MXPA98001957A - Promoter of the cdc25b gene, its preparation and - Google Patents

Abstract

The present invention relates to the promoter of the cdc25B gene, to a method for finding cdc25B promoters and to their use for preparing a pharmaceutical product.

Description

Promoter of the cdc25B gene, its preparation and use The invention relates to the promoter of the cdc25B gene, to a method for finding cdc25B promoters and to the use of the cdc25B promoter to prepare a pharmaceutical product. The division of the cell is subdivided into consecutive phases G0 or G1 # S, G2 and M. The S phase is the phase of DNA synthesis; it is followed by the transition phase G2 (phase G2) which, in turn, is followed by the mitosis phase (phase M), in which the progenitor cell divides into two daughter cells. The resting phase G0 (phase G0) or the transition phase Gx (phase G is located between the M phase and the S phase. Cell division is driven by a group of protein kinases, ie the cyclin / cdk complexes. These comprise a catalytic subunit [cyclin dependent kinase (cdk, eg cdkl, 2, 3, 4, 5, 6, 7 or 8) and a regulatory subunit, ie cyclin (eg cyclin A, B1-B3, D1 -D3, E, H or C.] The different complexes of cdk are particularly active in each phase of the cell cycle, for example the complexes of cdk cdk4 / cyclin Dl-3 and cdkd / cyclin Dl-3 in the middle phase Gl t the complex of cdk cdk2 / cyclin E in the tar phase Gl t the complex of cdk cdk2 / cyclin A in the S phase and the complexes of cdk cdkl / cyclin Bl-3 and cdkl / cilia A in the transition phase G2 / M The activity of cyclin / cdk complexes comprises phosphorylating and, consequently, activating or inactivating proteins that are directly or indirectly involved in the regulation of e DNA synthesis and mitosis. In correspondence with its function in the cell cycle, the genes for some cyclins and cdk's are periodically transcribed and / or periodically activated or inhibited, for example by means of the sonrolated degradation of cyclins, by means of the thickened binding of the cycle phase inhibitor cell (for example pl6IMK4A, pl5INK4A, p21Clpl, p27Kxpl, pl8INK4C, pl9INK D and P57) or by means of an activating modification (for example phosphatases cdc25 or cdk7 / cyclin H) or by inhibiting (for example ee 1 kinase) enzymes ( Reviews in Zwicker and Müller, Progr. Cell Cycle Res., 1, 91 (1995); La Thangue, Curr. Opin. Cell Biol., 6, 443 (1994), MacLachlan et al., Crit. Rev. Eukaryotic Gene Expr., 5, 127 (1995)). The higher eukaryotes possess at least three cdc25 phosphatases, namely cdc25A, cdc25B and cdc25C. The cDNAs of the genes for these phosphatases have already been cloned and analyzed (Okazaki et al., Gene 178, 111 (1996); Galaktiono et al., Cell 67, 1181 (199D) .The three phosphatases appear periodically in the However, the activating functions of these cdc25 phosphatases are clearly different (Jinno et al., EMBO J. 13, 1549 (1994), Honda et al., FEBS Lett. 318, 331 (1993), Hoffmann et al. ., EMBO J. 13, 4302 (1994)): cdc25A is predominantly expressed in the late phase G, and, in particular, regulates the transition from the Gt phase to the S phase (cell cycle initiation) by activating de-cdk complexes Cyclin is properly regulated by Myc (transcription) and Raf (activity), cds25B dephosphorylates tyrosine (tyrosine 14 and tyrosine 15) in the sdkl ATP binding pocket, thereby triggering its activation; from cyclin B (1-3) independently of cdkl, and its expression is deregulated and increases in cells infected with Rus (SV40 or PVH). sds25s dephosphorylates tyrosines (tyrosine 14 and tyrosine 15) in the ATP binding pocket of cdkl, thereby leading to its activation; it is expressed, in particular, in the G2 phase and regulates the entry into the M phase. The periodic expression of cdc25C in the G2 phase of the cell cycle is essentially regulated by an element (CDE-CHR) in the promoter region of cdc25C, element that is occupied by a repressor protein in the Go / G ^ phase and is free in the G2 phase. Even though the nucleotide sequence of this promoter element has been identified, it has also been found in promoters of the genes for cyclin A and cdkl, a nucleotide sequence (E2FBS-CHR) that differs to some extent has been detected in the promoter for Bmyb. The investigation of the cell cycle-dependent mode of operation of these promoter elements has shown that their blocking in the GQ / GL phase is followed by upregulation of the transcription of the relevant gene, supraregulation that takes place particularly at an early stage (in the Gí half phase) in the case of the B-myb gene, in the transition phase G / S in the case of cyclin A, in the S phase in the case of the cdkl gene, and only in the late phase S in the case of of the cdc25C gene (Zwicker and Müller, Progress in Cell Cycle Res. 1, 91 (1995); Lucibello et al., EMBO J. 14, 132 (1995); Liu et al., Nucí. Acids Res. 24, 2905 (1995), -Z icker et al., Nucí. Acids Res. 23, 3822 (1995); EMBO J. 14, 4514 (1995)). It has also been found that the CDE-CHR element (of the promoter for cyclin 25C, cyclin A and the cdkl gene) and the element E2FBS-CHR (of the promoter for the B-myb gene) are not only capable of inhibiting activation and the transcription of the homologous genes in the GQ / GL phase, but they are also capable of inhibiting the activation and transcription of other genes (see, for example, WO96 / 06943, DE19605274.2, DE19617851.7, WO96 / 06940, O96 / 06938, O96 / 06941 and O96 / 06939). These patent applications describe the combination of a cell cycle-dependent promoter with a non-specific, cell-specific, virus-specific or metabolically activatable promoter in order to activate the transcription of an effector gene that encodes a protein for prophylaxis and / or therapy of a disease, in a regulated manner. Diseases of this type can be, for example, tumor diseases, leukemias, autoimmune diseases, arthritis, allergies, inflammasiones, reshaping of transplanted organs, diseases of the blood syndromic system or of the blood coagulation system, or infections or lesions of the central nervous system. . The so-called chimeric promoter is a particular example of this possibility of combining different promoters with an element of the cell cycle specific promoter. In this chimeric promoter, the activity of a non-specific, cell-specific, virus-specific or metabolically activatable activating sequence (or promoter sequence) is largely restricted to the S and G2 phases of the cell cycle by the cell element. promoter CDE-CHR or E2FBS-CRH that immediately binds to it more ab jo. Subsequent investigations on the mode of action of the CDE-CHR promoter element revealed, in particular, that the cell cycle-dependent regulation by the CDE-CHR element of a trigger sequence located above depends, to a large extent, on whether the Activation sequence is activated by transcription factors that have activation domains rich in glutamine (Zicker et al., Nucí Acids Res. 23, 3822 (1995)). Examples of these transcription factors are Spl and NF-Y This therefore limits the use of the CDE-CHR promoter element for chimeric promoters. The same must be assumed to be true for the E2F-BS-CHB promoter element of the B-myb gene (Zwicker et al., Nucí Acids Res. 23, 3822 (1995)). Therefore, the object of the present invention is to find cell-cycle-specific promoters and promoter elements whose G0-specific and G_-specific repression depends on circumstances other than that under which the CDE-CHR promoter element is subjected. When the nucleotide sequence of the promoter of the urinary cdc25B gene and the nucleotide sequence of the 5 'non-coding region located immediately below were analyzed, including the initiation (or starting) region of the cdc25B gene (nucleotide sequence -950 to + 167), it was found that the functional regions of the cdc25B promoter sequence contain two E boxes, two E2F binding sites (putative), four SP1 binding sites (putative), an NF-Y binding site (putative ) and a TATA box. Surprisingly, it was not possible to find any nucleotide sequence with a homology with CDE-CHR or E2FBS-CHR. Accordingly, the functional regions of the cdc25B promoter sequence are clearly different from the functional regions of the cdc25C promoter, cyclin A, cdkl gene and B-myb gene. Furthermore, it is surprising that until now there is no report of a promoter of the cell cycle gene that contains a functional TATA box. Therefore, the present invention relates to the promoter of the cdc25B gene, a promoter that contains a sequence that is inhibited, under stringent conditions, with a sequence, as described in Table 1 (SEQ ID No: 7) or a functional part thereof, in particular to the promoter having the sequence described in Table 1 (SEQ ID No: 7) or a functional part thereof. The entire sequence, or fragments, of the cdc25B promoter were cloned into a plasmid located above a luciferase gene, and these plasmids were transfused into resting and proliferating fibroblasts, mouse or human, and the amount of lusiferase expressed was measured. . It was found that (as opposed to the position in proliferating cells) the cloned cdc25B sequence (promoter and 5 'non-coding region, approximately -950 to approximately +167) led to a strong suppression of luciferase gene expression in resting cells. , this deletion being reduced in a staggered manner by deletions at the 5 'end of the cdc25B promoter (from about -950 to about +167 to about -30 to about +167). Deletion fragments that were less than about -180 to about +167 led to the promoter activity also being reduced in proliferating cells.

Within the meaning of the present invention, the functional parts are to be understood, therefore, to be, in particular, the transcription factor binding sites detailed above, especially when they encompass more than about 50% of the entire promoter. Particular preference is given to the functional parts containing the TATA box, at least one SP1 binding site, at least one NFY binding site and, if appropriate, at least one E2F binding site and, when appropriate, at least one box E, so that it is possible to achieve an expression, dependent on the cell cycle, of an effector gene. These promoter sequences include, in particular, promoter sequences of the murine cdc25B gene, - however, they also include the promoter sequences of the human cdc25B gene. Other preferred parts of the new promoter are, according to Table 1, the nucleotides from about -950 to about +167, from about -950 to about +3, from about -930 to about +3, from about -720 to about +3, from about -340 to about +3, from about -180 to about +3, from about -100 to about +3, from about -80 to about +3, from about -60 to about +3 or from about -30 to about +3, and also parts of they contain the corresponding cis-functional regulatory elements according to Figure 6, in particular 5 'deletions and / or 3' deletions. The present invention also relates to a method for finding cdc25B promoters, wherein a new promoter or a part thereof is labeled, preferably radioactively labeled, and genomic DNA libraries, preferably from mammals, are they track by means of hybridization under stringent conditions. The person skilled in the art is familiar, for example, from Sambrook, J. et al. (1989) Molecular Cloning. A laboratory manual, Cold Spring Harbor Laboratory, New York, preparation of genomic DNA libraries and hybridization under stringent conditions. Hybridization conditions can be optimized, for example, as described by Szostak, J.W. et al. (1979) Hybridization with synthetic oligonucleotides, Methods in Enzymol. 68, 419-482. For example, in order to isolate the murine cdc25B promoter, a murine genomic phage library that, for example, was obtained from the mouse strain 129 FVJ, Stratagene, can be screened with a probe that contains a part of the sequence described in Table 1 (SEQ ID NO: 7), preferably containing the sequence SEQ ID NO: 4. The present invention further relates to a nucleic acid construct containing at least one new promoter. Preferably, the sequence of the new promoter of the cdc25B gene is combined with a structural gene, i.e. in general with a gene encoding a protein or an RNA in the form of an active compound. In the simplest case, this combination may constitute a nucleic acid construct containing the nucleotide sequence of the new promoter for the cdc25B gene and a structural gene, the promoter activating transcription of the structural gene, preferably in a cell cycle dependent manner. . The new promoter is preferably disposed above the structural gene. In another preferred embodiment, the 5 'non-coding region of the cdc25B gene (nucleotide sense from +1 to approximately +167) is inserted between the new promoter and the strutural gene. In another preferred embodiment, the new promoter is combined with at least one additional non-specific, virus-specific, metabolically specific, cell-specific, cycle-specific and / or cell proliferation-dependent activation sequence in order to regulate the expression of a strutural gene. Examples are promoters that are activated in endothelial cells, perito-neal cells, pleural cells, epithelial cells of the skin, lung cells, cells of the gastrointestinal tract, kidney cells and drainage routes of urine, muscle cells, tissue cells sonjuntivo , hematopo-yétisas cells, macrophages, lymphocytes, 'leukemia cells, tumor cells or glia cells; viral promoter sesuensias, such as HBV, HSV, HPV, EBV, HTLV, CMV or HIV; promoter or enhancer sequences that are activated by hypoxia, cell cycle-specific activation sequences of the genes encoding cdc25C, cyclin A, cdc2, E2F-1, B-myb and DHFR, and / or binding sequences, such as monomers or Myc E box multimers, for transcription factors that appear or are activated in a manner dependent on cell proliferation. • Various techniques can be used to combine the new promoter with at least one additional promoter. These techniques are described, for example, in DE19617851.7, DE19639103.2 and DE19651443.6. In another preferred embodiment of this invention, a nucleic acid construct is selected for the combination of the new promoter with at least one additional promoter or enhancer that contains the new promoter in a form in which it is mutated in at least one binding site for a transcription factor. This mutation blocks the initiation of transcription of the structural gene. Other components of the nucleic acid construct are, when appropriate, the structural gene, at least one additional promoter sequence or boost sequencer that can be engineered in a non-specific, cell-specific or virus-specific manner, by part of tetracycline and / or in a specific manner of the cell cycle, and which activates the transcription of at least one additional structural gene encoding at least one transcription factor that is mutated, so that it binds to the imitated binding site (s) of the new promoter and activates this promoter, and / or the structural gene that encodes a transcription factor.

