RU2197992C2 - Method for applying genetic therapy of vascular diseases by means of active substance specific to cell and dependant on cellular cycle - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves applying DNA structure which essential element is activating sequence, proportion modulus and active substance gene. The activating sequence is excited in cell- specific way in smooth muscle cells, activated endothelium cells, activated macrophages or activated lymphocytes. This activation process is cell cycle-specifically controlled by promotion modulus. The active substance is a smooth muscle cells growth inhibitor and/or blood coagulation inhibitor. The described DNA- sequence is inserted into virus or non-virus vector completed with a ligand agent possessing affinity to target cell. EFFECT: enhanced effectiveness of treatment. 34 cl, 11 dwg

Description

FIELD OF THE INVENTION
The DNA sequence for the gene therapy of vascular diseases is described. The essential elements of the DNA sequence are the activator sequence, the promoter module and the gene of the active substance. The activator sequence specific to the cell is activated in smooth muscle cells, activated endothelial cells, activated macrophages or activated lymphocytes. This activation specific to the cell cycle is regulated by the promoter module. The active substance is an inhibitor of smooth muscle cell growth and / or coagulation. The described DNA sequence is included in a viral or non-viral vector, complemented by a ligand with affinity for the target taphole.

1). Vascular diseases due to smooth muscle cells
Smooth muscle vascular cells are predominantly localized in the middle membrane of arterial blood vessels and are involved in local and systemic regulation of blood pressure. In the case of an intact, healthy vessel, these smooth muscle cells are at rest during cell division (R. Ross, Nature, 362, 801 (1993)). Vascular damage leads to the movement of smooth muscle cells into the inner membrane of the vessel wall, where they proliferate (the formation of a new inner membrane) and form extracellular matrix components.

 Proliferation of smooth muscle cells related to the inner membrane is considered as an essential component in the occurrence of arteriosclerosis (J. S. Forrester et al., Am. Coll. Cardiol., 17, 758 (1991)). Further, this proliferation of smooth muscle cells leads to repeated stenosis (narrowing) of the vessels after angioplasty operations, as well as after an increase in the size of the cavity of the narrowed vessels (RS Schwartz et al., Am. Coll. Cardiol., 20, 1284 (1992), MW Liu and et al., Circulation, 79, 1374 (1989)).

 As you know, arteriosclerosis, as well as stenosis and repeated stenosis of blood vessels, ultimately leads to vascular thrombosis and, thus, often to a life-threatening heart attack.

 Until now, however, there is still no therapy that, by inhibiting the growth of smooth muscle cells, would prevent vascular stenosis. True, it is known that heparin can inhibit the proliferation of smooth muscle cells (Cochran et al., J. Cell. Physiol., 124, 29 (1995)), but stenosis cannot be sufficiently prevented with heparin. Thus, there is a need for new methods to prevent the growth of smooth muscle cells in damaged vessels and thereby eliminate the risk of heart attack. They use knowledge about genes and molecules that regulatory interfere with the growth of smooth muscle cells.

 Thus, it is known that the c-myb proto-oncogen, as well as the cdc2 kinase and the "proliferating cell nuclear antigen" (PCNA), is involved in the proliferation of smooth muscle cells. By introducing antisense oligonucleotides to c-myb (Simons et al., Nature, 359, 67 (1992)), as well as oligonucleotides antisense oligonucleotides to cdc2 kinase in combination with PCNA antisense oligonucleotides (Morishita et al., Proc. Natl Acad. Sci., 90, 8474 (1993)) immediately after and locally at the site of vascular damage, proliferation of smooth muscle cells in rats can be prevented.

 Similar results are achieved in the case of rats and pigs by introducing the retinoblastoma (Rb) gene known as an oncogenic suppressor, also here immediately after and locally at the site of vascular damage. In order to prevent inactivation of the product produced by the retinoblastoma gene by phosphorylation, a point mutated Rb gene is used (exchange of Thr-246, Ser-601, Ser-605, Ser-780, Ser-786, Ser-787, Ser-800 to Ala; Thr -350 on Arg and Ser-804 on Glu (Hamel et al., Mol. Cell. Biol., 12, 3431 (1992)), which encodes a non-phosphorylated constitutively active form of Rb protein. This mutated Rb gene is inserted into a recombinant adenovirus with incomplete replication and this vector is administered locally (Chang et al., Science, 267, 518 (1995)).

 In another experiment, a recombinant incomplete replication adenovirus is used in which the gene is introduced into the herpes simplex virus thymidine kinase (AV-HS-TK). The product produced by the kinase gene is able to phosphorylate the ganciclovir precursor of the active substance ("preparation") and thus turn it into a non-nucleoside analog that inhibits DNA synthesis.

 The gene vector AV-HS-TK is introduced 7 days after the damage to the vessel, however, it is also locally at the site of injury, and then ganciclovir is intraperitoneally injected daily for 14 days. By this treatment, in the case of rats, a distinct inhibition of smooth muscle cell growth is achieved (Guzman et al., Proc. Natl. Acad. Sci., 91, 10732 (1994)). By the same kind of treatment, similar results are also achieved in the case of a pig (Ohno et al., Science, 265, 781 (1994)). However, here the vector is administered immediately after damage to the vessel, and ganciclovir is administered daily for 6 days.

 In general, these experiments show the possibility, with the help of gene therapy measures, which are interventions in the processes of smooth muscle cell division, to prevent stenosis after vascular damage.

 A disadvantage of the methods known from the literature, however, is that effective substances (vectors) need to be introduced locally at the site of vascular damage, and perhaps even from time to time it is necessary to obstruct the corresponding section of the vessel in order to prevent the “washing off” of the vectors. True, invasive interventions of this kind are performed in the usual way as part of an increase in the size of the cavity of the narrowed vessels, however, they require significant expenses and, for their part, pose a significant threat to the patient due to the danger of thrombosis and embolism.

 Incidentally, despite the local introduction of vectors, there is no guarantee that they transduce only proliferating smooth muscle cells. Transduction of nearby or removed cells can lead to side effects (including cell transformation and induction of tumors), as is currently discussed by a wide range of experts (Friedmann, Science, 244, 1275 (1989); Plummer, Scrip Magazine, III / 29 (1995)).

 As an alternative to the administration of the above vectors, systemic (e.g., intravenous or oral) administration of cytostatic drugs to inhibit the proliferation of smooth muscle cells at the site of vascular disease has only a minor and temporary effect, on the other hand, it causes the risk of endothelial damage and leads to significant acute as well as chronic side effects.

2). Thrombosis
Thrombosis is still a difficult to treat, partly life-threatening complication of metabolic diseases, such as arteriosclerosis, arterial and venous vascular diseases, and local, as well as systemic immuno-reactive syndromes (see reviews by Philipps et al., Blood, 71, 831 (1988) ); Harker, Biomed. Progr., 8, 17 (1995)).

 Although a number of anticoagulants, antithrombotic agents, fibrinolytic drugs and platelet aggregation inhibitors have been used in clinical practice for a long time and new substances are undergoing clinical trials, until now life-threatening complications of thrombosis cannot be adequately prevented or controlled (White, Scrip Magazine, 4 6 (1994); Antiplatelet Trialists' Collaboration BMJ, 308, 81 (1994)).

 Thus, there is a need for new drugs for the prevention and treatment of thrombosis (BMJ, 305, 567 (1992); Vinazzer, Biomedical Progress, 6, 17 (1993)). A significant part of thrombosis is caused by activated or damaged endothelial cells. They themselves or stimulated for proliferation due to growth factors in the blood smooth muscle cells alone or in combination with activated macrophages, lymphocytes, platelets and granulocytes cause activation of the coagulation system (Nemerson, Blood, 71, 1 (1988)).

 This activation ultimately leads to the formation of fibrin, the activation and aggregation of platelets and the formation of fibrin-rich or platelet-rich constricting or clogging blood vessels blood clots, thromboses. Thromboses of this kind in the arterial vascular system lead to heart attacks, which, for example, in the case of the heart or brain, are life-threatening.

 The currently used therapy with antithrombotic agents (like heparin or heparin fractions), anticoagulants (like coumarin), platelet aggregation inhibitors (like aspirin) and fibrinolotic drugs (like streptokinase, urokinase or tissue plasminogen activators (tPA)), however It has a confirmed by numerous clinical trials prophylactic effect on threatening thrombosis and therapeutic effect on existing thrombosis, this action, however, is insufficient. The reason for this is largely the fact that the effect of the therapeutic agents used is not limited to the site of the disease, that is, thrombosis, but they act systemically. Thus, bleeding thus limits both the increase in dose and the duration of use.

3). General Description of the Invention
The subject of the invention is an active substance (i.e. a medicine) that can be administered to a patient both locally and also systemically and which:
- affects only smooth muscle cells in the division stage, inhibits the proliferation of smooth muscle cells after vascular damage or vascular lesions, and thus prevents stenosis or repeated stenosis of blood vessels;
- inhibits blood coagulation only at the site of thrombosis, that is, at the site of activated and proliferating endothelial cells, smooth muscle cells of the inner lining of blood vessels, macrophages and / or lymphocytes; or
- inhibits both proliferation of smooth muscle cells and locally inhibits thrombosis there.

Основной элемент этого активного вещества представляет собой регулируемый клеточным циклом промоторный модуль.The main component of this active substance is the DNA construct, which consists of the following elements:
(DNA throughout the text of this application is used as a general concept for both complementary (cDNA) and genomic DNA sequences)

The main element of this active substance is a promoter module regulated by the cell cycle.

 Under the regulated cell cycle, the promoter module must be understood, for example, the nucleotide sequence of CDE-CHR-Inr- (see below). An essential function of the promoter module is the inhibition of the function of the activator sequence in the GO / Gl phase of the cell cycle and expression specific to the cell cycle in the S / G2 phase and, thus, in proliferating cells.

 The CDE-CHR-Inr promoter module was detected by a detailed study of the G2-specific expression of the human cdc25C promoter. The starting point was the finding of a repressor element (cell cycle dependent CDE element), which is responsible for the “shutdown” of the promoter in the Gl phase of the cell cycle (Lucibello et al., EMBO J., 14, 132 (1995)). By genomic dimethyl sulfate (DMS) footprinting and functional analysis (Fig. 1, 2), it can be shown that CDE Gl-specific binds a repressor (“CDE-binding factor”, CDF) and thereby leads to inhibition of transcription in non-proliferating (GO) cells . Localized in the region of the basal promoter CDE in its replication function depends on the "reverse activating (against the course of transcription) sequence" (UAS). This leads to the conclusion that the CDE-binding factor suppresses the transcriptional activating effect of the 5 ′ end-bound activator proteins in a cell cycle dependent manner, that is, in non-proliferating cells, as well as in the Gl phase of the cell cycle (FIG. 3).

