the same or different promoters, the technologies that have already been described in detail in British patent applications GB9417366.3; European EP97101507.8; EP97102547.3; and German DE19710643.9; DE19617851.7; DE19639103.2 and DE-19651443.6. These patent applications are expressly referred to in the context of this invention. Examples of such technologies are 7. 1) Chimeric promoters A chimeric promoter constitutes the combination of an upstream activator sequence, cell-specific, metabolic or virus-specific, with a downstream promoter module, containing the nucleotide sequence CDE-CHR or E2FBS-CHR , to which suppressor proteins bind, which can thereby inhibit the activation of the upstream activator sequence in the G0 and Gx phases of the cell cycle (British Patent Application GB9417366.3; Lucibello et al., EMBO J., 12 (1994)). Further investigations into the mode of function, especially of the CDE-CHR promoter element, established that the regulation, dependent on the cell cycle by the CDE-CHR element, of an upstream activator sequence is largely dependent on whether the sequence Activation is activated by transcription factors with glutamine-rich activation domains (Zwicker et al., Nuci, Acide Ree., 3. 822 (1995)). To such transcription factors belong, for example, Spl and NF-Y. This factor consequently limits the use of the CDE-CHR promoter element for chimeric promoters. The same can be assumed for the E2F-DS-CHB promoter element of the B-myb gene (Zwicker et al., Nucí.

Ree. 23, 3. 822 (1995)). 7. 2) Hybrid promoters The hybrid promoters have already been described in the German patent application DE19639103.2. For the combination of the promoter specific for endothelial cells with at least one other promoter, for example an artificial gene structure is chosen, which contains in total the following components: The nucleotide sequence of the promoter specific for endothelial cells, in a form in which that at least one binding site for a transcription factor has been mutated. The initiation of the transcription of the effector gene is blocked by this mutation. A transgene that as an effector gene encodes an active substance. At least one other sequence of promoter or intensifier that can be activated non-specifically, specifically for cells, specifically for viruses, by tetracycline and / or specifically for cell cycles, which activates the transcription of at least one gene for at least one transcription factor, which has been mutated from such that it can bind to the mutant binding site (s) at the promoter specific for endothelial cells and can activate it (or these). In an embodiment of this invention, given by way of example, the mutation in the promoter sequence can constitute p. ex. a mutation of the TATA box of the cdc25B promoter. The mutation of the TATA box can be for example TGTATAA. Through this mutation, the DNA binding site of the normal protein (TBP) that binds to the TATA box is no longer recognized and the effector gene can no longer be transcribed efficiently. Correspondingly, the nucleic acid sequence encoding the TBP may have a co-mutation. Through this co-mutation, TBP binds to the mutated TATA box (eg to TGTATAA) and consequently leads to efficient transcription of the effector gene. Such co-mutations of the TBP gene have been described for example by Strubin and Struhl (Cell, 721 (1922) and by Heard et al. (EMBO J. 3.519 (1993)). 7. 3) Multiple promoters in combination with a nuclear retention signal and a nuclear export factor.

This technology has already been described in detail in the German patent application DE-19617851.7. This patent application is expressly referred to. Such a promoter contains, according to the invention, the following components: an activatable promoter or intensifier sequence, specific for endothelial cells, which activates the basal transcription of a transgene, a transgene, which encodes an active substance as an effector gene, a nuclear retention signal (NRS), whose cDNA is directly or indirectly linked at the 5 'end with the 3' end of the structural gene (b). Preferably, the transcription product of the nuclear retention signal has a binding structure for nuclear export factor. - Another sequence of promoter or intensifier non-specifically activatable, specifically for cells, specifically for viruses, specifically for metabolites and / or specifically for cell cycles, which activates the basal transcription of an export factor nuclear, a nucleic acid encoding a nuclear export factor (NEF), which binds to the transcription product of the nuclear retention signal and thereby mediates the transport of the transcription product of the transgene from the cell nucleus. Preferably, the gene encoding the nuclear retention signal is selected from the group comprising the Rev-responsive element (RRE) of HIV-1 or HIV-2, the retention signal equivalent to retrorevirus RRE, or the retention signal equivalent to RRE of HBV. The nuclear export factor is preferably a gene selected from the group comprising the Rev gene of HIV-i viruses, HIV-2, Maedi-Visna virus, encephalitis-goat arthritis virus, infectious equine anemia, feline immunodeficiency virus, retrovirus, HTLV, or the hnRNP-Al protein gene or the TFIII-A transcription factor gene. 7. 4) Activator-sensitive promoter unit Activator-sensitive promoter units have already been described in detail in the German patent application DE19617851.7. This reference is made to the patent application. An activator-responsive promoter unit consists of the following components: one (or several) promoter sequence (s) or enhancer (s) same or different, which is (or is) activatable (s) specific of cell cycles, in a cell-dependent manner, metabolically, specifically for endothelial cells or specifically for viruses, or both specifically for cell cycles as well as metabolically, specifically for endothelial cells or specifically for viruses (so-called chimeric promoters) one (or several) subunit (s) of the same or different activator (s), which in each case are located downstream from the promoter or enhancer sequences, and is (or are) activated by them in their basal transcript - a promoter responsive to activators, which is activated by the expression products of one (or several) subunit (s) of activator (s). In a preferred embodiment, the promoter-sensitive promoter units according to the invention can constitute binding sequences for chimeric transcription factors from DNA binding domains, interaction domains between a protein and another protein and transactivation domains. All the binding sites of transcription factors mentioned in the application can be presented once (monomers) or in several copies (multimers for example up to 10 copies). An example of an activator-responsive promoter, activated by two activator subunits, constitutes the LexA operator in conjunction with the SV40 promoter. - The first activator subunit comprises the cDNA for the DNA and LexA binding protein encoding amino acids 1-81 or 202, whose 3 'end is linked to the 5' end of the cDNA for the Gal80 protein (amino acids 1-435). The second activator subunit comprises the cDNA of the Gal80 binding domain of the Gal4b protein, which encodes amino acids 851-881, whose 3 'end is linked to the 5' end of the SV40 large T antigen cDNA, which encodes amino acids 126-132, whose 3 'end is linked to the 5' end of the cDNA for the transactivation domain of HSV-1 VP16, which encodes amino acids 406-488. Another example of an activator-sensitive promoter, activated by two activator subunits, constitutes the binding sequence for the Gal4 protein in association with the SV40 promoter. - The first activation unit comprises the cDNA for the DNA binding domain of the Gal4 protein (amino acids 1-147), whose 3 'end is linked to the 5' end of the cDNA for the Gal80 protein (amino acids 1-435) . The second activation subunit comprises the cDNA for the Gal80 binding domain of Gal4 (amino acids 851-881), whose 3 'end is linked to the 5' end of the SV40 nuclear localization signal cDNA (SV40 large T; amino acids 126-132), whose 3 'end is linked to the 5' end of the cDNA for the transactivation domain of HSV-1 VP16 encoding amino acids 406-488. Another example of two activator subunits, which activate the activator-sensitive promoter, which consists of the binding sequence for the Gal4 protein and the SV40 promoter, constitute it: a first activation unit, comprising the cytoplasmic domain of the antigen of CD4 T cells (amino acids 397-435), whose 5 'end is linked to the 3' end of the cDNA for the transactivation domain of HSV-1 VP16 (amino acids 406-488), whose 5 'end is in turn linked to the 3 'end of the SV40 nuclear localization signal cDNA (SV40 large T, amino acids 16-132) and the second activation unit comprising the cDNA of the SV40 nuclear localization signal (large T of SV40; amino acids 126-132), the cDNA for the DNA binding domain of the Gal4 protein (amino acids 1-147), whose 3 'end is linked to the 5' end of the cDNA for the CD4 binding sequence of the protein of Ick p56 (amino acids 1-71). 8) Selection of the effector gene In the meaning of the invention, the nucleotide sequence according to the invention contains at least one effector gene (component e), which codes for a pharmacologically active substance for the prophylaxis and / or therapy of a disease. This active substance is selected from a group comprising cytokines, growth factors, antibodies or fragments of antibodies, receptors for cytokines or growth factors, proteins that act in an antiproliferative, apoptotic or cytostatic manner, inhibitors of angiogenesis, inhibitors of coagulation , substances that act in a fibrinolytic manner, plasma proteins, complement activating proteins, peptide hormones, virus envelope proteins, bacterial antigens and parasitic antigens, proteins that act on blood circulation and ribozymes. Preferably, in the case of the transgene it is a structural gene, which encodes a ribozyme, which inactivates the mRNA, which encodes a protein selected from the group comprising control proteins of cell cycles, especially cyclin A, cyclin B, cyclin DI, cyclin E, E2F1-5, cdc2, cdc25C or DPI, or virus proteins or cytokines growth factors or their receptors. In yet another embodiment, the effector gene can encode an enzyme, which dissociates a precursor compound from a drug in a drug. In yet another embodiment, the effector gene can encode a fusion protein between a ligand and an effector, the ligand being an antibody, an antibody fragment, a cytokine, a growth factor, an adhesion molecule or a hormone. peptide, and the effector being able to be a pharmacologically active substance as described above or an enzyme. For example, the structural gene can encode a fusion protein between a ligand and an enzyme, by dissociating the enzyme to a precursor compound of a drug in a drug and binding the ligand to a cell surface, preferably to endothelial cells or tumor cells. In the sense of the invention, the choice of the effector gene and of the additional promoter element that must eventually be combined with the promoter specific for endothelial cells, is oriented to the type of prophylaxis and / or therapy of the respective disease. For example, in the cases of the following diseases the following combinations of promoter sequences and effector genes should be chosen (a detailed description was already made in the patent applications EP97101507.8 EP97102547.3; DE19710643.9; DE197704301.1; DE19617851.7 DE19639103.2; DE19651443.6; EP95931204.2; EP95930524.4 EP95931205.9; EP95931933.6 and DE197011441.1, to which reference is made). 8. 1) Tumor therapy 8. 1.1.) Additional promoters non-specifically activated and / or - specifically for cell cycles and / or metabolically 8. 1.2.) Effector genes for inhibitors of cell proliferation, for example for - the retinoblastoma protein (pRb / pllO) or the related proteins pl07 and pl30 the retinoblastoma protein (pRb / pllO) or the related proteins pl07 and pl30 are inactivated by phosphorylation. Preferably, the genes of these cell cycle inhibitors, which have mutations for the sites of inactivation of the expressed proteins, should be used, without this being impaired in their function. Examples of these mutations were described for pllO. In an analogous manner, the DNA sequence for the pl07 protein or the pl30 protein is mutated. The p53 protein The p53 protein is inactivated in the cell either by binding to special proteins, such as for example the MDM 2, or by oligomerization of the p53 through the dephosphorylated serine, located at the end of C. Preferably, It therefore uses a DNA sequence for a p53 protein, which is shortened at the C terminus by serine 392. - p21 (WAF-1) p6 protein Other cdk inhibitors GADD45 protein Bak protein.