The arrangement of the individual components is represented, by way of example, by the diagram of Figure 1. In an exemplary embodiment of this invention, the mutation may be a mutation of the TATA box of the new pro-motor. The TATA box (TATAAA or TATATAA) is recognized as a binding site for the initiation complex of the RNA-po-limerases II and III which are present in the nucleus of the cell. The initiation of transcription, approximately 30 bases below the TATA box, is effected by binding the TATA box binding protein (TBP) that is involved in the transcription reaction of all RNA polymerases (I, II and III) that are present in the nucleus of the cell. An example of a promoter strictly dependent on the TATA box is the promoter for the U6 gene, which is transcribed by RNA polymerase III and whose gene product is involved in mRNA splicing. An example of a mutation of the sequence of the TATA box can be TGTATAA. As a result of this mutation, the DNA binding site of normal TBP is no longer recognized and the coding gene is not transcribed more efficiently. In the case of a mutation of this type, the gene encoding the transcription factor is a nucleic acid sequence encoding a commutated TBP. As a result of this connection, TBP binds to the mutated TATA box (for example to TGTATAA) and, thereby, leads to efficient transcription of the structural gene. Commanders of this type of the TBP gene have been described, for example, by Strubin and Struhl (Cell, 68, 721 (1992)) and by Heard et al. (EMBO J., 12, 3519 (1993)). A particularly preferred embodiment is a construction of a robust nusleiso, comprising (1) the new promoter of the TATA box including the cdc25B gene, the sequence of the TATA box being mutated in TGTA, (2) the GCCACC sequence, ( 3) the cDNA for the immunoglobulin signal peptide (nucleotide sequence = 63 a = 107), (4) the cDNA for the immunoglobulin signal peptide (nucleotide sequence = 93 a = 1982), (5) the gene promoter vWF (nucleotide sequence -487 to +247), and (6) the cDNA for the TATA box binding protein (nucleic acid sequence from 1 to 1001, which is mutated at the positions of nucleic acid 862 (A replaced by T), 889 and 890 (GT replaced by AC) and 895 (C replaced by G)). In another preferred embodiment of this invention, a nucleic acid construct is selected for the combination of the new promoter with at least one additional promoter which is referred to as a multiple promoter having a nuclear retention signal and an export factor and comprising the following components: a) a first sequence (I) of promoter or non-specific promoter, specific of the cell or specific of the virus that can be activated metabolically and / or in a specific way of the cell cycle that activates the basal transcription of a structural gene, b ) a structural gene, c) a nuclear retention signal (NRS), whose cDNA is linked, directly or indirectly, at its 5 'end, to the 3' end of the structural gene, preferably having the transcription product of the signal of nuclear retention a structure for joining a nuclear export factor, d) an additional promoter or reinforcer sequence (II), which activates the trans basal rate of a nuclear export factor (NEF), and e) a nucleic acid encoding a nuclear export factor (NEF) that binds to the transcription product of the nuclear retention signal and, therefore, intervenes in the transport of the nuclear Transcription product of the structural gene outside the nucleus of the cell. Within the meaning of this invention, at least one of the components of the promoter constitutes the new promoter. The first (I) promoter or enhancer sequence (a) and the second (II) promoter or enhancer sequence (d) may be identical or different and, when appropriate, may be non-specifically activatable, specifically of the cell, in a virus-specific manner or metabolically, in particular by hypoxia, or constitute a specific promoter of the additional cellular cislo. The arrangement of the individual components is represented, for example, in Figure 2. In the new nucleic acid constructs, components d) and e) can be located higher or lower than components a), b) and c) ( see also figure 2). Preferably, the gene encoding the nuclear retention signal (NRS) is selected from the element responsive to rev (RRE) of HIV-1 or HIV-2, the retention signal equivalent to RRE of retrovirus or the retention signal equivalent to RRE of HBV. The gene encoding the nuclear export factor (NEF) is preferably a gene that is selected from the rev gene of the HIV-1 or HIV-2 viruses, visna-maedi virus, caprine arthritis-encephalitis virus, anemia virus infectious equine, feline immunodeficiency virus, retrovirus or HTLV, the hnRNP-Al protein encoding the gene or the transcription factor TFIII-A encoding the gene. In another preferred embodiment, at least one promoter or enhancer sequence (component a) or d)) in the new nucleic acid constructs is a construct of a gene that is referred to as a promoter unit that responds to the activator and preferably comprises the following somponents: f) one or more identical or different promoter or enhancer sequences that can be activated, for example, in a cell-cycle-specific manner, in a cell-dependent manner, metabolically, in a cell-specific manner or in a cell-specific manner; virus-specific form, either specifically cell-cycle-like or metabolically, cell-specific or virus-specific form (so-called chimeric promoters), g) one or more identical or different activator subunits that are in each case located below the promoter or enhancer sequences and whose basal transcription is activated by these sequences, h) u n promoter that responds to the activator that is activated by the expression products of one or more activator subunits. The arrangement of the individual components of a promoter unit responsive to the preferred promoter is illustrated in Figure 3. The insertion of a promoter unit that responds to the preferred activator in a new nucleic acid construct is illustrated, for example, in Figure 4. In these promoter units that respond to the activator, which are exemplified in Figures 3 and 4, at least one promoter (I, II, III or IV) can constitute the new promoter. In a preferred embodiment, the promoter units responsive to the activator can form binding sequences for chimeric transcription factors that are comprised of DNA binding domains, protein / protein interaction domains and transactivation domains. All binding sites of the transcription phantom that are mentioned in the present invention can be present once (monomers) or in several copies (multimers, for example up to about 10 copies). The operator LexA in combination with the SV40 promoter is an example of a promoter that responds to the activator (h) which is activated by two activator subunits (g and g '). This promoter contains, for example, the following activator subunits: (1) the first activator subunit (g) contains the cDNA encoding amino acids 1-81 or 1-202 of the LexA DNA binding protein, the end of which 3 'is linked to the 5' end of the cDNA encoding the Gal80 protein (amino acids 1-435), and (2) the second activator subunit (g ') contains the cDNA encoding the Gal80 binding domain of the Gal4 protein encoding the aminoasides 851-881, whose 3 'end is linked to the 5' end of the cDNA encoding amino acids 126-132 of the large SV40 T antigen, whose 3 'end is linked to the 5' end of the cDNA encoding the amino acids 406-488 of the VP16 transactivation domain of HSV-1. The binding sequence for the Gal4 protein in combination with the SV40 promoter is another example of a promoter that responds to the activator that is activated by two activator subunits (g and g '). This promoter contains, for example, the following activator subunits: (1) the first activator subunit (g) contains the cDNA encoding the DNA binding domain of the Gal4 protein (aminoasides 1-147), its 3 'end is linked to the 5 'end of the cDNA for the Gal80 protein (amino acids 1-435), and (2) the second activator subunit (g') contains the cDNA encoding the Gal80 binding domain of Gal4 (amino acids 851 to 881), whose 3 'end is linked to the 5' end of the cDNA encoding the SV40 nuclear localization signal (SV40 large T, amino acids 126- -132), whose 3 'end is linked to the 5' end of the cDNA encoding amino acids 406 -488 of the VP16 transactivation domain of HSV-l. Another example of two activator subunits (g and g ') that activate the promoter that responds to the activator that contains the sequence to bind the Gal4 protein and the SV40 promoter is (1) the activation unit (g) that contains the DNA encoding the cytoplasmic domain of the CD4 antigen of T cells (amino acids 397-435), whose 5 'end is linked to the 3' end of the cDNA for the transactivation domain VP16 of HSV-1 (amino acids 406-488), whose 5 'end is linked, in turn, to the 3 'end of the cDNA for the SV40 nuclear localization signal (SV40 large T, amino acids 16-132), and (2) the activation unit (g') containing the cDNA encoding the SV40 nuslear localization signal (SV40 large T, amino acids 126-132), and 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 p56 Ick protein (amino acids 1-71). Therefore, a preferred embodiment is a nucleic acid construct that contains (1) one or more identical or different activator subunits, whose basal transcription is activated by a promoter or enhancer, and (2) a promoter that responds to the activator that is activated by the expression product of said activator subunit, and a particularly preferred embodiment is a nucleic acid construct that contains, as activator subunit (A), (1) the new promoter, (2) the signal from Nuclear localization (NLS) of SV40 (SV40 large T, amino acids 126-132; PKKKRKV), (3) the acid-binding transactivation domain (TAD) VP16 of HSV-1 (amino acids 406 to 488), and (4) the cDNA encoding the cytoplasmic part of the CD4 glycoprotein (amino acids 397-435); and, as another activator subunit (B), (1) the promoter of the cdc25C gene (nucleic acids -290 to (2) the SV40 nuclear localization signal (NLS) (SV40 large T, amino acids 126-132, PKKKRKV ), (3) the cDNA for the DNA binding domain of the Gal4 protein (amino acids 1 to 147), and (4) the cDNA for the CD4 binding sequence of the p56 Ick protein (amino acids 1-71) and also the promoter responsive to the activator containing up to about 10 copies of the binding sequence for the Gal4 binding protein, which has the nucleotide sequence 5 '-CGGACAATGTTGACCG-3', and the SV40 promoter basal (sequence of nucleotides 48 to 5191) and, if appropriate, a structural gene, preferably a complete cDNA encoding an active compound, an enzyme or a fusion protein that is constituted by a ligand and an active compound or by a ligand and an enzyme As a rule, the structural gene is a gene that encodes a pharmacologically compound active preferably selected from enzymes, fusion proteins, cytokines, chemokines, growth factors, receptors for cytokines, receptors for chemokines, receptors for growth factors, peptides or proteins with an antiproliferative or cytostatic or apoptotic effect, antibodies or fragments of antibodies, inhibitors of angiogenesis, hormones pep-tídisas, factors of coagulation, inhibitors of coagulation, peptides or fibrinolytic proteins, peptides or proteins that have an efesto on blood circulation, proteins of blood plasma, antigens of infectious agents, such as bacterial antigens and parasite antigens, cell antigens or tumor antigens, carrying out the antigen an immune action, thrombosis inducing substances, complement activating proteins, virus coating proteins and / or ribozymes. In the case of a ribozyme, the structural gene is preferably a gene encoding a ribozyme that inactivates mRNA that encodes a protein that is selected from proteins for cell cycle control, in particular, cyclin A, cyclin B, cyclin DI, cyclin E, E2F1-5, cdc2, cdc25C or DPI, virus proteins, cytokines, growth factors or their receptors. In another preferred embodiment, the structural gene may be a gene encoding an enzyme that is a precursor of a drug in a drug. In another preferred embodiment, the structural gene can encode a ligand / effector fusion protein, it being possible for the ligand to be an antibody, an antibody fragment, a cytokine, a growth factor, an adhesion molecule or a peptide hormone, and the effector a pharmacologically active compound as described above, or an enzyme. For example, the structural gene can encode a ligand / enzyme fusion protein, the enzyme cleaving a precursor of a drug in a drug and binding the ligand to the surface of the cell, preferably to endothelial cells or tumor cells. The nucleic acid constructs are preferably constituted by DNA. The term "nucleic acid constructs" is to be understood in general as artificial scaffolds consisting of nucleic acids that can be transcribed in the target cells. Preferably, they are inserted into a vector, with plasmid vectors or viral vectors being particularly preferred. In general, these vectors are administered to patients externally or internally, locally, orally, intravesically, nasally, intrabronchially, intramuscularly, subcutaneously in a body cavity, in an organ, in the blood circulation, in the respiratory tract, in the gastrointestinal tract and / or in the urogenital tract, and are used for the prophylaxis or therapy of a disease. Using the new nucleic acid constructs, a structural gene can be expressed in a cell-specific manner, in a manner specific to the virus, under designated metabolic conditions and / or in a cell cycle-specific manner, the structural gene preferably being a gene encoding a pharmacologically active compound or an enzyme that cleaves an inactive precursor of a drug in an active drug. The structural gene can be selected such that the pharmacologically active compound or the enzyme is expressed together with a ligand as a fusion protein, and this ligand binds to the cell surface, for example proliferating endothelial cells or tumor cells. The present invention also relates to a method for preparing a novel nucleic acid construct, in which the individual components of the nucleic acid construct are connected to each other. The connection of the individual components can be carried out using generally known methods, for example enzymatically or using ligases. The present invention further relates to cells, in particular yeast or mammalian cells, which harbor a novel nucleic acid construct. In a particularly preferred embodiment, the nucleic acid constructs are introduced into the cell lines that can be used, after transfection, to express the transgene. Accordingly, these cells can be used to provide a drug for patients. A preferred use of the new nucleic acid construct comprises treating a disease, with the provision of the drug comprising introducing a nucleic acid construct into a target cell and expressing the construct in a virus-specific manner or specific to the target or specific cell. in a metabolic or non-specific and specific way of the cell cycle. In general, the administration is carried out precisely as in the case of the new nucleic acid constructs and is also used for the prophylaxis or therapy of diseases. In order to prepare a drug, the endothelial cells can be isolated, for example, from the blood and transfected in vitro with the new nucleic acid construct, after they are reinjected into the patient, for example intravenously. Cells of this type that have been transfected in vitro can also be administered to patients in comination with a new vector. This combination has the advantage that the cells and the vectors can be administered or injected in each case simultaneously or at different times, in the same or different places. Therefore, the present invention also relates to the use of a novel nucleic acid construct or of a new cell to prepare a drug for the treatment of a disease that is selected from tumor diseases, leukemias, autoimmune diseases, allergies, arthritis, inflammations, organ rejections, graft reactions to host, diseases of the blood circulation, circulatory diseases, anemia, infections, hormonal diseases and / or damage to the central nervous system. Particular preference is given to using an endothelial cell to prepare a new drug. The new nucleic acid constructions do not occur in this way in nature, ie the structural gene does not combine naturally with the new promoter. The application in each case determines the choice of the promoters and the structural gene. The following examples serve as a guide in this regard. WO96 / 06940, WO96 / 06938, WO96 / 06941, WO96 / 06939, DE19605274.2, DE19617851.7, DE19639103.2 and DE19651443.6 provide a detailed breakdown of the individual somponents.