 This conclusion can be confirmed by the following experiment: the fusion of the virus-specific, non-regulatory cell cycle of the SV40 early enhancer with the minimal cdc25 promoter (consisting of CDE and located at the 3'-end of the starting region) leads to a clear regulation of the chimeric promoter by the cell cycle (figure 4) . Subsequent studies of the cdc25C enhancer show that NF-Y (CBF) (Dom et al., Cell, 50, 863 (1987); Van Hujisduijnen et al., EMBO J. 11, 3119 (1990); Coustry et al., J. Biol. Chem. 270, 468 (1995)), Sp1 (Kadonaga et al., TIBS, 11, 10 (1986)) and, possibly, a new binding CBS 7 transcription factor (CIF). Another conclusion of interest in this study is the discovery that NF-Y within the cdc25C enhancer effectively activates transcription only in combination with at least one other NF-Y complex or CIF. Both NF-Y and Sp1 belong to the class of glutamine-enriched activators, which is an important indication of the mechanism of repression (for example, interaction, respectively, interference with certain basal transcription factors or TAFS).

 A comparison of the promoter sequences of cdc25C, cyclin A and cdc2 shows homology in several areas (Fig. 5). In all three promoters (the existing deviations are functionally irrelevant), not only CDEs are preserved, but also neighboring Us units. All these regions, as expected, show protein binding in vivo in the case of CDE in a cell cycle dependent manner. In addition, it can be seen that all three promoters are deregulated due to a CDE mutation (Table 1). A similar similarity was observed when comparing cdc25C, cyclin A and cdc2.

 The sequences are also distinct (Figure 5) in the region immediately at the 3'end of the CDE ("region homologous to the cell cycle gene"; CHR). This area is functionally as important as CDE (Table 1), but it is not detected in vivo in dimethyl sulfate footprinting experiments. A possible explanation for this is the interaction of the factor with a small portion of DNA. The results of the Electrophoretic Mobility Shift Assay (EMSA) experiments indicate that CDE and CHR bind together to form a protein complex, CDF. These observations indicate that repression via CDF of glutamine-enriched activators proceeds via the frequently encountered mechanism of cell-transcriptional regulation.

 For regulation of the cdc25C promoter, however, not only the CDE-CHR region is important, but also one of the initiation sites (position + 1) within the nucleotide sequence of the basal promoter (positions ≤ -20 to ≥ +30, see Fig. 1) . Mutations in this region, which includes the in vitro YY-1 transcription factor binding site (Seto and Shenk, Nature, 354, 241 (1991); Usheva and Shenk, Cell, 76, 1115 (1994)), lead to complete deregulation. Given the proximity of CDE-CHR to the basal promoter, thus, the interaction of CDF with the basal transcription complex is very likely.

 As the activator sequence (reverse activating (against the transcription) sequence = UAS), it is necessary to understand the nucleotide sequence (promoter or enhancer sequence) with which transcription factors formed or active in the target cell interact. As the activator sequence, a CMV enhancer, a CMV promoter (European patent 073177 B1), SV40 promoter, or any other promoter or enhancer sequence known to those skilled in the art can be used. According to the present invention, however, the preferred activator sequences include such gene regulatory sequences, respectively, elements of genes that especially encode proteins formed in smooth muscle cells, activated endothelial cells, or in activated macrophages or lymphocytes.

 By active substance is meant the DNA sequence of a protein that can produce a therapeutic effect at the site of formation, i.e., inhibition of smooth muscle cell proliferation, coagulation or (in the case of two active substances) inhibition of proliferation and coagulation at the same time. In selecting the nucleotide sequence for the activator sequence and the active substance, they are guided by the target cell and the desired active substance.

 The DNA construct according to the invention is complemented in a manner known per se to a vector; for example, they are introduced into a viral vector (see D. Jolly, Cancer Gene Therapy, 1, 51 (1994)), or, however, supplemented with a plasmid. Viral vectors or plasmids can be combined with colloidal dispersions, for example, with liposomes (Farhood et al., Annal. Of the New York Academy of Sciences, 716, 23 (1994)) or, however, with polylysine ligand conjugates (Curiel et al., Annals of the New York Academy of Sciences, 716, 36 (1994)). In the same way, it is possible to obtain drugs using conventional auxiliary substances for the preparation of drugs.

 Such viral or non-viral vectors can be supplemented with a ligand that has an affinity for binding the membrane structure to a selected target cell. When choosing a ligand, in this way, they are guided by the choice of the target cell (see section 4.4 and subsequent and section 5.4 and subsequent). The active substance according to the invention is explained in more detail in accordance with the following examples.

4). Active substance for inhibiting smooth muscle cell proliferation:
4.1. Selection of activator sequence of smooth muscle cells
As activator sequences according to the present invention, it is preferable to select gene-regulatory sequences, respectively, elements of genes that encode the proteins formed especially in smooth muscle cells. These genes, for example, are as follows:
- tropomyosin (Tsukahara et al., Nucleic Acid Res., 22, 2318 (1994); Novy et al., Cell Motility and Cytosceleton, 25, 267 (1993); Wilton et al., Cytogenetics and Cell Genetics, 68, 122 (1995));
- α-actin (Sartorelli et al., Gens and Developm., 4, 1811 (1990); Miwa et al., Nucleic Acids Res, 18, 4263 (1990));
- α-myosin (Kelly et al., Can. J. Physiol. and Pharm., 72, 1351 (1994); Moussavi et al., Mol. Cell. Biochem. 128, 219 (1993));
- growth factor receptors, such as, for example, growth factor derived from platelets (PDGF), FGF (Rubin et al., Int. Congress Ser., 925, 131 (1990); Ross, Ann. Rev. Med., 38, 71 (1987);
- acetylcholine receptors (Dutton et al., PNAS USA, 90, 2040 (1993); Durr et al., Eur. J. Biochem., 224, 353 (1994));
- phosphofructokinase-A (Gekakis et al., Biochemistry, 33, 1771, (1994); Tsujino et al., J. Biol. Chem., 264, 15334 (1989); Castella-Escola et al., Gene, 91, 225 (1990));
- phosphoglyceratmutase (Nakatsuji et al., Mol. Cell. Biol., 12, 4384 (1992));
- troponin C (Lin et al., Mol. Cell. Biol., 11, 267 (1991));
- desmin (Li et al., J. Biol. Chem., 266, 6562 (1991); Neuromuscular Disorders, 3, 423 (1993));
- myogenin (Funk et al., PNAS USA, 89, 9484 (1992); Olson, Symp. Soc. Exp. Biol., 46, 331 (1992); Zhon et al., Mol. Cell. Biol., 14, 6232 (1994); Atchley et al., PNAS USA, 9JL, 11522 (1994));
endothelin A receptors (Hosoda et al., J. Biol. Chem., 26 7, 18797 (1992); Orelly et al., J. Cardiovasc, Pharm., 22, 18 (1993); Hayzer et al., Am . J. Med. Sci. 304, 231 (1992); Haendler et al., J. Cardiovasc. Pharm., 20, 1 (1992));
- VEGF; VEGF is produced by smooth muscle cells, especially under hypoxic conditions (Berse et al., Mol. Biol. Cell, 3, 211 (1992); Finkenzeller et al., VVRS, 208, 432 (1995); Tischer et al., VVRS, 165 , 1198 (1989); Leung et al., Science, 246, 1306 (1989); Ferrara et al., Endoc. Fev., 13, 18 (1992)).

The promoter sequences of genes for these proteins are indicated in the following works:
tropomyosin (Gooding et al., Embo. J., 13, 3861 (1994));
- α-actin (Shimizu et al., J. Biol. Chem., 270, 7631 (1995); Sartorelli et al., Genes Dev., 4, 1811 (1999));
- α-myosin (Kurabayashi et al., J. Biol. Chem., 265, 19271 (1990); Molkentin et al., Mol. Cell. Biol., 14, 4947 (1994));
- PDGF receptor (Pistritto et al., Antibiot. Chemother., 46, 73 (1994));
- FGF receptor (Myers et al., J. Biol. Chem., 270, 8257 (1995); Johnson et al., Adv. Cencer Res., 60, 1 (1993); Chellaiah et al., J. Biol. Chem., 269, 11620 (1994); Yu et al., Hum. Mol. Genetics, 3, 212 (1994); Wang et al., WWRS, 203, 1781 (1994); Murgue et al., Cancer Res. , 54, 5206 (1994); Avraham et al., Genomics, 21, 656 (1994); Burgess et al., Ann. Rev. Biochem. 58, 575 (1989));
- MRF-4 (Naidu et al., Mol. Cell. Biol., 15, 2707 (1995));
- phosphofructokinase A (Gekakis et al., Biochem., 33, 1771 (1994));
- phosphoglyceratmutase (Makatsuji et al., Mol. Cell. Biol., 12, 4384 (1992));
- troponin C (Ip et al., Mol. Cell. Biol., 14, 7517 (1994); Parmacek et al., Mol. Cell. Biol., 12, 1967 (1992));
myogenin (Salminen et al., J. Cell. Biol., 115, 905 (1991); Durr et al., Eur. J. Biochem. 224, 353 (1994); Edmondson et al., Mol. Cell. Biol., 12, 3665 (1992));
- endothelin A receptors (Hosoda et al., J. Biol. Chem., 267, 18797 (1992); Li and Paulia, J. Biol. Chem., 266, 6562 (1991));
Desmin (Li et al., Neuromisc. Dis., 3, 423 (1993); Li and Capetanaki, Nucl. Acids Res. 21, 335 (1993));
- VEGF.

