WO1996006938A1 - Genetic therapy of vascular diseases with a cell-specific active substance which is dependent on the cell cycle - Google Patents

Genetic therapy of vascular diseases with a cell-specific active substance which is dependent on the cell cycle Download PDF

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WO1996006938A1
WO1996006938A1 PCT/EP1995/003368 EP9503368W WO9606938A1 WO 1996006938 A1 WO1996006938 A1 WO 1996006938A1 EP 9503368 W EP9503368 W EP 9503368W WO 9606938 A1 WO9606938 A1 WO 9606938A1
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active ingredient
sequence
ingredient according
promoter
protein
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PCT/EP1995/003368
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German (de)
French (fr)
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Hans-Harald Sedlacek
Rolf Müller
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Hoechst Aktiengesellschaft
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Priority claimed from GB9417366A external-priority patent/GB9417366D0/en
Priority claimed from GBGB9506466.3A external-priority patent/GB9506466D0/en
Application filed by Hoechst Aktiengesellschaft filed Critical Hoechst Aktiengesellschaft
Priority to JP8508477A priority Critical patent/JPH10507626A/en
Priority to AU33874/95A priority patent/AU690733B2/en
Priority to DK95930524T priority patent/DK0777739T3/en
Priority to PCT/EP1995/003368 priority patent/WO1996006938A1/en
Priority to DE59508144T priority patent/DE59508144D1/en
Priority to AT95930524T priority patent/ATE191506T1/en
Priority to EP95930524A priority patent/EP0777739B1/en
Publication of WO1996006938A1 publication Critical patent/WO1996006938A1/en
Priority to GR20000401227T priority patent/GR3033535T3/en

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4736—Retinoblastoma protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2830/00—Vector systems having a special element relevant for transcription
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
    • C—CHEMISTRY; METALLURGY
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • a DNA sequence for the gene therapy of vascular diseases is described.
  • Essential elements for the DNA sequence are the activator sequence, the promoter module and the gene for the active substance.
  • the activator sequence is activated cell-specifically in smooth muscle cells, activated endothelial cells, activated macrophages or activated lymphocytes. This activation is regulated in a cell cycle-specific manner by the promoter module.
  • the active substance is an inhibitor for the growth of smooth muscle cells and / or for coagulation.
  • the DNA sequence described is inserted into a viral or non-viral vector, supplemented by a ligand with affinity for the target cell.
  • Vascular smooth muscle cells are predominantly located in the arterial tunica media and are involved in local and systemic blood pressure regulation. In the uninjured, healthy vessel, these smooth muscle cells are in the resting state of cell division (R. Ross, Nature 3J52, 801 (1993)). Vascular injuries cause smooth muscle cells to migrate into the intimal layer of the vessel wall, where they proliferate (formation of neointima) and form extracellular matrix components.
  • Intimal proliferation of smooth muscle cells is considered an essential component in the development of arteriosclerosis (J.S. Forrester et al., Am. Coll. Cardiol. 17, 758 (1991)). Furthermore, this proliferation of smooth muscle cells leads to restenosis of the vessels after angioplasty operations as well as after balloon dilation of narrowed vessels (RS Schwartz et al., Am. Coll. Cardiol. 2Ü, 1284 (1992), MW Liu et al., Circulation 72 , 1374 (1989)).
  • a replication-deficient recombinant adenovirus in which the gene for the herpes simplex virus thymidine kinase was inserted (AV-HS-TK).
  • the kinase gene product is in the Able to phosphorylate the prodrug ganciclovir and thereby convert it into a nucleoside analog that inhibits DNA synthesis.
  • the gene vector AV-HS-TK was administered 7 days after the vascular damage, but also here locally at the site of the damage, and ganciclovir was subsequently injected intraperitoneally daily for 14 days.
  • This treatment significantly inhibited the growth of smooth muscle cells in the rat (Guzman et al., Proc. Natl. Acad. Sei. 9., 10732 (1994)).
  • Similar results were also achieved in pigs (Ohno et al., Science 2_2L5_, 781 (1994)).
  • the vector was administered immediately after the vascular injury and the ganciclovir administration daily for 6 days.
  • a disadvantage of the methods known from the literature is that the active substances (vectors) have to be applied locally at the site of the vascular injuries, and even the relevant vessel section may have to be temporarily closed in order to prevent the vectors from washing away.
  • Such invasive interventions are routinely performed as part of balloon dilatation of stenosed vessels, but they require considerable effort and in turn represent a considerable risk to the patient due to the risk of thrombosis and embolism.
  • the systemic (eg intravenous or oral) administration of cytostatics for the inhibition of proliferation of smooth muscle cells at the site of the vascular disease is only minor and temporary effect, on the other hand has the risk of damage to endothelia and leads to considerable acute and chronic side effects.
  • Thromboses are still a difficult to treat, sometimes life-threatening complication of metabolic diseases, such as arteriosclerosis, arterial and venous vascular diseases and local as well as systemic immunoreactive syndromes (reviews by Philipps et al., Blood 71, 831 (1988), Harker, Biomed. Progr. 8_, 17 (1995)).
  • metabolic diseases such as arteriosclerosis, arterial and venous vascular diseases and local as well as systemic immunoreactive syndromes (reviews by Philipps et al., Blood 71, 831 (1988), Harker, Biomed. Progr. 8_, 17 (1995)).
  • thromboses There is therefore a great need for new drugs for the prevention and therapy of thromboses (BMJ 305, 567 (1992), Vinazzer, Biomedical Progress ü, 17 (1993)).
  • a significant portion of thrombosis is due to activated or damaged endothelial cells. These themselves or the smooth muscle cells stimulated by proliferation in the blood cause an activation of the coagulation system alone or in association with activated macrophages, lymphocytes, thrombocytes and granulocytes (Nemerson, Blood ZI, 1 (1988)).
  • antithrombotics such as heparin or fractions of heparin
  • anticoagulants such as coumarin
  • platelet aggregation inhibitors such as aspirin
  • fibrinolytics such as streptokinase, urokinase or tissue plasminogen activators (tPA)
  • tPA tissue plasminogen activators
  • the invention now relates to an active ingredient (i.e. a medicament) which can be given locally and systemically to patients and which
  • the central component of this active ingredient is a DNA construct, which consists of the following elements:
  • DNA is used throughout the text of this application as a common term for both a complementary (cDNA) and a genomic DNA sequence).
  • Activator sequence cell cycle regulated active substance (UAS) promoter module is used throughout the text of this application as a common term for both a complementary (cDNA) and a genomic DNA sequence.
  • UAS Activator sequence cell cycle regulated active substance
  • the central element of this active ingredient is the cell cycle-regulated promoter module.
  • the nucleotide sequence - CDE-CHR-Inr- (see below) is to be understood as a cell cycle-regulated promoter module.
  • the essential function of the promoter module is the inhibition of the function of the activator sequence in the G0 / G1 phase of the cell cycle and a cell cycle-specific expression in the S / G2 phase and thus in proliferating cells.
  • the promoter module CDE-CHR-Inr was discovered in the context of a detailed investigation of the G2-specific expression of the human cdc25C promoter.
  • the starting point was the discovery of a repressor element ("cell cycle dependent element”; CDE), which is responsible for switching off the promoter in the G1 phase of the cell cycle (Lucibello et al., EMBO J. 14, 132 (1995)) / für Genomic dimethyl sulfate (DMS) footprinting and functional analyzes (FIGS. 1, 2) showed that the CDE binds a repressor ("CDE binding factor"; CDF) G1 -specifically and thereby inhibits transcription in non- proliferating (G0) cells.
  • CDE binding factor CDE binding factor
  • the CDE located in the area of the basal promoter is dependent on an "upstream activating sequence" (UAS) in its repressing function.
  • UAS upstream activating sequence
  • cdc25C promoter Of importance for the regulation of the cdc25C promoter is not only the CDE-CHR region, 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 area, which includes the in vitro binding site for the transcription factor YY-1 (Seto and Shenk, Nature 3_5_, 241 (1991), Usheva and Shenk, Cell 7_6_, 1115 (1994)) lead to complete deregulation. In Given the proximity of the CDE-CHR to the basal promoter, an interaction of the CDF with the basal transcription complex is very likely.
  • activator sequence UAS
  • upstream activator sequence UAS
  • CMV enhancer the CMV promoter (EP 073.177.B1)
  • SV40 promoter any other promoter or enhancer sequence known to the person skilled in the art
  • the preferred activator sequences include those gene regulatory sequences or elements from genes which encode proteins formed particularly in smooth muscle cells, in activated endothelial cells or in activated macrophages or lymphocytes.
  • the active substance is to be understood as the DNA sequence for a protein which can have the therapeutic effect at the point of origin - that is to say the inhibition of the proliferation of smooth muscle cells, coagulation or (in the case of two active substances) both.
  • the selection of the nucleotide sequence for the activator sequence and for the active substance depends on the target cell and the desired active substance.
  • the DNA construct according to the invention is completed to a vector in a manner familiar to the person skilled in the art; for example, it is inserted into a viral vector (see, for this, D. Jolly, Cancer Gene Therapy 1, 51 (1994)), or supplemented to form a plasmid.
  • Viral vectors or plasmids can be complexed with colloidal dispersions, for example with liposomes (Farhood et al., Annais of the New York Academy of Sciences 7_16_, 23 (1994)) or with polylysine-ligand conjugates (Curiel et al., Annais of the New York Academy of Sciences 716. 36 (1994)).
  • a pharmaceutical preparation can also be carried out with the usual pharmaceutical excipients.
  • Such viral or non-viral vectors can be supplemented by a ligand which has binding affinity for a membrane structure on the selected target cell.
  • the choice of ligand thus depends on the selection of the target cell (see p. 21, 4.4 ff and p. 34, 5.4ff).
  • the active ingredient according to the invention is explained in more detail using the following examples: 4 ⁇ Active ingredient for the inhibition of smooth muscle cell proliferation
  • gene regulatory sequences or elements from genes are preferred which encode proteins formed, particularly in smooth muscle cells. Examples of these genes are:
  • VEGF is formed by smooth muscle cells, especially under hypoxic conditions (Berse et al., Mol. Biol. Cell 3, 211 (1992), Finkenzeller et al., BBRC 223, 432 (1995), Tischer et al., BBRC 135 , 1198 (1989), Leung et al., Science 246, 1306 (1989), Ferrara et al., Endoc. Rev. 13, 18 (1992)).
  • the gene regulatory sequences for the VEGF gene are:
  • HHL helix-loop-helix
  • MyoD Myf-5, myogenin, MRF4 (overview in Olson and Klein, Genes Dev. 3, 1 (1994)
  • muscle-specific transcription activators muscle-specific transcription activators
  • the zinc finger protein GATA-4 Zinci et al., Mol Cell. Biol. 13, 2235 (1993); Ip et al., Mol. Cell. Biol. 14, 7517 (1994)
  • GATA-4 Zinci et al., Mol Cell. Biol. 14, 7517 (1994)
  • MEF-2 Transcription factors Yu et al., Gene Dev. 5, 1783 (1992)
  • the HLH proteins and GATA-4 show muscle-specific transcription not only with promoters of muscle-specific genes, but also in a heterologous context, including with artificial promoters.
  • artificial promoters are for example:
  • An active substance in the sense of the invention is a DNA sequence whose expressed protein inhibits the proliferation of smooth muscle cells.
  • These cell cycle inhibitors include, for example, the DNA sequences for the following proteins:
  • the retinoblastoma protein (pRb / p110) and the related p107 and p130 proteins are inactivated by phosphorylation.
  • a pRb / p110 -, p107 - or p130 cDNA sequence is thus preferably used which is mutated in such a way that the phosphorylation sites of the encoded protein are exchanged for non-phosphorylatable amino acids.
  • the cDNA sequence for the retinoblastoma protein (p110) can no longer be phosphorylated by replacing the amino acids in positions 246, 350, 601, 605, 780, 786, 787, 800 and 804
  • its binding activity with the large T antigen is not impaired, for example the amino acids Thr-246, Ser-601, Ser-605, Ser-780, Ser-786, Ser-787 and Ser-800 with Ala, the amino acid Thr-350 exchanged with Arg and the amino acid Ser-804 with Glu.
  • the DNA sequence for the p107 protein or the p130 protein is mutated in an analogous manner.
  • the protein p53 is inactivated in the cell either by binding to special proteins such as MDM2 or by oligomerizing the p53 via the dephosphorylated C-terminal serine 392 (Schikawa et al., Leukemia and Lymphoma H, 21 (1993) and Brown , Annais of Oncology 4, 623 (1993)).
  • a DNA sequence is therefore preferably used for a p53 protein which is shortened at the C-terminal by the serine 392.
  • the active substance is also to be understood as a DNA sequence that expresses a cytostatic or cytotoxic protein.
  • cytostatic or cytotoxic protein include, for example
  • the active substance is also to be understood as the DNA sequence for an enzyme which converts an inactive precursor of a cytostatic into a cytostatic.
  • DNA sequence of one of the following enzymes should be used:
  • CB carboxy peptidase
  • Oxidase especially * human lysyl oxidase
  • the homologous signal sequence contained in the DNA sequence can be replaced by a heterologous signal sequence which improves the extracellular discharge.
  • the signal sequence of the ⁇ -glucuronidase (DNA position ⁇ 27 to 93; Oshima et al., PNAS 64, 685 (1987)) can be replaced by the signal frequency for the human immunoglobulin (DNA position ⁇ 63 to> 107; Riechmann et al., Nature 332, 323 (1988).
  • the invention further relates to an active substance in which a combination of the DNA sequences of several identical active substances (A, A) or different active substances (A, B) is present.