I) Promoter sequences Within the meaning of the present invention, nucleotide sequences which, in general, after binding transcription factors, activate the transcription of a structural gene that binds to the 3 'end have to be used as promoter sequences [components a ), c), f) or f ')] • The choice of promoter sequence (s) to be combined with the cdc25B promoter depends on the disease to be treated and on the target cell to be transduced. Thus, it is possible that the additional promoter sequence is unlimitedly scalable, in a manner specific to the target cell, under defined metabolic conditions, in a specific manner of the cell cycle or in a specific manner of the virus. In addition, identical or different promoter sequences can be used in components a), c), f) and / or f '). Examples of the promoter sequences to be selected are promoter and activator sequences that can be activated in an unlimited manner, such as the RNA polymerase III promoter, the RNA polymerase II promoter, the CMV promoter and the enhancer. of CMV, or the SV40 promoter; promoter sequences and viral activator sequences, such as those of HBV, HCV, HSV, HPV, EBV, HTLV or HIV. When using the HIV promoter, preference is given to using the complete LTR sequence including the TAR sequence [position -453 to -80, Rosen et al., Cell 41, 813 (1985)] as a virus-specific promoter. . Promoter sequences also include metabolically activatable promoter and enhancer sequences, such as the hypoxia-inducible enhancer (Semenza et al., PNAS 88, 5680 (1991); McBurney et al., Nuci. Acids Res. , 5755 (1991)]; cellular-specific promoters, such as the promoter of the cdc25C gene, the cislina A gene, the cdc2 gene, the B-myb gene, the DHFR gene or the E2F-1 gene, or binding sequences for transcription factors that appear or are activated during cell proliferation, such as binding sequences for c-myc proteins, including these binding sequences monomers or multimers of the nucleotide sequence that is designated the Myc E box [5'-GGAAGCAGACCACGTGGTCTGCTTCC-3 '; Blackwood and Eisenmann, Sciense 251, 1211, (1991)]; promoters astivable by tetra-cyclin, such as the tetracycline operator in combination with an appropriate repressor, - chimeric promoters that constitute a combination of an activator sequence located above that can be activated in a cell-specific manner, metabolically or in a virus-specific manner, with a downstream promoter module containing, for example, the nucleotide sequence CDE-CHR or E2FBS-CHR, to which suppressor proteins bind and, thereby, are capable of inhibiting activation of the activator sequence located higher in the G0 phase and in the G2 phase of the cell cycle (WO96 / 06943; Lucibello et al., EMBO J. 14, 12 (1994)); promoters that can be activated in a cell-specific manner, such as promoter or activator sequences from promoters or enhancers of the genes that encode proteins that are preferably formed in the selected cells. Examples of cell-specific promoter promoters are promoter and activator sequences that are activated in endothelial cells, such as the promoter and activator sequences of the genes encoding the brain-specific endothelial glucose-1 transporter, endoglin, VEGF receptor 1 (flt-1), VEGF receptor 2 (flk-1 or KDR), til-1 or til-2, B61 receptor (Esk receptor), B61, endothelin, especially endothelin B or endothelium 1 , endothelin resepters, in particular the endothelin B receptor, mannose 6-phosphate receptors, von Willebrand factor, IL-la receptor, IL-1/3, IL-1, vascular cell adhesion molecule (VCAM-1) or synthetic activator sequences comprising oligomerized sites to bind transcription factors that are preferentially or selectively active, for example endothelial cells. An example is the transcription factor GATA 2, whose binding site in the endothelin 1 gene is 5'-TTATCT-3 '[Lee et al., Biol. Chem. 266, 16188 (1991), Dor ann et al., J. Biol. Chem. 267, 1279 (1992) and Wilson et al., Mol. Cell Biol. 10, 4854 (1990)]. Additional cell-specific activatable promoters are promoter or astivator sequences which are activated in cells in the vicinity of activated endothelial cells, such as the promoter and activator sequences of the genes encoding VEGF, the gene regulatory sequences being the VEGF gene the 5 '-flanking region, the 3' -flanking region, the c-Src gene or the v-Src gene, or steroid hormone receptors and their promoter elements (Truss and Beato, Endocr. Rev. 14, 459 (1993)), in particular the promoter of the mammary tumor virus in mice. Examples of promoter or activator sequences that are activated in muscle cells, in particular in smooth muscle cells, are promoter sequences and activator of the genes encoding tropomyosin, α-actin, α-myosin, receptor PDGF, FGF receptor, MRF-4, phosphofructokinase A, phosphoglycerate mutase, troponin C, myogenin, endothelin A receptors, desmin, VEGF, the gene regulatory sequences for the VEGF gene, or promoters, have already been described. artificial ". Examples of artificial promoters of this type are multiple copies of the binding site (to DNA) for muscle-specific helix-loop-helix (HLH) proteins, such as box E (Myo D) (eg 4x AGCAGGTGTTGGGAGGC, SEQ ID N0.:1) or multiple copies of the GATA 4 binding site of the zinc finger protein of the a-myosin heavy chain gene DNA (for example 5 '-GGCCGATGGGCAGATAGAGGGGGCCGATGGGCAGATAGAGG3', SEQ ID NO .: 2) . Examples of HLH proteins are MyoD, Myf-5, myogenin or MRF4. The HLH proteins and, also, GATA 4, exhibit muscle-specific transcription not only with promoters of muscle-specific genes, but also in a heterologous context, that is also with artificial promoters. The promoter and activator sequences that are activated in glia cells are, in part, the sequences or regulatory elements of genes that, for example, encode the following proteins: the specific protein of Schwann cells, periaxin, glutamine synthetase, the protein specific glia cells (glial fibrillary acidic protein = GFAP), glia cell protein SlOOb, IL-6 (CNTF), receptors 5-HT, TNF?!, IL-10, insulin-like growth factor receptor 1 and II or VEGF, the gene regulatory sequences for the VEGF gene having already been listed. The promoter and activator sequences that are activated in hematopoietic cells are promoter sequences for genes for a cytokine or its receptor that are expressed in hematopoietic cells or in adjacent cells, such as the stroma. These promoter sequences include, for example, promoter sequences for the following cytokines and their receptors: stem cell factor receptor, stem cell factor, IL-Ia, IL-1 receptor, IL-3 receptor IL-3 (ot subunit), IL-3 receptor (β subunit), IL-6, IL-6 receptor, GM-CSF, GM-CSF receptor (a chain), interferon-1 regulatory factor (IRF- 1), the promoter of IRF-1 being activated in equal measure by IL-6 and by IFN? or IFN3, erythropoietin or erythropoietin receptor. Examples of promoter and activator sequences that are activated in lymphocytes and / or macrophages are the promoter and gene activator sequences for cytokines, cytokine receptors and adhesion molecules and receptors for the Fc fragment of antibodies. Examples are the promoter sequences for the following proteins: IL-1 receptor, IL-lar, IL-1/3, IL-2, IL-2 receptor, IL-3, IL-3 receptor (subunit a), IL-3 receptor (subunit β), IL-4, receptor of IL-4, IL-5, IL-6, IL-6 receptor, interferon-1 regulatory factor (IRF-1), with the IRF-1 promoter being activated in equal measure by IL-6 as part of of IFN? or IFNJ, promoter that responds to IFN ?, IL-7, IL-8, IL-10, IL-11, IFN ?, GM-CSF, GM-GSF receptor (a chain), IL-13, LIF, receptor of the macrophage colony stimulating factor (M-CSF), type I and type II macrophage purifying receptors, MAC-1 (leukocyte function antigen), LFA-la (leukocyte function antigen) or p50.96 (antigen of the leukocyte function). The promoter and activator sequences that are activated in synovial cells are, for example, the promoter sequences for matrix metalloproteinases (MMP), for example for MMP1 (interstitial collagenase) or MMP3 (strome-lysine / transin). In addition, they include promoter sequences for metalloproteinase (TIMP) tissue inhibitors, for example TIMP-1, TIMP-2 or TIMP-3. Examples of promoter and activator sequences that are activated in leukemia cells are promoters for c-myc, HSP70, bcl-1 / cyclin DI, bsl-2, IL-6, IL-10, TFNa, TNF / 3, HOX11, BCR-Abl, E2A-PBX1, PML-RARA (promyelocytic leukemia retinoic acid receptor) or c-myc, the c-myc proteins being joined to multimers of the nucleotide sequence called a Myc E saga (5 '-GGAAGCAGACCAGCTGGTCTGCTTCC-3 ', SEQ ID NO .: 3) and activating them. An example of a promoter or activator sequence that is activated in tumor cells is a gene regulatory nucleotide sequence with which transcription factors that are formed or active in tumor cells interact. Within the meaning of this invention, preferred promoter or activator sequences include geneside regulators or elements from genes that, in particular, encode proteins that are formed in cancer cells or sarcoma cells. Thus, preference is given to using the promoter of the N-CAM protein in the case of small cell bronchial carcinomas, to using the promoter of the recipient of the growth factor of hepatitis or of L-plastin in the case of ovarian carcinomas. and to use the promoter of L-plastin or polymorphic epithelial mucin (PEM) in the case of pancreatic carcinomas.