The gene regulatory sequences of the VEGF gene are as follows:
the promoter sequence of the VEGF gene (5'-flanking region) (Michenko et al., Cell. Mol. Biol. Res., 40, 35 (1994); Tischer et al., J. Biol. Chem., 266, 11947 (1991)); or
- enhancer sequence of the VEGF gene (3'-flanking region) (Michenko et al., Cell. Mol. Biol. Res., 40, 35 (1994)); or
- c-Src gene (Mukhopadhyay et al., Nature, 375, 577 (1995) 7 Bonham et al., Oncogene, 8, 1973 (1993); Parker et al., Mol. Cell. Biol., 5, 831 (1985); Anderson et al., Mol. Cell. Biol., 5. 1122 (1985)); or
- V-Src gene (Mukhodpadhyay et al., Nature, 375, 577 (1995); Anderson et al., Mol. Cell. Biol., 5, 1122 (1985); Gibbs et al., J. Virol., 53, 19 (1985));
- "artificial"promoters; helix-loop-helix (HLH) family factors (MyoD, Myf-5, myogenin, MRF4 (reviewed by Olson and Klein, Genes Dev., 8, 1 (1994)) are described as muscle-specific transcription activators. Further, to specific muscle transcription factors include the zinc finger protein GATA-4 (Arceci et al., Mol. Cell. Biol., 13, 2235 (1993); Ip et al., Mol. Cell. Biol., 14, 7517 (1994 )), as well as groups of transcription factors MEF-2 (Yu et al., Gene Dev., 6, 1783 (1992)).

) (Molkentin и др., Mol. Cell. Biol., 14, 4947 (1994)).HLH-proteins, as well as GATA-4, exhibit muscle-specific transcription not only with promoters of muscle-specific genes, but also in heterologous environments, as well as with artificial promoters. Such artificial promoters, for example, are the following:
- multiple copies of the binding sites (DNA) of muscle-specific HLH proteins, such as the E-block (MyoD)
(e.g. 4xAGCAGGTGTTGGGAGGC) (Weintraub et al., PNAS 87, 5623 (1990));
- multiple copies of the GATA-4 DNA binding site of the Franklin Syndrome α-myosin gene
(eg,

) (Molkentin et al., Mol. Cell. Biol., 14, 4947 (1994)).

4.2. Selection of the active substance of smooth muscle cells
As the active substance according to the invention, it is necessary to understand the DNA sequence, the expressed protein of which inhibits the proliferation of smooth muscle cells. These cell cycle inhibitors include, for example, the DNA sequences of the following proteins:
a) Inhibiting proteins:
- retinoblastoma protein (pRb = pllO) or related proteins p107 and p130 (La Thangue, Curr. Opin. Cell Biol., 6, 443 (1994));
- p53 protein (Prives et al., Genes Dev., 1, 529 (1993));
p21 (WAF-1) -protein (El-Deiry et al., Cell, 75, 817 (1993));
p16 protein (Serrano et al., Nature, 366, 704 (1993); Kamb et al., Science, 264, 436 (1994); Nobori et al., Nature, 368, 753 (1994));
- other cdK inhibitors (review by Pines, TIBS, 19, 143 (1995));
- GADD45 protein (Papathanasiou et al., Mol. Cell. Biol., 11, 1009 (1991); Smith et al., Science, 266, 1376 (1994));
- bak protein (Farrow et al., Nature, 374, 731 (1995); Chittenden et al., Nature, 374, 733 (1995); Kiefer et al., Nature, 374, 736 (1995)).

 In order to prevent the rapid intracellular inactivation of these cell cycle igniters, it is preferable to use genes that have mutations in the inactivation sites of the expressed proteins, without compromising their function.

 Retinoblastoma protein (pRb / pllO) and related proteins p107 and p130 are inactivated by phosphorylation. Thus, a pRb / p110, p107, or p130 cDNA sequence is preferably used that is mutated so that the phosphorylation sites of the encoded protein are replaced by non-phosphorylated amino acids.

 According to Hamel et al. (Mol. Cell. Biol., 12, 3431 (1992)), the cDNA sequence of the retinoblastoma protein (p110) due to the exchange of amino acids at positions 246, 350, 601, 605, 780, 786, 787, 800 and 804 becomes more non-phosphorylated, however, its binding activity to the large T-antigen does not deteriorate. For example, amino acids Thr-246, Ser-601, Ser-605, Ser-780, Ser-786, Ser-787 and Ser-800 are replaced by Ala, amino acid Thr-350 is replaced by Arg and amino acid Ser-804 is replaced by Glu.

 Similarly, the DNA sequence of protein p107 or protein p130 is mutated.

 The p53 protein is inactivated in the cell either by binding to special proteins, such as MDM2, or by oligomerization of p53 via the dephosphorylated C-terminal serine-392 (Schikawa et al., Leukemia and Lymphoma, 11, 21 (1993); Brown, Annals of Oncology, 4, 623 (1993)). Thus, the DNA sequence of the p53 protein, which is shortened at the C-terminus to serine-392, is preferably used.

b) Cytostatic or cytotoxic proteins
As an active substance, further, it is necessary to understand the DNA sequence that expresses (expresses) a cytostatic or cytotoxic protein.

Such proteins include, for example, the following:
- perforin (Lin et al., Immunol. Today, 16, 194 (1995));
- granzyme (Smyth et al., Immunol. Today, 16, 202 (1995));
- tumor necrosis factor (Porter, Tib Tech., 9, 158 (1991); Sidhu et al., Pharmac. Ther., 57, 79 (1993)); especially
the tumor necrosis factor α (Beutler et al., Nature, 320, 584 (1986); Kriegler et al., Cell, 53, 45 (1988));
β-tumor necrosis factor (Gray et al., Nature, 312, 721 (1984); Li et al., J. Immunol., 138, 4496 (1987); Aggarwal et al., J. Biol. Chem., 260, 2334 (1985)).

c) Enzymes
As an active substance, however, it is also necessary to understand the DNA sequence of the enzyme, which converts the inactive precursor of the cytostatic preparation into a cytostatic preparation.

 Such enzymes that break down inactive precursors of substances (drugs) to active cytostatic drugs (drugs), and possible drugs and drugs have already been clearly described by Deonarain et al., (Br. J. Cancer, 70, 786 (1994)), Mullen (Pharmac. Ther., 63, 199 (1994)) and Harris et al. (Gene Ther., 1, 170 (1994)).

For example, you can use the DNA sequence of one of the following enzymes:
- herpes simplex virus thymidine kinase
(Garapin et al., PNAS USA, 76, 3755 (1979); Vile et al., Cancer Res., 53, 3860 (1993); Wagner et al., PNAS USA, 78, 1441 (1981); Moelten et al. ., Cancer Res., 46, 5276 (1986); J. Natl. Cancer Inst., 82, 297 (1990));
- chickenpox virus thymidine kinase
(Huber et al., PNAS USA, 88, 8039 (1991); Snoeck, Int. J. Antimicrob. Agents, 4, 211 (1994));
- bacterial nitroreductase
(Michael et al., FEMS Microbiol. Letters, 124, 195 (1994); Bryant et al., J. Biol. Chem., 266, 4126 (1991); Watanabe et al., Nucleic Acids Res., 18, 1059 (1990));
- bacterial β-glucuronidase
(Jefferson et al., PNAS USA, 83, 8447 (1986));
- plant β-glucuronidase from dry crops
(Schulz et al., Phytochemistry, 26, 933 (1987));
- human β-glucuronidase
(Bosslet et al., Br. J. Cancer, 65, 234 (1992); Oshima et al., PNAS USA, 84, 685 (1987));
- human carboxypeptidase (CB), for example:
CB-A mast cell
(Reynolds et al., J. Clin. Invest, 89, 273 (1992));
SV-In the pancreas
(Yamamoto et al., J. Biol. Chem., 267, 2575 (1992);
Catasus et al., J. Biol. Chem., 270, 6651 (1995));
- bacterial carboxypeptidase
(Hamilton et al., J. Bacteriol., 174, 1626 (1992); Osterman et al., J. Protein Chem., 11, 561 (1992));
- bacterial β-lactamase
(Rodriges et al., Cancer Res., 55, 63 (1995); Hussain et al., J. Bacteriol., 164, 223 (1985); Coqe et al., Embo J., 12, 631 (1993)) ;
- bacterial cytosine deaminase
(Mullen et al., PNAS USA, 89, 33 (1992); Austin et al., Mol. Pharmac., 43, 380 (1993); Danielson et al., Mol. Microbiol., 6, 1335 (1992)) ;
- human catalase, respectively peroxidase
(Ezurum et al., Nucl. Acids Res. 21, 1607 (1993));
- phosphatase, in particular
human alkaline phosphatase
(Gum et al., Cancer Res., 50, 1085 (1990));
human acid prostate phosphatase
(Sharieff et al., Am. J. Hum. Gen., 49, 412 (1991); Song et al., Gene, 129, 291 (1993); Tailor et al., Nucl. Acids Res., 18, 4928 (1990));
- acid phosphatase type 5
(Gene, 130, 201 (1993));
- oxidase, in particular
human lysyl oxidase
(Kimi et al., J. Biol. Chem., 270, 7176 (1995));
human acid D-amino oxidase
(Fukui et al., J. Biol. Chem., 267, 18631 (1992));
- peroxidase, in particular
human glutathione peroxidase
(Chada et al., Genomics, 6, 268 (1990); Ishida et al., Nucl. Acids Res., 15, 10051 (1987));
human eosinophilic peroxidase
(Ten et al., J. Exp. Med., 169, 1757 (1989); Sahamaki et al., J. Biol. Chem., 264, 16828 (1989));
- human thyroid peroxidase
(Kimura, PNAS USA, 84, 5555 (1987)).

 To facilitate the secretion of these enzymes, it is possible that the homologous signal sequence contained in the DNA sequence can be replaced by a heterologous extracellular-enhancing signal sequence.

 For example, the signal sequence of β-glucuronidase (position in DNA ≤ 27 to 93; Oshima et al., PNAS, 84, 685 (1987)) can be replaced by the signal sequence of human immunoglobulin (position in DNA ≤ 63 to ≥ 107; Riechmann and et al., Nature, 332, 323 (1988)).

 Further, it is preferable to select cDNAs of such enzymes that, due to point mutations, accumulate to a lesser extent in lysosomes. Point mutations of this kind are described, for example, for β-glucuronidase (Shipley et al., J. Biol. Chem., 268, 12193 (1993)).

4.3. The combination of the same or different active substances of smooth muscle cells
The subject of the invention, further, is an active substance in which there is a combination of DNA sequences of several identical active substances (A, A) or different active substances (A, B). For expression of two DNA sequences, preferably the cDNA of the “internal ribosome aminoacyl site” (IRES) cDNA is included as a regulatory element. Such IRESs are described by Mountford and Smith (TIG, 11, 179 (1995)), Kaufman et al., Nucl. Acids Res., 19, 4485 (1991); Morgan et al., Nucl. Acids Res. 20, 1293 (1992) and Dirks et al., Gene, 129, 247 (1993); Pelletier and Sonenberg, Nature, 334, 320 (1988); et al., Bio Techn., 12, 694 (1994).