.) Effector genes for coagulation-inducing factors and inhibitors of angiogenesis, for example: Plasminogen activator inhibitor-1 (PAI-1) PAI-2 PAI-3 Angiostatin Interferons (IFNa, IFN / S or IFN?) - Platelet factor 4 TIMP-l TIMP-2 TIMP-3 Leukemia inhibitor factor (LIF) - Tissue factor and its active fragments for coagulation Factor X or factor X mutations , corresponding to the German patent application DE19701141.1, to which "reference is expressly made. 8. 1.4.) Effector genes for cytostatic and cytotoxic proteins, for example for The perforin The granzyme The IL-2 - The IL-4 The IL-12 The interferons, such as for example IFN-OI, IFN / S or IFN? TNFs, such as TNFa or TNF / S - Oncostatin M Sphingomyelinase, Magainin and magainin derivatives. 8. 1.5.) Effector genes for cytostatic or cytotoxic antibodies and for fusion proteins between fragments of antibodies that bind to antigens, with proteins or cytostatic, cytotoxic or excitatory enzymes of inflammation. Cytostatic or cytotoxic antibodies include those which are directed against membrane structures of endothelial cells as described, for example, by Burrows et al. (Pharmac. Ther., £ 4, 155 (1994)), Hughes et al. (Cancer Res. £, 6.214 (1989)) and Maruyama et al. (PNAS USA 87 5744 (1990)). In particular, antibodies against VEGF receptors are counted among them. Otherwise belong to them cytostatic or cytotoxic antibodies that are directed against membrane structures in tumor cells. Such antibodies were described, for example, by Sedlacek et al., Contrib. to Oncol 2, Karger Verlag, München (1998) and Contrib. to Oncol. 43. Karger Verlag, Manchen (1992). Other examples are the antibodies against sialyl Lewis; against peptides in tumors, which are recognized by T cells; against proteins expressed by oncogenes; against gangliosides such as GD3, GD2, GM2, 9-0-acetyl GD3, fucosyl-GM1; against antigens of blood groups and precursors of these against antigens existing in the polymorphic epithelial mucin; against existing antigens in heat shock proteins (Heat Shock Proteinen = HSP) 15 - Moreover, they belong to them antibodies directed against membrane structures of leukemia cells. Monoclonal antibodies of this type have already been described in large numbers for diagnostic and therapeutic procedures (Compendiums in articles by Krieteneen, Danieh Medical Bulletin 41 52 (1994), Schranz, Therapy Hungarica 28, 3 (1990), Drexler and collaborator, Leuk, Ree.10, 2. 179 (1986), Naeim, Die. Markere 7, 1 (1989), Stickne and collaborator, Curr. Opin.

Oncol. 4, 847 (1992); Drexler and collaborator, Blut 7, 327 (1988); Freedman et al., Cancer Invest, 69 (1991)). Depending on the type of leukemia, monoclonal antibodies or their fragments are suitable as ligands. of antibodies that bind to antigens, which are directed against the following membrane antigens: Table 2: Membrane antigen cells AML CD1 3 CD1 5 CD33 CAMAL Sialosil-Le B-CLL CD5 CD1 c CD23 Idiotypes and isotypes of membrane immunoglobulins TCLL CD33 M38 Receptors of IL-2 T Cell Receptors ALL CALLA CD19 Lymphoma "not Hodgkin's' The humanization of murine antibodies, the preparation and optimization of genes for Fab and recombinant Fv fragments is carried out in a manner corresponding to the technique known to a person skilled in the art (Winter et al., Nature 349, 293 (1991); Hoogenboome et al. collaborators, Rev. Tr. Transm. Hemobiol. £, 19 (1993), Girol.Immunol., 28, 1-37 (1991) or Huston and collaborator, Intern. Rev. Immunol., 10., 195 (1993). The fusion of the recombinant Fv fragments with genes for proteins or cytostatic, cytotoxic or inflammatory excitatory enzymes is carried out in the same way according to the state of the art known to a person skilled in the art. 8. 1.6.) Effector genes for fusion proteins of other ligands that bind to endothelial cells or tumor cells, with proteins or cytostatic or cytotoxic enzymes. The ligands include, for example, all substances that bind to membrane structures or membrane receptors on endothelial cells. For example, they belong to them: Antibodies or antibody fragments Cytokines such as for example IL-1 or growth factors or fragments or partial sequences thereof that bind to receptors expressed by endothelial cells, such as for example PDGF, bFGF, VEGF, TGF. Moreover, they belong to them adhesion molecules, which bind to activated and / or proliferating endothelial cells. They belong to them for example SLex, LFA-1, MAC-1, LECAM-1, VLA-4 or vitronectin. They also belong to the substances that bind to membrane structures or membrane receptors of tumor cells or leukemia. For example, they belong to them the growth factors or their fragments, or partial sequences of these, that bind to receptors expressed by leukemia cells or tumor cells. Such growth factors have already been described (Compendiums in the articles of Cross and collaborators, Cell £ 4, 271 (1991), Aulitzky and colaboradoree, Druge 48 667 (1994), Moore, Clin. Cancer Ree. 1, 3 (1995), Van Kooten and collaborator, Leuk. Lymph. 12, 27 (1993). The fusion of the genes of these ligands that bind to the target cell, with proteins or cytostatic enzymes, cytotoxic or excitatory of inflammations, is carried out correspondingly to the state of the art with the methods known by a person skilled in the art.