  • the cDNA of an "internal ribosome entry site" IRES
  • IRES interposed as a regulatory element.
  • IRES have been described by Mountford and Smith (TIG 11, 179 (1995), Kaufman et al., Nucl. Acids Res. 12, 4485 (1991), Morgan et al., Nucl. Acids Res. 22, 1293 (1992) and Dirks et al., Gene 122, 247 (1993), Pelletier and Sonenberg, Nature 334, 320 (1988), Sugitomo et al., BioTechn. 12, 694 (1994).
  • the cDNA of the IRES sequence of the poliovirus (position ⁇ 140 to> 630 of the 5 'UTR (Pelletier and Sonenberg, Nature 334, 320 (1988)) for linking the DNA of the antithrombotic substance A (at the 3' end) and the DNA of the antithrombotic substance B (at the 5 'terminus) can be used.
  • Activator cell cycle active substance intally active substance sequence regulated (cell cycle ribosome (cell cycle (UAS) promoter inhibitor) entry inhibitor)
  • such an active ingredient has an additive or synergistic effect in the sense of the invention.
  • the coagulation system can be activated and thromboses can occur.
  • thrombosis can be prevented by prophylactic administration of an anticoagulant (aspirin, heparin or another antithrombotic).
  • the anticoagulant is administered systemically, i.e. orally or parenterally.
  • the side effects of the anticoagulant often prevent sufficient concentration at the site of the intimal growth of the smooth muscle cell.
  • the prophylaxis of thrombosis by such anticoagulants is uncertain (Pukac, Am. J. Pathol. 132, 1501 (1991)). It is a further object of the invention that the active substance claimed in the sense of the invention contains, in addition to an active substance which is a cell cycle inhibitor, the DNA sequence for an active substance which is an anticoagulant as a further element.
  • the expression of the anticoagulant is controlled in the same way as the expression of the cell cycle inhibitor by the activator sequence and the cell cycle-regulated repressor module.
  • the simultaneous expression of both the cell cycle inhibitor and the anticoagulant is preferably regulated by an "internal ribosome entry site” (IRES) gene element.
  • IRS internal ribosome entry site
  • PA plasminogen activators
  • tissue PA tissue PA
  • uPA urokinase-like PA
  • protein C antithrombin III
  • tissue factor pathway inhibitor tissue factor pathway inhibitor
  • Urokinase-type plasminogen activator uPA
  • Serine proteinase inhibitors such as
  • TFPI Tissue Factor Pathway Inhibitor
  • ligands which bind to the surface of smooth muscle cells are preferred as ligands in colloidal dispersions, for example polylysine-ligand conjugates.
  • ligands include antibodies or antibody fragments, directed against membrane structures of smooth muscle cells, such as, for example
  • the murine monoclonal antibodies are preferably used in humanized form. Humanization takes place in the method described by Winter et al. (Nature 349, 293 (1991) and Hoogenbooms et al. (Rev. Tr. Transfus. Hemobiol. 35, 19 (1993). Antibody fragments are prepared according to the prior art, for example in the manner described by Winter et al., Nature 349, 293 (1991), Hoogenboom et al., Rev. Tr. Transfus. Hemobiol. 36, 19 (1993), Girol, Mol. Immunol. 23, 1379 (1991) or Huston et al., Int. Rev. Immunol 12, 195 (1993).
  • the ligands also include all active substances which bind to membrane structures or membrane receptors on smooth muscle cells (review by Pusztai et al., J. Pathol. 162, 191 (1993), Harris, Current Opin. Biotechnol. 2, 260 (1991)). For example, this includes growth factors or their fragments or partial sequences of them, which bind to receptors expressed by smooth muscle cells, for example
  • the human myogenin promoter (pos. ⁇ -210 to> +54, the DNA sequence published by Salmin et al., J. Cell Biol. 115. 905 (1991)) is connected at its 3 'end with the 5'- Term of the CDE-CHR-Inr module of the human cdc25C gene (pos. ⁇ -20 to> +121, linked to the sequence published by Lucibello et al., EMBO J. 14, 132 (1995)) (see FIG. 6) .
  • the linkage takes place with the aid of enzymes known and commercially available to the person skilled in the art.
  • Various fragments of the myogenin promoter sequence are also used (see FIG. 6).
  • the DNA sequence of the myogenin promoter containing the TATA box is used. However, the promoter sequence position ⁇ -210 to> -40 can also be used come (see Fig. 6).
  • the chimeric myogenin promoter module transcription control unit thus produced is labeled at its 3 'end with the 5' terminus of a DNA which encompasses the complete coding region of the human ⁇ -glucuronidase (DNA pos. ⁇ 27-> 1982, which was described by Oshima et al., PNAS USA 64, 684 (1987) published sequence), linked (see Fig. 6).
  • This DNA also contains the signal sequence necessary for secretion (22 N-terminal amino acids). In order to facilitate cellular removal, this signal sequence should preferably be exchanged for the signal sequence of the immunoglobulin (position ⁇ 63 to> 107; Riechmann et al., Nature 332, 323 (1988) (FIG. 7).
  • the transcription control units thus produced and the DNA for human ⁇ -glucuronidase, clones are cloned into pUC18 / 19 or Bluescript-derived plasmid vectors which can be used directly or in colloidal dispersion systems for in vivo application.
  • the chimeric genes can be transferred into viral vectors or other suitable vectors and injected.
  • the myogenin repressor module- ⁇ -glucuronidase element produced as under 2) becomes at its 3 'end with the 5' terminus of the cDNA the "internal ribosome entry site" of the poliovirus (position ⁇ 140 to> 630 of the 5 'UTR element , Pelletier and Sonenberg, Nature 334, 320 (1988)).
  • the 5' terminus of the DNA of the tissue plasminogen activator position ⁇ 85 to> 1753, Pennica et al., Nature 301, 214 (1983) is again linked (FIG. 7).
  • the entire construct is then cloned into pUC17 / 19 or Bluescript-derived plasmid vectors, which can be used directly or in colloidal dispersion systems for in vivo transfer.
  • the chimeric genes can be transferred to viral vectors or other suitable vectors (Fig. 8). 5. Active ingredient for inhibiting coagulation
  • gene regulatory sequences or elements from genes are preferably used which encode detectable proteins in smooth muscle cells, in activated endothelial cells, in activated macrophages or in activated lymphocytes.
  • activator sequences for genes in smooth muscle cells are already under 4.1. listed.
  • proteins which are formed particularly in activated endothelial cells are described by Burrows et al. (Pharmac. Therp. 64, 155 (1994)) and Plate et al. (Brain Pathol. 4, 207 (1994)).
  • these proteins which increasingly occur in endothelial cells, include:
  • Brain endothelial cells are characterized by a very strong expression of this transporter to accomplish the transendothelial transport of D-glucose into the brain (Gerhart et al., J. Neurosci. Res. 22, 464 (1989)).
  • the promoter sequence was developed by Murakami et al. (J. Biol. Chem. 267, 9300 (1992)).
  • Endoglin appears to be a non-signal-transmitting receptor for TGFß (Gougos et al., J. Biol. Chem. 265, 8361 (1990), Cheifetz, J. Biol. Chem. 26Z, 19027 (1992), Moren et al., BBRC 132, 356 (1992)). It occurs in small amounts on normal endothelium, but is increasingly expressed on proliferating endothelium (Westphal et al., J. Invest. Derm. 122, 27 (1993), Burrows et al., Pharmac. Ther. 64, 155 (1994 )). The promoter sequence was developed by Bellon et al. (Eur. J. Immunol. 23, 2340 (1993)) and Ge et al. (Gene 133, 201 (1994)).
  • VEGF receptor-2 (flk-1, KDR) (Terman et al., BBRC 1SZ, 1579 (1992))
  • the B61 molecule represents the ligand for the B61 receptor.
  • Endothelin receptors in particular the endothelin B receptor (Webb et al., Mol. Pharmacol. 4Z, 730 (1995), Haendler et al., J. Cardiovasc. Pharm. 22, 1 (1992)).
  • the promoter sequences are from Ludwig et al. (Gene 142, 311 (1994)), Oshima et al. (J. Biol. Chem. 263, 2553 (1988)) and Pohlmann et al. (PNAS USA 64, 5575 (1987)).
  • IL-1 is produced by activated endothelial cells (Warner et al., J. Immunol. 132, 1911 (1987)).
  • VCAM-1 Vascular Cell Adhesion Molecule
  • VCAM-1 VCAM-1 in endothelial cells is activated by lipopolysaccharides, TNF- ⁇ (Neish et al., Mol. Cell. Biol. 3, 2558 (1995)), IL-4 (lademarko et al., J. Clin. Invest 25, 264 (1995)) and IL-1 (Marni et al., J. Clin. Invest. 22, 1866 (1993)).
  • VCAM-1 The promoter sequence of VCAM-1 was described by Neish et al., Mol. Cell. Biol. 15, 2558 (1995), Ahmad et al., J. Biol. Chem. 2Z2, 8976 (1995), Neish et al., J. Exp. Med. 1Z6, 1583 (1992), lademarco et al., J. Biol. Chem. 26Z, 16323 (1992) and Cybulsky et al., PNAS USA 66, 7859 (1991).
  • synthetic activator sequences can also be used which consist of oligomerized binding sites for transcription factors which are preferentially or selectively active in endothelial cells.
  • GATA-2 whose binding site in the endothelin-1 gene is 5'-TTATCT-3 '(Lee et al., Biol. Chem. 266, 16188 (1991), Dorfmann et al., J. Biol Chem. 26Z, 1279 (1992) and Wilson et al., Mol. Cell. Biol. 12, 4854 (1990)).
  • the activator sequence is also to be understood as meaning promoter sequences of the genes for proteins which are formed to an increased extent in the immune reaction in macrophages and / or in lymphocytes. These include, for example:
  • Adhesion proteins such as integrin beta2 protein (Nueda et al., J. Biol. Chem. 268. 19305 (1993))
  • M-CSF Macrophage Colony Stimulating Factor
  • Interferon regulator / factor 1 whose promoter is stimulated by IL-6 as well as by IFN ⁇ or beta
  • a DNA sequence which encodes a protein which directly or indirectly inhibits platelet aggregation or a blood coagulation factor or stimulates fibrinolysis is to be used as active substance in the sense of this invention.
  • Such an active substance is called an anticoagulant.
  • Genes for, for example, plasminogen activators (PA), such as tissue PA (tPA) or urokinase-like PA (uPA) or protein C, antithrombin III, C-1S inhibitor, ⁇ 1-antitrypsin, the tissue factor are anticoagulants Use pathway inhibitor (TFPI) or hirudin.
  • PA plasminogen activators
  • tPA tissue PA
  • uPA urokinase-like PA
  • TFPI anticoagulants
  • hirudin anticoagulants Use pathway inhibitor
  • the invention further relates to an active ingredient in which there is a combination of the DNA sequences of two identical anticoagulant substances (A, A) or two different anticoagulant substances (A, B).
  • A, A identical anticoagulant substances
  • A, B two different anticoagulant substances
  • To express both DNA sequences preferably the cDNA of an "internal ribosomal entry site" (IRES) interposed as a regulatory element.
  • IRS internal ribosomal entry site
  • IRES IRES have been described, for example, by Montford and Smith (TIG H, 179 (1995), Kaufman et al., Nucl. Acids Res. 12, 4485 (1991), Morgan et al., Nucl. Acids Res. 22, 1293 (1992, Dirks et al., Gene 126, 247 (1993), Pelletier and Sonenberg, Nature 334, 320 (1988) and Sugitomo et al., BioTechn. 12, 694 (1994).
  • the cDNA of the IRES sequence of the poliovirus (position ⁇ 140 to> 630 of the 5 'UTR (Pelletier and Sonenberg, Nature 334, 320 (1988)) for linking the DNA of the antithrombotic substance A (at the 3' end) and the DNA of the antithrombotic substance B (at the 5 'terminus) can be used.
  • Such a combination of two identical or different genes produces an additive effect (with the same genes) or a synergistic effect of the selected antithrombotic substances.
  • Substances which bind to the cell surface of smooth muscle cells or of proliferating endothelial cells or of activated macrophages and / or lymphocytes are preferred as ligands for viral or non-viral vectors, for example in colloidal dispersions containing polylysine-ligand conjugates.
  • this includes antibodies or antibody fragments, directed against membrane structures of endothelial cells, as described, for example, by Burrows et al. (Pharmac. Ther. 64, 55 (1994)), Hughes et al. (Cancer Res. 42, 6214 (1989) and Maruyama et al. (PNAS-USA 6Z, 5744 (1990).
  • these include antibodies against the VEGF receptors.
  • the murine monoclonal antibodies are preferably used in humanized form. Humanization takes place in the section 4.4. illustrated way. Antibody fragments are produced according to the state of the art, for example in section 4.4. described way.
  • the ligands also include all active substances which bind to membrane structures or membrane receptors on endothelial cells.
  • these include substances that contain mannose, IL-1 or growth factors or their fragments or partial sequences thereof that bind to receptors expressed by endothelial cells, such as PDGF, bFGF, VEGF, TGFß (Pusztain et al., J. Pathol. 162, 191 (1993)).
  • Adhesion molecules that bind to activated and / or proliferating endothelial cells.
  • Adhesion molecules of this type 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. 2, 175 (1990), Honn et al., Cancer Metast. Rev. H, 353 (1992)).