 Thus, cDNA of poliovirus IRES sequences (position ≤ 140 to ≥ 630 5'-UTR (Pelletier and Sonenberg, Nature, 334, 320 (1988)) can be used to bind DNA of antithrombotic substance A (at the 3'-end) and DNA of antithrombotic substance B (at the 5'-end).

Такого рода активное вещество в зависимости от сочетания обладает аддитивным или синергическим действием согласно изобретению.

This kind of active substance, depending on the combination, has an additive or synergistic effect according to the invention.

 Due to the growth of smooth muscle cells related to the inner shell, as well as due to their apoptosis or necrosis, the coagulation system can be activated as a result of exposure to the cycle inhibitor and thrombosis can occur. Thromboses of this kind must be prevented by the prophylactic administration of a coagulation inhibitor (aspirin, heparin or other antithrombotic agent). The coagulation inhibitor is administered systemically, i.e., orally or parenterally.

 However, often the side effects of the coagulation inhibitor prevent sufficient concentration at the site of smooth muscle cell growth belonging to the inner membrane.

 Thus, the prevention of thrombosis due to such coagulation inhibitors is unreliable (Pukac, Am. J. Pathol., 139, 1501 (1991)).

 A further subject of the invention is that the active substance claimed in the invention, in addition to the active substance, which is a cell cycle inhibitor, contains, as another element, the DNA sequence of the active substance, which is a coagulation inhibitor.

Экспрессия ингибитора свертывания регулируется таким же образом, как и экспрессия ингибитора клеточного цикла, за счет активаторной последовательности и регулируемого клеточным циклом репрессорного модуля. Одновременная экспрессия как ингибитора клеточного цикла, так же как ингибитора свертывания, регулируется предпочтительно элементом гена "внутренним рибосомным аминоацильным сайтом" (IRES).

The expression of the coagulation inhibitor is regulated in the same manner as the expression of the cell cycle inhibitor, due to the activator sequence and the cell cycle regulated repressor module. Simultaneous expression as a cell cycle inhibitor, as well as a coagulation inhibitor, is preferably regulated by the internal ribosome aminoacyl site (IRES) gene element.

As a coagulation inhibitor, genes, for example plasminogen activators (PA), such as tissue PA (tPA) or urokinase-like PA (uPA), protein C, antithrombin-III, tissue factor metabolic pathway inhibitor or hirudin can be used. The DNA sequences of these coagulation inhibitors are described below:
- tissue plasminogen activator (tPA)
(Sasaki et al., Nucl. Acids Res., 16, 5695 (1988); Pennica et al., Nature, 301, 214 (1983); Wei et al., DNA, 4, 76 (1985); Harris et al. ., Mol. Biol. Med., 3, 279 (1986));
- plasminogen activator urokinase type (uRA)
(Miyake et al., J. Biochem., 104, 643 (1988); Nelles et al., J. Biol. Chem., 262, 5682 (1987));
- tPA and uPA hybrids
(Kalyan et al., Gene, 68, 205 (1988); Devries et al., Biochem. 27, 2565 (1988));
- protein C (Foster et al., PNAS, 82, 4673 (1985));
- hirudin
(Maerki et al., Semin. Thromb. Hemostas., 17, 88 (1991); De Taxis du Poet et al., Blood Coag. Fibrin., 2, 113 (1991); Harvey et al., PNAS USA, 83 1084 (1986); Sachhieri et al., European patent 0324712 B1; European patent 0142860 B1);
serine proteinase inhibitors (serpins), such as, for example, a C-lS inhibitor
(Bock et al., Biochem., 25, 4292 (1986); Davis et al., PNAS USA, 83, 3161 (1986); Que, BBPC, 137, 620 (1986); Rauth et al., Proteine Sequences and Data Analysis, 1, 251 (1988); Carter et al., Eur. J. Biochem., 173, 163 (1988); Tosi et al., Gene, 42, 265 (1986); Carter et al., Eur. J. Biochem., 197, 301 (1991); Eldering et al., J. Biol. Chem., 267, 7013 (1993));
- 1-antitrypsin
(Tosi et al., Gene, 42, 265 (1986); Graham et al., Hum. Genetics, 85, 381 (1990); Hafeez et al., J. Clin. Invest., 89, 1214 (1992); Tikunova et al., Bioorganic Chemistry, 17, 1694 (1991); Kay et al., Human Gene Ther., 3, 641 (1992); Lemarchand et al., Mol. Biol., 27, 1014 (1993); Lambach et al., Human Mol. Gen., 7, 1001 (1993));
- antithrombin-III
(Stackhouse et al., J. Biol. Chem., 258, 703 (1983); Olds et al., Bioshem. 32, 4216 (1993); Laue et al., Nucl. Acids Res., 22, 3556 ( 1994));
- tissue factor metabolic pathway inhibitor
(TFPI)
(Enjyoji et al., Genomics, 17, 423 (1993); Wun et al., J. Biol. Chem., 263, 6001 (1988); Girard et al., Thromb. Res., 55, 37 (1989) )

4.4. Smooth muscle cell ligand selection
As a ligand in colloidal dispersions, for example, polylysine ligand conjugates, substances that bind to the surface of smooth muscle cells are preferred. This includes antibodies or antibody fragments directed against the membrane structures of smooth muscle cells, such as:
- antibody 10F3
(Printseva et al., Exp. Cell Res., 169, 85 (1987); Am. J. Path., 134, 305 (1989)); or
anti-actin antibody
(Desmonliere et al., Comptes Rendus des Seances de la Soc. De Biol. And de ses Filiales, 182, 391 (1988)); or
- antibodies against angiotensin-II receptors
(Butcher et al., WWRA, 196, 1280 (1993)); or
- antibodies against growth factor receptors
(review by Mendelsohn, Prog. All., 45, 147 (1988); Sato et al., J. Nat. Canc. Inst., 81, 1600 (1989); Hynes et al., BBA, 1198, 165 (1994) );
or antibodies directed, for example, against
- EGF receptors
(Fan et al., Cancer Res., 53, 4322 (1993); Bender et al., Cancer Res., 52, 121 (1992); Aboud-Pirak et al., J. Nat. Cancer Inst., 80, 1605 (1988); Sato et al., Mol. Biol. Med., 1, 511 (1983); Kawamoto et al., PNAS, 80, 1337 (1983));
- or against PDGF receptors
(Yu et al., J. Biol. Chem., 269, 10668 (1994); Kelly et al., J. Biol. Chem., 266, 8987 (1991); Bowen-Pope et al., J. Biol. Chem., 257, 5161 (1982));
- or against FGF receptors
(Vanhalteswaran et al., J. Cell. Biol., 115, 418 (1991); Zhan et al., J. Biol. Chem., 269, 20221 (1994));
- or antibodies against endothelin-A receptors.

 Mouse monoclonal antibodies should preferably be used in humanized form. Humanization is carried out by the method described by Winter et al., Nature, 349, 293 (1991), and Hoogenbooms et al., Rev. Tr. Transfus Hemobiol., 36, 19 (1993). Antibody fragments are prepared according to the prior art, for example, by the method described by Winter et al., Nature. 349, 293 (1991); Hoogenboom et al., Rev. Tr. Transfus Hemobiol., 36, 19 (1993); Girol, Mol. Immunol. 28, 1379 (1991) or Huston et al., Int. Res. Immunol., 10, 195 (1993).

Ligands include, further, all active substances that bind to membrane structures or membrane receptors of smooth muscle cells (review by Pusztai et al., J. Pathol., 169, 191 (1993); Harris, Current Opin. Biotechnol., 2, 260 (1991)). For example, this includes growth factors or fragments thereof, respectively, their partial sequences that bind to expressed smooth muscle cells by receptors, such as:
- PDGF
(Westermark et al., Cancer Res., 51, 5087 (1991); Ponten et al., J. Invest. Dermatol., 102, 304 (1994));
- EGF
(Modjtahedi et al., Int. J. Oncol., 4, 277 (1994);
Carpentar et al., J. Biol. Chem., 265, 7709 (1990);
- TGF-β
(Segarini, BBA, 1155, 269 (1993));
- TGF-α
(Salomon et al., Cancer Cells, 2, 389 (1990));
- FGF
(Burgess et al., Annu. Rev. Biochem., 58, 575 (1989));
- endothelin-A
(Oreilly et al., J. Cardiovasc. Pharm., 22, 18 (1993)).

4.5. Obtaining biologically active substances of smooth muscle cells
The preparation of the active substance according to the invention is described in more detail in accordance with the following examples:
a). Design of the chimeric promoter myogenin promoter CDE-CHR-Inr
The human myogenin promoter (position ≤ -210 to ≥ +54 of the DNA sequence published by Salmin et al., J. Cell. Biol., 115, 905 (1991)) binds to the 5'-end of CDE-CHR by its 3 ′ end -Inr module of the human cdc25C gene (position ≤ -20 to ≥ +121 of the sequence published by Lucibello et al., EMBO J., 14, 132 (1995)) (see FIG. 6). The binding is carried out using known to the specialist and commercially available enzymes. In addition, various fragments of the myogenin promoter sequence are used (see FIG. 6). Thus, a DNA sequence of the myogenin promoter is used containing a TATA block. However, the promoter sequence at position ≤ -210 to ≥ -40 can also be used in the same way (see FIG. 6).

b) The construction of a plasmid containing the main (Central) component of the active substance
The thus obtained chimeric control unit for the transcription of the myogenin promoter module by its 3'-end is linked to the 5'-end of the DNA, which contains the complete coding region of human β-glucuronidase (position in DNA ≤ 27 - ≥ 1982 of the sequence published by Oshima et al., PNAS USA 84, 684 (1987)). (see Fig.6).