) Effector genes for inflammation inducers, for example for IL-1 IL-2-RANTES (MCP-2) a monocyte activating and chemotactic factor (MCAF) IL-8 inflammatory protein macrophage-1 (MIP-la, -β) neutrophil activating protein-2 (NAP-2) IL-3 IL-5 a human leukemia inhibitory factor (LIF) - IL -7 IL-5 eotaxin IL-13 GM-CSF-G-CSF M-CSF the cobra venom factor (CVF, cobra venom factor) or partial sequences of the FVC, which correspond functionally to the human complement factor C3b, that is, they can bind to the complement factor B and after their dissociation by the factor D constitute a C3 convertase the human complement factor C3 or its partial sequence C3b dissociation products of the human complement factor C3, which resemble functional and structurally to the CVF bacterial proteins, which activate the complement or cause inflammations, such as for example porins of Salmonella typhimurium, factors "conglomerators = dumping" of Staphylococcus aureue, modulins especially of gram-negative bacteria, the "outer membrane protein" major = major outer membrane protein Legionellas or Hae ophilus influenza type B or Klebeiellae, or M molecules of group G. Streptococcus. 8. 1.8.) Effector genes for enzymes intended for the activation of cytostatic agent precursors, for example for enzymes that dissociate inactive precursor substances (prodrugs = prodrugs) in active cytostatic agents (drugs = drugrs). Such substances and the respective prodrugs and drugs have already been described comprehensively by Deonarain et al. (Br. J. Cancer ZQ, 786 (1994)) Mullen (Pharmac. Ther. 6, 199 (1994)) and Harris and collaborators (Gene Ther.1, 170 (1994)). For example, the DNA sequence of one of the following enzymes should be used: thymidine kinase from herpes simplex virus thymidine kinase from varicella Zoster virus - bacterial nitro-reductase jS-glucuronidase bacterial / plant 3-glucuronidase from Sécale cereale Human 3-glucuronidase human carboxypeptidases (CB), for example CB-A of the priming cells, CB-B of the pancreas or bacterial carboxy-peptidase /? -lactamase bacterial cytosine-deaminase bacterial catalase or human peroxidase phosphatases, in special human alkaline phosphatase, prostate-human acid phosphatase or acid phosphatase of type 5 oxidases, especially human lysyl oxidase or human D-amino-oxidase human acid peroxidases, especially human glutathione peroxidase, human eosinophil peroxidase or thyroid peroxidase human galactosidase 8. 2.) Therapy of autoimmune diseases and inflammations 8. 2.1.) Additional promoters that can be activated non-specifically and / or specifically for cell cycles and / or metabolically 8. 2.2.) Effector genes for allergy therapy, for example for IFN / 8 IFN? IL-10 - antibodies or fragments of antibodies against IL-4 soluble IL-4 receptors IL-12 TGFjS 8. 2.3.) Effector genes for the avoidance of rejection of transplanted organs, for example for IL-10 TGFjS soluble IL-1 receptors - IL-2 receptors soluble IL-1 receptor antagonists soluble IL-6 receptors immunosuppressive antibodies or its fragments that contain VH and VL or their fragments of VH and VL bound through a linker. Immunosuppressant antibodies are for example antibodies specific for the T cell receptor or its CD3 complex, against CD4 or CD8, in addition against the IL-2 receptor, the IL-1 receptor or the IL-4 receptor or against the molecules of adhesion CD2, LFA-1, CD28 or CD40.

.) Effector genes for the therapy of autoimmune diseases mediated by antibodies, for example for TGF / J IFNa - IFN / S IFN? IL-12 soluble IL-4 receptors soluble IL-6 receptors - immunosuppressive antibodies or their fragments containing VH and VL .) Effector genes for the therapy of cell-mediated autoimmune diseases, for example for - IL-6, IL-9 IL-10 IL-13 TNFc. or TNFjS - an immunosuppressant antibody or its fragments containing VH and VL ) Effector genes for inhibitors of cell proliferation, cytostatic or cytotoxic proteins, inducers of inflammations and enzymes for the activation of cytostatic agent precursors. Examples of genes encoding such proteins have already been discussed in the "structural genes for tumor therapy" paragraph. In the same way as already described therein, structural genes encoding fusion proteins based on antibodies or recombinant Fab or Fv fragments of these antibodies or other ligands specifically for the target cell and cytokines can be used in the sense of the invention. previously mentioned, growth factors, receptors, proteins and cytostatic or cytotoxic enzymes.

.) Structural genes for the therapy of arthritis In the sense of the invention, structural genes are selected whose expressed protein directly or indirectly inhibits inflammation for example in a joint and / or promotes the reconstitution of extracellular matrix (cartilages, connective tissues) in the joint To them belong, for example: - an IL-1 receptor antagonist (IL-1-RA), • an IL-1-RA inhibits IL-1 binding; a soluble IL-1 receptor, a soluble IL-1 receptor binds and inactivates IL-1-IL-6, IL-6 increases the secretion of TMIP and superoxides and decreases the secretion of IL-1 and TNF! by synovial cells and chondrocytes • 'a soluble TNF receptor a TNF receptor binds and inactivates TNF IL-4 IL-4 inhibits the formation and secretion of IL-1, TNFa and MMP IL-10 inhibits IL-10 the formation and secretion of IL-1, TNFQÍ and MMP, and increases the secretion of TIMP the insulin-like growth factor (IGF-1) IGF-1 stimulates the synthesis of extracellular matrix a TGF / d, especially TGFjSl and TGF / 32 a TGF / 3 stimulates the synthesis of extracellular matrix superoxide dismutase TIMP, especially TIMP-1, TIMP-2 or TIMP-3.