  • the ligands also include substances which specifically bind to the surface of immune cells. These include antibodies or antibody fragments directed against membrane structures of immune cells, as described, for example, by Powelson et al., Biotech. Adv. H, 725 (1993). Furthermore, the ligands also include monoclonal or polyclonal antibodies or antibody fragments which bind with their constant domains to Fc ⁇ or ⁇ receptors of immune cells (Rojanasakul et al., Pharm. Res. 11, 1731 (1994)).
  • murine monoclonal antibodies are preferably used in humanized form (see section 4.4.) And fragments, for example with the one in section 4.4. cited methodology.
  • the ligands also include all substances that bind to membrane receptors on the surface of immune cells.
  • examples include growth factors such as cytokines, EGF, TGF, FGF or PDGF, or their fragments or partial sequences thereof, which bind to receptors expressed by such cells.
  • the human endothelin-1 promoter (position ⁇ -170 to> -10, Wilson et al., Mol. Cell. Biol. 12, 4854 (1990)) or a variant shortened by the TATA box (position ⁇ -170 to> -40) are at their 3 'end with the 5' terminus of the CDE-CHR-Inr module (position ⁇ -20 to> +121) of the human cdc25C gene (Lucibello et al., EMBO J., 14, 132 (1995)) linked (Fig. 9).
  • the linkage takes place with the aid of enzymes known and commercially available to the person skilled in the art.
  • the chimeric endothelin-1 promoter module transcription unit described is at its 3 'ends with the 5' terminus of a DNA which encompasses the entire coding region of the tissue plasminogen activator (Position ⁇ 85 to> 1753, Pennica et al., Nature 221, 214 (1983)), linked (Fig. 9).
  • This PNA also contains the signal sequence necessary for secretion.
  • Transcription control units and the DNA for tissue plasminogen activator are cloned into pUC19 / 19 or Bluescript-derived plasmid vectors which can be used directly or in colloidal dispersion systems for in vivo application.
  • the chimeric genes can be transferred into viral vectors or other suitable vectors and injected.
  • the human myogenin promoter (pos. ⁇ -210 to> +54, that of Salmin et al., J. Cell Biol. 115. 905 (published in 1991) is labeled with the 5'- at its 3 'end.
  • Term of the CDE-CHR-Inr module of the human cdc25C gene (pos. ⁇ -20 to> +121, linked to the sequence published by Lucibello et al., EMBO J. 14, 132 (1995)) (see FIG. 10)
  • the linkage takes place with the aid of enzymes known and commercially available.
  • various fragments of the myogenin promoter sequence are used (see FIG. 10).
  • the DNA sequence of the myogenin promoter containing the TATA box is used.
  • the promoter sequence position ⁇ -210 to> -40 are used.
  • the chimeric myogenin promoter module transcription control unit produced in this way is linked at its 3 'end to the 5' terminus of a DNA which contains the complete coding region of the tissue plasminogen activator (see FIG. 10).
  • This DNA also contains the signal sequence necessary for secretion.
  • Transcription control units and the DNA for the tissue plasminogen activator are cloned into pUC18 / 19 or Bluescript-derived plasmid vectors which can be used directly or in colloidal dispersion systems for in vivo application.
  • the chimeric genes can be transferred into viral vectors or other suitable vectors and injected.
  • the myogenin-CDE-CHR-Inr transcription unit is at its 3 'end with the 5' end of the DNA for the tissue factor pathway inhibitor (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)).
  • TFPI tissue factor pathway inhibitor
  • the linkage takes place with the aid of enzymes known and commercially available to the person skilled in the art.
  • the 3 'end of the DNA for TFPI is now linked to the 5 * end of the cDNA of the internal ribosome entry site (position ⁇ 140 to>630; Pelletier and Finberg, Nature 334, 320 (1988)) and, exclusively, its 3' end linked to the 5 'end of the DNA for the tissue plasminogen activator (see FIG. 11).
  • This active ingredient thus produced is cloned exclusively into puc18 / 19 or into Bluescript-derived plasmid vectors which can be used directly or in colloidal dispersion systems for in vivo application.
  • the chimeric genes can be transferred into viral vectors or other suitable vectors and injected.
  • An active substance according to the present invention after local or systemic, preferably intravenous or intra-arterial, administration has a predominant, if not exclusive, effect on such smooth muscle cells, which are caused by damage or injuries to the vessel (in particular the endothelial layer) and possibly after migration into the intima of the vessel volume are directly accessible from here.
  • tissue-specific activator sequence and cell cycle-regulated repressor module ensures that the cell cycle inhibitor is activated predominantly or exclusively in dividing smooth muscle cells.
  • mutant cell cycle inhibitors according to the invention ensures their longer-term proliferation-inhibiting effect.
  • An active ingredient according to the present invention further enables that after local (for example in tissue, body cavities or tissue spaces) or after systemic, preferably intravenous or intraarterial administration, predominantly, if not exclusively, smooth muscle cells, activated proliferating endothelial cells, activated lymphocytes or activated macrophages express the antithrombotic substance and this is thus released at the site of thrombosis.
  • the active ingredient promises a high degree of safety due to its cell and cell cycle specificity, it can also be used in high doses and, if necessary, several times in intervals of days or weeks for the prophylaxis or therapy of vascular occlusions, caused by proliferating smooth muscle cells and / or used for the prophylaxis and / or therapy of thromboses.
  • Nucleotide sequence of the cdc25C promoter region with the protein binding sites found in vivo (genomic DMS footprinting; ⁇ (filled circles): complete constitutive protection; o (open circles: partial constitutive protection; * (asterisk): cell cycle-regulated, G1-specific protection).
  • Gray areas indicate the Y c boxes (NF-Y binding points). Starting points are marked by filled squares.
  • Chimeric constructs consisting of different portions of the human endothelin-1 promoter, the 3 'fused promoter module with the CDE and CHR repressor elements and a DNA for the human tissue plasminogen activator (complete coding area, Pennica et al., Nature 301, 214 (1983)) ) as an effector.
  • Positions refer to the information provided by Wilson et al., Mol. Cell. Biol. IQ, 4854 (1990) for the endothelin-1 gene or on the system used by Lucibello et al., EMBO J. 14, 132 (1995) for cdc25C.
  • Chimeric constructs consisting of different portions of the human myogenin (Myf-4) promoter, the 3 'fused promoter module with the CDE and CHR repressor elements and a DNA for the human tissue plasminogen activator (complete coding area) as effector.
  • Positions refer to the information from Salminen et al., J. Cell Biol. 115, 905 (1991) for the myogenin gene and to the system used by Lucibello et al., EMBO J. 14, 132 (1995) for cdc25C and on the specifications of Pennica et al., Nature 301, 214 (1983) for the tissue plasminogen activator.
  • Results of transient transfections in HIH3T3 cells are shown as RLUs / 1000.
  • mCDE mutated CDE (pos. -13: G ⁇ T);
  • mCHR mutated CHR (pos. -6 to -3).

Abstract

A DNA sequence is disclosed for the genetic therapy of vascular diseases. The essential components of the DNA sequence are the activator sequence, the promoter module and the active substance coding gene. The activator sequence is specifically activated in smooth muscle cells, activated endothelial cells, activated macrophages or activated lymphocytes. Activation is cell cycle-regulated by the promoter module. The active substance represents an inhibitor of the growth of smooth muscle cells and/or coagulation. The disclosed DNA sequence is inserted into a viral or non-viral vector, supplemented with a ligand with affinity for the target cells.

Description

Gentherapeutische Behandlung von Gefäßerkrankungen durch einen zellspezifischen, zellzyklusabhängigen WirkstoffGene therapy treatment of vascular diseases using a cell-specific, cell cycle-dependent agent
Technisches GebietTechnical field
Es wird eine DNA-Sequenz für die Gentherapie von Gefäßerkrankungen beschrieben. Wesentliche Elemente für die DNA-Sequenz sind die Aktivatorsequenz, das Promotormodul und das Gen für die Wirksubstanz. Die Aktivatorsequenz wird zellspezifisch aktiviert in glatten Muskelzellen, aktivierten Endothelzellen, aktivierten Makrophagen oder aktivierten Lymphozyten. Diese Aktivierung wird zellzyklusspezifisch reguliert durch das Promotormodul. Die Wirksubstanz stellt einen Inhibitor für das Wachstum glatter Muskelzellen und/ oder für die Gerinnung dar. Die beschriebene DNA-Sequenz wird eingefügt in einen viralen oder nicht-viralen Vektor, ergänzt um einen Liganden mit Affinität für die Zielzelle.A DNA sequence for the gene therapy of vascular diseases is described. Essential elements for the DNA sequence are the activator sequence, the promoter module and the gene for the active substance. The activator sequence is activated cell-specifically in smooth muscle cells, activated endothelial cells, activated macrophages or activated lymphocytes. This activation is regulated in a cell cycle-specific manner by the promoter module. The active substance is an inhibitor for the growth of smooth muscle cells and / or for coagulation. The DNA sequence described is inserted into a viral or non-viral vector, supplemented by a ligand with affinity for the target cell.
1 ) Gefäßerkrankungen durch glatte Muskelzellen1) Vascular diseases caused by smooth muscle cells
Glatte Muskelzellen der Gefäße sind vorwiegend in der arteriellen Tunica media lokalisiert und an der lokalen und systemischen Blutdruckregelung beteiligt. Beim unverletzten, gesunden Gefäß sind diese glatten Muskelzellen im Ruhezustand der Zellteilung (R. Ross, Nature 3J52, 801 (1993)). Verletzungen der Gefäße führen zur Wanderung von glatten Muskelzellen in die Intimaschicht der Gefäßwand, wo sie proliferieren (Neointimabildung) und extrazelluläre Matrixkomponenten bilden.Vascular smooth muscle cells are predominantly located in the arterial tunica media and are involved in local and systemic blood pressure regulation. In the uninjured, healthy vessel, these smooth muscle cells are in the resting state of cell division (R. Ross, Nature 3J52, 801 (1993)). Vascular injuries cause smooth muscle cells to migrate into the intimal layer of the vessel wall, where they proliferate (formation of neointima) and form extracellular matrix components.
Die intimale Proliferation von glatten Muskelzellen wird als eine wesentliche Komponente bei der Entstehung der Arteriosklerose angesehen (J.S. Forrester et al., Am. Coll. Cardiol. 17, 758 (1991)). Des weiteren führt diese Proliferation glatter Muskelzellen zur Restenose der Gefäße nach angioplastischen Operationen wie auch nach der Ballondilatation von eingeengten Gefäßen (R.S. Schwartz et al., Am. Coll. Cardiol. 2Ü, 1284 (1992), M.W. Liu et al., Circulation 72, 1374 (1989)).Intimal proliferation of smooth muscle cells is considered an essential component in the development of arteriosclerosis (J.S. Forrester et al., Am. Coll. Cardiol. 17, 758 (1991)). Furthermore, this proliferation of smooth muscle cells leads to restenosis of the vessels after angioplasty operations as well as after balloon dilation of narrowed vessels (RS Schwartz et al., Am. Coll. Cardiol. 2Ü, 1284 (1992), MW Liu et al., Circulation 72 , 1374 (1989)).
Bekanntermaßen führt die Arteriosklerose wie auch die Stenose und Restenose von Gefäßen zur schlußendlichen Thrombose des Gefäßes und damit häufig zu einem lebensbedrohlichen Infarkt. Bislang ist jedoch noch keine Therapie verfügbar, welche durch Hemmung des Wachstums von glatten Muskelzellen Stenosen von Gefäßen verhindert. Zwar ist bekannt, daß Heparin die Proliferation glatter Muskelzellen zu hemmen vermag (Cochran et al., J. Cell Physiol. 124, 29 (1995), mit Heparin konnte jedoch die Entstehung von Stenosen nicht ausreichend verhindert werden. Es war somit naheliegend, daß neue Verfahren geprüft wurden, das Wachstum von glatten Muskelzellen in den verletzten Gefäßen zu verhindern und hierdurch das Infarkt¬ risiko zu beheben. Hierbei wurde die Kenntnis von den Genen und Molekülen genutzt, welche in das Wachstum von glatten Muskelzellen regulierend eingreifen.As is known, arteriosclerosis as well as the stenosis and restenosis of vessels lead to the eventual thrombosis of the vessel and thus often to a life-threatening infarction. So far, however, no therapy is available which prevents stenosis of vessels by inhibiting the growth of smooth muscle cells. Although it is known that heparin is able to inhibit the proliferation of smooth muscle cells (Cochran et al., J. Cell Physiol. 124, 29 (1995), the development of stenoses could not be sufficiently prevented with heparin. It was therefore obvious that New methods have been tested to prevent the growth of smooth muscle cells in the injured vessels and thereby eliminate the risk of infarction, using knowledge of the genes and molecules that regulate the growth of smooth muscle cells.
So ist bekannt, daß das Protoonkogen c-myb wie auch die cdc2-Kinase und das "proliferating cell nuclear antigen (PCNA)" an der Proliferation von glatten Muskelzellen beteiligt sind. Durch Verabreichung von Antiseπse c-myb Oligonukleotiden (Simons et al., Nature 359. 67 (1992)) wie auch von Antisense cdc2-Kinase Oligonukleotiden in Kombination mit Antisense PCNA Oligonukleotiden (Morishita et al., Proc. Natl. Acad. Sei. 9_0_, 8474 (1993)) unmittelbar nach und lokal am Ort der Gefäßschädigung konnte eine Proliferation von glatten Muskelzellen in der Ratte verhindert werden.It is known that the proto-oncogene c-myb as well as the cdc2 kinase and the "proliferating cell nuclear antigen (PCNA)" are involved in the proliferation of smooth muscle cells. By administration of antiseptic c-myb oligonucleotides (Simons et al., Nature 359, 67 (1992)) as well as of antisense cdc2-kinase oligonucleotides in combination with antisense PCNA oligonucleotides (Morishita et al., Proc. Natl. Acad. Sei. 9_0_, 8474 (1993)) immediately after and locally at the site of the vascular damage, a proliferation of smooth muscle cells in the rat could be prevented.