 This DNA also contains the signal sequence necessary for secretion (22 N-terminal amino acids). To facilitate cell isolation, this signal sequence is preferably exchanged for an immunoglobulin signal sequence (position ≤ 63 to ≥ 107; Riechmann et al., Nature, 332, 323 (1988)) (FIG. 7). Thus obtained transcriptional control units and human β-glucuronidase DNA are cloned into pUC18 / 19 or from plasmid vectors produced from Bluescript, which can be used directly or in colloidal dispersion systems for in vivo administration. Alternatively, chimeric genes can be transformed into viral vectors or other suitable vectors and injected.

in). Construction of a plasmid containing β-glucuronidase genes, as well as tissue plasminogen activator
Obtained as in claim 2), the β-glucuronidase element of the myogenin repressor module with its 3 ′ end is connected to the 5 ′ end of the cDNA of the “internal ribosomal aminoacial site” of the poliovirus (position ≤ 140 to ≥ 630 of the 5′-UTR element of Pelletier and Sonenberg, Nature, 334, 320 (1988)). The 5 ′ end of the DNA of the tissue plasminogen activator (position ≤ 85 to ≥ 1753; Pennica et al., Nature, 301, 214 (1983)) is again linked to its 3′-end (FIG. 7). The whole construct is then cloned into pUC17 / 19 or from Bluescript-derived plasmid vectors, which can be used directly or in colloidal dispersion systems for in vivo transfer. Alternatively, chimeric genes can be transformed into viral vectors or other suitable vectors (FIG. 8).

5). Coagulation Inhibition Active
5.1. The choice of the activator sequence for inhibition or coagulation
As activator sequences according to the invention, it is preferable to use gene-regulatory sequences, respectively, elements from genes that encode proteins found in smooth muscle cells, in activated endothelial cells, in activated macrophages or in activated lymphocytes.

a) Smooth muscle cells
Examples of activator sequences of genes in smooth muscle cells are already indicated in Section 4.1.

b) Activated Endothelial Cells
Examples of proteins that are especially formed in activated endothelial cells are described by Burrows et al. (Pharmac. Therp., 64, 155 (1994)) and Plate et al. (Brain Pathol., 4, 207 (1994)). In particular, these proteins, which are intensively formed in endothelial cells, include, for example, the following:
- brain-specific endothelial carrier of glucose-I; endothelial cells of the brain are characterized by very strong expression of this carrier in order to carry out transendothelial transport of D-glucose to the brain (Gerhart et al., J. Neurosci. Res., 22, 464 (1989)). The promoter sequence is described by Murakami et al., J. Biol. Chem., 267, 9300 (1992);
- endoglin; endoglin is apparently a TGF-β receptor (Gougos et al., J. Biol. Chem., 265, 8361 (1990); Cheifetz, 1. Biol. Chem., 267, 19027 (1992); Moren et al., VVRS, 189, 356 (1992)). In small amounts, it is found in normal endothelium, but is strongly expressed by the proliferating endothelium (Westphal et al., J. Invest. Derm., 100, 27 (1993); Burrows et al., Pharmac. Ther., 64, 155 (1994) ) The promoter sequence is described by Bellon et al. (Eur. J. Immunol., 23, 2340 (1993)) and Ge et al. (Gene, 138, 201 (1994)).

- VEGF receptors:
Two receptors are distinguished (Plate et al., Int. J. Cancer, 59, 520 (1994)):
VEGF receptor-1 (flt-1)
(Vries et al., Science, 255, 989 (1992))
(in the cytostatic part contains fms-like tyrosine kinase); and
VEGF receptor-2 (flk-1, KDR)
(Terman et al., VVRS, 187, 1579 (1992))
(contains tyrosine kinase in the cytoplasmic part).

и Risau, Development, 119, 957 (1993); Dumont и др., Oncogene, 7, 1471 (1992));
- В61-рецептор (Eck-рецептор)
(Bartley и др., Nature, 368, 558 (1994); Pandey и др., Science, 268, 567 (1995); van der Geer и др., Ann. Rev. Cell Biol., 10, 251 (1994));
- B61
Молекула B61 представляет собой лиганд В61-рецептора
(Hoizman и др., J. Am. Soc. Nephrol., 4, 466 (1993); Bartley и др., Nature, 368, 558 (1994));
- эндотелии, в особенности:
- эндотелин В
(Oreilly и др., J. Cardiovasc. Pharm., 22, 18 (1993); Benatti и др., J. Clin. Invest., 91, 1149 (1993); O'Reilly и др., ВВРС, 193, 834 (1993)). Промоторная последовательность описывается Benatti и др., J. Clin. Invest., 91, 1149 (1993);
- эндотелин-1
(Yanasigawa и др. , Nature, 332, 411 (1988)). Промоторная последовательность описывается Wilson и др., Mol. Cell. Biol., 10, 4654 (1990);
- рецепторы эндотелина, в особенности рецептор эндотелина-В
(Webb и др. , Mol. Pharmacol., 47, 730 (1995); Haendler и др., J. Cardiovasc. Pharm., 20, 1 (1992));
- манноза-6-фосфат-рецепторы
(Perales и др., Eur. J. Biochem., 226, 225 (1994)).Both receptors are almost exclusively found in endothelial cells (Senger et al., Cancer Metast. Rev., 12, 303 (1993));
- other endothelial specific receptor tyrosine kinases:
- til-1 or til-2
(Partanen et al., Mol. Cell Biol., 12, 1698 (1992);

and Risau, Development, 119, 957 (1993); Dumont et al., Oncogene, 7, 1471 (1992));
- B61 receptor (Eck receptor)
(Bartley et al., Nature, 368, 558 (1994); Pandey et al., Science, 268, 567 (1995); van der Geer et al., Ann. Rev. Cell Biol., 10, 251 (1994) );
- B61
The B61 molecule is a B61 receptor ligand
(Hoizman et al., J. Am. Soc. Nephrol., 4, 466 (1993); Bartley et al., Nature, 368, 558 (1994));
- endothelium, in particular:
- endothelin B
(Oreilly et al., J. Cardiovasc. Pharm., 22, 18 (1993); Benatti et al., J. Clin. Invest. 91, 1149 (1993); O'Reilly et al., VVRS, 193, 834 (1993)). The promoter sequence is described by Benatti et al., J. Clin. Invest., 91, 1149 (1993);
- endothelin-1
(Yanasigawa et al., Nature, 332, 411 (1988)). The promoter sequence is described by Wilson et al., Mol. Cell. Biol., 10, 4654 (1990);
- endothelin receptors, in particular the endothelin-B receptor
(Webb et al., Mol. Pharmacol., 47, 730 (1995); Haendler et al., J. Cardiovasc. Pharm., 20, 1 (1992));
- mannose-6-phosphate receptors
(Perales et al., Eur. J. Biochem., 226, 225 (1994)).

 Promoter sequences are described by Ludwig et al.

(Gene, 142, 311 (1994)), Oshima et al., (J. Biol. Chem., 263, 2553 (1988)) and Pohlmann et al., (PNAS USA, 84, 5575 (1987));
- von Willebrand factor;
the promoter sequence is described by Jahroudi and Lynch (Mol. Cell. Biol., 14, 999 (1994); Ferreira et al., Biochem. J., 293, 641 (1993)) and Aird et al. (PNAS USA, 92, 4567 (1995));
- interleukin-lα, interleukin-1β.

Interleukin-1 is produced by activated endothelial cells (Wamer et al., J. Immunol., 139, 1911 (1987)). Promoter sequences are described by Hangen et al., Mol. Carcinog., 2, 68 (1986), Turner et al., J. Iimnunol. 143, 3556 (1989); Fenton et al., J. Immunol., 138, 3972 (1987); Bensi et al., Cell. Growth. Diff., 1, 491 (1990); Hiscott et al., Mol. Cell. Biol., 13, 6231 (1993); and Mori et al., Blood, 84, 1688 (1994);
- receptor for interleukin-1;
the promoter sequence is described by Ye et al., PNAS USA, 90, 2295 (1993);
- vascular cell adhesion molecule (VCAM-1). The expression of VCAM-1 in endothelial cells is activated by lipopolysaccharides, the tumor necrosis factor α (Neish et al., Mol. Cell. Biol., 15, 2558 (1995)); interleukin-4 (Iademarko et al., J. Clin. Invest., 95, 264 (1995)) and interleukin-1 (Marni et al., J. Clin. Invest., 92, 1866 (1993)).

The promoter sequence of VCAM-1 is described by Neish et al., Mol. Cell. Biol., 15, 2558 (1995); Ahmad et al., J. Biol. Chem., 270, 8976 (1995); Neish et al., J. Exp. Med., 176, 1583 (1992); Iademarco et al., J. Biol. Chem. , 267, 16323 (1992) and Cybulsky et al., PNAS USA, 88, 7859 (1991);
- synthetic activator sequence. Synthetic activator sequences that consist of oligomerized binding sites of transcription factors, preferably or selectively active in endothelial cells, can also be used as alternatives to natural endothelial specific promoters. An example of this is the transcription factor GATA-2, the binding site of which in endothelin-1 is the 5'-TTATST-3 'gene (Lee et al., Biol. Chem., 266, 16188 (1991); Dorfmann et al., J. Biol. Chem., 267, 1279 (1992) and Wilson et al., Mol. Cell. Biol. 10, 4854 (1990)).

in). Activated macrophages and / or activated lymphocytes
As the activator sequence according to the present invention, in addition, it is necessary to understand the promoter sequences of the protein genes that are intensively formed during the immune response in macrophages and / or lymphocytes. This includes, for example:
- interleukin-1
(Bensi et al., Gene, 52, 95 (1987); Fibbe et al., Blut., 59, 147 (1989));
- receptor for interleukin-1
(Colotta et al., Immunol. Today, 15, 562 (1994); Sims et al., Clin. Immunol. Immunopath. 72, 9 (1994); Ye et al., PNAS USA, 90, 2295 (1993) );
- interleukin-2
(Jansen et al., Cell, 39, 207 (1994); Ohbo et al., J. Biol. Chem. 270, 7479 (1995));
- receptor for interleukin-2 (Semenzato et al., Int. J. Clin. Lab. Res., 22, 133 (1992));
- γ-interferon
(Kirchner, DMW, 111, 64 (1986); Lehmann et al., J. Immunol., 153, 165 (1994));
- interleukin-4
(Paul, Blood, 77, 1859 (1991); te Velde et al., Blood, 76, 1392 (1990));
- receptor for interleukin-4
(Vallenga et al., Leukemia, 7, 1131 (1993); Gelizzi et al., Int. Immunol., 2, 669 (1990));
- interleukin-3
(Frendl, Int. J. Immunopharm., 14, 421 (1992));
- interleukin-5
(Azuma et al., Nucl. Acid Res., 14, 9149 (1986); Yokota et al., PNAS, 84, 7388 (1987));
- interleukin-6
(Brack et al., Int. J. Clin. Lab. Res., 22, 143 (1992);
- LIF
(Metcalf, Int. J. Cell Clon., 9, 95 (1991); Samal, BBA, 1260, 27 (1995));
- interleukin-7
(Joshi et al., 21, 681 (1991));
- interleukin-10
(Benjamin et al., Leuk. Lymph., 12, 205 (1994); Fluchiger et al., J. Exp. Med., 179, 91 (1994));
- interleukin-11
(Yang et al., Biofactors, 4, 15 (1992));
- interleukin-12
(Kiniwa et al., J. Clin. Invest., 90, 262 (1992), Gatelay, Cancer Invest., 11, 500 (1993));
- interleukin-13
(Punnonen et al., PNAS, 90, 3730 (1993); Muzio et al., Blood, 83., 1738 (1994));
- GM-CSF
(Metcalf, Cancer, 15, 2185 (1990));
GM-CSF receptor
Nakagawa et al., J. Biol. Chem., 269, 10905 (1994));
- adhesion proteins like integrin, beta-2 protein
(Nueda et al., J. Biol. Chem., 268, 19305 (1993)).