Therapy of defective formation of blood cells .) Additional promoters - activatable non-specifically and / or specifically for cell cycles and / or metabolically .) Effector genes for the therapy of anemia, for example for erythropoietin .) Effector genes for the therapy of leukopenia, for example for - G-CSF GM-CSF M-CSF ) Effector genes for the therapy of thrombocytopenia, for example for IL-3 leukemia inhibitory factor (LIF) IL-11 thrombopoietin Therapy of nervous system injuries .) Additional promoters non-specifically activated and / or - specifically for cell cycles and / or metabolically .) Effector genes for neuronal growth factors, for example - FGF nerve growth factor (NGF) brain-derived neurotrophic factor (BDNF) neurotrophin-3 (NT-3) neurotrophin-4 (NT-4) - ciliary neurotrophic factor ( CNTF) .) Effector genes for enzymes, for example for tyrosine hydroxylase dopa decarboxylase .) Effector genes for cytokines and their inhibitors, which inhibit or neutralize the neurotoxic effect of TNFa, for example for TGF3 - soluble TNF receptors TNF receptors neutralize TNFa IL-10 IL-10 inhibits the formation of IFN ?, TNFa, IL-2 e IL-4 - IL-1 receptors soluble IL-1 receptor I an IL-1 receptor soluble IL-l receptors neutralize the activity of IL-1 - an IL-1 receptor antagonist IL receptors -6 soluble Therapy of disorders of the blood coagulation system and blood circulation .) Additional promoters - activatable non-specifically and / or specifically for cell cycles and / or metabolically .) Effector genes for the inhibition of coagulation or for the promotion of fibrinolysis, for example for a tissue-type plasminogen activator (tPA) a urokinase-type plasminogen activator (uPA) hybrids of tPA and uPA-protein C hirudin serine protease inhibitors (serpins), such as for example the C-1S inhibitor, al-antitrypsin or antithrombin-III - a tissue factor pathway inhibitor (TFPI) .) Effector genes for the promotion of coagulation, for example for - F VII F IX von Willebrand factor F XIII PAI-1 - PAI-2 a tissue factor and fragments thereof ) Effector genes for angiogenesis factors, for example for - VEGF FGF 8.5.5.) Effector genes for blood pressure decrease, for example for kallikrein "nitric oxide synthase" of endothelial cells 8. 5.6.) Effector genes for the inhibition of smooth muscle cell proliferation after endothelial layer injury, for example for an antiproliterative, cytostatic or cytotoxic protein or an enzyme for the dissociation of cytostatic agent precursors in cytostatic agents such as already stated above (when treating tumors) or - a fusion protein of one of these active substances with a ligand, for example with an antibody or antibody fragments specific for muscle cells 8. 5.7.) Effector genes for other blood plasma proteins, for example for an albumin an inactivator of serum cholinesterase Cl - transferrin 1-antitrypsin 8. 6.) Vaccinations 8. 6.1.) Additional nonspecific and / or specific promoters for cell cycles 8. 6.2.) Effector genes for the prophylaxis of infectious diseases The possibilities of preparing effective vaccines by conventional means are limited.

As a result, the technology of DNA vaccines was developed. These DNA vaccines, however, raise questions about the intensity of the activity (Fynan et al., Int. J. Immunopharm, 17, 79 (1995), Donnelly et al., Immunol., 2, 20 (1994)). According to this invention, it is necessary to have a higher activity of DNA vaccines. As the active substance, the DNA of a protein formed by the pathogenic agent of infection must be chosen which, by provocation of an immunological reaction, ie by binding to antibodies and / or cytotoxic T lymphocytes, leads to the neutralization and / or the annihilation of the agent pathogen. Such so-called neutralization antigens are already used as vaccine antigens (see the compendium in the article by Ellis, Adv. Exp. Med. Biol. 227, 263 (1992)). Preferred in the sense of the invention is DNA encoding antigens for neutralization of following pathogens: influenza A virus HIV (human immunodeficiency virus or HIV) rabies virus HSV (herpes simplex virus) - RSV (respiratory syncytial virus) parainfluenza virus a rotavirus VZV (varicella Zoster virus) ) CMV (cytomegalo-virus) - measles virus HPV (human papillomavirus) HBV (hepatitis B virus) HCV (hepatitis C virus) HDV (hepatitis D virus) - HEV (hepatitis E virus) HAV (hepatitis A virus) the antigen of Vibrio Cholera Borrelia burgdorferi Helicobacter pylori a malaria antigen To such active substances belongs in the sense of the invention however also the DNA of an anti-idiotype antibody or of its fragments that bind to antigens, whose binding structures * antigens (the "complementary determining regions = complementary discovery regions") constitute copies of the protein or carbohydrate structure of the neutralizing antigen of the pathogen of infections. Such anti-idiotype antibodies can especially replace carbohydrate antigens in the case of pathogens of bacterial infections. Such anti-idiotype antibodies and their cleavage products were described comprehensively by Hawkine and collaborators (J. Immunother, 14, 274 (1993)) and Weeterink and Apicella (Springer).

Seminare Immunopathol. 1, 227 (1993)) 8. 6.3.) Effector genes for "tumor vaccines" They belong to antigens located in tumor cells. Such antigens were described, for example, by Sedlacek and collaborator, Contrib. to Oncol. 22. , Karger Verlag, München (1988) and Contrib. to Oncol 43 Karger Verlag München (1992). Other examples are genes for the following antigens or for the following anti-idiotype antibodies: Sialil-Lewis, peptides in tumors, which are recognized by T cells proteins expressed by oncogenes - blood group antigens and their antigen precursors in mucin epithelial polymorphic antigens in heat shock proteins 8.7.) Therapy of chronic infectious diseases 8. 7.1.) Additional promoters specific for viruses and / or - specific for cell cycles and / or nonspecific. 8. 7.2.) Effector genes, for example for a protein, which has cytostatic, apoptotic or cytotoxic effects. an enzyme that dissociates a precursor compound of an antiviral or cytotoxic substance in the active substance 8. 7.3.) Effector genes for antiviral proteins cytokines and growth factors that are antivirally active. These include for example IFNar, IFNJ, IFN ?, TNF3, TNFa, IL-1 or TGF0 - antibodies with a specificity, which inactivates the respective virus or its fragments containing VH and VL, or its VH and VL fragments bound to through a linker, prepared as already described.

Antibodies against virus antigens are for example: anti-HBV anti-HCV anti-HSV anti-HPV anti-HIV anti-EBV anti-HTLV anti-Coxsackie virus anti-Hantaan virus a protein that binds to Rev. These proteins bind to Rev RNA and inhibit post-transcriptional steps, dependent on Rev, of retrovirus gene expression. Examples of proteins that bind to Rev are: RBP9-27 RBP1-8U RBP1-8D Pseudogenes of RBP1-8. - for ribozymes, which digest the mRNA of genes for control proteins of the cell cycle or the mRNA of viruses. Catalytic ribozymes for HIV were described, for example, by Christoffereen and collaboraree, J. Med. Chem. 38 2. 033 (1995). 8. 7.4) Effector genes for antibacterial proteins Antibacterial proteins belong, for example, to antibodies, which neutralize bacterial toxins or opsonize bacteria. For example, they belong to them antibodies against meningococci C or B coli Bor relia Peeudomonae Helicobacter pylori Staphylococcal aureue 9) Combination of the same or different structural genes: The object of the invention is, moreover, an artificial nucleic acid structure, in which a combination of the DNA sequences of identical structural genes or of two different structural genes is presented. For the expression of both DNA sequences, another promoter sequence is inserted or preferably the cDNA of an "internal ribosome entry site" (IRES of internal! Riboeome entry site) as a regulatory element between both structural genes.

An IRES makes possible the expression of two DNA sequences linked together through an IRES. Such IRES were described for example by Montford and Smi th (TIG 11, 179 (1995); Kaufman et al., Nucí, Acide, Res. 11, 4.485 (1991); Morgan et al., Nucí.

Acide Ree. 2, 1, 293 (1992); Dirke and colaboradoree, Gene 128. 247 (1993); Pelletier and Sonnenberg, Nature 334, 320 (1988) and Sugi tome and colaboradoree, Biotech. 12, 694 (1994)).