Ähnliche Ergebnisse wurden in der Ratte und beim Schwein erzielt mit der Verabreichung des als Onkogensuppressor bekannten Retinoblastoma(Rb)genes, auch hier unmittelbar nach und lokal am Ort der Gefäßschädigung gegeben. Um eine Inaktivierung des Rb-Genproduktes durch Phosphorylierung zu verhindern, wurde ein punktmutiertes Rb-Gen verwendet (Austausch von Thr-246, Ser-601 , Ser-605, Ser-780, Ser-786, Ser-787, Ser-800 mit Ala, von Thr-350 mit Arg und von Ser-804 mit Glu (Hamel et al., Mol. Cell Biol. 12, 3431 (1992)), welches eine nicht phosphorylierbare, konstitutiv aktive Form des Rb-Proteins kodiert. Dieses mutierte Rb-Gen wurde eingebaut in einen replikationsdefizienten rekombinanten Adenovirus und dieser Vektor lokal verabreicht (Chang et al., Science 267. 518 (1995)).Similar results were obtained in rats and pigs with the administration of the retinoblastoma (Rb) gene known as an oncogene suppressor, also given immediately after and locally at the site of the vascular damage. To prevent inactivation of the Rb gene product by phosphorylation, a point mutated Rb gene was used (exchange of Thr-246, Ser-601, Ser-605, Ser-780, Ser-786, Ser-787, Ser-800 with Ala, of Thr-350 with Arg and of Ser-804 with Glu (Hamel et al., Mol. Cell Biol. 12, 3431 (1992)), which encodes a non-phosphorylatable, constitutively active form of the Rb protein, which mutated Rb gene was inserted into a replication-deficient recombinant adenovirus and this vector was administered locally (Chang et al., Science 267, 518 (1995)).
In einem anderen Versuchsansatz wurde ein replikationsdefizientes rekombinantes Adenovirus verwendet, in welchem das Gen für die Herpes simplex-Virus- Thymidinkinase eingefügt war (AV-HS-TK). Das Kinase-Gen-Produkt ist in der Lage, die Wirkstoffvorstufe ("Prodrug") Ganciclovir zu phosphorylieren und hierdurch in ein Nukleosidanalog umzuwandeln, welches die DNA-Synthese inhibiert.In another experimental approach, a replication-deficient recombinant adenovirus was used, in which the gene for the herpes simplex virus thymidine kinase was inserted (AV-HS-TK). The kinase gene product is in the Able to phosphorylate the prodrug ganciclovir and thereby convert it into a nucleoside analog that inhibits DNA synthesis.
Der Genvektor AV-HS-TK wurde 7 Tage nach der Gefäßschädigung, jedoch auch hier lokal am Ort der Schädigung verabreicht und nachfolgend täglich über 14 Tage Ganciclovir intraperitoneal injiziert. Durch diese Behandlung wurde bei der Ratte eine deutliche Hemmung des Wachstums der glatten Muskelzellen erzielt (Guzman et al., Proc. Natl. Acad. Sei. 9 ., 10732 (1994)). Mit einer gleichartigen Behandlung wurden ähnliche Ergebnisse auch am Schwein erzielt (Ohno et al., Science 2_2L5_, 781 (1994)). Die Gabe des Vektors erfolgte hier jedoch unmittelbar nach der Gefäßverletzung und die Ganciclovir-Verabreichung täglich über 6 Tage.The gene vector AV-HS-TK was administered 7 days after the vascular damage, but also here locally at the site of the damage, and ganciclovir was subsequently injected intraperitoneally daily for 14 days. This treatment significantly inhibited the growth of smooth muscle cells in the rat (Guzman et al., Proc. Natl. Acad. Sei. 9., 10732 (1994)). With a similar treatment, similar results were also achieved in pigs (Ohno et al., Science 2_2L5_, 781 (1994)). However, the vector was administered immediately after the vascular injury and the ganciclovir administration daily for 6 days.
Insgesamt verdeutlichen diese Versuche die Möglichkeit, durch unterschiedliche gentherapeutische Maßnahmen, welche in die Zellteilungsvorgänge von glatten Muskelzellen eingreifen, die Stenose nach Verletzungen der Gefäße zu verhindern.Overall, these experiments clarify the possibility of preventing the stenosis after injury to the vessels by means of various gene therapy measures which intervene in the cell division processes of smooth muscle cells.
Nachteil der literaturbekannten Verfahren ist jedoch, daß die wirksamen Substanzen (Vektoren) lokal am Ort der Gefäßverletzungen appliziert werden müssen, wobei gegebenenfalls sogar der betreffende Gefäßabschnitt zeitweise verschlossen sein muß, um ein Abschwemmen der Vektoren zu verhindern. Derartige invasive Eingriffe werden im Rahmen der Ballondilatation von stenosierten Gefäßen zwar routinemäßig durchgeführt, benötigen jedoch einen beträchtlichen Aufwand und stellen ihrerseits eine erhebliche Gefährdung des Patienten durch das Risiko von Thrombosen und Embolien dar.A disadvantage of the methods known from the literature, however, is that the active substances (vectors) have to be applied locally at the site of the vascular injuries, and even the relevant vessel section may have to be temporarily closed in order to prevent the vectors from washing away. Such invasive interventions are routinely performed as part of balloon dilatation of stenosed vessels, but they require considerable effort and in turn represent a considerable risk to the patient due to the risk of thrombosis and embolism.
Zum anderen ist trotz der lokalen Applikation der Vektoren nicht gesichert, daß sie nur glatte, proliferierende Muskelzellen transduzieren. Die Transduktion anderer nahe oder entfernt gelegener Zellen könnte zu Nebenwirkungen (u.a. Transformation von Zellen und Tumorinduktionen) führen, wie sie heute in der Fachwelt diskutiert werden (Friedmann, Science 244. 1275 (1989), Plummer, Scrip Magazine Hl/29 (1995)).On the other hand, despite the local application of the vectors, it is not certain that they only transduce smooth, proliferating muscle cells. The transduction of other nearby or distant cells could lead to side effects (including transformation of cells and tumor induction), as are discussed today in the specialist world (Friedmann, Science 244, 1275 (1989), Plummer, Scrip Magazine HL / 29 (1995) ).
Als Alternative zur Gabe der oben beschriebenen Vektoren ist die systemische (z.B. intravenöse oder orale) Gabe von Zytostatika für die Proliferationshemmung von glatten Muskelzellen am Ort der Gefäßerkrankung nur von geringer und vorübergehender Wirkung, weist andererseits das Risiko einer Schädigung von Endothelien auf und führt zu erheblichen akuten wie auch chronischen Nebenwirkungen.As an alternative to the administration of the vectors described above, the systemic (eg intravenous or oral) administration of cytostatics for the inhibition of proliferation of smooth muscle cells at the site of the vascular disease is only minor and temporary effect, on the other hand has the risk of damage to endothelia and leads to considerable acute and chronic side effects.
2^ Thrombosen2 ^ thrombosis
Thrombosen stellen eine immer noch schwer zu behandelnde, zum Teil lebensbedrohende Komplikation von metabolischen Erkrankungen, wie Arteriosklerose, arteriellen und venösen Gefäßerkrankungen und lokalen wie auch systemischen immunreaktiven Syndromen dar (Übersichten bei Philipps et al., Blood 71, 831 (1988), Harker, Biomed. Progr. 8_, 17 (1995)).Thromboses are still a difficult to treat, sometimes life-threatening complication of metabolic diseases, such as arteriosclerosis, arterial and venous vascular diseases and local as well as systemic immunoreactive syndromes (reviews by Philipps et al., Blood 71, 831 (1988), Harker, Biomed. Progr. 8_, 17 (1995)).
Obwohl eine Reihe von Antikoagulantien, Antithrombotika, Fibrinolytika und Thrombozytenaggregationsinhibitoren bereits seit längerem in klinischer Anwendung und neue Substanzen in klinischer Erprobung sind, lassen sich die lebensbedrohenden Komplikationen der Thrombose bislang weder in ausreichendem Maße verhüten noch beherrschen (White, Scrip Magazine 4, 6 (1994), Antiplatelet Trialists' Collaboration BMJ 3jQ8_, 81 (1994)).Although a number of anticoagulants, antithrombotics, fibrinolytics and platelet aggregation inhibitors have been in clinical use for a long time and new substances have been in clinical trials, the life-threatening complications of thrombosis have so far not been adequately prevented or controlled (White, Scrip Magazine 4, 6 (1994 ), Antiplatelet Trialists' Collaboration BMJ 3jQ8_, 81 (1994)).
Es besteht somit ein großer Bedarf nach neuen Arzneimitteln zur Verhütung und zur Therapie von Thrombosen (BMJ 305. 567 (1992), Vinazzer, Biomedical Progress ü, 17 (1993)). Ein beträchtlicher Teil der Thrombosen hat seine Ursache in aktivierten oder geschädigten Endothelzellen. Diese selbst oder die durch Wachstumsfaktoren im Blut zur Proliferation stimulierten glatten Muskelzellen bewirken alleine oder in Gesellschaft mit aktivierten Makrophagen, Lymphozyten, Thrombozyten und Granulozyten eine Aktivierung des Gerinnungssystems (Nemerson, Blood ZI, 1 (1988)).There is therefore a great need for new drugs for the prevention and therapy of thromboses (BMJ 305, 567 (1992), Vinazzer, Biomedical Progress ü, 17 (1993)). A significant portion of thrombosis is due to activated or damaged endothelial cells. These themselves or the smooth muscle cells stimulated by proliferation in the blood cause an activation of the coagulation system alone or in association with activated macrophages, lymphocytes, thrombocytes and granulocytes (Nemerson, Blood ZI, 1 (1988)).
Diese Aktivierung führt letztendlich zur Bildung von Fibrin, zur Aktivierung und Ag¬ gregation von Thrombozyten und zur Bildung von Fibrin-reichen oder Thrombo- zyten-reichen blutgefäßverengenden oder verschließenden Blutgerinnseln, den Thrombosen. Derartige Thrombosen führen im Bereich des arteriellen Gefäßsy¬ stems zu Infarkten, welche beispielsweise am Herzen oder im Gehirn lebensbe¬ drohlich sind. Die bislang etablierte Therapie mit Antithrombotika (wie Heparin oder Fraktionen von Heparin), Antikoagulantien (wie Coumarin), Thrombozytenaggregationsinhibi- toren (wie Aspirin) und Fibrinolytika (wie Streptokinase, Urokinase oder Gewebe- plasminogenaktivatoren (tPA)) bewirkt zwar eine durch zahlreiche klinische Studien belegte prophylaktische Wirkung auf drohende Thrombosen und therapeutische Wirkung auf bestehende Thrombosen, diese Wirkung ist jedoch unzulänglich. Der Grund hierfür liegt zu einem beträchtlichen Teil in der Tatsache, daß die Wirkung der verwendeten Therapeutika nicht beschränkt ist auf den Ort der Erkrankung, d.h. der Thrombose, sondern daß sie systemisch wirken. Die hierdurch bedingten Blutungen beschränken somit sowohl die Dosiserhöhung als auch die Zeitdauer der Anwendung.This activation ultimately leads to the formation of fibrin, to the activation and aggregation of thrombocytes and to the formation of clots which are rich in fibrin or thrombocytes and which clog or close the blood vessels, the thromboses. Such thromboses lead to infarcts in the arterial vascular system, which are life-threatening, for example, in the heart or in the brain. The previously established therapy with antithrombotics (such as heparin or fractions of heparin), anticoagulants (such as coumarin), platelet aggregation inhibitors (such as aspirin) and fibrinolytics (such as streptokinase, urokinase or tissue plasminogen activators (tPA)) does indeed result in numerous clinical studies Proven prophylactic effect on impending thrombosis and therapeutic effect on existing thrombosis, but this effect is insufficient. The reason for this lies to a considerable extent in the fact that the effect of the therapeutic agents used is not limited to the location of the disease, ie the thrombosis, but that they act systemically. The bleeding caused by this thus limits both the dose increase and the duration of use.
3) Allgemeine Beschreibung der Erfindung3) General description of the invention
Gegenstand der Erfindung ist nunmehr ein Wirkstoff (d. h. ein Arzneimittel), welcher lokal wie auch systemisch Patienten gegeben werden kann und welcherThe invention now relates to an active ingredient (i.e. a medicament) which can be given locally and systemically to patients and which
- weitgehend nur glatte, in Zellteilung befindliche Muskelzellen beeinflußt, die Proliferation von glatten Muskelzellen nach Gefäßverletzungen oder Gefä߬ schäden hemmt und damit die Stenose oder Restenose von Gefäßen verhindert oderlargely affects only smooth muscle cells in cell division, inhibits the proliferation of smooth muscle cells after vascular injuries or vascular damage and thus prevents the stenosis or restenosis of vessels or
- weitgehend nur am Ort einer entstehenden Thrombose, d.h. am Ort von aktivierten und proliferierenden Endothelzellen, glatten Muskelzellen der Intima, Makrophagen und/oder Lymphozyten die Blutgerinnung inhibiert oder- largely only at the site of an emerging thrombosis, i.e. inhibits blood clotting at the site of activated and proliferating endothelial cells, smooth muscle cells of the intima, macrophages and / or lymphocytes or
- welcher sowohl die Proliferation glatter Muskelzellen hemmt als auch dort die Thrombose lokal inhibiert.- Which both inhibits smooth muscle cell proliferation and locally inhibits thrombosis there.