The promoter sequences of these proteins are known from the following publications:
- receptor for interleukin-1
(Ye et al., PNAS USA, 90, 2295 (1993));
- interleukin-lα.

(Hangen et al., Mol. Carcinog., 2, 68 (1986); Turner et al., J. Immunol., 143, 3556 (1989); Mori et al., Blood, 84, 1688 (1994));
- interleukin-lβ
(Fenton et al., J. Immunol., 138, 3972 (1987); Bensi et al., Cell Growth. Diff., 1, 491 (1990); Turner et al., J. Immunol., 143, 3556 ( 1989); Hiscott et al., Mol. Cell. Biol., 13, 6231 (1993));
- interleukin-2
(Fujita et al., Cell, 46, 401 (1986); Nama et al., J. Exp. Med. 181, 1217 (1995); Kant et al., Lymph. Rec. Interact., 179 (1989) ; Kamps et al., Mol. Cell. Biol., 10, 5464 (1990); Williams et al., J. Immunol., 141, 662 (1988); Brunvand, FASEB J., 6, A 998 (1992) );
- receptor for interleukin-2
(Ohbo et al., J. Biol. Chem., 270, 7479 (1995); Shibuya et al., Nucl. Acids Res., 18, 3697 (1990); Lin et al., Mol. Cell. Biol., 13, 6201 (1993); Williams et al., J. Immunol., 141, 662 (1988));
- γ-interferon
(Ye et al., J. Biol. Chem., 269, 25728 (1994));
- interleukin-4
(Rooney et al., EMBO J., 13, 625 (1994); Hama et al., J. Exp. Med., 181, 1217 (1995); Li-Weber et al., J. Immunol, 153, 4122 (1994); Min et al., J. Immunol., 148, 1913 (1992); Abe et al., PNAS, 89, 2864 (1992));
- receptor for interleukin-4
(Beckmann et al., Chem. Immunol., 51, 107 (1992); Ohara et al., PNAS, 85, 8221 (1988));
- interleukin-3
(Mathey-Prevot et al., PNAS USA, 87, 5046 (1990); Cameron et al., Blood, 83, 2851 (1994); Arai et al., Lymphokine Res., 9, 551 (1990));
- receptor for interleukin-3 (α-subunit);
Miyajima et al., Blood, 85, 1246 (1995); Rapaport et al., Gene, 137, 333 (1993); Kosugi et al., VVRS, 208, 360 (1995));
- receptor for interleukin-3 (β-subunit)
(Gorman et al., J. Biol. Chem., 267, 15842 (1992); Kitamura et al., Cell, 66, 1165 (1991); Hayashida et al., PNAS USA, 87, 9655 (1990));
- interleukin-5
(Lee et al., J. Allerg. Clin. Immunol., 94, 594 (1994); Kauhansky et al., J. Immunol., 152, 1812 (1994); Staynov et al., PNAS USA, 92, 3606 (1995));
- interleukin-6
(Lu et al., J. Biol. Chem., 270, 9748 (1995); Gruss et al., Blood, 80, 2563 (1992); Ray et al., PNAS, 85, 6701 (1988); Droogmans and al., DNA Sequence, 3, 115 (1992); Mori et al., Blood, 84, 2904 (1994); Liberman et al., Mol. Cell. Biol. 10, 2327 (1990); Ishiki et al. ., Mol. Cell. Biol., 10, 2757 (1990));
- interleukin-7
(Pleiman et al., Mol. Cell. Biol., 11, 3052 (1991); Lapton et al., J. Immunol. 144, 3592 (1990));
- interleukin-8
(Chang et al., J. Biol. Chem., 269, 25277 (1994); Sprenger et al., J. Immunol., 153, 2524 (1994));
- interleukin-10
(Kim et al., J. Immunol., 148, 3616 (1992); Platzer et al., DNA Sequence, 4, 399 (1994); Kube et al., Cytokine, 7, 1 (1995));
- interleukin-11
(Yang et al., J. Biol. Chem., 269, 32732 (1994));
- GM-CSF
(Nimer et al., Mol. Cell. Biol., 10, 6084 (1990); Staynov et al., PNAS USA, 92, 3606 (1995); Koyano-Nakayawa et al., Int. Immunol., 5, 345 (1993); Ye et al., Nucl. Acids Res. 22, 5672 (1994));
GM-CSF receptor (α chain)
(Nakagawa et al., J. Biol. Chem., 269, 10905 (1994));
- macrophage colony stimulating factor receptor (M-CSF)
(Yue et al., Mol. Cell. Biol., 13, 3191 (1993); Zhang et al., Mol. Cell. Biol., 14, 373 (1994));
- type I and type II macrophage saprophytic receptors
(Moulton et al., Mol. Cell. Biol., 14, 4408 (1994));
- interleukin-13
(Staynov et al., PNAS USA, 92, 3606 (1995));
- LIF
(Gough et al., Ciba Found. Symp., 167, 24 (1992); Stahl et al., Cytokine, 5, 386 (1993));
- interferon regulatory factor-1, the promoter of which is stimulated by interleukin-6, also gamma or beta interferon
(Harrock et al., EMBO J., 13, 1942 (1994));
- reactive promoter of γ-interferon
(Lamb et al., Blood, 83, 2063 (1994));
- γ-interferon
(Hardy et al., PNAS, 82, 8173 (1985));
- MAC-1
(Dziennis et al., Blood, 85, 319 (1995); Bauer et al., Hum. Gene Ther., 5, 709 (1994); Hickstein et al., PNAS USA, 89, 2105 (1992));
- LFA-1
(Nueda et al., J. Biol. Chem., 268, 19305 (1993); Agura et al., Blood, 79, 602 (1992); Cornwell et al., PNAS USA, 90, 4221 (1993));
- p150, 95
(Noti et al., DNA and Cell. Biol., 11, 123 (1992); Lopezcabrera et al., J. Biol. Chem., 268, 1187 (1993)).

5.2. The choice of active substance to inhibit coagulation
As the active substance according to the present invention, it is necessary to understand a DNA sequence that encodes a protein that directly or indirectly inhibits platelet aggregation, or blood coagulation factor, or stimulates fibrinolysis.

 Such an active substance is designated as a coagulation inhibitor. As coagulation inhibitors, you can use genes, for example, plasminogen activators (RA), such as tissue PA (tPA), or urokinase-like PA (uPA), or protein C, antithrombin-III, C-IS-inhibitor, α-1-antitrypsin , tissue factor metabolic pathway inhibitor (TFPI) or hirudin. The DNA sequences of these coagulation inhibitors are already described in section 4.3.

5.3. The combination of two identical or different active substances to inhibit coagulation
The subject of the invention, further, is an active substance in which a combination of DNA sequences occurs: two identical, coagulation-inhibiting substances (A, A), or two different coagulation-inhibiting substances (A, B). For expression of both DNA sequences, cDNAs of the “internal ribosome aminoacyl site” (IRES) cDNA are preferably included as a regulatory element.

Такого рода IRES описываются, например, Montford и Smith (TIG, 11, 179 (1995); Kaufman и др. , Nucl. Acids Res., 19, 4485 (1991); Morgan и др., Nucl. Acids Res. , 20, 1293 (1992); Dirks и др., Gene, 128, 247 (1993); Pelletier и Sonenberg, Nature, 334, 320 (1988) и Sugitomo и др., Bio-Techn., 12, 694 (1994).

IRES of this kind are described, for example, by Montford and Smith (TIG, 11, 179 (1995); Kaufman et al., Nucl. Acids Res., 19, 4485 (1991); Morgan et al., Nucl. Acids Res., 20 , 1293 (1992); Dirks et al., Gene, 128, 247 (1993); Pelletier and Sonenberg, Nature, 334, 320 (1988) and Sugitomo et al., Bio-Techn., 12, 694 (1994).

 Thus, cDNA of poliovirus IRES sequences (position ≤ 140 to ≥ 630 5'-UTR (Pelletier and Sonenberg, Nature, 334, 320 (1988)) can be used to bind DNA of antithrombotic substance A (at the 3'-end) and DNA of antithrombotic substance B (at the 5'-end).

 This kind of combination of two identical or different genes promotes the additive effect (in the case of identical genes) or the synergistic effect of the selected antithrombotic substances.

5.4. The choice of ligand for inhibition of coagulation
As the ligand of viral or non-viral vectors, for example, in colloidal dispersions containing polylysine ligand conjugates, substances that bind to the surface of smooth muscle cells, or proliferating endothelial cells, or activated macrophages and / or lymphocytes are preferred.

a). Smooth muscle cell ligands
Examples of ligands that bind to smooth muscle cells are already listed in section 4.4.

b) Activated Endothelial Cell Ligands
These include according to the invention antibodies or antibody fragments directed against the membrane structures of endothelial cells, which are 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)). In particular, antibodies against VEGF receptors are included.

 Mouse monoclonal antibodies should preferably be used in humanized form. Humanization is carried out as described in section 4.4. way. Antibody fragments are prepared according to the prior art, for example, as described in section 4.4.