II) Nuclear export signals and nuclear export factors In a preferred embodiment, the nuclear retention signal (NRS) is a nucleotide sequence that prevents the transport of a pre-messenger RNA, which is linked thereto, through the membrane nuclear, but that, on the other hand, also constitutes a structure to join an export protein. This export protein intervenes in the transport of a pre-messenger or messenger RNA containing NRS outside the nucleus of the cell to the interior of the cytoplasm. A pre-messenger RNA containing the NRS is therefore secreted outside the nucleus of the cell when bound to the export protein (Fischer et al., Cell, 82, 475 (1995)). The nuclear export signals (NES) are preferably the sequence of the re-troviral response element (RRE). In the case of HIV-1, this RRE is a sequence in the env gene that spans 243 nucleotides (nucleotides 7362--7595). However, the nuclear export signal (NES) may also be any homologous and / or functionally similar (analogous) nucleotide sequence, such as the RRE equivalent element of the HBV virus (Huang et al., Mol. Cell Biol., 13, 7476 (1993)). In new nucleic acid constructs, the nuclear export factor (NEF) is a nucleotide sequence that encodes a protein that binds to the NRS mRNA and intervenes in the transport of pre-messenger RNA or messenger RNA containing NRS outside the nucleus. of the cell and to the interior of the cytoplasm (or outside the cytoplasm and to the interior of the nucleus of the cell). In particular, the rev gene from retroviruses is used, especially the HIV-1 or HIV-2 virus. The rev protein from the retroviral rev gene is linked by its N-merminal domain to the RRE in the pre-mRNA. The binding between the RRE and the rev protein allows the unseparated pre-messenger RNA, and also any other RNA containing an RRE, to be transported outside the nucleus of the cell and into the cytoplasm and, thereby, substantially increase translation.

Within the meaning of the present invention, nucleotide sequences encoding proteins that are homologous and functionally similar to the HIV-1 rev protein (Bogerd et al., Cell, 82, 485 (1995)), such as the rev gene of the visna-maedi virus (VMV) or the rev gene of caprine arthritis-encephalitis virus (CAEV) can also be used as NEF's. However, genes encoding proteins can also be used which, even when they only have a slight homology or no homology with the rev protein, are functionally similar to the rev protein of HIV-1. Examples are the rev gene of HTLV-1 and the rev genes of equine infectious anemia virus (EIAV) and feline immunodeficiency virus (FIV). In an alternative embodiment, the NEF's can also be nucleotide sequences for proteins that carry out the secretion of RNA outside the nucleus, without this RNA being retained in the nucleus by means of an NRS. Examples of these proteins are the TFIIIA transcription factor or the heterogeneous nuclear ribonuclear protein Al (hnRNPAl protein). In a broader sense, nuclear transport proteins also include heat shock protein 70 (hsc70) and the protein kinase inhibitor CPKI. Characteristics shared in common by the NEF and its homologous and analogous proteins are a domain that is further forward of the amino terminus, the monomeric protein being linked to the NRS RNA, and a domain that is usually rich in leucine (hnRNPAl). it is an exseption of it) and that is required for the transport function of the NEF. Within the meaning of this invention, the expression of the NEF gene is under the control of a sequencer of promoter that is located upstream at the 5 'end of the NEF gene, as already described above in detail.

III) Structural genes Within the meaning of the invention, the structural genes [component b)] encode an active compound for the prophylaxis and / or therapy of a disease. Structural genes and promoter sequences have to be selected with respect to the nature of disease therapy and taking into consideration the target cell to be transduced. For example, the following combinations of promoter sequences and structural genes are to be selected in association with the following diseases. A detailed description has already been given in patent applications WO96 / 06940, DE19605274.2, DE19617851.7, DE19639103.2 and DE19651443.6, which are incorporated herein by reference. Examples of target cells that are selected for tumor therapy are: proliferating endothelial cells, stromal cells and muscle cells that bind to the endothelial cell, or tumor cells or leukemia cells. Promoters are endothelial cell thickeners and cell-system-specific or non-cell-specific or muscle-cell-specific and cell-cycle specific or tumor cell-specific (solid tumors and leukemias) and cell cycle-specific. When selecting structural genes for inhibitors of cell proliferation, for example for the retinoblastoma protein (pRb = pllO) or the related pl07 and pl30 proteins, the following strategy can be chosen: The retinoblastoma protein (pRb / pllO) and the related pl07 and pl30 proteins are inactivated by phosphorylation. Preference is given to using the genes of these cell cycle inhibitors that exhibit mutations for the sites of inactivation of the expressed proteins, without thereby impairing their function. Examples of these mutations have been described for pllO. The DNA sense for the pl07 protein or the pl30 protein can be mutated in an analogous manner. The p53 protein is another inhibitor of cell proliferation. The p53 protein is inactivated in the cell by binding to special proteins, such as MDM2, or by oligomerization of p53 by dephosphorylated C serine-C. Accordingly, preference is given to using a DNA sequence for a p53 protein that has been truncated by the loss of serine 392 at the C-terminus. Other inhibitors are p21 (WAF-1), the p6 protein, other cdk inhibitors, the GADD45 protein or the bak protein. Structural genes for coagulation-inducing factors and inhibitors of angiogenesis encode, for example, the plasminogen activator inhibitor l (PAI-1), PAI-2, PAI-3, angiostatin, interferons (IFNo;, IFNß or IFN), platelet factor 4, IL-12, TIMP-1, TIMP-2, TIMP-3, leukemia inhibitory factor (LIF) or tissue factor (TF) and its fragments that are active in the coagulation. Structural genes for cytostatic and cytotoxic proteins encode, for example, perforin, granzyme, IL-2, IL-4, IL-12, interferons, such as IFNa, IFNS or IFN, TNF, such as TNFa or TNF / 3, oncostatin M, sphingomyelinase or magainin and magainin derivatives. Structural genes that encode cytostatic or cytotoxic antibodies and fusion proteins between fragments of antibodies that bind antigen and cytostatic, cytotoxic or inflammatory proteins or enzymes can be chosen according to the following strategy: Cytostatic or cytotoxic antibodies include , for example, those directed against endothelial cell membrane structures, as described, for example, by Burrows et al. (Pharmac. Ther., 64, 155 (1994)), Hughes et al., (Cancer Res., 49, 6214 (1989)) and Maruyama et al., (PNAS USA, 87, 5744 (1990)). These antibodies include, in particular, antibodies against the VEGF receptors. In addition, they include cytostatic or cytotoxic antibodies that are directed against membrane structures in tumor cells. These antibodies have been reviewed, for example, by Sedlacek et al., Contrib. to Oncol. , 32, editorial Karger, Munich (1988) and Contrib. to Onsol. , 43, editorial Karger, Munish (1992). Other examples are antibodies against Lewis sialyl, against peptides in tumors that are recognized by T cells; against proteins that are expressed from oncogenes; against gangliosides, such as GD3, GD2, GM2, 9-0-acetyl GD3 and fucosyl GM1, against blood group antigens and their precursors, against antigens in polymorphic epithelial mucin, and against antigens in heat shock proteins. In addition, they include antibodies that are directed against membrane structures of leukemia cells. A large number of monoclonal antibodies of this type have already been described for diagnostic and therapeutic processes (reviews in Kristensen, Danish Medical Bulletin, 47, 52 (1994), Schranz, Therapia Hungarica, 38, 3 (1990), Drexler et al. , Leuk, Res., 10, 279 (1986), Naeim, Dis., Markers, 71 (1989), Stickney et al., Curr .. Opin. Oncol., 4, 847 (1992); Drexler et al. , Blut, 57, 327 (1988), Freedman et al., Cancer Invest., 9, 69 (1991)). Depending on the type of leusemia, monoclonal antibodies or their antibody fragments that bind to the antigen, which are directed against the following membrane antigens, are suitable for use as a ligand, for example: Antícreno cells of the AML membrane CD13 CD15 CD33 CAMAL sialosilo-Le B-CLL CD5 CDlc CD23 idiotypes and isotypes of the immunoglobulins of the membrane T-CLL CD33 M38 receptors of IL-2 T-cell receptors ALL CALLA CD19 non-Hodgkin's lymphoma The humanization of murine antibodies, and the preparation and optimization of genes for recombinant Fab and Fv fragments are carried out according to the technique sonosida by the person skilled in the art (Winter et al., Nature, 349, 293 (1991); Hoogenbooms et al., Rev. Tr. Transfus, Hemobiol., 36, 19 (1993), Girol, Mol. Immunol., 28, 1379 (1991) or Huston et al., Intern. Rev. Immunol., 10, 195 (1993)). The recombinant Fv fragments are likewise fused with genes for cytostatic, cytotoxic or inflammatory proteins or enzymes according to the state of the art 'known to the person skilled in the art. Structural genes that encode fusion proteins between ligands that bind to target cells and cytostatic and cytotoxic proteins can be selected according to the following strategy. Ligands include, for example, all substances that bind to membrane structures or membrane receptors on endothelial cells. Examples of these substances are cytokines, such as IL-1, or growth factors, or their fragments or partial sequences thereof, which bind to receptors that are expressed by endothelial cells, for example PDGF, bFGF, VEGF, TGF. In addition, they include adhesion molecules that bind to activated and / or proliferating endothelial cells. Examples of these are SLex, LFA-1, MAC-1, LECAM-1, VLA-4 or vitronectin. In addition, substances that bind to membrane structures or receptors on the membrane of tumor cells or leukemia are included. Examples are growth factors or their fragments or partial sequences thereof that bind to receptors that are expressed by leukemia cells or tumor cells. Growth factors of this type have already been described (reviews in Cross et al., Cell, 64, 271 (1991), Aulitzky et al., Drugs, 48, 667 (1994), Moore, Clin.

Cancer Res. , 1, 3 (1995), Van Kooten et al., Leuk. Lymph., 27 (1993)). The genes for these ligands that bind to the target cell are fused with the genes for cytostatic, cytotoxic or inflammatory proteins or enzymes according to the state of the art using methods that are known to the person skilled in the art. Structural genes for inflammation inducers encode, for example, IL-1, IL-2, RANTES (MCP-2), monocyte activating and chemotactic factor (MCAF), IL-8, macrophage inflammatory protein 1 (MIP-la , MIP-1/3), neutrophil activating protein 2 (NAP-2), IL-3, IL-5, human leukemia inhibitory factor (LIF), IL-7, IL-11, IL-13, GM -CSF, G-CSF, M-CSF, cobra venom factor (FVC) or partial sequelae of FVC that functionally correspond to the human complement factor C3b, that is, they are able to bind complement B factor and, after the cleavage by factor D, constitute a C3 convertase, a human complement C3 or its partial sequence C3b, cleavage products of the human complement factor C3 that are functionally and structurally similar to FVC, or bacterial proteins that activate a complement or induce inflammations, for example porins of Salmonella typhimurium, agglutination scars of Staphylosoccus aureus, mod ulinas, particularly from Gram-negative bacteria, major outer membrane protein from legionellas or from Haemophilus influenzae type B or from Klebsiellas or M molecules derived from Group G Streptococci. Structural genes that encode enzymes to activate precursors of cytostatic agents, for example, they encode enzymes that cleave inactive precursors (prodrugs) in active cytostatic agents (drugs) and the relevant prodrugs and drugs in each case have already been reviewed by Deonarain et al. (British Journal Cancer, 70, 786 (1994)), Mullen, Pharmas. Ther., 63, 199 (1994)) and Harris et al. (Gene Ther., 1, 170 (1994)). For example, DNA sesuensia can be used for one of the following enzymes: thymidine kinase virus herpes simplex, thymidine kinase varicella zoster virus, bacterial nitroreductase, bacterial / 3-glucuronidase, / 3-glucuronidase plant prosedente of Sécale cereale, human 3-glucuronidase, human carbo-xipeptidase (CB), for example CB-A from mast cells, CB-B, pancreatic or bacterial carboxypeptidase, bacterial β-lactamase, bacterial cytosine deaminase, catalase or human peroxidase , phosphatase, in particular human alkaline phosphatase, human prostatic acid phosphatase or acid type 5 phosphatase, oxidase, in particular human lysyl oxidase or human acid D-aminooxidase, peroxidase, in particular human glutathione-peroxidase, human eosinophil peroxidase or human thyroid peroxidase, or galactosidase. In addition, the therapy of autoimmune diseases and inflammations is described in WO / 06941 and DE19651443.6 which are incorporated herein by reference. Examples of suitable target cells are proliferating endothelial cells, macrophages and / or lymphosites or synovial cells. The promoters are, for example, specific for endothelial and specific cell cycle or macrophage-specific and / or lymphocyte-specific and / or cell-cycle-specific or specific synovial and / or cell-cycle cell-specific cells. Structural genes for allergy therapy encode, for example, IFN / 3, IFN ?, IL-10, antibodies or fragments of antibodies against IL-4, soluble IL-4 receptors, IL-12 or TGF3. Structural genes for preventing rejection of transplanted organs encode, for example, IL-10, TGF0, soluble IL-1 receptors, soluble IL-2 receptors, IL-1 receptor antagonists, soluble IL-6 receptors or immunosuppressive antibodies or their fragments containing VH and containing VL or their VH and VL fragments that are connected in the manner of a linker. Examples of immunosuppressive antibodies are antibodies that are specific for the T cell receptor or its CD3 complex, or are directed against CD4 or CD8 and, in addition, against the IL-2 receptor, the IL-1 receptor or the receptor. of IL-4 or against adhesion molecules CD2, LFA-1, CD28 or CD40. Structural genes for the therapy of autoimmune diseases involving antibodies encode, for example, TGF0, IFNa, IFN / 3, IFN ?, IL-12, soluble IL-4 receptors, soluble IL-6 receptors or immunosuppressive antibodies. -pressors or their fragments containing VH and containing VL.