Zentraler Bestandteil dieses Wirkstoffes ist ein DNA-Konstrukt, welches aus folgenden Elementen besteht:The central component of this active ingredient is a DNA construct, which consists of the following elements:
(DNA wird im gesamten Text dieser Anmeldung als gemeinsamer Begriff sowohl für eine komplementäre (cDNA) als auch für eine genomische DNA-Sequenz benutzt). Aktivatorsequenz zellzyklusreguliertes Wirksubstanz (UAS) Promotormodul(DNA is used throughout the text of this application as a common term for both a complementary (cDNA) and a genomic DNA sequence). Activator sequence cell cycle regulated active substance (UAS) promoter module
Das zentrale Element dieses Wirkstoffes stellt das zellzykiusregulierte Promotorrnodul dar.The central element of this active ingredient is the cell cycle-regulated promoter module.
Als zellzyklusreguliertes Promotorrnodul ist beispielsweise die Nukleotidsequenz - CDE-CHR-Inr- (siehe unten) zu verstehen. Die wesentliche Funktion des Promotormoduls ist die Hemmung der Funktion der Aktivatorsequenz in der G0/G1 Phase des Zellzyklus und eine Zellzyklus-spezifische Expression in der S/G2 Phase und damit in proliferierenden Zellen.The nucleotide sequence - CDE-CHR-Inr- (see below) is to be understood as a cell cycle-regulated promoter module. The essential function of the promoter module is the inhibition of the function of the activator sequence in the G0 / G1 phase of the cell cycle and a cell cycle-specific expression in the S / G2 phase and thus in proliferating cells.
Das Promotorrnodul CDE-CHR-Inr wurde im Rahmen einer detaillierten Untersuchung der G2-spezifιschen Expression des menschlichen cdc25C Promotors entdeckt. Ausgangspunkt war die Auffindung eines Repressorelementes ("cell cycle dependent element"; CDE), das für die Abschaltung des Promotors in der G1 -Phase des Zellzyklus verantwortlich ist (Lucibello et al., EMBO J. 14, 132 (1995))/ Durch genomisches Dimethylsulfat (DMS)-Footprinting und funktionelle Analysen (Fig. 1 , 2) konnte gezeigt werden, daß das CDE einen Repressor ("CDE- binding factor"; CDF) G1 -spezifisch bindet und hierdurch zu einer Inhibition der Transkription in nicht-proliferierenden (G0) Zellen führt. Das im Bereich des Basalpromotors lokalisierte CDE ist in seiner reprimierenden Funktion abhängig von einer "upstream activating sequence" (UAS). Dies führte zu dem Schluß, daß der CDE-bindende Faktor die transkriptionsaktivierende Wirkung 5' gebundener Aktivatorproteine in einer zellzyklusabhängigen Weise, d.h. in nicht- proliferierenden Zellen sowie in der G1 -Phase des Zellzyklus, hemmt (Fig. 3).The promoter module CDE-CHR-Inr was discovered in the context of a detailed investigation of the G2-specific expression of the human cdc25C promoter. The starting point was the discovery of a repressor element ("cell cycle dependent element"; CDE), which is responsible for switching off the promoter in the G1 phase of the cell cycle (Lucibello et al., EMBO J. 14, 132 (1995)) / Durch Genomic dimethyl sulfate (DMS) footprinting and functional analyzes (FIGS. 1, 2) showed that the CDE binds a repressor ("CDE binding factor"; CDF) G1 -specifically and thereby inhibits transcription in non- proliferating (G0) cells. The CDE located in the area of the basal promoter is dependent on an "upstream activating sequence" (UAS) in its repressing function. This led to the conclusion that the CDE-binding factor affects the transcription activating activity of 5 'bound activator proteins in a cell cycle dependent manner, i.e. in non-proliferating cells as well as in the G1 phase of the cell cycle, (Fig. 3).
Diese Schlußfolgerung konnte durch ein weiteres Experiment bestätigt werden: Die Fusion des viralen, nicht-zellzyklusregulierten frühen SV40-Enhancers mit einem cdc25 Minimalpromotor (bestehend aus CDE und den 3' gelegenen Startstellen) führte zu einer klaren Zellzyklusregulation des Chimären Promotors (Fig. 4). Nachfolgende Untersuchungen des cdc25C Enhancers haben gezeigt, daß es sich bei den vom CDF zellzyklusabhängig regulierten Transkriptionfaktoren um NF-Y (CBF) (Dorn et al., Cell 50, 863 (1987), Van Hujisduijnen et al., EMBO J. 9, 3119 (1990), Coustry et al., J. Biol. ehem. 270, 468 (1995)), Sp1 ( Kadonaga et al., TIBS 11, 10 (1986)) und einen möglicherweise neuen an CBS7 bindenden Transkriptionsfaktor (CIF) handelt. Ein weiterer interessanter Befund dieser Studie war die Beobachtung, daß NF-Y innerhalb des cdc25C Enhancers nur in Ko¬ operation mit mindestens einem weiteren NF-Y Komplex oder mit CIF effizient die Transkription aktiviert. Sowohl NF-Y als auch Sp1 gehören zur Klasse der glutaminreichen Aktivatoren, was wichtige Hinweise auf den Mechanismus der Repression (z.B. Interaktion bzw. Interferenz mit bestimmten basalen Transkrip¬ tionsfaktoren oder TAFs) liefert.This conclusion could be confirmed by another experiment: The fusion of the viral, non-cell cycle-regulated early SV40 enhancer with a cdc25 minimal promoter (consisting of CDE and the 3 'starting sites) led to a clear cell cycle regulation of the chimeric promoter (FIG. 4) . Subsequent investigations of the cdc25C enhancer have shown that the transcription factors regulated by the CDF cell cycle-dependent are NF-Y (CBF) (Dorn et al., Cell 50, 863 (1987), Van Hujisduijnen et al., EMBO J. 9, 3119 (1990), Coustry et al., J. Biol. Former 270, 468 (1995)), Sp1 (Kadonaga et al., TIBS 11, 10 (1986)) and a possibly new CBS7 binding transcription factor (CIF). Another interesting finding of this study was the observation that NF-Y within the cdc25C enhancer only efficiently activated the transcription in cooperation with at least one other NF-Y complex or with CIF. Both NF-Y and Sp1 belong to the class of activators rich in glutamine, which provides important information on the mechanism of repression (eg interaction or interference with certain basic transcription factors or TAFs).
Ein Vergleich der Promotorsequenzen von cdc25C, cyclin A und cdc2 zeigte Homologien in mehreren Bereichen (Fig. 5). Nicht nur das CDE ist in allen 3 Promotoren konserviert (die vorhandenen Abweichungen sind funktionell nicht relevant), sondern auch die benachbarten Y -Boxen. Alle diese Bereiche zeigten wie erwartet Proteinbindung in vivo, im Falle des CDE in zellzyklusabhängiger Weise. Außerdem konnte gezeigt werden, daß alle 3 Promotoren durch eine Mutation des CDE dereguliert werden (Tabelle 1). Eine bemerkenswerte Ähnlichkeit wurde bei dem Vergleich der cdc25C, cyclin A und cdc2A comparison of the promoter sequences of cdc25C, cyclin A and cdc2 showed homologies in several areas (FIG. 5). Not only is the CDE conserved in all 3 promoters (the existing deviations are not functionally relevant), but also the neighboring Y boxes. All of these areas showed protein binding in vivo as expected, in the case of CDE in a cell cycle dependent manner. It could also be shown that all 3 promoters are deregulated by a mutation in the CDE (Table 1). A remarkable similarity was found when comparing the cdc25C, cyclin A and cdc2
Sequenzen auch im Bereich unmittelbar 3' vom CDE ("cell cycle genes homology region"; CHR) deutlich (Fig. 5). Dieser Bereich ist funktioneil ebenso bedeutsam wie das CDE (Tabelle 1), wird in den in vivo Dimethylsulfat (DMS)-Footprinting Experimenten jedoch nicht sichtbar. Eine mögliche Erklärung hierfür ist eine Interaktion des Faktors mit der kleinen Furche der DNA. Ergebnisse aus "electro- phoretic mobility shift assay" (EMSA) Experimenten deuten darauf hin, daß CDE und CHR gemeinsam einen Proteinkomplex, den CDF, binden. Diese Beobachtungen weisen daraufhin, daß die CDF-vermittelte Repression glutamin- reicher Aktivatoren ein häufig vorkommender Mechanismus zellzyklusregulierter Transkription ist.Sequences clearly in the area immediately 3 'from the CDE ("cell cycle genes homology region"; CHR) (FIG. 5). This area is functionally as important as the CDE (Table 1), but is not visible in the in vivo dimethyl sulfate (DMS) footprinting experiments. A possible explanation for this is an interaction of the factor with the small groove of the DNA. Results from "electrophoretic mobility shift assay" (EMSA) experiments indicate that CDE and CHR bind together a protein complex, the CDF. These observations indicate that CDF-mediated repression of glutamine-rich activators is a common mechanism for cell cycle-regulated transcription.
Von Bedeutung für die Regulation des cdc25C Promotors ist jedoch nicht nur der CDE-CHR-Bereich, sondern auch eine der Initiationsstellen (Position +1) innerhalb der Nukleotidsequenz des basalen Promotors (Positionen < -20 bis > +30, siehe Fig. 1). Mutationen in diesem Bereich, welcher die in vitro Bindestelle für den Transkriptionsfaktor YY-1 (Seto und Shenk, Nature 3_5_ , 241 (1991), Usheva und Shenk, Cell 7_6_, 1115 (1994)) einschließt, führen zu einer völligen Deregulation. In Anbetracht der Nähe des CDE-CHR zum basalen Promotor ist somit eine Interaktion des CDF mit dem basalen Transkriptionskomplex sehr wahrscheinlich.Of importance for the regulation of the cdc25C promoter is not only the CDE-CHR region, 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 area, which includes the in vitro binding site for the transcription factor YY-1 (Seto and Shenk, Nature 3_5_, 241 (1991), Usheva and Shenk, Cell 7_6_, 1115 (1994)) lead to complete deregulation. In Given the proximity of the CDE-CHR to the basal promoter, an interaction of the CDF with the basal transcription complex is very likely.
Als Aktivatorsequenz (upstream activator sequence = UAS) ist eine Nukleotidsequenz (Promotor- oder Enhancersequenz) zu verstehen, mit der Transkriptionsfaktoren gebildet oder aktiv in der Zielzelle interagieren. Als Aktiva¬ torsequenz kann der CMV-Enhancer, der CMV-Promotor (EP 073.177.B1), der SV40 Promotor oder jede andere, dem Fachmann bekannt Promotor- oder Enhancersequenz verwendet werden. Im Sinne dieser Erfindung zählen jedoch zu den bevorzugten Aktivatorsequenzen solche genregulatorischen Sequenzen bzw. Elemente aus Genen, die besonders in glatten Muskelzellen, in aktivierten Endo- thelzellen oder in aktivierten Makrophagen oder Lymphozyten gebildete Proteine kodieren.An activator sequence (upstream activator sequence = UAS) is to be understood as a nucleotide sequence (promoter or enhancer sequence) with which transcription factors are formed or actively interact in the target cell. The CMV enhancer, the CMV promoter (EP 073.177.B1), the SV40 promoter or any other promoter or enhancer sequence known to the person skilled in the art can be used as the activator sequence. For the purposes of this invention, however, the preferred activator sequences include those gene regulatory sequences or elements from genes which encode proteins formed particularly in smooth muscle cells, in activated endothelial cells or in activated macrophages or lymphocytes.
Als Wirksubstanz ist die DNA-Sequenz für ein Protein zu verstehen, welches am Ort der Entstehung den therapeutischen Effekt - das heißt die Inhibition der Proliferation der glatten Muskelzellen, der Gerinnung oder (im Falle zweier Wirksubstanzen) beides - bewirken kann. Die Auswahl der Nukleotidsequenz für die Aktivatorsequenz und für die Wirksubstanz richtet sich nach der Zielzelle und der gewünschten Wirksubstanz.The active substance is to be understood as the DNA sequence for a protein which can have the therapeutic effect at the point of origin - that is to say the inhibition of the proliferation of smooth muscle cells, coagulation or (in the case of two active substances) both. The selection of the nucleotide sequence for the activator sequence and for the active substance depends on the target cell and the desired active substance.
Das erfindungsgemäße DNA-Konstrukt wird in einer dem Fachmann geläufigen Weise zu einem Vektor vervollständigt; so wird es beispielsweise in einen viralen Vektor eingefügt (siehe hierzu D. Jolly, Cancer Gene Therapy 1, 51 (1994)), oder aber zu einem Plasmid ergänzt. Virale Vektoren oder Plasmide können mit kolloidalen Dispersionen komplexiert werden, so beispielsweise mit Liposomen (Farhood et al., Annais of the New York Academy of Sciences 7_16_, 23 (1994)) oder aber mit Polylysin-Ligand-Konjugaten (Curiel et al., Annais of the New York Academy of Sciences 716. 36 (1994)). Ebenso mit den gebräuchlichen Arzneimittelhilfsstoffen kann eine Arzneimittelzubereitung erfolgen.The DNA construct according to the invention is completed to a vector in a manner familiar to the person skilled in the art; for example, it is inserted into a viral vector (see, for this, D. Jolly, Cancer Gene Therapy 1, 51 (1994)), or supplemented to form a plasmid. Viral vectors or plasmids can be complexed with colloidal dispersions, for example with liposomes (Farhood et al., Annais of the New York Academy of Sciences 7_16_, 23 (1994)) or with polylysine-ligand conjugates (Curiel et al., Annais of the New York Academy of Sciences 716. 36 (1994)). A pharmaceutical preparation can also be carried out with the usual pharmaceutical excipients.