Thus, for example, the cDNA of the IRES sequence of the poliovirus can be used (position = 140 to = 630 of the 5'UTR). Preferably, structural genes having an additive effect can be linked in the sense of the invention, via other promoter sequences or an IRES sequence. In the sense of the invention, combinations of structural genes for example for 9. 1.) The therapy of tumors - cytostatic, apoptotic, cytotoxic or inflammation-stimulating proteins, same or different enzymes equal or different for the dissociation of the precursor compound of a cytostatic agent 9. 2.) Therapy of autoimmune diseases different cytokines or receptors with synergistic effect for the inhibition of cellular and / or humoral immunological reaction or TIMP's different or equal 9. 3.) The therapy of the defective formation of blood cells different cytokines, which occur hierarchically, such as for example IL-1, IL-3, IL-6 or GM-CSF and erythropoietin, G-CSF or thrombopoietin 9.4 .) The nerve cell injury therapy a neuronal growth factor and a cytokine or the inhibitor of a cytokine 9. 5.) Therapy of disorders of the blood coagulation system and blood circulation an antithrombotic agent and a fibrinolytic agent (eg tPA or uPA) or a cytostatic, apoptotic or cytotoxic protein and an antithrombotic or fibrinolytic agent several different blood coagulation factors acting synergistically, for example F VIII and vWF or F VIII and F IX 9. 6.) Vaccinations an antigen and an immunostimulatory cytokine, such as for example an IL-la receptor, Ih-lß, IL-2, GM-CSF, IL-3 or IL-4 different antigens from a pathogen of infections or different pathogens from infections or different antigens from a type of tumors or from different types of tumors 9. 7.) The therapy of viral infectious diseases an antiviral protein and a cytostatic protein, apoptotic or cytotoxic antibodies against different surface antigens of a virus or of several viruses 9. 8.) Therapy of infectious bacterial diseases antibodies against different surface antigens and / or toxins of a germ 10) Insertion of signal sequences and transmembrane domains A detailed description of the technology has already been made in patent applications DE19639103.2 and DE19651443.6 to which reference is expressly made. . 1.) For translational reinforcement, the nucleotide sequence GCCACC can be inserted at the 3 'end of the promoter sequence and immediately next to the 5' end of the initiation signal (ATG) of the signal or transmembrane sequence. or GCCGCC (Kozak, J. Cell Biol. 108. 299 (1989)). . 2.) To facilitate the secretion of the product from expression of the structural gene, the homologous signal sequence optionally contained in the DNA sequence of the structural gene can be replaced by a heterologous signal sequence, which improves the output from the intracellular region.

Thus, for example, the signal sequence for the immunoglobulin can be inserted (positions in the DNA = 63 to 2107; Riechmann et al.

Nature 222., 323 (1988)) or the signal sequence for CEA (position in DNA = 33 up to = 134; Schrewe and collaborator, Mol. Cell. Biol. 1 £ L, 2. 738 (1990); ing and collaborating, Cancer Ree. Q. 6. 534 (1990) or the human respiratory syncytial virus glycoprotein signal sequence (amino acid cDNA = 38 to = 50 or 48 to 65; Lichtenetein et al., J. Gen. Virol 72, 109 (1996)) . 3.) For the anchoring of the active substance in the cell membrane of the transduced cell that forms the active substance, a sequence for a transmembrane domain can be introduced, alternatively or additionally to the signal sequence. Thus, for example, the transmembrane sequence of the human macrophage colony stimulating factor can be introduced (position in the DNA from = 1,485 to = 1,554, Coeman and collaborator, Behring Inet, My tt. £ 2, 5 (1988)) or the DNA sequence for the signal and transmembrane region of the human respiratory syncytial virus (RSV) glycoprotein G virus (amino acids 1 to £ 2 or its partial sequences, amino acids 38 to 63; Vijaya and collaborator, Mol. Cell. Biol. £ 1.709 (1988), Lichtenstein et al., J. Gen. Virol. 22, 109 (1996)) or the DNA sequence for the signal and transmembrane region of influenza virus neuraminidase (amino acids 7). up to 35 or the partial amino acid sequence 7 to 27; Brown and collaborator, J. Virol. £ 2, 3,824 (1988)) between the promoter sequence and the structural gene sequence. . 4.) For the anchoring of the active substance in the cell membrane of the transduced cells that form the active substance, however, the nucleotide sequence can also be introduced for an anchoring of glycophospholipids. The introduction of a glycophospholipid anchor is carried out at the 3 'end of the nucleotide sequence for the structural gene and can be carried out in addition to the introduction of a signal sequence. Glycophospholipid anchors have been described for example for CEA, for N-CAM and for other membrane proteins, such as for example Thy-l (see the compendium of Ferguso, et al., Ann.Rev. Biochem. 57, 285 ( 1988)). . 5.) Another possibility for the anchoring of active substances to the cell membrane corresponding to the present invention is the use of a DNA sequence for a fusion protein between a ligand and an active substance. The ligand specificity of this fusion protein is directed towards a membrane structure in the cell membrane of the targeted target cells. . 5.1.) The ligands, which bind to the cell surface, belong for example antibodies or fragments of antibodies, directed towards structures located on the surface of for example endothelial cells. Especially among these are antibodies against the VEGF receptors or against quinine or muscle cell receptors, such as antibodies against actin or antibodies against angiotensin-II receptors or antibodies against receptors for growth factors, such as for example against recipients of EGF or against PDGF receptors or against FGF receptors or antibodies against endothelin A receptors. Ligands also include antibodies or their fragments, which are directed against tumor-specific or tumor-associated antigens, present in the membrane of tumor cells . Such antibodies have already been described. - The murine monoclonal antibodies are preferably used in a humanized form. The recombinant FAB and Fv fragments and their fusion proteins are prepared as already described, with the technology known to a person skilled in the art. . 5.2.) To the ligands belong in addition all active substances such as for example cytokines or adhesion molecules, growth factors or their fragments or partial sequences thereof, mediators or peptide hormones, which bind to membrane structures or receptors of membranes in the respective selected cell. For example, ligands for endothelial cells, such as IL-1, PDGF, bFGF, VEGF, TGG / 8 (Pusztain et al., J. Pathol 169. 191 (1993)) or quinine and derivatives of, or analogous compounds, belong to them. a, quinine. They also belong to them adhesion molecules. Adhesion molecules of this type; such as for example SLex, LFA-1, MAC-1, LeCAM-i, VLA-4 or vitronectin and derivatives of, or compounds analogous to, vitronectin, have already been described for endothelial cells (compilations in the Augustin-Voss articles and collaboradoree, J. Cell. Biol. 119. 483"'(1992), Pauli et al., Cancer Metast. Rev., 175 (1990); Honn et al., Cancer Metast., Rev. 11, 353 (1992); Varner and collaborator, Cell Adh. Commun. 2, 367 (1995)).

The invention is explained in more detail by the following Examples, without being limited thereto. 11) Preparation and use of the artificial nucleic acid structure Artificial structures (also known as constructs) of nucleic acid preferably consist of DNA. Nucleic acid constructs are understood as meaning artificial structures based on a nucleic acid, which can be transcribed in the target cells. These are preferably inserted into a vector, with plasmid or plasmid vectors complexed with non-viral supports being particularly preferred (Fritz et al., Hum. Gene Ther.7: 1395 (1996)).; Solodin and colaboradoree, Biochem. 3. 4; 13,537 (1995); Abdallak et al., Hum. Gene Ther. 7: 1,947 (1996); Ledley, Hum. Gene Ther. 6: 1129 (1995); Scho field and colaboradoree, Br. Med. Bull. 51: 56 (1995); Behr, Bioconj. Chem. 5: 382 (1994); Cotten and colaboradoree, Curr. Opin. Biotechnol. 4: 705 (1993); Hodgeon and collaborator, Nature Biotechnol. 14: 339 (1996). The vectors are introduced with the technologies known to a person skilled in the art (Cotten et al., Curr Opin, Biotechnol 4: 705 (1993), Schef field and collaboraree, Br.