Structural genes for the therapy of autoimmune diseases involving cells encode, for example, IL-6, IL-9, IL-10, IL-13, TNFa or TNF / 3, IL-13 or an antibody in suppressor or its fragments containing VH and containing VL. Structural genes for inhibitors of cell proliferation, cytotoxic or cytotoxic proteins and enzymes for activating cytostatic agent precursors have already been listed above in relation to tumor therapy. In the same way to that already described at that point, within the meaning of the present invention can be made use of structural genes encoding fusion proteins comprising antibodies or recombinant Fab or Fv fragments of these antibodies, or other ligands that are specific for the target cell, and the cytokines, the growth factors, receptors, the cytostatic or cytotoxic proteins and enzymes mentioned above. Within the meaning of the invention, structural genes whose expressed protein directly or indirectly inhibits inflammation, for example in a joint, and / or promotes reconstitution of the extracellular matrix (cartilage and connective tissue) in the joint are selected for the therapy of arthritis. Examples of these proteins are the IL-1 receptor antagonist (IL-1-RA), since IL-1-RA inhibits the binding of IL-1! and IL-3, the soluble IL-1 receptor, since the soluble IL-1 receptor binds and inactivates IL-1, IL-6, since IL-6 increases the secretion of TIMP and superoxides and decreases the secretion of IL-1 and TNFa by synovial cells and chondrocytes, soluble TNF receptor, since the soluble TNF receptor binds and inactivates TNF, IL-4, since IL-4 inhibits the formation and secretion of IL -1, TNFa and MMP, IL-10, since IL-10 inhibits the formation and secretion of IL-1, TNFa and MMP and increases the sesssion of TIMP, the insulin-like growth fastor (IGF-1), that IGF-1 stimulates the synthesis of the extracellular matrix, TGFβ, especially TGFßl and TGF / 32, since TGF / 3 stimulates the synthesis of the extracellular matrix and superoxide dismutase or TIMP, specifically TIMP-1, TIMP-2 or TIMP -3. The therapy of the deficient formation of blood cells has already been described in detail in WO96 / 06941 which is incorporated herein by reference. Examples of suitable target cells are immature proliferating cells of the hematopoietic system or stromal cells that are adjacent to the hematopoietic cells. Promoters are, for example, specific for hematopoietic cells and / or are cell-specific or non-cell-specific and cell-cycle specific. A structural gene for the therapy of anemia codes, for example, erythropoietin. Structural genes for the therapy of leukopenia encode, for example, G-CSF, GM-CSF or M-CSF. Stricture genes for the therapy of thrombocytopenia encode, for example, IL-3, the leukemia inhibitory factor (LIF), IL-11 or thrombopoietin. Target cells suitable for the therapy of the nervous system lesion are: glia cells or proliferating endothelial cells. In this case, the promoters are specific to the glia cells and specific to the cell cycle or specific to endothelial and cell cycle-specific or non-specific and specific cell cycle cells. Structural genes for neuronal growth factors encode, for example, FGF, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT- 4) or ciliary neurotrophic factor (CNTF). Structural genes for enzymes encode, for example, tyrosine hydroxylase or dopa decarboxylase. Structural genes for cytokines and their inhibitors that inhibit or neutralize the neutro-rot effect of TNFa 'encode, for example, TGF / 3, soluble TNF receptors, TNFa receptors neutralize TNFa, IL-10, since IL-10 inhibits the formation of IFN ?, TNFa, IL-2 and IL-4, soluble IL-1 receptors, IL-1 receptor I, IL-1 receptor II, given that IL receptors -1 soluble neutralize the activity of IL-1, IL-1 receptor antagonist or soluble IL-6 receptors. The therapy of disorders of the blood coagulation system and the blood circulatory system has already been described in detail in patent applications WO96 / 06938, DE19617851.7 and DE19639103.2 which are incorporated herein by reference. Examples of suitable target cells are endothelial cells, proliferating endothelial cells, somatic cells in the vicinity of endothelial cells and smooth muscle cells or macrophages. Promoters are, for example, non-cell-specific and cell-cycle specific or specific for endothelial cells, smooth muscle or macrophage cells and cell cycle-specific. Structural genes for the inhibition of coagulation or to promote fibrinolysis encode, for example, the tissue plasminogen activator (tPA), the urokinase-type plasminogen activator (uPA), hybrids of tPA and uPA, protein C, hirudin, serine proteinase inhibitors (serpins), such as C-1S inhibitor, al-antitrypsin or antithrombin III or the tissue factor pathway inhibitor (TFPI). Structural genes for promoting coagulation encode, for example, F VIII, F IX, von Willebrand factor, F XIII, PAI-1, PAI-2 or tissue factor and fragments thereof. Structural genes for the factors of angiogenesis encode, for example, VEGF or FGF. Structural genes for reducing blood pressure encode, for example, kallikrein or nitric oxide-synthase of endothelial cells. Structural genes for inhibiting the proliferation of smooth muscle cells after injury to the endothelial layer encode, for example, an anti-proliferative, cytostatic or cytotoxic protein or an enzyme for cleaving precursors of cytostatic agents in cytostatic agents, as already described. previously mentioned in the therapy of tumors, or a fusion protein of one of these active compounds with a ligand, for example an antibody or fragments of antibodies that are or are specific for muscle cells. Structural genes for other blood plasma proteins encode, for example, albumin, Cl inactivator, serum cholinesterase, transferrin or 1-antitrypsin. The use of nucleic acid constructs for vaccines has already been described in detail in patent applications WO96 / 06941, DE19617851.7, DE19639103.2 and DE19651443.6, which are incorporated herein by reference. Examples of suitable target cells are muscle cells, macrophages and / or lymphocytes or endothelial cells. Promoters are, for example, non-specific and specific to the cell cycle or specific to the target cells and specific to the cell cycle. The DNA for a protein that is formed by an infectious agent and that leads, by inducing an immune reaction, that is by means of binding to the antibody and / or by means of cytotoxic T lymphocytes, to the neutralization and / or destruction of the agent it is used, for example, as a structural gene for the prophylaxis of infectious diseases. The so-called neutralization agents are already used as vaccination agents (see review in Ellis, Adv. Exp. Med. Biol., 327, 263 (1992)). However, the possibilities of preparing effective vaccines are conventionally limited. In addition, DNA vaccines raise issues with respect to efficacy (Fynan et al., Int. J. Immunopharm., 17, 79 (1995).; Donnelly et al., Immunol. 2, 20 (1994)). An advantage of the present invention is that it is possible to rely on the efficiency being greater. Therefore, preference is given, within the meaning of the present invention, to a DNA encoding neutralizing antigens from the following pathogens: influenza A virus, HIV, rabies virus, HSV (herpes simplex virus) ), RSV (respiratory syncytial virus), parainfluenza virus, rotavirus, VZV (varicella zoster virus), CMV (cytomegalovirus), measles virus, HPV (human papillomavirus), HBV (hepatitis B virus), HCV (human hepatitis C), HDV (hepatitis D virus), HEV (hepatitis E virus), HAV (hepatitis A virus), vibrio cholera antigen, Borrelia burgdorferi or Helicobacter pylori or malaria antigen. However, active substances of this nature also include DNA for an anti-idiotype antibody, or its antigen-binding fragments, whose antigen-binding structures (the determinant regions of complementarity) constitute copies of the structure protein or the carbohydrate structure of the neutralizing antigen of the infectious agent. Anti-idiotype antibodies can, in particular, replace carbohydrate antigens in the case of bacterial infectious agents. Anti-idiotype antibodies and their cleavage products have been reviewed by Hawkins et al. (J. Immunother., 14, 273 (1993)) and Westerink and Apicella (Springer Seminars in Immuno-pathol., 15, 227 (1993)). Examples of structural genes for "tumor vaccines" are genes that encode antigens in tumor cells. These antigens have been reviewed, for example, by Sedlacek et al., Contrib. To Oncol., 32, Karger Publishing, Munich (1988) and Contrib. To Oncol, 43, Karger Publishing House, Munich (1992). Other examples are genes that encode the following antigens or the following anti-idiotype antibodies: sialyl Lewis, peptides in tumors that are recognized by T cells, proteins that are expressed from oncogenes, blood group antigens and their precursors, antigens in polymorphic epithelial mucin or antigens in heat shock proteins. The therapy of chronic infectious diseases has already been described in detail in patent applications WO96 / 06941, Del9617851.7, DE19639103.2 and DE19651443.6, which are incorporated herein by reference. A suitable target cell is a liver cell, a lymphocyte and / or macrophage, an epithelial cell or an endothelial cell. Promoters are, for example, virus-specific or cell-specific and cell-cycle-specific. Structural genes encode, for example, a protein that exhibits cytostatic, apoptotic or cytotoxic effects, or an enzyme that cleaves a precursor of an antiviral or cytotoxic substance of this type in the active substance. Examples of structural genes encoding antiviral proteins are genes for cytokines and growth factors that have an antiviral effect, for example IFNa, IFN / J, IFN ?, TNFβ, TNFα, IL-1 or TGFα, or antibodies that have an antiviral effect. specificity that inactivates the relevant virus, or its fragments containing VH and containing VL, or its VH and VL fragments that are linked by means of a linker, as already described. Examples of antibodies against the virus antigen are: anti-HBV, anti-HCV, anti-HSV, anti-HPV, anti-HIV, anti-EBV, anti-HTLV, anti-Coxsackie virus or Hantaan anti-virus. Another example of an antiviral protein is the rev binding protein. This protein binds to the rev RNA and inhibits the post-transcriptional steps dependent on rev in the expression of retrovirus genes. Examples of these proteins that bind to rev are RBP9-27, RBP1-8U, RBP1-8D or pseudogenes of RBP1-8. Another viral structural gene encodes ribozymes that digest the mRNA of genes for the cell cycle control proteins or virus mRNA. Ribozymes that are catalytic for HIV have been reviewed, for example, by Christof-fersen et al., J. Med. Chem., 38, 2033 (1995). Examples of structural genes that encode antibacterial proteins are genes for antibodies that neutralize bacterial toxins or opsonize bacteria. Examples of these antibodies are antibodies against meningococci C or B, E. coli, Borrelia, Pseudomonas, Helicobacter pylori or Staphylococcus aureus.

IV) Combination of identical or different structural genes A detailed description has already been given in documents WO96 / 06941, WO96 / 06939, WO96 / 06940, WO96 / 06938, DE19639103.2 and DE19651443.6 which are incorporated herein by reference. An example of a combination of structural genes is a self-reinforcing expression system, if appropriate pharmacologically controllable, in which there is a combination of the DNA sequences of two identical structural genes or of two different structural genes [component c) and e ' )]. An additional promoter sequence or, preferably, cDNA for an internal ribosome entry site (IRES) is interspersed, as a regulatory element, between the two structural genes in order to express the two DNA sequences. An IRES makes it possible to express two DNA sequences that are linked to one another by means of an IRES. IRES's of this type have been described, for example, by Montford and Smith (TIG, 11, 179 (1995), Kaufman et al., Nucí Acids Res., 19, 4485 (1991), - Morgan et al., Nucí Acids Res., 20, 1293 (1992), Dirks et al., Gene, 128, 247 (1993), Pelletier and Sonenberg, Nature, 334, 320 (1988) and Sugitomo et al., BioTeshn., 12, 694 (1994)). Thus, for example, the cDNA for the IRES sequence of the virus of the poly (position = 140 = 630 of 5 'UTR) can be used.