Derartige virale oder nicht-virale Vektoren können durch einen Liganden ergänzt werden, welcher Bindungsaffinität für eine Membranstruktur auf der ausgewählten Zielzelle hat. Die Auswahl des Liganden richtet sich somit nach der Auswahl der Zielzelle (siehe S. 21 , 4.4 ff und S. 34, 5.4ff). Der erfindungsgemäße Wirkstoff wird anhand folgender Beispiele näher erläutert: 4^ Wirkstoff zur Inhibition der Proliferation glatter MuskelzellenSuch viral or non-viral vectors can be supplemented by a ligand which has binding affinity for a membrane structure on the selected target cell. The choice of ligand thus depends on the selection of the target cell (see p. 21, 4.4 ff and p. 34, 5.4ff). The active ingredient according to the invention is explained in more detail using the following examples: 4 ^ Active ingredient for the inhibition of smooth muscle cell proliferation
4.1. Auswahl der Aktivatorsequenz für glatte Muskelzellen4.1. Selection of the activator sequence for smooth muscle cells
Als Aktivatorsequenzen im Sinne der Erfindung sind bevorzugt genregulatorische Sequenzen bzw. Elemente aus Genen zu verwenden, die, besonders in glatten Muskelzellen, gebildete Proteine kodieren. Diese Gene sind beispielsweise:As activator sequences in the sense of the invention, gene regulatory sequences or elements from genes are preferred which encode proteins formed, particularly in smooth muscle cells. Examples of these genes are:
- Tropomyosin- tropomyosin
(Tsukahara et al., Nucleic Acid Res. 22, 2318 (1994), Novy et al., Cell Motility and the Cytosceleton 25, 267 (1993), Wilton et al., Cytogenetics and Cell Genetics 6_8_, 122 (1995))(Tsukahara et al., Nucleic Acid Res. 22, 2318 (1994), Novy et al., Cell Motility and the Cytosceleton 25, 267 (1993), Wilton et al., Cytogenetics and Cell Genetics 6_8_, 122 (1995))
- α-Actin- α-actin
(Sartorelli et al., Gens and Developm. 4, 1811 (1990), Miwa et al., Nucleic Acids Res. 13, 4263 (1990))(Sartorelli et al., Gens and Developm. 4, 1811 (1990), Miwa et al., Nucleic Acids Res. 13, 4263 (1990))
- α-Myosin- α-myosin
(Kelly et al., Can. J. Physiol. and Pharm. Z2, 1351 (1994), Moussavi et al., Mol. Cell. Biochem. 12S, 219 (1993)(Kelly et al., Can. J. Physiol. And Pharm. Z2, 1351 (1994), Moussavi et al., Mol. Cell. Biochem. 12S, 219 (1993)
- Rezeptoren für Wachstumsfaktoren wie beispielsweise PDGF, FGF (Rubin et al., Int. Congress Ser. 225, 131 (1990), Ross, Ann. Rev. Med. 2S, 71 (1987)- Receptors for growth factors such as PDGF, FGF (Rubin et al., Int. Congress Ser. 225, 131 (1990), Ross, Ann. Rev. Med. 2S, 71 (1987)
- Rezeptoren für Acetylcholin- Acetylcholine receptors
(Dutton et al., PNAS USA 9jQ, 2040 (1993), Dürr et al., Eur. J. Biochem. 224. 353 (1994))(Dutton et al., PNAS USA 9jQ, 2040 (1993), Dürr et al., Eur. J. Biochem. 224, 353 (1994))
- Phosphofructokinase-A- Phosphofructokinase-A
(Gekakis et al., Biochemistry 3_3_ 1771 (1994), Tsujino et al., J. Biol. Chem. 264. 15334 (1989), Castella-Escola et al., Gene 21, 225 (1990)) Phosphoglyceratemutase(Gekakis et al., Biochemistry 3, 1771 (1994), Tsujino et al., J. Biol. Chem. 264, 15334 (1989), Castella-Escola et al., Gene 21, 225 (1990)) Phosphoglycerate mutase
(Nakatsuji et al., Mol. Cell Biol. 12, 4384 (1992))(Nakatsuji et al., Mol. Cell Biol. 12, 4384 (1992))
Troponin CTroponin C
(ün et al., Mol. Cell Biol. 11, 267 (1991))(ün et al., Mol. Cell Biol. 11, 267 (1991))
DesminDesmin
(Li et al., J. Biol. Chem. 26_6_, 6562 (1991), Neuromuscular Disorders 3_, 423(Li et al., J. Biol. Chem. 26_6_, 6562 (1991), Neuromuscular Disorders 3_, 423
(1993))(1993))
MyogeninMyogenin
(Funk et al., PNAS USA S2, 9484 (1992), Olson, Symp. Soc. Exp. Biol. 46_, 331 (1992), Zhon et al., Mol. Cell. Biol. 4, 6232 (1994), Atchley et al., PNAS USA 21, 11522 (1994))(Funk et al., PNAS USA S2, 9484 (1992), Olson, Symp. Soc. Exp. Biol. 46_, 331 (1992), Zhon et al., Mol. Cell. Biol. 4, 6232 (1994), Atchley et al., PNAS USA 21, 11522 (1994))
Rezeptoren für Endothelin AReceptors for endothelin A
(Hosoda et al., J. Biol. Chem. 2SZ, 18797 (1992), Oreilly et al., J. Cardiovasc. Pharm. 22, 18 (1993), Hayzer et al., Am. J. Med. Sei. 3Ω4_, 231 (1992), Haendler et al., J. Cardiovasc. Pharm. 22, 1 (1992))(Hosoda et al., J. Biol. Chem. 2SZ, 18797 (1992), Oreilly et al., J. Cardiovasc. Pharm. 22, 18 (1993), Hayzer et al., Am. J. Med. 3Ω4_, 231 (1992), Haendler et al., J. Cardiovasc. Pharm. 22, 1 (1992))
VEGFVEGF
VEGF wird von glatten Muskelzellen, besonders unter hypoxischen Bedingungen, gebildet (Berse et al., Mol. Biol. Cell 3, 211 (1992), Finkenzeller et al., BBRC 223, 432 (1995), Tischer et al., BBRC 135, 1198 (1989), Leung et al., Science 246, 1306 (1989), Ferrara et al., Endoc. Rev. 13, 18 (1992)).VEGF is formed by smooth muscle cells, especially under hypoxic conditions (Berse et al., Mol. Biol. Cell 3, 211 (1992), Finkenzeller et al., BBRC 223, 432 (1995), Tischer et al., BBRC 135 , 1198 (1989), Leung et al., Science 246, 1306 (1989), Ferrara et al., Endoc. Rev. 13, 18 (1992)).
Die Promotorsequenzen der Gene für diese Proteine sind durch folgende Arbeiten zugänglich:The promoter sequences of the genes for these proteins are accessible through the following work:
- Tropomyosin- tropomyosin
(Gooding et al., Embo J. 13, 3861 (1994))(Gooding et al., Embo J. 13, 3861 (1994))
- α-Actin- α-actin
(Shimizu et al., J. Biol. Chem. 222, 7631 (1995), Sartorelli et al., Genes Dev. 4, 1811 (1999)) α-Myosin(Shimizu et al., J. Biol. Chem. 222, 7631 (1995), Sartorelli et al., Genes Dev. 4, 1811 (1999)) α-myosin
(Kurabayashi et al., J. Biol. Chem. 235, 19271 (1990), Molkentin et al., Mol.(Kurabayashi et al., J. Biol. Chem. 235, 19271 (1990), Molkentin et al., Mol.
Cell. Biol. 14, 4947 (1994))Cell. Biol. 14, 4947 (1994))
Rezeptor für PDGFReceptor for PDGF
(Pistritto et al., Antibiot. Chemother. 43, 73 (1994))(Pistritto et al., Antibiot. Chemother. 43, 73 (1994))
Rezeptor für FGFFGF receptor
(Myers et al., J. Biol. Chem. 270, 8257 (1995), Johnson et al., Adv. Cancer Res. 52, 1 (1993), Chellaiah et al., J. Biol. Chem. 232, 11620 (1994), Yu et al., Hum. Mol. Genetics 3, 212 (1994), Wang et al., BBRC 223, 1781 (1994), Murgue et al., Cancer Res. 54, 5206 (1994), Avraham et al., Genomics 21, 656 (1994), Burgess et al., Ann. Rev. Biochem. 53, 575 (1989))(Myers et al., J. Biol. Chem. 270, 8257 (1995), Johnson et al., Adv. Cancer Res. 52, 1 (1993), Chellaiah et al., J. Biol. Chem. 232, 11620 (1994), Yu et al., Hum. Mol. Genetics 3, 212 (1994), Wang et al., BBRC 223, 1781 (1994), Murgue et al., Cancer Res. 54, 5206 (1994), Avraham et al., Genomics 21, 656 (1994), Burgess et al., Ann. Rev. Biochem. 53, 575 (1989))
MRF-4MRF-4
(Naidu et al., Mol. Cell. Biol. 1^, 2707 (1995))(Naidu et al., Mol. Cell. Biol. 1 ^, 2707 (1995))
Phosphofructokinase APhosphofructokinase A
(Gekakis et al., Biochem. 33, 1771 (1994))(Gekakis et al., Biochem. 33, 1771 (1994))
PhosphoglyceratemutasePhosphoglycerate mutase
(Makatsuji et al., Mol. Cell. Biol. 12, 4384 (1992))(Makatsuji et al., Mol. Cell. Biol. 12, 4384 (1992))
Troponin CTroponin C
(Ip et al., Mol. Cell. Biol. 14, 7517 (1994), Parmacek et al., Mol. Cell. Biol. 12,(Ip et al., Mol. Cell. Biol. 14, 7517 (1994), Parmacek et al., Mol. Cell. Biol. 12,
1967 (1992))1967 (1992))
MyogeninMyogenin
(Salminen et al., J. Cell Biol. 115, 905 (1991), Dürr et al., Eur. J. Biochem.(Salminen et al., J. Cell Biol. 115, 905 (1991), Dürr et al., Eur. J. Biochem.
224. 353 (1994), Edmondson et al., Mol. Cell. Biol. 12, 3665 (1992))224: 353 (1994), Edmondson et al., Mol. Cell. Biol. 12, 3665 (1992))
Rezeptoren für Endothelin AReceptors for endothelin A
(Hosoda et al., J. Biol. Chem. 23Z, 18797 (1992), Li and Paulia, J. Biol.(Hosoda et al., J. Biol. Chem. 23Z, 18797 (1992), Li and Paulia, J. Biol.
Chem. 233, 6562 (1991)) - Desmin (Li et al., Neuromusc. Dis. 3, 423 (1993), Li and Capetanaki, Nucl. Acids Res. 21, 335 (1993)).Chem. 233, 6562 (1991)) - Desmin (Li et al., Neuromusc. Dis. 3, 423 (1993), Li and Capetanaki, Nucl. Acids Res. 21, 335 (1993)).
-VEGF-VEGF
Die genregulatorischen Sequenzen für das VEGF-Gen sindThe gene regulatory sequences for the VEGF gene are
* die Promotorsequenz des VEGF-Gens (5' flankierende Region) (Michenko et al., Cell Mol Biol. Res. 42, 35 (1994), Tischer et al., J. Biol. Chem. 253, 11947 (1991)) oder * the promoter sequence of the VEGF gene (5 'flanking region) (Michenko et al., Cell Mol Biol. Res. 42, 35 (1994), Tischer et al., J. Biol. Chem. 253, 11947 (1991)) or
* die Enhancersequenz des VEGF-Gens (3' flankierende Region) (Michenko et al., Cell Mol. Biol. Res. 42, 35 (1994) oder * the enhancer sequence of the VEGF gene (3 'flanking region) (Michenko et al., Cell Mol. Biol. Res. 42, 35 (1994) or
* das c-Src-Gen* the c-Src gene
(Mukhopadhyay et al., Nature 315, 577 (1995), Bonham et al., Oncogene 3, 1973 (1993), Parker et al., Mol. Cell. Biol. 5, 831 (1985), Anderson et al., Mol. Cell. Biol. 5, 1122 (1985)) oder(Mukhopadhyay et al., Nature 315, 577 (1995), Bonham et al., Oncogene 3, 1973 (1993), Parker et al., Mol. Cell. Biol. 5, 831 (1985), Anderson et al., Mol. Cell. Biol. 5, 1122 (1985)) or
* das V-Src-Gen* the V-Src gene
(Mukhodpadhyay et al., Nature 3Z5, 577 (1995), Anderson et al., Mol. Cell. Biol. 5, 1122 (1985), Gibbs et al., J. Virol. 53, 19 (1985))(Mukhodpadhyay et al., Nature 3Z5, 577 (1995), Anderson et al., Mol. Cell. Biol. 5, 1122 (1985), Gibbs et al., J. Virol. 53, 19 (1985))
* "artifizielle" Promotoren* "artificial" promoters
Faktoren der Helix-Loop-Helix (HLH)-Familie (MyoD, Myf-5, Myogenin, MRF4 (Übersicht in Olson and Klein, Genes Dev. 3, 1 (1994)) sind als muskelspezifische Transkriptionsaktivatoren beschrieben. Des weiteren gehören zu den muskelspezifischen Transkriptionsaktivatoren das Zinkfingerprotein GATA-4 (Arceci et al., Mol Cell. Biol. 13, 2235 (1993); Ip et al., Mol. Cell. Biol. 14, 7517 (1994)) sowie die Gruppen der MEF-2 Transkriptionsfaktoren (Yu et al., Gene Dev. 5, 1783 (1992)).Factors of the helix-loop-helix (HLH) family (MyoD, Myf-5, myogenin, MRF4 (overview in Olson and Klein, Genes Dev. 3, 1 (1994)) are described as muscle-specific transcription activators muscle-specific transcription activators, the zinc finger protein GATA-4 (Arceci et al., Mol Cell. Biol. 13, 2235 (1993); Ip et al., Mol. Cell. Biol. 14, 7517 (1994)) and the groups of MEF-2 Transcription factors (Yu et al., Gene Dev. 5, 1783 (1992)).