 Ligands further include all active substances that bind to membrane structures or membrane receptors of endothelial cells. For example, this includes substances that contain terminal mannose, further, interleukin-1 or growth factors or fragments thereof, respectively, their partial sequences that bind to receptors expressed by endothelial cells, such as PDGF, bFGF, VEGF, TGF-β (Pusztain et al., J. Pathol., 169, 191 (1993)).

 Further, such ligands include adhesive molecules that bind to activated and / or proliferating endothelial cells. Adhesion molecules of this kind, such as SLex, LFA-1, MAC-1, LECAM-1 or VLA-4, have already been described (reviews by Augustin-Voss et al., J. Cell. Biol., 119, 483 (1992 ); Pauli et al., Cancer Metast. Rev., 9, 175 (1990); Honn et al., Cancer Metast. Rev., 11, 353 (1992)).

in). Ligands of activated macrophages and / or activated lymphocytes
The ligands according to the invention further include substances that specifically bind to the surface of immune cells. This includes antibodies or antibody fragments directed against the membrane structures of immune cells, which are described, for example, Powelson et al., Biotech. Adv., 11, 725 (1993).

 Further, ligands also include monoclonal or polyclonal antibodies or antibody fragments that, through their constant domains, bind to Fc or μ receptors of immune cells (Rojanasakul et al., Pharm. Res., 11, 1731 (1994)).

 Mouse monoclonal antibodies in humanized form are also preferably used (see section 4.4.) And fragments are obtained, for example, using the procedure indicated in section 4.4.

 Ligands, further, include all substances that bind to membrane receptors on the surface of immune cells. For example, these include growth factors such as cytokines, EGF, TGF, FGF or PDGF or fragments thereof, respectively, their partial sequences that bind to receptors expressed by such cells.

5.5. Obtaining an active substance to inhibit coagulation
The preparation of the active substance according to the invention is described in more detail in accordance with the following examples.

a). The design of the chimeric promoter of endothelin-1-CDE-CHR-Inr
The human endothelin-1 promoter (position ≤ -170 to ≥ -10; Wilson et al., Mol. Cell. Biol., 10, 4854 (1990)) or a variant shortened by the TATA block (position ≤ -170 to ≥ -40 ) at its 3'-end is associated with the 5'-end of the CDE-CHR-Inr module (position ≤ -20 to ≥ +121) of the human cdc25C gene (Lucibello et al., EMBO J., 14, 132 (1995) ) (Fig. 9). The binding is carried out using known to the specialist and commercially available enzymes.

b) The construction of a plasmid containing the chimeric promoter endothelin-1-CDE-CHR-Inr in the Central component of the active substance
The described transcriptional unit of the chimeric promoter module of endothelin-1 is linked at its 3'-end to the 5'-end of DNA, which contains the complete coding region of tissue plasminogen activator (position ≤ 85 to ≥ 1753; Pennica et al., Nature, 301, 214 ( 1983)) (Fig. 9). This DNA also contains the signal sequence necessary for secretion. Transcriptional control units and tissue plasminogen activator DNA are cloned into pUC19 / 19 or from Bluescript-derived plasmid vectors, which can be used directly or in colloidal dispersion systems for in vivo administration. Alternatively, chimeric genes are transformed into viral vectors or other suitable vectors and injected.

in). Design of the chimeric promoter CDE-CHR-Inr myogenin
The human myogenin promoter (position ≤ -210 to ≥ +54 of the DNA sequence published by Salmin et al., J. Cell. Biol., 115, 905 (1991)) at its 3 ′ end is linked to the 5′-end of CDE- CHR-Inr module of the human cdc25C gene (position ≤ -20 to ≥ +121 of the sequence published by Lucibello et al., EMBO J., 14, 132 (1995)) (see FIG. 10). The binding is carried out using known to the specialist and commercially available enzymes. In addition, various fragments of the myogenin promoter sequence are used (see FIG. 10). Thus, a DNA sequence containing the myogenin promoter containing a TATA block is used. However, the promoter sequence in position ≤ -210 to ≥ -40 can also be used in the same way.

d). The construction of a plasmid containing the chimeric promoter CDE-CHR-Inr myogenin in the Central component of the active substance
Thus obtained control transcriptional unit of the chimeric promoter module of myogenin at its 3'-end is associated with the 5'-end of the DNA, which contains the complete coding region of tissue plasminogen activator (see Fig. 10). This DNA also contains the signal sequence necessary for secretion. Transcriptional control units and tissue plasminogen activator DNA are cloned into pUC18 / 19 or from Bluescript-derived plasmid vectors that can be used directly or in a colloidal dispersion system for in vivo administration. Alternatively, chimeric genes can be transformed into viral vectors or other suitable vectors and injected.

e). Construction of a plasmid containing two genes of active substances
The transcriptional unit of CDE-CHR-Inr myogenin described in paragraph c) is linked at its 3'-end to the 5'-end of the tissue factor metabolic pathway inhibitor DNA (TFPI; position ≤ 133 to ≥ 957; Wun et al., J. Biol Chem., 263, 6001 (1988); or position ≤ 382 to ≥ 1297; Girard et al., Thromb. Res., 55, 37 (1989)). The binding is carried out using known to the specialist and commercially available enzymes.

 The 3 ′ end of TFPI DNA is now linked to the 5 ′ end of the cDNA of the internal ribosomal aminoacyl site (position ≤ 140 to ≥ 630; Pelletier and Sonnenberg, Nature., 334, 320 (1988)) and then its 3 ′ end is linked to 5 the 'end of DNA of a tissue plasminogen activator (see Fig. 11). This thus obtained active substance is finally cloned into pUC18 / 19 or from plasmid vectors produced from Bluescript, which, directly or in a colloidal dispersion system, can be used for in vivo administration. Alternatively, chimeric genes can be transformed into viral vectors or other suitable vectors and injected.

6. The effect of the active substance on smooth muscle cells and / or on coagulation
The active substance according to the present invention, after local or systemic, preferably intravenous or intraarterial administration, exerts a predominant, if not exceptional, effect on smooth muscle cells that are directly accessible due to damage or trauma to the vessel (especially the endothelium layer) and, possibly, after entering the internal the sheath of the volume of a blood vessel.

 By combining the tissue-specific activator sequence and the cell cycle-regulated repressor module, it is ensured that the cell cycle inhibitor is activated predominantly or exclusively in dividing smooth muscle cells.

 Thanks to the use of mutated cell cycle inhibitors according to the invention, their longer proliferation-inhibiting effect is ensured.

 The active substance according to the present invention, further, allows for the fact that after local (for example, in tissue, body cavities or intermediate spaces between tissues) or after systemic, preferably intravenous or intraarterial administration, predominantly, if not exclusively, in smooth muscle cells activated proliferating endothelial cells, activated lymphocytes, or activated macrophages express an antithrombotic substance, and so it is released at the site of thrombosis.

 Since the active substance, both due to its specificity to the cell and also due to its specificity to the cell cycle, guarantees a high degree of safety, it can also be used in high dosages and, if necessary, repeatedly at intervals of days or weeks for the prevention or therapy of blockage vessels caused by proliferating smooth muscle cells, and / or for the prevention and / or therapy of thrombosis.

Explanation of Figures 1-11
Figure 1:
The nucleotide sequence of the cdc25C promoter region with in vivo protein binding sites (genomic dimethyl sulfate footprinting; filled circle indicates complete constitutive protection; open circle represents partial constitutive protection; asterisk indicates Gl-specific cell regulation; CBS constitutive binding site; CDE cell cycle dependent element. The gray-stained areas indicate Y _C blocks (NF-Y binding sites). Starting sites are marked with filled squares.

Figure 2:
Derepression of the cdc25C promoter specifically at G ₀ by mutation of cdc.

Figure 3:
Schematic representation of the regulation of the cdc25C enhancer by CDE.

Figure 4:
G ₀ / G ₁ - specific repression of the 8Y40 enhancer due to CDE.

Figure 5:
Homology in the CDE-CHR region and located at the 5'-ends of the Y _C blocks in cdc25C, cyclin-A and cdc2 promoters.

6:
Chimeric construct consisting of various parts of the human myogenin promoter (Myf-4), fused at the 3'-end of the promoter module with CDE and CHR repressor elements, as well as human β-glucuronidase DNA (full coding region, position ≤ 27 to ≥ 1982) as an effector (Oshima et al., PNAS USA, 84, 685 (1987)). Location data refer to Salminen et al., J. Cell. Biol., 115, 905 (1991) for the myogenin gene, respectively, to the cdc25C system used by Lucibello et al., EMBO J., 14, 132 (1995).

7:
Replacing the homologous signal sequence of β-glucuronidase with a heterologous signal sequence (human immunoglobulin). Data on the location of the signal sequence (MGWSCIILFLVATAT) of immunoglobulin (HuVH-cAMP) refers to the data of Riechmann et al., Nature, 332, 323 (1988).

 Alternative: Embedding an immunoglobulin signal peptide for better extracellular isolation of β-glucuronidase; see (B).

Fig. 8:
A chimeric construct consisting of various parts of the human myogenin promoter (Myf-4) fused at the 3'-end of the promoter module with CDE and CHR repressor elements, as well as human β-glucuronidase DNA, an internal ribosome aminoacial site as a regulatory nucleotide sequence and DNA tissue plasminogen activator. Location data refers to those of Salminen et al., J. Cell. Biol., 115, 905 (1991) for myogenin; Lucibello et al., EMBO J., 14, 132 (1995) for the element CDE / CHR-Inr; Oshima et al., PNAS USA, 84, 685 (1987) for β-glucuronidase; Riechmann et al., Nature, 332? 323 (1988) for the immunoglobulin signal sequence; Pelletier and Sonenberg, Nature, 334, 320 (1988) for the internal ribosomal aminoacial site of poliovirus and Pennica et al., Nature, 301, 214 (1983) for human tissue plasminogen activator.

Fig.9:
A chimeric construct consisting of various parts of the human endothelin-1 promoter fused at the 3'-end of the promoter module with CDE and CHR repressor elements, as well as human tissue plasminogen activator DNA (full coding region; Pennica et al., Nature, 301, 214 (1983)) as an effector. Location data are those of Wilson et al., Mol. Cell. Biol., 10, 4854 (1990) for the endothelin-1 gene, respectively, used by Lucibello et al., EMBO J., 14, 132 (1995), cdc25C system.