Med. Bull. 51: 56 (1995), Ledley, Hum. Gene Ther. 6: 129 (1995)) in the endothelial cell precursor cell, or in endothelial cells. In another embodiment, the artificial nucleic acid structures according to the invention are introduced into a viral vector (Weir and collaborators, Hum. Gene Ther 7: 1331 (1996); Flotte et al., Gene Ther. 2: 357 (1995), Efstathion and collaborators, Br. Med. Bull. 51: 45 (1995), Kremer and collaborator, Br. Med. Bull. 51: 31 (1995), Vile and collaborator Br. Med. Bull. 51: 12 (nineteen ninety five)); Randrianarieon and collaborator, Biologicale 23: 145 (1995), Jolly Cancer Gene Ther. 1:51 (1994)), and η transfected with these endothelial cells. Cells transduced in this way are administered to patients externally or internally, locally, within a body cavity, within an organ, in the bloodstream, in the respiratory tract, in the gastro-intestinal tract, in the tract urogenital, in a cavity with wounds, either intramuscularly or subcutaneously, by means of the artificial nucleic acid structures according to the invention, or a structural gene can be expressed in endothelial cells or in endothelial cell precursor cells in a specific manner for cells and possibly also specifically for viruses, under certain metabolic conditions and / or specifically for cell cycles and / or in a drug-induced manner, in the case of the structural gene preferably of a gene, which encodes a substance activates pharmacologically active or also an enzyme that dissociates an inactive precursor compound from a drug or in an active drug. The structural gene can be selected such that the pharmacologically active substance or enzyme is expressed as a fusion protein with a ligand and binds this ligand to the cell surface, e.g. ex. proliferating endothelial or tumor cells. The invention is now described in more detail with the help of the Figure and the Examples, without being limited thereto.

Legend of the Figure: Figure 1: Artificial nucleic acid structure for cell transformation 12) Examples for explaining the idea of the invention 12. l. Cultivation of endothelial cells from CD34-positive blood cells, without the use of fibronectin or bovine brain. CD34-positive blood cells, isolated as described by Asahara et al., Science. 275: 964 (1997), were cultured either - in batch a), as published by Asahara, in plastic bottles, coated with fibronectin and with the addition of a bovine brain extract (100 μg / ml), or also (in batch) corresponding to this invention, in plastic bottles not coated with fibronectin and without the addition of bovine brain extract, but with the addition of VEGF and bFGF (entity Sigma, in each case 1% v / v) in each case with the addition of fetal calf serum (FKS, at 20%) in a culture medium (Medium 199) a 37 ° C and mediating gassing with 5% C02. After 6 days the proportion of cells that grew adhesively, were spindle-shaped and formed structures similar to capillaries was determined with a microscope, and the proportion of endothelial cells was determined by labeling with antibodies specific for endothelial cells (anti-CD31, anti-CD31). cWF, anti-Flik 1) with the help of FACS analysis. No difference was found in the number or morphology of these cells between batch a) and batch b). In series of experiments of both batches, the proportion of endothelial cells fluctuated between 1 and 10%, which shows that the addition of the growth factors VEGF and bFGF can replace the coating of the culture flask with fibronectin and the addition of extract of the brain.

) Culturing of endothelial cells from mononuclear cells of blood From 120 ml of blood mononuclear cells were isolated with the help of centrifugation through a Ficoll gradient and the non-adherent mononuclear blood cells were separated by incubation for 1 hour in the cell culture flask and by subsequent decanting. These cells were seeded in culture flasks corresponding to batch b) and cultured for 6 days at 37 ° C and gassed with 5% C02. After 6 days the proportion of endothelial cells was determined, as described in section 12.1). This was between 2 and 20% in different series of experiments. 12. 3.) Culturing of endothelial cells from blood cells positive for CD14 From 120 ml of blood from a healthy donor mononuclear cells were isolated with the help of centrifugation through a Ficoll gradient (Ficoll-Paque, Pharmacia, Uppsala) and the non-adherent mononuclear blood cells were separated by incubation for 60 min in a cell culture flask and subsequent decanting. The non-adherent mononuclear cells (NMZ), thus isolated, contained 0.3-0.05% of CD34 positive cells and 5-10% of CD14 positive cells (monocytes and monocyte-like cells). 1 x 106 NMZ were adjusted in 1 x 106 cells / ml of Medium 199 containing 20% fetal calf serum (both from Gibco) and 100 μg ECGS (Harbor Bioproducts, Norwood, MA) or VEGF (Pepro Techn., London, England) and incubated at 37 ° C for 1-3 hours in plastic containers lined with fibronectin (Harbor Bioproducts, Norwood, MA). Through this incubation, the proportion of CD14-positive cells was increased from 5-10% to 25-30%. The NMZ were separated by careful washing and the CD14 positive or CDII positive cells were isolated with magnetic beads, coated with anti-CD14 or anti-CDll (CD14 / CD11 Micro Beads, Miltenyi Biotec, Bergisch Gladbach, Germany), correspondingly to the manufacturer's data. NMZ cells, containing approximately 80% of CD14-positive cells were incubated in the above-mentioned medium 199, supplemented with FKS and ECGS or VEGF, in plastic containers lined with fibronectin at 37 ° C with 5% C02 in one cell. humid atmosphere. The cells existing in the culture were investigated after 6 hours, 3 days and 5 days with the help of monoclonal antibodies and with the help of RT-PCR. After 6 hours, small mononuclear cells positive for CD14 were observed, which were positive for specific markers of endothelial cells, acetyl-LDL receptors, CD34, Flk-1 and von Willebrand factor. On the 3rd day, these cells showed intense signs of proliferation. On the 5th day, large granular oval cells and fusiform cells could be observed, all of which bore the endothelial cell markers that are indicated but no longer the CD14 marker. As soon as these endothelial cells had grown together, they additionally expressed VE-C adhein. After 1 to 2 weeks more than 80% of the cells in the culture were endothelial cells. 12. 4.) Transformation specific for endothelial cells, and culture of endothelial cells from mononuclear cells of the blood. From 120 ml of blood mononuclear cells were isolated by centrifugation through a Ficoll gradient, and by incubation for 1 hour in a cell culture flask and subsequent decanting the non-adherent mononuclear blood cells were separated. These blood cells are adjusted to a concentration of lxl07 / ml of the culture medium, seeded in 60 mm culture dishes and incubated for 10 min at 37 ° C with a complex based on the plasmid of the invention and Superfect. (entity Quiagen). The preparation of the complex is carried out according to the data of the manufacturer of Superfect.

The plasmid according to the invention contains, in the reading direction from 5 'to 3', the following DNA sequences: the promoter of the human endoglin gene (NS1- 2415; patent application D19704301.1) the cDNA of kinase 4 human cyclin-dependent (cdk-4) with a mutation in codon 24 [exchange of an arginine (CGT) for a cysteine (TGT), W? lfel and collaborator, Science 269: 1 .281 (1997)]. The nuclear localization signal (NLS) of SV40 [SV40 large T; amino acids 126 to 132; PKKKRKV (SEQ ID NO .: 3); Dingwall et al., TIBS 16: 478 (1991)]. The link of the individual components of the artificial structure is made through appropriate restriction sites, which are treated together by PCR amplification at the ends of the different elements. The binding is carried out with the aid of specific enzymes for the restriction sites, known to a person skilled in the art, and of DNA ligases. These enzymes can be purchased commercially. With the help of these enzymes, the nucleotide thus prepared was incorporated into the cloning. After incubation of the mononuclear blood cells with the Superfect and plasmid complex, the blood cells are washed and cultured in a cell culture medium, as described in paragraph 12.2). After 6 days, the proportion of endothelial cells is determined as described in paragraph 12.1.). This fluctuates between 10 and 60% in different series of experiments. 12. 5.) Preparation and use of transduced endothelial cells as vectors Isolated, transduced and reproduced endothelial cells, as described in paragraph 12.4.) Are seeded into 60 mm culture dishes and incubated for 10 min at 37 ° C with a complex of another plasmid according to the invention and Superfect (Quiagen entity). The preparation of this complex is carried out according to the data of the manufacturer of Superfect. The plasmid according to the invention contains in the reading direction from 5 'to 3' the following DNA sequences: Subunit A) of activator promoter of the cdc25C gene (nucleic acids 290 to +121; Zwicker and collaborator, EMBO J. 14, 4. 514 (1995); Zwicker and collaborator, Nuci.