Die HLH-Proteine sowie GATA-4 zeigen muskelspezifische Transkription nicht nur mit Promotoren von muskelspezifisehen Genen, sondern auch im heterologen Kontext, so auch mit artifiziellen Promotoren. Derartige artifizielle Promotoren sind beispielsweise:The HLH proteins and GATA-4 show muscle-specific transcription not only with promoters of muscle-specific genes, but also in a heterologous context, including with artificial promoters. Such artificial promoters are for example:
* multiple Kopien der (DNA) Bindestelle für muskelspezifische HLH- Proteine wie der E-Box (Myo D)* multiple copies of the (DNA) binding site for muscle-specific HLH proteins such as the E-Box (Myo D)
(z.B. 4x AGCAGGTGTTGGGAGGC) (Weintraub et al., PNAS SZ, 5623 (1990))(e.g. 4x AGCAGGTGTTGGGAGGC) (Weintraub et al., PNAS SZ, 5623 (1990))
* multiple Kopien der DNA Bindungsstelle für GATA-4 des oc-Myosin- Heavy Chain Genes * multiple copies of the DNA binding site for GATA-4 of the oc-myosin heavy chain gene
(z.B. δ^GGCCSAJiaGGCAG L^GAGGGGGCCSAIGGGCASAIΔGAGG-(e.g. δ ^ GGCCSAJiaGGCAG L ^ GAGGGGGCCSAIGGGCASAIΔGAGG-
3')3 ')
(Molkentin et al., Mol. Cell. Biol. 14, 4947 (1994))(Molkentin et al., Mol. Cell. Biol. 14, 4947 (1994))
4.2. Auswahl der Wirksubstanz für glatte Muskelzellen4.2. Selection of the active substance for smooth muscle cells
Als Wirksubstanz in Sinne der Erfindung ist eine DNA-Sequenz zu verstehen, deren exprimiertes Protein die Proliferation von glatten Muskelzellen inhibiert. Zu diesen Zelizyklusinhibitoren gehören beispielsweise die DNA-Sequenzen für folgende Proteine:An active substance in the sense of the invention is a DNA sequence whose expressed protein inhibits the proliferation of smooth muscle cells. These cell cycle inhibitors include, for example, the DNA sequences for the following proteins:
a) inhibitorische Proteinea) inhibitory proteins
- das Retinoblastomprotein (pRb=p110) oder die verwandten p107 und p130 Proteine (La Thangue, Curr. Opin. Cell Biol. 5, 443 (1994))- the retinoblastoma protein (pRb = p110) or the related p107 and p130 proteins (La Thangue, Curr. Opin. Cell Biol. 5, 443 (1994))
- das p53 Protein (Prives et al., Genes Dev. Z, 529 (1993))- the p53 protein (Prives et al., Genes Dev. Z, 529 (1993))
- das p21 (WAF-1) Protein (El-Deiry et al., Cell Z5, 817 (1993))- the p21 (WAF-1) protein (El-Deiry et al., Cell Z5, 817 (1993))
- das p16 Protein (Serrano et al., Nature 353, 704 (1993), Kamb et al., Science 234, 436 (1994), Nobori et al., Nature 353, 753 (1994))- the p16 protein (Serrano et al., Nature 353, 704 (1993), Kamb et al., Science 234, 436 (1994), Nobori et al., Nature 353, 753 (1994))
- andere cdK-lnhibitoren (Übersicht bei Pines, TIBS 13, 143 (1995))- other CDK inhibitors (overview from Pines, TIBS 13, 143 (1995))
- das GADD45 Protein (Papathanasiou et al., Mol. Cell. Biol. 11, 1009 (1991), Smith et al., Science 253, 1376 (1994))- the GADD45 protein (Papathanasiou et al., Mol. Cell. Biol. 11, 1009 (1991), Smith et al., Science 253, 1376 (1994))
- das bak Protein (Farrow et al„ Nature 3Z4, 731 (1995), Chittenden et al., Nature 3Z4, 733 (1995), Kiefer et al., Nature 3Z4, 736 (1995)). Um eine schnelle intrazelluläre Inaktivierung dieser Zelizyklusinhibitoren zu verhindern, sind bevorzugt solche Gene zu verwenden, welche Mutationen für die Inaktivierungsstellen der exprimierten Proteine aufweisen, ohne daß diese hierdurch in ihrer Funktion beeinträchtigt werden.- the bak protein (Farrow et al “Nature 3Z4, 731 (1995), Chittenden et al., Nature 3Z4, 733 (1995), Kiefer et al., Nature 3Z4, 736 (1995)). In order to prevent a rapid intracellular inactivation of these cell cycle inhibitors, it is preferable to use genes which have mutations for the inactivation sites of the expressed proteins without their function being impaired thereby.
Das Retinoblastomprotein (pRb/p110) und die verwandten p107 und p130 Proteine werden durch Phosphorylierung inaktiviert. Bevorzugt wird somit eine pRb/p110 -, p107 - oder p130 cDNA-Sequenz verwendet, die derart mutiert ist, daß die Phosphorylierungsstellen des kodierten Proteins gegen nicht phosphorylierbare Aminosäuren ausgetauscht sind.The retinoblastoma protein (pRb / p110) and the related p107 and p130 proteins are inactivated by phosphorylation. A pRb / p110 -, p107 - or p130 cDNA sequence is thus preferably used which is mutated in such a way that the phosphorylation sites of the encoded protein are exchanged for non-phosphorylatable amino acids.
Entsprechend Hamel et al. (Mol. Cell Biol. 12, 3431 (1992)wird die cDNA-Sequenz für das Retinoblastomprotein (p110) durch Austausch der Aminosäuren in den Positionen 246, 350, 601, 605, 780, 786, 787, 800 und 804 nicht mehr phosphorylierbar, seine Bindungsaktivität mit dem großen T-Antigen wird jedoch nicht beeinträchtigt. Beispielsweise werden die Aminosäuren Thr-246, Ser-601 , Ser-605, Ser-780, Ser-786, Ser-787 und Ser-800 mit Ala, die Aminosäure Thr-350 mit Arg und die Aminosäure Ser-804 mit Glu ausgetauscht.According to Hamel et al. (Mol. Cell Biol. 12, 3431 (1992), the cDNA sequence for the retinoblastoma protein (p110) can no longer be phosphorylated by replacing the amino acids in positions 246, 350, 601, 605, 780, 786, 787, 800 and 804 However, its binding activity with the large T antigen is not impaired, for example the amino acids Thr-246, Ser-601, Ser-605, Ser-780, Ser-786, Ser-787 and Ser-800 with Ala, the amino acid Thr-350 exchanged with Arg and the amino acid Ser-804 with Glu.
In analoger Weise wird die DNA-Sequenz für das p107 Protein oder das p130 Protein mutiert.The DNA sequence for the p107 protein or the p130 protein is mutated in an analogous manner.
Das Protein p53 wird in der Zelle inaktiviert entweder durch Bindung an spezielle Proteine, wie beispielsweise MDM2 oder durch Oligomeri- sierung des p53 über das dephosphorylierte C-terminale Serin 392 (Schikawa et al., Leukemia und Lymphoma H, 21 (1993) und Brown, Annais of Oncology 4, 623 (1993)). Bevorzugt wird somit eine DNA- Sequenz für ein p53 Protein verwendet, welches C-terminal verkürzt ist um das Serin 392.The protein p53 is inactivated in the cell either by binding to special proteins such as MDM2 or by oligomerizing the p53 via the dephosphorylated C-terminal serine 392 (Schikawa et al., Leukemia and Lymphoma H, 21 (1993) and Brown , Annais of Oncology 4, 623 (1993)). A DNA sequence is therefore preferably used for a p53 protein which is shortened at the C-terminal by the serine 392.
zytostatische oder zvtotoxische Proteinecytostatic or zvtotoxic proteins
Als Wirksubstanz ist des weiteren eine DNA-Sequenz zu verstehen, die ein zytostatisches oder zytotoxisches Protein exprimiert. Zu derartigen Proteinen zählen beispielsweiseThe active substance is also to be understood as a DNA sequence that expresses a cytostatic or cytotoxic protein. Such proteins include, for example
- Perforin (Lin et al., Immunol. Today Q, 194 (1995))- Perforin (Lin et al., Immunol. Today Q, 194 (1995))
- Granzym (Smyth et al., Immunol. Today 15, 202 (1995))Granzyme (Smyth et al., Immunol. Today 15, 202 (1995))
- TNF (Porter, TibTech 2, 158 (1991), Sidhu et al., Pharmac. Ther. 5Z, 79 (1993)), im speziellenTNF (Porter, TibTech 2, 158 (1991), Sidhu et al., Pharmac. Ther. 5Z, 79 (1993)), in particular
* TNFα (Beutler et al., Nature 322, 584 (1986), Kriegler et al., Cell 53, 45 (1988) * TNFα (Beutler et al., Nature 322, 584 (1986), Kriegler et al., Cell 53, 45 (1988)
* TNFß (Gray et al., Nature 312, 721 (1984), Li et al., J. Immunol. 138. 4496 (1987), Aggarwal et al., J. Biol. Chem. 252, 2334 (1985) * TNFß (Gray et al., Nature 312, 721 (1984), Li et al., J. Immunol. 138, 4496 (1987), Aggarwal et al., J. Biol. Chem. 252, 2334 (1985)
c) Enzymec) enzymes
Als Wirksubstanz ist jedoch auch die DNA-Sequenz für ein Enzym zu verstehen, welches eine inaktive Vorstufe eines Zytostatikums in ein Zytostatikum umwandelt.However, the active substance is also to be understood as the DNA sequence for an enzyme which converts an inactive precursor of a cytostatic into a cytostatic.
Derartige Enzyme, welche inaktive Vorsubstanzen (Prodrugs) in aktive Zytostatika (Drugs) spalten und die jeweils zugehörigen Prodrugs und Drugs sind bereits von Deonarain et al. (Br. J. Cancer Z2, 786 (1994), von Müllen, Pharmac. Ther. 53, 199 (1994) und Harris et al., Gene Ther. 1, 170 (1994)) übersichtlich beschrieben worden.Such enzymes, which cleave inactive substances (prodrugs) into active cytostatics (drugs) and the associated prodrugs and drugs have already been described by Deonarain et al. (Br. J. Cancer Z2, 786 (1994), von Müllen, Pharmac. Ther. 53, 199 (1994) and Harris et al., Gene Ther. 1, 170 (1994)).
Beispielsweise ist die DNA-Sequenz eines der folgenden Enzyme zu verwenden:For example, the DNA sequence of one of the following enzymes should be used:
- Herpes Simplex Virus Thymidinkinase- Herpes simplex virus thymidine kinase
(Garapin et al., PNAS USA ZS, 3755 (1979), Vile et al., Cancer Res. 53, 3860 (1993), Wagner et al., PNAS USA ZS, 1441 (1981), Moelten et al., Cancer Res. 45, 5276 (1986), J. Natl. Cancer Inst. 32, 297 (1990))(Garapin et al., PNAS USA ZS, 3755 (1979), Vile et al., Cancer Res. 53, 3860 (1993), Wagner et al., PNAS USA ZS, 1441 (1981), Moelten et al., Cancer Res. 45, 5276 (1986), J. Natl. Cancer Inst. 32, 297 (1990))
- Varizella Zoster Virus Thymidinkinase- Varizella zoster virus thymidine kinase
(Huber et al., PNAS USA SS, 8039 (1991), Snoeck, Int. J. Antimicrob. Agents 4 211 (1994))(Huber et al., PNAS USA SS, 8039 (1991), Snoeck, Int. J. Antimicrob. Agents 4 211 (1994))
- bakterielle Nitroreduktase (Michael et al., FEMS Microbiol. Letters 124, 195 (1994), Bryant et al., J.- bacterial nitroreductase (Michael et al., FEMS Microbiol. Letters 124, 195 (1994), Bryant et al., J.
Biol. Chem. 265, 4126 (1991), Watanabe et al., Nucleic Acids Res. 13,Biol. Chem. 265, 4126 (1991), Watanabe et al., Nucleic Acids Res. 13,
1059 (1990)) bakterielle ß-Glucuronidase1059 (1990)) bacterial β-glucuronidase
(Jefferson et al., PNAS USA 33, 8447 (1986) pflanzliche ß-Glucuroniase aus Seeale cereale(Jefferson et al., PNAS USA 33, 8447 (1986) vegetable β-glucuroniase from Seeale cereale
(Schulz et al., Phytochemistry 26, 933 (1987)) humane ß-Glucuroniase(Schulz et al., Phytochemistry 26, 933 (1987)) human β-glucuroniase
(Bosslet et al., Br. J. Cancer 55, 234 (1992), Oshima et al., PNAS USA(Bosslet et al., Br. J. Cancer 55, 234 (1992), Oshima et al., PNAS USA
64, 685 (1987)) humane Carboxy peptidase (CB) z.B.64, 685 (1987)) human carboxy peptidase (CB) e.g.