Preference is given to structural genes that exhibit an additive efesto and that are linked by means of additional promoter sequences or an IRES sesuensia. Preferred combinations of strutural genes for tumor therapy sodify, for example, identical or different cytostatic, apoptotic, cytotoxic or inflammatory proteins and / or identical or different enzymes for cleavage of the precursor of a cytostatic agent; Preferred combinations for the therapy of autoimmune diseases encode different cytokines or receptors with a synergistic efesto, to inhibit the cellular and / or humoral immune reaction or TIMP's different or identical; Preferred combinations for the therapy of the deficient formation of blood cells encode different hierarchically consecutive cytokines, such as IL-1, IL-3, IL-6 or GM-CSF and erythropoietin, G-CSF or thrombopoietin, - preferred combinations for the nerve cell injury therapy encodes a neuronal growth factor and a cytokine or the inhibitor of a cytokine; Preferred combinations for the therapy of disturbances of the blood sperculation system and the blood circulatory system encode an antithrombotic agent and a fibrinolytic agent (for example tPA or uPA) or a cytostatic, apoptotic or cytotoxic protein and an antithrombotic agent or a fibrinolytic agent, or several different blood coagulation factors acting synergistically, for example F VIII and vWF or F VIII and F IX; Preferred combinations for vaccines encode an antigen and an immunostimulatory cytokine, for example IL-1 receptor, IL-1/3, IL-2, GM-CSF, IL-3 or IL-4, different antigens from an infectious agent or of different infectious agents, or different antigens of a tumor type or of different types of tumors, - preferred combinations for the therapy of viral infested diseases sodifisan an antiviral protein and a cytostatic, apoptotic or cytotoxic protein, or antibodies different antigen of surface of a virus or several viruses; and preferred combinations for the therapy of bacterial infectious diseases encode antibodies against different surface antigens and / or toxins from a sausative organism.

V) Insertion of signal sequences and domains of t ansme frog A detailed description has already been given in patent applications DE19639103.2 and DE19651443.6 which are incorporated herein by reference. In order to improve translation, the nucleotide sequence GCCACC or GCCGCC (Kozak, J. Cell Biol., 108, 299 (1989)) can be inserted at the 3 'end of the promoter sequence and directly at the 5' end of the initiation signal (ATG) of the signal or transmembrane sequence. In order to facilitate the secretion of the structural gene expression product, the homologous signal sequence that can be presented in the DNA sequence of the structural gene can be replaced by a heterologous signal sequence that improves extracellular secretion. Thus, for example, the signal sequence for immunoglobulin can be inserted (position 63 to 107 of DNA; Riechmann et al., Nature, 332, 323 (1988)) or the signal sequence for CEA (position 33 to 134 of DNA, Schre et al., Mol Cell Biol., 10, 2738 (1990), Berling et al. al., Cancer Res., 50, 6534 (1990)) or the signal sequence of the human respiratory syncytial virus glycoprotein (cDNA for amino acids 38 to 50 or 48 to 65; Lishtenstein et al., J. Gen. Virol ., 77, 109 (1996)).

A sequence for a transmembrane domain can be inserted, alternatively or, in addition to the signal sequence, in order to anchor the active compound in the cell membrane of the transduced cell that is forming the active compound. For example, the transmembrane sequence of the human macrophage colony-stimulating phantom (position 1485 to 1554 of DNA, Cosman et al., Behring Inst. "Mitt., 77, 15 (1988)) or the DNA sequence. for the signal and transmembrane regions of glycoprotein G of human respiratory syncytial virus (RSV) (amino acids 1 to 63 or their partial sequences, amino acids 38 to 63; Vijaya et al., Mol. Cell Biol., 8, 1709 ( 1988), Lichtenstein et al., J. Gen. Virol., 77, 109 (1996)) or the DNA sequence for the signal and transmembrane regions of the influenza virus neuroaminidase (amino acid 7 to 35). or amino acids 7 to 27 of the partial sequence, Brown et al, J. Virol, 62, 3824 (1988)) can be inserted, for example, between the promoter sequence and the structural gene sequence. of nucleotides for an anchoring of glycophospholipids can also be inserted in order to anchor the active compound in the cell membrane of the The transduced cells that are forming the active compound. A glycophospholipid anchor is inserted at the 3 'end of the nucleotide sequence for the structural gene, it being possible for this insertion to take place in addition to inserting a signal sequence. The glycophospholipid anchors have been described, for example, for CEA, for N-CAM and for other membrane proteins, for example Thy-1 (see review in Ferguson et al., Ann.Rev. Biochem., 57, 285 (1988)). Another option for anchoring active compounds to the cell membrane according to the present invention is to use a DNA sequence for a ligand / active compound fusion protein. The ligand specificity of this fusion protein is directed against a structure of the membrane in the cell membrane of the selected target cell. Ligands that bind to the surface of cells include, for example, antibodies or fragments of antibodies that are directed against structures on the surface of, for example, endothelial cells. These antibodies or antibody fragments include, in particular, antibodies against VEGF receptors or against quinine receptors. They may also be directed against muscle cells, such as antibodies against actin or antibodies against angiotensin II receptors or antibodies against receptors for growth factors, such as against EGF receptors or sontra receptors of PDGF or against FGF receptors, or antibodies against endothelin A receptors. Ligands also include antibodies or their fragments that are directed against tumor-specific or tumor-associated antigens in the tumor cell membrane. Antibodies of this nature have already been described. The murine monoclonal antibodies are preferably used in a humanized form. The recombinant Fab and Fv fragments and their fusion products are prepared using methods that are known to the person skilled in the art, as already described. The ligands further include all active compounds such as cytokines or adhesion molecules, growth factors or their fragments or partial sequences thereof, mediators or peptide hormones that bind to the membrane structures or membrane receptors in the relevant selected cell. Examples are ligands for endothelial cells, such as IL-1, PDGF, bFGF, VEGF, TGGβ (Pusztain et al., J. Pathol., 169, 191 (1993)) or quinine and quinine derivatives or analogues. These ligands also include adhesion molecules. Adhesion molecules of this nature, such as SLex, LFA-1, MAC-1, LeCAM-1, VLA-4 or vitronectin or vitronectin derivatives or analogs, have already been described for endothelial cells (reviews in Augustin-Voss et al. ., J. Cell Biol., 119, 483 (1992), Pauli et al., Canser Metast, Rev., 9, 175 (1990), Honn et al., Cancer Metast. Rev., 11, 353 (1992).; Varner et al., Cell Adh. Commun., 3, 367 (1995)). The invention is clarified with the help of the following figures, tables and examples without being limited thereto.

Description of figures and tables Figure 1: Diagrammatic description of a new nucleic acid construct comprising components a) -d) Figure 2: Diagrammatic description of a new nucleic acid construct comprising components a) -e) Figure 3 .- Diagrammatic description of a promoter unit that responds to the activator Figure 4: Diagrammatic description of a new nucleic acid construct comprising a promoter unit that responds to the activator Figure 5: Genomic structure of the murine cdc25B promoter / enhancer region . In carrying out restriction digestion with various enzymes, a map of the genomic site was prepared from three isolated phage clones. A 4.6 kb fragment directly bordering the 5 'region of the cDNA was excised from phage VI and subcloned into the Bluescript SKII vector (Stratagene). a) = phage clone b) = subcloned fragment Figure 6: Deletion mutants of the murine cdc25b promoter. The figure describes different 5 'deletions and a 3' deletion of the promoter, and the putative transcription phantom binding sites that are located in this region of the promoter. The designation of the individual deletion constructions is based on the position of the 5 'end in sequencing. The fragments were purified through QIAquick® rotating columns (Qiagen) and cloned into the vestige pGL3 (Promega). Table 1: Region sesuenciada of murine cds25B promoter. The region that directly binds to the 5 'end of the published cDNA sequence (Kakizuka et al., Genes Dev. 6, 587 (1992)) was sequenced. The table shows the arrangement of putative binding sites and the start of transcription. On the one hand, putative binding sites are activators, such as those that occur in many cell cycle-specific promoters that are regulated by repression. In addition, there are putative E2F binding sites and, in the 5 'region, two E boxes to which the repressor activity is set. The TATA box that is described is an element of the sequence that is unusual for genes that are regulated in a specific manner of the cell cycle and that is obviously functionally important in this promoter since it specifies the position of the start of transcription. Table 2: Promoter activity of the different deletion constructs. The table shows the relative luciferase activity in growing and serum-free NIH-3T3 cells of the constructs described in Figure 6. Induction of the promoter cell cycle, which is determined from the quotient of cell values in development versus exhausted cells, occurs in the final column. In this respect, the value for the longest construction in developing cells is set at 100, the remaining values being compared with this reference value Table 2a: Deletions of a relatively large size to determine functional regions in the promoter, and mutation Punctual of the TATA saja. Table 2b: Sequential deletion of the proximal Spl binding site and the NF-Y binding site, and point mutation of the NF-Y binding site. In this case, the activity of developing cells of the construction that are all these sites of union to the astivador is stable again, for reasons of simplicity, in 100 (the real astivity does not correspond with that of the construction B -950). a) Deletion constructions tested (see Figure 6) b) Promoter activity in developing cells c) Promoter activity in resting cells (devoid of serum) d) Induction of cell cycle (Quotient of promoter activity in developing cells compared to cells at rest) Examples 1. Cloning and analysis of the urinary cdc25B promoter In order to clone the murine cdc25B promoter, approximately phage plaques from a murine genomic phage library (mouse strain 129 FVJ, Stratagene) were screened in A-Fix (Stratagene). The probe used in this screening was an 80 bp oligonucleotide that was directed against the outer 5 'region of the murine cdc25B cDNA (Kakizuka et al., Genes Dev., 6, 587 (1992)). The sequence is: Probe 1: 5'TCTAGCTAGCCTTTGCCCGCCCCGCCAC- GATGGAGGTACCCCTGCAGAAGTCTGCGCCGGGTT- CAGCTCTCAGTCCTGCC-3 '. SEQ ID NO .: 4) Three of the six resulting phage clones were isolated, ampld and plotted on a map by restriction digestion (Gibco enzymes) and two additional probes were used that were directed against other seses located 3 'of the murine cdc25B cDNA (figure 5). Probe 2: 5'GGTCATTCAAAATGAGCAGTTACCATAAAACGCTTCCGATC CTTACCAGTGAGGCTTGCTGGAACACAGTCCGGTGCTG-3 ' (SEQ ID NO .: 5) Probe 3: 5'GTTAAAGAAGCATTGTTATTATGGGGAGGGGGGAGCAACCT CTGGGTTCAGAATCTACATATGCTGGAAGGCCCCAATGA-3 '(SEQ ID NO .: 6) Finally, a 4.6 kb fragment from the proximal enhancer region bordering the published cDNA (Kakizuka et al., See above) was excised from phage VI using the EcoRI and Sal I enzymes (Gibco) purd by electrophoresis in agarose gel and using QIAquick® rotating columns (Qiagen) and inserted into a Bluescript SKII vector (Stratagene) (Figure 5). 1.5 kb of the 3 'region of the cloned 4.6 kb fragment was identd and identd by comparing the sequence with sdc25B cDNA sequences from various species, which was the murine homologue of the cdc25B gene. Various fragments were excised from this sequenced region, cloned into a luciferase reporter vector pGL3 (Promega) and assayed for promoter activity in mouse fibroblasts NIH-3T3 (ATCC). Transfection (transient) was carried out using the DEAE / dextran method (modd later by Sompayrac et al., PNAS, 78, 7575 (1981)). The controls used in these experiments were the SV40 basal promoter in the vector pGL3 (Promega), promoter which is not subject to any regulation of the signant cell cycle and, as a positive control, a fragment of the human cdc25B promoter (C290, Lucibello et al., EMBO J., 14, 132 (1995)), which was also cloned into the pGL3 vector. Luciferase activity was determined as described (Herber et al., Oncogene, 9, 1295 (1994)). The nucleotide sequence -950 +167 was found to be the promoter of the murine c25B gene (SEQ ID NO: 7, see Table 1). Various deletion fragments were excised from the promoter of the murine cdc25B gene (FIG. 6), cloned in a luciferase reporter mask pGL3 (Promega) and tested for promoter activity, as described above, in fibroblasts. of mouse NIH-3T3.