Acide Ree. 23, 3. 822 (1995)). the SV40 nuclear localization signal (NLS) (SV40 large T, amino acids 126-132; PKKKRKV (SEQ ID NO: 3), Dingwall et al., TIBS 16: 478 (1991)) the acid transactivation domain (TAD) of HSVL1 VP16 (amino acids 406 to 488; Triezenberg and collaborator, Genee Develop 2, 718 (1988); Triezenberg, Curr. Opin. Gen. Developm. 190 (1985)) the cDNA for the cytoplasmic part of the CD4 glycoprotein (amino acids 397-435; Si pson et al, Oncogene 4, 1, 141 (1989); Maddon et al., Cell 42, 93 (1985)) Activator Subunit B) The promoter of the human endoglin gene (nucleic acids I up to 2415; patent application D19704301.1) the SV40 nuclear localization signal (NLS) (SV40 large T, amino acids 126-132; PKKKRKV ( SEQ ID NO: 3), Dingwall et al., TIBS 16: 478 (1991)) the cDNA for the DNA binding domain of the Gal4 protein (amino acids 1 to 147, Chaeman and Kornberg, Mol. Cell. Biol. , 2916 (1990)) the cDNA for the CD4-binding sequence of the Ick protein p56 (amino acids 1-71; Shaw et al., Cell 59, 627 (1989); Turner et al., Cell 60, 755 (1990); Perl utter et al., J. Cell. Biochem. 38, 117 (1988) Activator-sensitive promoters lOx the binding sequence for the Gal4 binding protein with the nucleotide sequence 5'-CGGACAATGTTGACCG-3 '(SEQ ID NO .: 4, Chaeman and Kornberg Mol. Cell. Biol. 10, 2. 916 (1989)). the basal promoter of SV40 (nucleic acids 48 to 5191; Tooze (compiler) DNA tumor virus (Cold Spring Harbor New York, New York, Cold Spring Harbor Laboratory) Effector gene cDNA for human jS-glucuronidase (nucleotide sequence 93 to 1982; Oehima and collaborator PNAS USA 84, 65 (1987)) The operating mode of the trigger sequence that is described is the following: The cdc25B promoter specifically regulates for the cell cycle the transcription of the combined cDNAs for the activation domain of VP16 and the cytoplasmic part (activation A subunit).

The promoter of the human endoglin gene specifically regulates for endothelial cells the transcription of the combined cDNAs for the Gal4 DNA binding protein and the CD4 binding part of the Ick p56 protein (activation B subunit) .

The expression products of activator subunits A and B are dimerized by binding of the CD4 domain to the Ick domain p56.

The dimeric protein constitutes a chimeric transcription factor for the promoter-responsive promoter (DNA sequence for the Gal4 binding domains and the SV40 promoter) for the transcription of the effector gene (= luciferase gene).

The binding of the individual constituents of the artificial structure is effected through appropriate restriction sites, which are treated together by PCR amplification at the ends of the different elements. The binding is carried out with the help of specific enzymes for the restriction sites, known to a person skilled in the art, and of DNA ligases. These enzymes can be purchased commercially. With the help of these enzymes, the artificial nucleotide structure thus produced is incorporated into the cloning vector of plasmid pXP2 (Nordeen, BioTechniquee 6, 454 (1988)). After incubation of the mononuclear blood cells with the Superfect and plasmid complex, the blood cells are washed and cultured in a cell culture medium as described in paragraph 12.2.).

After 6 days, the amount of / S-glucuronide-sa produced by the endothelial cells is measured with the aid of 4-methyl-umbelliferyl-jS-glucuronide as a substrate.

To verify the specificity for cell cycles, endothelial cells are synchronized by subtraction of methionine over 48 hours in G0 / Gx. The DNA content of the cells is determined after staining with Hoechst 33258 in the cell sorter with fluorescence activation (Lucibello et al., EMBO J. 14, 132 (1995)).

The following results are achieved: In transfected endothelial cells no increase in /? -glucuronidase can be determined in comparison with untransfected fibroblasts. Transfected endothelial cells clearly express more amount of | S-glucuronidase than non-transfected endothelial cells. Proliferating endothelial cells (DNA> 2S; S = simple set of chromosomes) clearly secrete more jß-glucuronidase than in endothelial cells synchronized by GQ / G-L (DNA = 2S). Accordingly, the promoter-responsive, promoter unit that is described leads to a cell-specific expression dependent on cell cycles of the 0-glucuronidase structural gene.

The endothelial cells according to the present invention make possible, after local application, by example at the tumor site or after intracranial or subarachnoidal administration, or administration by systemic route, preferably intravenously or intra-arterially. , that these endothelial cells are colonized preferably in regions with vascular lesions, and by specificity for cell cycles and for endothelial cells of the activator-sensitive promoter unit 3-glucuronidase are predominantly, if not exclusively, secreted by endothelial cells in proliferation. This 3-glucuronidase dissociates to a well-compatible doxorubicin, then injected. This inhibits the proliferation of endothelial cells and acts cytostatic on these cells as well as on contiguous tumor cells. This leads to the inhibition of tumor growth.

SEQUENCE PROTOCOL (1) GENERAL DATA: (i) APPLICANT: (A) NAME: Hoechst Marion Roussel Deutsc land GbmH (B) STREET: (C) LOCALITY: Frankfurt (D) REGION: (E) COUNTRY: Germany (F) POSTAL CODE NUMBER: 65926 (G) TELEPHONE: 069-305-3005 (H) TELEFAX: 069-35-7175 (I) TELEX: (ii) TITLE OF THE INVENTION: Genetically modified cells and their use in the prophylaxis or therapy of diseases (iii) NUMBER OF SEQUENCES: 4 (iv) LEGIBLE FORM BY COMPUTER: (A) DATA SUPPORT: Diskette < B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) LOGIC SYSTEM - SOFTWARE: Patentin Relay n ° 1.0, version n ° 1.25 (EPA) (2) INFORMATION ABOUT SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (C) CHAIN FORM: individual (D) TOPOLOGY: linear (ii) TYPE OF THE MOLECULE: Peptide (ix) CHARACTERISTICS: (A) NAME / KEY: Peptides (B) SITUATION: 1..23 (D) OTHER DATA: / note = "Xaa - one of the 20 genetically encodable amino acids; Xaa * = chain of amino acids that consists of of 7-80 amino acids Xaa. (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: Leu Xaa Asp Xaa Leu Xaa Xaa Leu Xaa * Leu Xaa Cys Xaa Glu Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Ser Asp Asp Glu 20 (2) INFORMATION ABOUT SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) CHAIN FORM: double (D) TOPOLOGY: linear (ii) TYPE OF THE MOLECULE: DNA (ix) FEATURES: (A) NAME / KEY: Myc E-Box (B) SITUATION: 1..26 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: GGAAGCAGAC CACGTGGTTCT GCTTCC (2) INFORMATION ABOUT SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (OR CHAIN FORM: individual (D) TOPOLOGY: linear (ii) TYPE OF THE MOLECULE: Peptide (ix) CHARACTERISTICS: (A) NAME / KEY: NLS of SV 40 (B) SITUATION: 1..7 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: Pro Lys Lys Lys Arg Lys Val 1 5 (2) INFORMATION ABOUT SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) CHAIN FORM: individual (D) TOPOLOGY: linear (ii) TYPE OF THE MOLECULE: DNA (ix) CHARACTERISTICS: (A) NAME / KEY: Sequence of union for Gal 4 (B) SITUATION: 1..16 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: CGGACAATGT TGACCG