Figure 10:
A chimeric construct consisting of various parts of the human myogenin promoter (Myf-4) fused at the 3'-end of the promoter module with CDE and CHR repressor elements, as well as human tissue plasminogen activator DNA (full coding region) as an effector. Location data refers to those of Salminen et al., J. Cell. Biol., 115, 905 (1991) for the myogenin gene, respectively, to the cdc25C system used by Lucibello et al., EMBO J., 14, 132 (1995) and to Pennica et al., Nature, 301, 214 (1983) for tissue plasminogen activator.

11:
Chimeric construction with two effector genes.

Location data for tissue factor metabolic pathway inhibitor are data
*) Wun et al., J. Biol. Chem., 263, 6001 (1988);
**) Girard et al., Thromb. Res., 55, 37 (1989);
and data for the internal ribosomal aminoacial site (IRES) of the cDNA are based on those of Pelletier and Sonenberg, Nature, 334, 320 (1988).

Claims (34)

 1. A DNA construct consisting of an activator sequence regulated by the cell cycle of the promoter module, including elements of the CDE-CHR-Inr and containing positions ≤ -20 to ≥ +30 of the cdc25C-promoter region (nucleotide sequence GGCTGGCGG-AAGGTTTTGAATGGTCAACGCCTGCGG-CTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT represents a “cell cycle dependent element” (TGGCGG nucleotide sequence), CHR represents a “region homologous to the cell cycle gene (GTTTGAA nucleotide sequence), and Inr means an initiation site (position +1), as well as important to initiate adjacent sequences, and a DNA sequence encoding an active substance, which is a cell cycle inhibitor and / or coagulation inhibitor.  2. The DNA construct according to claim 1, characterized in that the activator sequence is represented by promoter or enhancer sequences and is regulated by transcription factors formed in smooth muscle cells.  3. The DNA construct according to claim 2, characterized in that it contains as an activator sequence a CMV enhancer, CMV promoter or SV40 promoter; or the promoter sequence of the tropomyosin, α-actin, α-myosin gene, PDGF receptors, FGF receptors, MRF-4, acetylcholine receptors, phosphofructokinase-A, phosphoglyceratmutase, troponin C, myogenin, endothelin or desmin receptors; or as an activator sequence, multiple copies of the muscle-specific HLH protein binding site (E-block) or GATA-4; or the VEGF promoter sequence, or the VEGF enhancer sequence, or the c-Src or v-Src DNA sequence that regulate the VEGF gene.  4. The DNA construct according to any one of claims 1 to 3, characterized in that the DNA sequence encodes the active substance of the cell cycle inhibitor, which is: retinoblastoma protein pRb / p110, protein p107 or protein p130; or p53 protein; or p21 protein, p16 protein, or another cyclin-dependent kinase (CDK) inhibitor; or GADD45 protein; or bak protein; or a cytostatic or cytotoxic protein; or an enzyme for cleaving the precursors of cytostatic agents to cytostatic agents.  5. The DNA construct according to claim 4, characterized in that the reginoblastoma protein (pRb / p110) due to the replacement of amino acids at positions 246, 350, 601, 605, 780, 786, 787, 800 and 804 becomes more non-phosphorylated, however, without impairing its binding activity to a large T-antigen; for example, because the amino acids Thr-246, Ser-601, Ser-605, Ser-780, Ser-786, Ser-787 and Ser-800 are replaced by A1a, the amino acid Thr-350 is replaced by Arg, and the amino acid Ser -804 is replaced by Glu; or protein p107 is mutated similarly to protein pRb / 110; or protein p130 is mutated similarly to protein pRb / 110.  6. The DNA construct according to claim 4, characterized in that the p53C protein at the C-terminus is truncated to serine-392.  7. The DNA construct according to any one of claims 1 to 3, characterized in that the cell cycle inhibitor is perforin, granzyme, tumor necrosis factor α or tumor necrosis factor β.  8. The DNA construct according to any one of claims 1 to 3, characterized in that the cell cycle inhibitor is an enzyme and this enzyme is herpes simplex virus thymidine kinase, cytosine deaminase, chickenpox virus thymidine kinase, nitroreductase, β-glucuronidase (especially human, plant or bacterial β-glucuronidase), carboxypeptidase (preferably bacteria of the genus Pseudomonas), lactase (preferably Bacillus cereus), pyroglutamate aminopeptidase, D-aminopeptidase, oxidase, peroxidase, phosphatase, hydroxynitrilease, p a rotase, esterase or glycosidase, the signal sequence of this enzyme being homologous or heterologous for better cell isolation.  9. The DNA construct of claim 8, characterized in that through point mutation to the DNA sequence of the enzyme, the accumulation of lysosomes is reduced and extracellular secretion is increased.  10. The DNA construct according to any one of claims 1 to 9, characterized in that it contains the DNA sequences of several identical or different cell cycle inhibitors, and possibly two DNA sequences are linked to each other via the DNA sequence of the internal ribosomal aminoacyl site.  11. DNA construct according to any one of claims 1 to 3, characterized in that the DNA sequence encodes the active substance of a coagulation inhibitor.  12. The DNA construct according to any one of claims 1 to 9, characterized in that the DNA sequence of the cell cycle inhibitor is complemented by the DNA sequence of a coagulation inhibitor, and these two DNA sequences are linked to each other via the DNA sequence of the internal ribosomal aminoacyl site.  13. DNA construct according to any one of claim 11 or 12, characterized in that the inhibitor DNA sequence encodes tPA, uPA, hybrid molecules of tPA and uPA, protein C, antithrombin III, TFPI, C-1 inhibitor, α-1 -antitrypsin or hirudin.  14. The DNA construct according to claim 1, characterized in that the activator sequence is represented by promoter or enhancer sequences and is regulated by transcription factors formed in proliferating endothelial cells.  15. The DNA construct according to 14, characterized in that it contains the promoter sequence of the endothelial carrier of glucose-1, endoglin, VEGF receptor-1 or -2, receptor tyrosine kinase til-1 or til-2, receptor B61, ligand B61, endothelin, especially endothelin-B-1, mannose-6-phosphate receptor, interleukin-1α or interleukin-1β, interleukin-1 receptor, VCAM-1, von Willebrand factor; or a synthetic activator sequence from oligomerized transcription factor binding sites that are preferably or selectively active in endothelial cells, such as GATA-2, whose binding site is 5'-TTATST-3 '.  16. The DNA construct according to claim 1, characterized in that the activator sequence is represented by promoter or enhancer sequences and is regulated by transcription factors generated upon activation by macrophages and in macrophages or lymphocytes.  17. The DNA construct according to clause 16, characterized in that it contains the promoter sequence of cytokines or their receptors, in particular interleukin-1α or interleukin-1β, interleukin-1 receptor, interleukin-2, interleukin-2 receptor, γ-interferon, interleukin-3, interleukin-3 receptor, interleukin-4, interleukin-4 receptor, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, M-CSF receptor, type I or type II saprophytic macrophage receptor, factor 1 regulation of interferon, γ-interferon, sensitive to the γ-interferon promoter, granulocyte macrophage colony stimulating factor (GM-CSF), GM-CSF receptor, granulocyte colony stimulating factor (G-CSF), adhesion proteins like LFA-1, Mac-1 or p150 / 95, or leukemia inhibiting factor (LIF).  18. The DNA construct according to any one of paragraphs.14-17, characterized in that the DNA sequence encodes the active substance of a coagulation inhibitor.  19. The DNA construct of claim 18, wherein the DNA sequence of the coagulation inhibitor encodes a tissue plasminogen activator (tPA), urokinase (uPA), a hybrid of tPA and uPA, protein C, antithrombin-III, a tissue factor metabolic pathway inhibitor, C-1-inhibitor, α-1-antitrypsin or hirudin.  20. The DNA construct according to any one of claims 1 to 3 and 14-19, characterized in that it contains the DNA sequence of several identical or different coagulation inhibitors, and possibly two DNA sequences are linked to each other through the DNA sequence of the internal ribosomal aminoacial site.  21. The DNA construct according to any one of claims 1 to 20, characterized in that it is inserted into the vector.  22. The DNA construct of claim 21, wherein the vector is a virus.  23. The DNA construct of claim 21, wherein the virus is a retrovirus, an adenovirus, an adeno-associated virus, a herpes simplex virus, or a smallpox vaccine virus.  24. The DNA construct according to any one of claims 1 to 23, characterized in that it is inserted into the plasmid.  25. The DNA construct according to any one of paragraphs.21-24, characterized in that it is obtained in a colloidal dispersion system.  26. The DNA construct according A.25, characterized in that the colloidal dispersion system are liposomes.  27. The DNA construct according to claim 25, wherein the colloidal dispersion system are polylysis ligands.  28. The DNA construct according to any one of paragraphs.21-27, characterized in that it is supplemented with a ligand that binds to the membrane structures of smooth muscle cells, activated endothelial cells, activated macrophages or activated lymphocytes.  29. The DNA construct according to claim 21, wherein the ligand is a polyclonal or monoclonal antibody or a fragment of this antibody, which (which) specifically binds with its variable domains to the membrane structure of smooth muscle cells, activated endothelial cells, activated macrophages or activated lymphocytes; or is a cytokine or growth factor or fragment thereof, respectively, a partial sequence thereof that (which) binds to a receptor for smooth muscle cells, activated endothelial cells, activated macrophages or activated lymphocytes; or the ligand is an adhesive molecule such as SLeX, LFA-1, MAC-1, LECAM-1 or VLA-4.  30. The DNA construct according to clause 29, wherein the membrane structure of the receptor is mannose, interleukin-1 or growth factor, such as PDGF, FGF, VEGF, TGF-β.  31. The DNA construct of claim 29, wherein the membrane structures are actin, angiotensin, interleukin receptors, EGF receptors, PDGF receptors, FGF receptors, or endothelin receptors.  32. The DNA construct according to p, characterized in that the membrane structures are receptors of cytokines, growth factors, interferons, chemokines, in particular receptors for stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-4 , interleukin-6, interleukin-8, interleukin-10, α-interferon, β-interferon, γ-interferon, GM-CSF, M-CSF, G-CSF or tumor necrosis factor α.  33. The DNA construct according to any one of claims 1 to 32, used as part of a drug for injection into tissue, such as muscle, connective tissue, liver, kidneys, spleen, lungs, skin; for local application to the skin or mucous membranes; for injection into the body cavity, such as joints, pleural cavity, subarachnoid space; or for injection into the blood vessel system, as an intra-arterial or intravenous injection.  34. DNA construct according to any one of claims 1 to 32, used for the prevention and treatment of diseases of blood vessels.

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