In order to analyze the reaction of the cell cycle of the different deletion constructs, in each case normal transiently transfected cells were compared with similar cells that were devoid of serum after transfection, as described (Lucibello et al., EMBO J., 14, 132 (1995)). The results are summarized in table 2. In this context, the longest construction, ie B-950 (sequencing of nucleotides -950 a +167) exhibited a cell cycle regulation of 10.1 which was comparable with that of the human cdc25C construct (not listed in table 2). The deletion of the region 3 'down to +3 (see Figure 6) did not result in any loss of activity or regulation of the promoter cell cycle, it being, therefore, possible to further delimit the region responsible for the regulation of the promoter. Deletions from the 5 'region of the promoter gave rise to two different effects; On the one hand, the delession of the longest construction, ie B-950, down to B-340 resulted in an increase in activity in G0 / G1 cells (corresponding to a deregulation.) Additional deletions determine a reduction of promoter activity until the last deletion, which then determines a renewed deregulation (table 2a) .The initiation site was determined by primer extension.To this end, RNA was isolated from NIH-3T3 mouse mouse fibroblasts normal and the reaction was carried out using different primers and MMLV reverse transcriptase (Gibco) .The initiation site depicted on the map is located on a sequence element similar to the primer, 24 base pairs in the 3 'position of the TATA box (SEQ ID NO .: 7, see table 3). If the activity of the promoter in the deletion constructs is observed against the background of the putative transcription factor binding sites listed in Table 3, then it is evident that these effects are mediated by the de-letion of specific binding sites: the deletion of the E boxes located in the 5 'region, as well as the deletion of the putative E2F binding site located in the vicinity of the TATA box leads to derepression of the promoter. On the other hand, deletions of putative activator binding sites (predominantly SP1 binding sites) and an NF-Y binding site decrease promoter activity (see Table 2b). In this context, the point mutation of the putative NF-Y site resulted in a loss of activity of more than 74% compared to the wild-type construct (see Table 2b). Electrophoretic mobility shift assays (EMSA's as described in Zwicker et al., Nucleic Acids Res. 23, No. 19, page 3822 et seq., 1995) using specific antibodies against Spl / Sp3 and NF-Y (Santa Cruz ) and cross-competitive experiments are Spl or NF-Y bona fide binding sites demonstrated a specific binding of Spl / Sp3 and NF-Y to the respective putative binding sites. While the shorter construction (B-30) containing the TATA saja and the inisium site shown on the map is largely unregulated, it exhibits an astivity that is two hundred times greater than the background activity of the pGL3 vector. In addition, the point mutation of the TATA box resulted in a loss of more than 25% of the activity of the promoter (see Table 2a), thereby confirming its functional role in the regulation of promoter activity. The transcription factor binding sites (mainly SP1 and NF-Y) correspond to the many described genes that are regulated in a cell cycle-specific manner by repression or activation (for review, see Zwicker and Müller, TiGS 14 , 3 (1997)); however, no promoter of the cell cycle gene containing a functional TATA box has been previously described. The promoter of the murine sdc25B gene therefore encompasses the nucleotides = -950 a = +167 or partial sequences of these nucleotide sequences, for example = -950 a = +1, -930 to +167, -720 to +167 , -340 to +167, -180 to 167, -100 to +167, -80 to +167, -60 to +167 or -30 to +167, and / or corresponding partial sesuencias to +3 or +1. Starting from the murine promoter sequences that the authors of this invention have ensontrado, it is a simple matter now for a person skilled in the art to find promoters of non-murine cdc25B that are homologous to the murine cdc25B promoter by labeling, preferably by radiolabelling the murine promoter and tracing genomic DNA genomes obtained from mammalian cells by means of hybridization in rigorous sonications. 2. Preparation of gene constructs using a multiple promoter technology a) Preparation of a promoter unit that responds to the activator The new promoter unit that responds to the activator comprises the following sequencies of different nucleotides that follow one another in the downward direction: Activator subunit A promoter of the cdc25B gene (nucleic acids -950 to +167) the localization signal Nucleic acid (NLS) of SV40 (SV40 large T, amino acids 126-132; PKKKRKV, Dingwall et al., TIBS, 16, 478 (1991)) the acid transactivation domain (TAD) of HSV-1 VP16 (amino acids 406 to 488; Triezenberg et al., Genes Developm., 2, 718 (1988); Triezenberg, Curr Opin. Gen. Develop., 5, 190 (1995)) - the cDNA for the cytoplasmic part of the CD4 glycoprotein (amino acids 397-435; Simpson et al., Oncogene, 4, 1141 (1989); Maddon et al., Cell, 93 (1985)) Subunit B activator promoter of the cdc25C gene (nucleic acids -290 to +121; Zwicker et al. al., EMBO J., 14, 4514 (1995); Zwicker et al., Nucí. Acids Res., 23, 3822 (1995)) the nuclear localization signal (NLS) of SV40 (SV40 large T amino acids 126-132; PKKKRKV, Dingwall et al., TIBS, 16, 478 (1991)) - the cDNA for the DNA binding domain of the Gal4 protein (amino acids 1 to 147, Chasman and Kornberg, Mol Cell. Biol., 10, 2916 (1990)) the cDNA for CD4-binding sequencing of the p56 Ick protein (amino acids 1-71; Shaw et al., Cell, 59, 627 (1989); Turner et al., Cell, 60, 755 (1990 Perlmutter et al., J. Cell. Biochem., 38, 117 (1988)) Promoters that respond to the activator lOx the binding sequence for the Gal4 binding protein having the nucleotide sequence 5'- CGGACAATGTTGACCG-3 ' (Chasman and Kornberg, Mol. Cell, Biol. 10, 2916 (1989)) the SV40 basal promoter (nucleic acids 48 to 5191; Tooze (comp), effector gene of DNA tumor viruses (Cold Spring Harbor New York, New York , Cold Spring Harbor Laboratory) the DNAs for luciferase (Nordeen BioTeshniques, 6, 454 (1988)) The described activator sesquence functions as follows: The cdc25B promoter regulates the transcription of the combined cDNAs for the astivation domain of VP16 and the sitoplasmic part of CD4 (activation subunit A) in a specific manner of the cell cycle. The cdc25C promoter regulates the transsription of the combined cDNAs for the Gal4 DNA binding protein and the CD4 binding portion of the p56 Ick protein (activation B subunit) in a specific cell cycle manner. The expression products of subunits A and B of the activator are dimerized by the binding of the CD4 domain to the p56 Ick domain. The dimeric protein constitutes a chimeric transcription factor for the promoter that responds to the activator (sequesion of DNA for the Gal4 binding domains / the SV40 promoter) for the transcription of the effector gene (= gene luciferase). The individual components of the construct are linked together by means of suitable restriction sites that are added at the ends of the different elements during PCR amplification. The link is made using enzymes that are known to the person skilled in the art and that are specific for restriction sites, and DNA ligases. These enzymes can be obtained comersially. The nucleotide construct that has been prepared in this way is cloned into the plasmid vector pXP2 (Nordeen, BioTechniques, 6, 454 (1988)), which is then used directly or in colloidal dispersion systems, for an application in alive. 3T3 fibroblasts maintained in cultures are transfected with the described plasmid using the method known to the person skilled in the art (Lucibello et al., EMBO J., 14, 132 (1995)) and the amount of luciferase produced by the fibroblasts, as described by Herber et al. (Oncogene, 9, 1295 (1994)) and Lucibello et al. (EMBO J., 14, 132 (1995)). In order to verify the specificity of the cell cycle, the fibroblasts are synchronized in GQ / GI separating serum over a period of 48 hours. The DNA content of the cells is determined in a fluorescence-activated cell sorter after staining with Hoechst 33258 (Lucibello et al., EMBO J., 14, 132 (1995)).

The following results are obtained: In the transfected fibroblast a marked increase in luciferase can be confirmed in comparison with untransfected fibroblasts. Proliferating fibroblasts (DNA> 2S) form essentially more luciferase than fibroblasts that are synchronized in G0 / Gx (DNA = 2S). Accordingly, the promoter unit that responds to the activator that has been dessrita leads to a cell cycle-dependent expression of the luciferase reporter gene. b) Preparation of a hybrid promoter The new hybrid promoter comprises the following different nucleotide sequences which follow each other in the downward direction: the promoter of the cdc25B gene (nusleic acids -950 to +167) The TATA box (TATATAA nucleic acids in the -30 a position) -23 are mutated in TGTATAA)). - the GCCACC sequence (Kodak, J. Cell Biol., 108, 229 (1989)) the cDNA for the immunoglobulin signal peptide (nucleotide sequence <63 to> 107; Riechmann et al., Nature 332, 323 ( 1988)) - the cDNA for -glucuronidase nucleotide sequence = 93 a = 1982; Oshima et al., PNAS USA, 84, 685 (1987)) the promoter of the von Willebrand factor gene (wWF) (nucleic acids -487 to +247, Jahroudi and Lynch, Mol.Cell Biol. 14, 999 (1994 )) the gene for the TATA box binding protein (sequelae of nusleic acids +1 to +1001, which is mutated in nucleic acids 862 (A replaced by T), 889 and 890 (GT replaced by AC) and 895 (C replaced by G) (Strubin and Struhl, Cell, 68, 721 (1992); Heard et al., EMBO J., 12, 3519 (1993)) The individual components of the construction are linked through sites of restriction that are introduced into the ends of the different elements during PCR amplification.The binding is carried out using enzymes that are known to the person skilled in the art and are specific for restriction sites, and DNA ligases. of nucleotides that has been prepared in this manner is cloned into a vector of plasmid pUC18 / l9 which is used directly or in colloidal dispersion systems for an in vivo application. Human umbilisal cord endothelial cells and fibroblasts (Wi-38) that are maintained in culture are transfected with the plasmid described using the method known to the person skilled in the art (Lucibello, et al., EMBO J., 14, 132 (1995 )), and the amount of / 3-glucuroni-dasa that is produced by endothelial cells is measured using 4-methylumbelliferyl-3-glucuronide as a substrate. In order to verify the specificity of the cell cycle, endothelial cells are synchronized in GQ / GJ ^ by separating methionine for a period of 48 hours. The DNA content of the cells is determined in a fluorescence activated cell sorter after staining with Hoechst 33258 (Lucibello et al., EMBO J., 14, 132 (1995)). The following results are obtained: In transfected fibroblasts, no increase in / 3-glucuronidase can be confirmed in comparison with non-transfibed fibroblasts. The transflated endothelial cells express more / 3-glusuronidase than do the non-transfected endothelial cells. Proliferating endothelial cells (DNA> 2S; S = single set of chromosomes) secrete essentially more / 3-glucuronidase than endothelial cells that are unskilled in GO / G-L (DNA = 2S). Accordingly, the multiple promoter unit that has been dessrita sonduse to a cell-specific and cell cycle-dependent expression of the structural gene? -glucuronidase. c) Preparation of a multiple promoter with a nuclear retention signal (NRS) and a nuclear export factor (NEF) The new multiple promoter comprises the following different nucleotide sequences which follow one after the other in the downward direction: the promoter of the cdc25B gene (nusleisosas -950 to +167) the GCCACC sequence; I know that. ID. No .: 1 (Kodak, J. Cell Biol., 108, 229 (1989)) the cDNA for the immunoglobulin signal peptide (nucleotide sequence = 63 a = 107; Rieshmann et al., Nature 332, 323 (1988)) cDNA for -glucuronidase (nucleotide sequence = 93 a = 1982; Oshima et al., PNAS USA, 84, 685 (1987)) - the RER cDNA of the HIV-1 virus as the nuclear retention signal (NRS) (nucleotide sequence 7357 to 7602; Ratner et al., Nature, 313, 277 (1985); Malim et al., Nature, 338, 254 (1989)) - the promoter of the von Willebrand factor gene (wWF) (Nucleic acids -487 to +247; Jahroudi and Lynch, Mol.

Cell Biol. 14, 999 (1994)) The cDNA for the REV of the HIV-1 virus as the nuclear export factor (NEF) (amino acid sessurance 1-117; Ratner et al., Nature, 313, 277 (1985)). The individual somponents of the construction are linked by means of suitable restriction sites that are introduced at the ends of the different elements during amplification by PCR. The link is made using enzymes that are detected by the person skilled in the art and which are specific for restriction sites, and DNA ligases. These enzymes can be obtained commercially. The nucleotide construct that has been prepared in this way is cloned into a plasmid vector pUC18 / l9 which is used directly or in colloidal dispersion systems for in vivo application. Human umbilical cord endothelial cells and fibroblasts (Wi-38) that are kept in culture are transfected with the plasmid described using the method known to the person skilled in the art (Lucibello, et al., EMBO J., 14, 132 (1995 )), and the amount of / 3-glucuronidase that is produced by endothelial cells is measured using 4-methylumbelliferyl-3-glucuronide as a substrate. In order to verify the specificity of the cell cycle, endothelial cells are synchronized in GQ / G-L by separating methionine for a period of 48 hours. The DNA content of the cells is determined in a fluorescence activated cell sorter after staining with Hoechst 33258 (Lusibello et al., EMBO J., 14, 132 (1995)). The following results are obtained: In the transfibed fibroblasts no increase in β-glucuronidase can be confirmed in comparison with untransfected fibroblasts. The transfected endothelial cells express essentially more / 3-glucuronidase than does the non-transfected endothelial cells. Proliferating endothelial cells (DNA> 2S; S = simple set of chromosomes) secrete essentially more -glucuronide-dasa than do endothelial cells that are synchronized in G0 / Gx (DNA = 2S). Accordingly, the multiple promoter unit described leads to cell-specific and cell cycle-dependent expression of the structural gene / 3-glucuronides.