Claims (26)

1. CLAIMS 1.- Cells for application in gene therapy, obtainable by a) isolation of non-adherent mononuclear cells obtained from blood or body fluids containing cells; b) cultivation of the cells obtained in step a) in a cell culture medium containing gangliosides, phospholipids, glycolipids and / or growth factors, growth factors for endothelial cells, including those factors that influence differentiation, survival , migration and / or vascularization; c) optionally, immortalization of the cells obtained in steps a) or b) by transformation with an oncogene, activation of an oncogene or inactivation of a suppressor gene; and d) optionally, transfecting the cells obtained in steps a) and b) or in step c) with an artificial nucleic acid structure for gene therapy, containing an effector gene, which can be activated by appropriate promoter systems specifically for target cells, specifically for cell cycles, specifically for viruses and / or hypoxia.
2. 2. Cells according to claim 1, the cells being positive for CD34, CD14, CDll, CDllb, CD13, CD64 or CD68 or endothelial cells.
3. 3. - Cells according to one of claims 1 or 2, these cells being characterized because they come from the blood in veins, capillaries, arteries, umbilical cord or placenta, bone marrow, spleen, lymph nodes, space peritoneal, pleural space, lymph, veins, arteries, capillaries and / or connective tissue fluid.
4. 4. - Cells according to claim 3, characterized in that the growth factor in step b) of claim 1 is selected from the group consisting of ECGF, FGF, FGF0, VEGF, ECAF, IGF-1; IGF-2; SC-3; EGF; SCF; TGF / 3, angiogenin, pleiotropin and ligand Flt-3.
5. 5. Cells according to one of claims 3 or 4, characterized in that the oncogene in step c) of the claim has been mutated in such a way that the gene product of this oncogene can still fully activate the cell cycle, but this activation of the Cell cycle can no longer be inhibited by cellular inhibitors.
6. 6. Cells according to claim 5, characterized in that the oncogene is selected from the group containing mutated cdk-4, cdk-6 and cdk-2.
7. 7. Cells according to claim 6, characterized in that the nucleotide sequence for cdk-4 has been mutated in position 24 in such a way that the properly encoded arginine has been changed by a cysteine.
8. 8. - Cells according to one of claims 3 or, characterized in that the inactivation of a suppressor gene according to step c) in claim 1 is achieved by transforming the cells with a nucleic acid sequence encoding a protein, which inactivates at least one gene product of the suppressor gene.
9. 9. Cells according to claim 8, characterized in that the inactivating protein of the gene product of the suppressor gene is selected from the group containing the E1A protein of the adenovirus, the E1B protein of the adenovirus, the large T antigen of the SV40 virus, the E6 protein of the papillomavirus, the E7 protein of the papillomavirus, the MDM-2 protein and a protein containing at least one amino acid sequence LXDLXXL-II-LXCXEXXXXXSDDE, where X means a variable amino acid and -II- means an arbitrary chain of amino acids of 7-80 amino acids.
10. 10. Cells according to one of claims 5 to 9, characterized in that the cell is an endothelial cell and the oncogene that has been used for the transformation or the nucleic acid sequence that has been used for the inactivation of the suppressor gene, has been linked to a specific activation sequence for the endothelium, which controls the transcription of the oncogene or of said nucleotide sequence.
11. 11. Cells according to one of claims 1 to 10, characterized in that the artificial nucleic acid structure in step d) of claim 1, • contains at least one activation sequence unlimitedly, a specific activation sequence for cells endothelial, virus-specific, a metabolically activatable activation sequence and / or an activation sequence specifically activatable for cell cycles; and • at least gene effector, whose expression is controlled by the activation sequence.
12. 12. Cells according to claim 11, characterized in that the expression of the effector gene is controlled by at least two identical or different activation sequences.
13. 13. Cells according to one of claims 11 or 12, characterized in that the activation of the activation sequence is self-reinforcing and / or pharmacologically controllable.
14. 14. Cells according to one of claims 12 or 13, characterized in that the second activator sequence is selected from the group containing sequences of virus promoters such as HBV, HCV, HSV, HPV, EBV, HTLV, CMV or HIV; sequences of promoters or intensifiers activated by hypoxia or specific activation sequences for cell cycles of the genes for cdc25C, cdc25B, cyclin A, cdc2, E2F-1, B-myb and DHFR; binding sequences for activation factors that appear or have been activated in a manner dependent on cell proliferation, such as monomers or multimers of the Myc E box.
15. 15. Cells according to one of claims 13 or 14, characterized in that in the case of of the effector gene is a gene, which codes for an active substance, which is selected from the group containing cytokines, chemokines, growth factors, receptors for cytokines, chemokines or growth factors, proteins that act in an antiproliferative way or either cytostatic or apoptotic, antibodies, antibody fragments, angiogenesis inhibitors, peptide hormones, coagulation factors, coagulation inhibitors, fibrinolytic proteins, peptides or proteins that act on blood circulation, blood plasma proteins and pathogen antigens of infections, either of cells or of tumors, producing the antigen chosen an immunological reaction.
16. 16. Cells according to one of claims 13 or 14, characterized in that in the case of the effector gene it is a gene encoding an enzyme, which dissociates a precursor compound of a drug in a drug.
17. 17.- Cells according to one of claims 13 or 14, characterized in that in the case of the effector gene it is a gene that encodes a fusion protein of a ligand and an active substance, or a fusion protein of a ligand and a enzyme, the ligand being selected from a group comprising cytokines, growth factors, antibodies, antibody fragments, peptide hormones, mediators, cell adhesion proteins and proteins that bind to LDL receptors.
18. 18. - Cells according to one of the preceding claims, characterized in that in the case of the artificial nucleic acid structure, introduced into the endothelial cell, it is a DNA.
19. 19. Cells according to claim 18, characterized in that the artificial nucleic acid structure is introduced into a vector.
20. 20. Cells according to claim 19, characterized in that it is a plasmid vector.
21. 21. Cells according to claim 19, characterized in that it is a viral vector.
22. 22. Cells according to claims 1 to 21, characterized in that they are administered externally, by the peroral, intravesical, nasal, intrabronchial routes or in the gastro-intestinal tract or within an organ, within a body cavity, in the musculature , subcutaneously or in the bloodstream, for the prophylaxis or therapy of a disease.
23. 23. - Use of a cell according to one of claims 1 to 22, for the preparation of a medicament for the treatment of a disease selected from the group consisting of tumors, leukemias, autoimmune diseases, allergies, arthritis, inflammations, rejections of organs, transplant reactions against host, blood coagulation diseases, diseases of the circulation, anemia, infections, hormonal diseases and injuries of the central nervous system.
24. 24. - Process for the production of a cell according to one of claims 1 to 22, characterized in that the following steps are carried out: a) isolation of cells from blood or body fluids containing cells; b) cultivation of the cells obtained in step a) in a culture medium containing gangliosides, phospholipids, glycolipids and / or growth factors; c) optionally, immortalization of the cells obtained in steps a) or b) by transformation with an oncogene, activation of an oncogene or inactivation of a suppressor gene; d) transfection of the cells obtained in steps a) and b) or in step c) with an artificial nucleic acid structure for gene therapy, containing an effector gene, which can be activated by appropriate promoter systems so specific for target cells, specifically for cell cycles, specifically for viruses and / or hypoxia.
25. 25. Drugs, which contain cells according to one of claims 1 to 22.
26. 26.- Cells obtainable according to claim 1 for the endothelialization of injured vessels.