Application An active compound according to the described examples ensures, after local administration, for example at the site of the tumor, or after intracranial or subarachnoid administration, or systemically, preferably intravenous or intraarterial administration which, as a result -of the specificity of the cell cycle and the specificity of the endothelial cells of the multiple promoter unit are mainly, if not exclusively, only the proliferating endothelial cells that secrete β -glucuronidase. This 3-glucuronidase cleaves a well-tolerated doxorubicin - / 3-glucuronide (Jasquesy et al., EPO 0 511 917 Al) which is now injected into doxorubicin, which has a cytostatism effect. Doxorubicin inhibits the proliferation of endothelial cells and exerts a cytostatism effect on these cells and also on adjacent tumor cells. This results in the inhibition of tumor development.

Claims (39)

1. A promoter of the cds25B gene, which results in a sequence that hybridizes with a sequencing as described in Table 1 (SEQ ID No: 7) or a functional part thereof under stringent conditions.

2. A promoter according to claim 1, wherein the promoter comprises the sesuence described in Table 1 (SEQ ID No: 7) or functional part thereof.

3. - A promoter according to claim 1 or 2, wherein the functional part comprises the TATA box, at least one Spl binding site and at least one NF-Y binding site and, where appropriate, at least an E2F-binding site and, also where appropriate, at least one E.sup.E

4. A promoter according to one of claims 1-3, comprising the sequence spanning the nucleotides from about -950 to about +167 of Table 1 or fragments thereof that still comprise all of the functional cis-regulatory elements of the nucleotide promoter sesuence from about -950 to about +167 as described in Figure 6.

5.- A promoter according to one of claims 1-3, comprising the sequencing spanning the nucleotides from about -950 to about +3 of Table 1 or fragments thereof which still comprise all of the cis-regulatory elements functionally The sequence of the promoter of the nucleotides is from about -950 to about +3 as described in Figure 6.

6. A promoter according to one of claims 1-3, which includes the sesuensia spanning the nucleotides from about -930 to about +3 of Table 1 or fragments thereof which still comprise all of the functional cis-regulatory elements of the promoter sequence of the nucleotides from about -930 to about +3 as described in the figure 6.

A promoter according to one of claims 1-3, comprising the sequence spanning the nucleotides from about -720 to about +3 of Table 1 or fragments thereof which still comprise all of the cis elements -functional regulators of the nucleotide promoter sequence from about -720 to about +3 as described in figure 6.

8. - A prom according to one of claims 1-3, comprising the sequence spanning the nucleotides from about -340 to about 3 of Table 1 or fragments thereof which still comprise all of the functional cis-regulatory elements of the sequence of the nucleotide promoter from about -340 to about +3 as described in figure 6.

9. - A promoter according to one of claims 1-3, comprising the sequence spanning the nucleotides from about -180 to about +3 of Table 1 or fragments thereof which still comprise all of the cis-regulatory functional elements of the promoter sequence of the nucleotides from about -180 to about +3 as described in Figure 6.

10. A promoter according to one of claims 1-3, comprising the sequence spanning the nucleotides from about - 100 to about +3 of Table 1 or fragments thereof which still comprise all of the functional cis-regulatory elements of the nucleotide promoter sequence from about -100 to about +3 as described in Figure 6.

11. A promoter according to one of claims 1-3, comprising the sequence spanning the nucleotides from about -80 to about +3 of Table 1 or fragments thereof which still comprise all of the functional cis-regulatory elements of the nucleotide promoter sequence from about -80 to about +3 as described in Figure 6.

12. - A promoter according to one of claims 1-3, comprising the sesuence spanning the nucleotides from about -60 to about +3 of Table 1 or fragments thereof which still comprise all of the cis-regulatory funtional elements of the promoter sequence of the nucleotides from about -60 to about +3 as described in figure 6.

13. - A promoter according to one of claims 1-3, comprising the sequence spanning the nucleotides from about -30 to about +3 of Table 1 or fragments thereof which still comprise all of the cis-functional elements of the promoter sequence of the nucleotides from about -30 to about +3 as disclosed in Figure 6.

14. - A promotion to find promoters of cdc25B, which comprises labeling, preferably radiolabeling a promoter according to one or more of Claims 1 to 13, and to screen genomic DNA libraries, preferably from mammalian cells, by hybridization under stringent conditions.

15. A method for isolating the murine cdc25B promoter, comprising a murine genomic phage library, obtained from the mouse strain 129FVJ, with a probe that is a part of the sesuence described in Table 1 (SEQ ID. NO: 7), preferably containing the SEQ sequence ID NO: 4.

16.- A nucleic acid construct comprising at least one promoter according to one of claims 1 to 13.

17. - A nucleic acid construct according to claim 16, further comprising a structural gene.

18. - A nucleic acid construct according to claim 16, wherein said promoter is disposed above the structural gene.

19. A nucleic acid construct according to claim 17 or 18, wherein the non-coding region 5 'of the cdc25B gene has the nucleotide sequence from +1 to approximately +167 inserted between said promoter and the structural gene.

20. A nucleic acid construct according to one of claims 16-19, wherein the promoter according to one of claims 1-13 is combined with at least one additional sequencing sequence, this sequence of additional activation of a sequence being selected. of non-specific activation, specific to the virus, metabolically specific, specific to the cell, specific to the cell cycle and / or dependent on cell proliferation.

21. A nucleic acid construct according to claim 20, wherein the additional activation sequence is selected from promoters that are activated in endothelial cells, peritoneal cells, pleurale cells, skin epithelial cells, lung cells, gastrointestinal tract cells, kidney cells and drainage routes of urine, muscle cells, connective tissue cells, hematopoietic cells, macrophages, lymphocytes, leukemia cells, tumor cells or glia cells; virus promoter sequences, such as HBV, HCV, HSV, HPV, EBV, HTLV, CMV or HIV; promoter or enhancer sequences that are activated by hypoxia, cell cycle-specific activation sequences of the genes encoding cdc25C, cyclin A, cdc2, E2F-1, B-myb and DHFR, and / or binding sequences, such as monomers or multimers of the Myc E box, for transsripsion fasters that appear or are activated in a manner dependent on cell proliferation.

22. - A nucleic acid construct according to one of claims 16-21, wherein the promoter according to one of claims 1-13 is present in a form in which at least one binding site is mutated for a factor of transcript

23. A construction of nusleic acid according to claim 22, in which the TATA sachet is mutated.

24. - A nucleic acid construct according to claim 22 or 23, wherein, in addition to a structural gene, an additional sequencer or promoter sequence that can be activated in a non-specific, specific manner is present. of the cell or virus-specific, by tetracycline and / or in a cell cycle-specific manner, and which activates the transcription of at least one additional structural gene encoding at least one transcription factor that is mutated, so that it binds to the mutant binding site (s) of the promoter according to claim 22 or 23 and activates this promoter, and / or the structural gene encoding a transcription factor.

25. A nucleic acid construct according to one of claims 22-24, wherein the transcription factor is a protein (TBP) that binds to a mutated TATA box.

26. A nucleic acid construct according to claim 25, comprising (1) the promoter according to one of claims 1-13, including the TATA box, the sequence of the TATA box being mutated in TGTA, (2) the sequence GCCACC, (3) the cDNA for the immunoglobulin signal peptide (nusleotide sequencing = 63 a = 107), (4) the / cDNA for / 3-glusuronidase (nucleotide sequence = 93 a >; 1982), (5) the vWF gene promoter (nucleotide sequencing -487 to +247), and (6) the cDNA for the TATA box binding protein (nucleic acid sequence 1 to 1001, which is mutated at positions of nucleic acid 862 (A replaced by T), 889 and 890 (GT replaced by AC) and 895 (C replaced by G)).

27.- A nucleic acid construct that somersates a promoter and a structural gene, to which a nanosal retention signal (NRS) is added to its 3 'end, and an adisional promoter that astivates the transcription of the gene that encodes a nuclear export factor (NEF), this NEF binding to the NRS mRNA, wherein at least one of said promoters is a promoter according to one of claims 1-13.

28. A nucleic acid construct according to one of claims 20-27, wherein at least one promoter or enhancer is replaced by a promoter unit that responds to the activator.

29. A nucleic acid construct according to claim 28, wherein the promoter unit that responds to the activator comprises at least the following components: (1) one or more activating subunits, identical or different, whose basal transcription is activated by a promoter or enhancer and (2) a promoter that responds to the activator that is activated by the expression product of said activator subunit.

30. A construction of nusleiso acid according to claim 28 or 29, comprising, as a subunit (A) of astivador, (1) the promoter according to one of claims 1-13, (2) the nuclear localization signal (NLS) of SV40 (SV40 large T, amino acids 126-132, PKKKRKV), (3) the acid-binding transactivation domain (TAD) VP16 of HSV-1 (amino acids 406 to 488), and (4) the cDNA encoding the cytoplasmic part of the CD4 glycoprotein (amino acids 397-435); and, as a second activator subunit (B), (1) the promoter of the sdc25C gene (nelic acids -290 to +121), (2) the SV40 nuclear localization signal (SV40) (SV40 large T; 126-132; PKKKRKV); (3) the cDNA for the DNA binding domain of the Gal4 protein (amino acids 1 to 147), and (4) the cDNA for the CD4 binding sequence of the p56 Ick protein (amino acids 1-71) and also the promoter responsive to the activator containing up to about 10 copies of the binding sequence for the Gal4 binding protein, which has the nucleotide sequence 5 '-CGGACAATGTTGACCG-3', and the SV40 promoter basal (nucleotide sequence 48 to 5191 ); and, if appropriate, a structural gene, preferably a complete cDNA encoding an active compound, an enzyme or a fusion protein that is constituted by a ligand and an active compound or by a ligand and an enzyme.

31. A nucleic acid construct according to one of claims 17 to 30, wherein the structural gene is a gene that encodes an active compound that is selected from enzymes, fusion proteins, cytokines, chemokines, growth factors, receptors for cytokines, chemokine receptors, receptors for growth factors, peptides or proteins with an antiproliferative or cytostatic or apoptotic effect, antibodies, fragments of anti-bodies, inhibitors of angiogenesis, peptide hormones, coagulation factors, coagulation inhibitors, fibrinolytic proteins, peptides or proteins that have an effect on blood circulation, blood plasma proteins, antigens of infectious agents, cell antigens or tumor antigens, thrombosis inducing substances, complement activating proteins, virus coating proteins and / or ribozymes.

32. A nucleic acid construct according to claim 31, wherein the structural gene is a gene that encodes an enzyme that cleaves a precursor of a drug in a drug.

33. A nucleic acid construct according to claim 31 or 32, wherein the structural gene is a gene encoding a ligand / active compound fusion protein or a ligand / enzyme fusion protein, the cytokine ligand being selected , growth factors, antibodies, antibody fragments, peptide hormones, mediators and / or cell adhesion proteins.

34. A nucleic acid construct according to claims 16-33 in which it is nucleic acid is DNA.

35. A nucleic acid construct according to one of claims 16-34, wherein the nucleic acid construct is inserted into a vector, preferably a plasmid vector or a viral vector.

36. A process for preparing a nucleic acid construct according to one of claims 16 to 35, comprising joining the individual components to one another.

37. A cell that houses a nucleic acid construct according to one of claims 16-35. 38.- The use of a nucleic acid construct according to one of claims 16-35, or of a cell according to claim 37, for preparing a pharmaceutical product for treating a disease that is selected from tumor diseases, leukemias, autoimmune diseases, allergies, arthritis, inflammations, organ rejections, graft reactions to host, diseases of blood circulation, diseases, anemia, infections, hormonal diseases and / or central nervous system injury. 39.- The use of a cell according to claim 38, wherein the cell is an endothelial cell.