* CB-A der Mastzelle * CB-A of the mast cell
(Reynolds et al., J. Clin. Invest. 32, 273 (1992))(Reynolds et al., J. Clin. Invest. 32, 273 (1992))
* CB-B des Pankreas * CB-B of the pancreas
(Yamamoto et al., J. Biol. Chem. 23Z, 2575 (1992), Catasus et al., J.(Yamamoto et al., J. Biol. Chem. 23Z, 2575 (1992), Catasus et al., J.
Biol. Chem. 2Z2, 6651 (1995)) bakterielle Carboxy peptidaseBiol. Chem. 2Z2, 6651 (1995)) bacterial carboxy peptidase
(Hamilton et al., J. Bacteriol. 1Z4, 1626 (1992), Osterman et al., J. Protein Chem. 11, 561 (1992)) bakterielle ß-Laktamase(Hamilton et al., J. Bacteriol. 1Z4, 1626 (1992), Osterman et al., J. Protein Chem. 11, 561 (1992)) bacterial β-lactamase
(Rodrigues et al., Cancer Res. 55, 63 (1995), Hussain et al., J. Bacteriol. 164. 223 (1985), Coque et al., Embo J. 12, 631 (1993) bakterielle Cytosine deaminase(Rodrigues et al., Cancer Res. 55, 63 (1995), Hussain et al., J. Bacteriol. 164, 223 (1985), Coque et al., Embo J. 12, 631 (1993) bacterial cytosine deaminase
(Müllen et al., PNAS USA 62, 33 (1992), Austin et al., Mol. Pharmac. 43, 380 (1993), Danielson et al., Mol. Microbiol. 6, 1335 (1992) humane Catalase bzw. Peroxidase (Ezurum et al., Nucl. Acids Res. 21, 1607 (1993)) Phosphatase, im besonderen(Müllen et al., PNAS USA 62, 33 (1992), Austin et al., Mol. Pharmac. 43, 380 (1993), Danielson et al., Mol. Microbiol. 6, 1335 (1992) human catalase and Peroxidase (Ezurum et al., Nucl. Acids Res. 21, 1607 (1993)) phosphatase, in particular
* humane alkalische Phosphatase* human alkaline phosphatase
(Gum et al., Cancer Res. 52, 1085 (1990))(Gum et al., Cancer Res. 52, 1085 (1990))
* humane saure Prostataphosphatase * human acid prostate phosphatase
(Sharieff et al., Am. J. Hum. Gen. 42, 412 (1991), Song et al., Gene 129. 291 (1993), Tailor et al., Nucl. Acids Res. l , 4928 (1990))(Sharieff et al., Am. J. Hum. Gen. 42, 412 (1991), Song et al., Gene 129, 291 (1993), Tailor et al., Nucl. Acids Res. L, 4928 (1990) )
* Typ 5 saure Phosphatase (Gene 132, 201 (1993)) * Type 5 acid phosphatase (Gene 132, 201 (1993))
Oxidase, im besonderen * humane LysyloxidaseOxidase, especially * human lysyl oxidase
(Kimi et al., J. Biol. Chem.2Z2, 7176 (1995))(Kimi et al., J. Biol. Chem. 2Z2, 7176 (1995))
* humane saure D-aminooxidase* human acidic D-aminooxidase
(Fukui et al., J. Biol. Chem. 26Z, 18631 (1992)) - Peroxidase, im besonderen(Fukui et al., J. Biol. Chem. 26Z, 18631 (1992)) - peroxidase, in particular
* humane Gluthation Peroxidase * Human glutation peroxidase
(Chada et al., Genomics 6, 268 (1990), Ishida et al., Nucl. Acids Res. 1^, 10051 (1987))(Chada et al., Genomics 6, 268 (1990), Ishida et al., Nucl. Acids Res. 1 ^, 10051 (1987))
* humane Eosinophilen Peroxidase * human eosinophil peroxidase
(Ten et al., J. Exp. Med. 13 ., 1757 (1989), Sahamaki et al., J. Biol. Chem. 264, 16828 (1989))(Ten et al., J. Exp. Med. 13, 1757 (1989), Sahamaki et al., J. Biol. Chem. 264, 16828 (1989))
* humane Schilddrüsen Peroxidase (Kimura, PNAS USA 64, 5555 (1987)). * Human thyroid peroxidase (Kimura, PNAS USA 64, 5555 (1987)).
Zur Erleichterung der Sekretion der aufgeführten Enzyme kann die jeweils in der DNA-Sequenz enthaltende homologe Signalsequenz ersetzt werden durch eine heterologe, die extrazelluläre Ausschleusung verbessernde Signalsequenz.To facilitate the secretion of the enzymes listed, the homologous signal sequence contained in the DNA sequence can be replaced by a heterologous signal sequence which improves the extracellular discharge.
So kann beispielsweise die Signalsequenz der ß-Glucuronidase (DNA Position < 27 bis 93; Oshima et al., PNAS 64, 685 (1987)) ersetzt werden durch die Signalfrequenz für das humane Immunglobulin (DNA Position < 63 bis > 107; Riechmann et al., Nature 332, 323 (1988).For example, the signal sequence of the β-glucuronidase (DNA position <27 to 93; Oshima et al., PNAS 64, 685 (1987)) can be replaced by the signal frequency for the human immunoglobulin (DNA position <63 to> 107; Riechmann et al., Nature 332, 323 (1988).
Des weiteren sind bevorzugt DNAs solcher Enzyme zu wählen, welche durch Punktmutationen in einem geringeren Maße in Lysosomen gespei¬ chert werden. Derartige Punktmutationen wurden beispielsweise für die ß- Glucuronidase beschrieben (Shipley et al., J. Biol. Chem. 268. 12193 (1993)).Furthermore, preference is given to choosing DNAs of those enzymes which are stored to a lesser extent in lysosomes by point mutations. Such point mutations have been described, for example, for β-glucuronidase (Shipley et al., J. Biol. Chem. 268, 12193 (1993)).
4.3. Kombination von gleichen oder unterschiedlichen Wirksubstanzen für glatte Muskelzellen4.3. Combination of the same or different active substances for smooth muscle cells
Gegenstand der Erfindung ist des weiteren ein Wirkstoff, in welchem eine Kombination der DNA-Sequenzen von mehreren gleichen Wirksubstanzen (A,A) oder unterschiedlichen Wirksubstanzen (A,B) vorliegt. Zur Expression von zwei DNA-Sequenzen ist vorzugsweise die cDNA einer "intemal ribosome entry site" (IRES) als regulatorisches Element zwischengeschaltet. Derartige IRES wurden von Mountford und Smith (TIG 11, 179 (1995), Kaufman et al., Nucl. Acids Res. 12, 4485 (1991), Morgan et al., Nucl. Acids Res. 22, 1293 (1992) und Dirks et al., Gene 122, 247 (1993), Pelletier und Sonenberg, Nature 334. 320 (1988), Sugitomo et al., BioTechn. 12, 694 (1994) beschrieben.The invention further relates to an active substance in which a combination of the DNA sequences of several identical active substances (A, A) or different active substances (A, B) is present. For expression of two DNA sequences, the cDNA of an "internal ribosome entry site" (IRES) is preferably interposed as a regulatory element. Such IRES have been described by Mountford and Smith (TIG 11, 179 (1995), Kaufman et al., Nucl. Acids Res. 12, 4485 (1991), Morgan et al., Nucl. Acids Res. 22, 1293 (1992) and Dirks et al., Gene 122, 247 (1993), Pelletier and Sonenberg, Nature 334, 320 (1988), Sugitomo et al., BioTechn. 12, 694 (1994).
So kann die cDNA der IRES-Sequenz des Poliovirus (Position < 140 bis > 630 des 5' UTR (Pelletier und Sonenberg, Nature 334, 320 (1988)) zur Verknüpfung der DNA der antithrombotischen Substanz A (am 3' Ende) und der DNA der antithrombotischen Substanz B (am 5' Terminus) verwendet werden.Thus, the cDNA of the IRES sequence of the poliovirus (position <140 to> 630 of the 5 'UTR (Pelletier and Sonenberg, Nature 334, 320 (1988)) for linking the DNA of the antithrombotic substance A (at the 3' end) and the DNA of the antithrombotic substance B (at the 5 'terminus) can be used.
Aktivator¬ zellzyklus¬ Wirksubstanz intemal Wirksubstanz sequenz reguliertes (Zellzyklus- ribosome (Zellzyklus- (UAS) Promotor¬ inhibitor) entry inhibitor) rnodul A site A oder BActivator cell cycle active substance intally active substance sequence regulated (cell cycle ribosome (cell cycle (UAS) promoter inhibitor) entry inhibitor) A site A or B module
Ein derartiger Wirkstoff weist je nach Kombination additive oder synergistische Wirkung im Sinne der Erfindung auf.Depending on the combination, such an active ingredient has an additive or synergistic effect in the sense of the invention.
Im Gefolge des intimalen Wachstums der glatten Muskelzellen wie auch durch deren Apoptose oder Nekrose als Ergebnis der Einwirkung des Zellzyklusin- hibitors kann das Gerinnungssystem aktiviert werden und können Thrombosen auftreten. Derartige Thrombosen sind durch eine prophylaktische Gabe eines Gerinnungshemmers (Aspirin, Heparin oder eines anderen Antithrombotikums) zu verhindern. Die Verabreichung des Gerinnungshemmers erfolgt systemisch, d.h. oral oder parenteral.As a result of the intimal growth of smooth muscle cells as well as their apoptosis or necrosis as a result of the action of the cell cycle inhibitor, the coagulation system can be activated and thromboses can occur. Such thrombosis can be prevented by prophylactic administration of an anticoagulant (aspirin, heparin or another antithrombotic). The anticoagulant is administered systemically, i.e. orally or parenterally.
Häufig verhindern jedoch die Nebenwirkungen des Gerinnungshemmers eine ausreichende Konzentration am Ort des intimalen Wachstums der glatten Muskelzelle.However, the side effects of the anticoagulant often prevent sufficient concentration at the site of the intimal growth of the smooth muscle cell.
Somit ist die Prophylaxe der Thrombose durch derartige Gerinnungshemmer unsicher (Pukac, Am. J. Pathol. 132, 1501 (1991)). Es ist nun ein weiterer Gegenstand der Erfindung, daß der im Sinne der Erfindung beanspruchte Wirkstoff zusätzlich zu einer Wirksubstanz, die einen Zelizyklusinhibitor darstellt, als weiteres Element die DNA-Sequenz für eine Wirksubstanz, die einen Gerinnungshemmer darstellt, enthält.Thus the prophylaxis of thrombosis by such anticoagulants is uncertain (Pukac, Am. J. Pathol. 132, 1501 (1991)). It is a further object of the invention that the active substance claimed in the sense of the invention contains, in addition to an active substance which is a cell cycle inhibitor, the DNA sequence for an active substance which is an anticoagulant as a further element.
Aktivator¬ zellzyklus¬ Wirksubstanz internal Wirksubstanz sequenz reguliertes (Zelizyklus¬ ribosome Gerinnungs¬ (UAS) Promotor¬ inhibitor) entry hemmer) rnodul siteActivator cell cycle active substance internal active substance sequence regulated (cell cycle ribosome coagulation (UAS) promoter inhibitor) entry inhibitor site
Die Expression des Gerinnungshemmers wird in gleicher Weise wie die Expression des Zellzyklusinhibitors durch die Aktivatorsequenz und das zellzykiusregulierte Repressormodul gesteuert. Die gleichzeitige Expression sowohl des Zellzyklusinhibitors als auch des Gerinnungshemmers wird vorzugsweise durch ein "internal ribosome entry site" (IRES) Genelement geregelt.The expression of the anticoagulant is controlled in the same way as the expression of the cell cycle inhibitor by the activator sequence and the cell cycle-regulated repressor module. The simultaneous expression of both the cell cycle inhibitor and the anticoagulant is preferably regulated by an "internal ribosome entry site" (IRES) gene element.
Als Gerinnungshemmer sind Gene für beispielsweise Plasminogenaktivatoren (PA), so der Gewebe-PA (tPA) oder der Urokinase-ähnliche PA (uPA), Protein C, Antithrombin-Ill, Tissue Factor Pathway Inhibitor oder Hirudin einzusetzen. Die DNA-Sequenzen für diese Gerinnungshemmer wurden wie folgt beschrieben:Genes for, for example, plasminogen activators (PA), such as tissue PA (tPA) or urokinase-like PA (uPA), protein C, antithrombin III, tissue factor pathway inhibitor or hirudin, are used as anticoagulants. The DNA sequences for these anticoagulants were described as follows:
- Tissue Plasminogen Activator (tPA)- Tissue plasminogen activator (tPA)
(Sasaki et al., Nucl. Acids Res. 16, 5695 (1988), Pennica et al., Nature 221, 214 (1983), Wei et al., DNA 4, 76 (1985), Harris et al., Mol. Biol. Med. 3, 279 (1986))(Sasaki et al., Nucl. Acids Res. 16, 5695 (1988), Pennica et al., Nature 221, 214 (1983), Wei et al., DNA 4, 76 (1985), Harris et al., Mol Biol. Med. 3: 279 (1986))
- Urokinase-type Plasminogen Activator (uPA)- Urokinase-type plasminogen activator (uPA)
(Miyake et al., J. Biochem. 124, 643 (1988), Nelles et al., J. Biol. Chem. 262, 5682 (1987))(Miyake et al., J. Biochem. 124, 643 (1988), Nelles et al., J. Biol. Chem. 262, 5682 (1987))
- Hybride von tPA und uPA- Hybrid of tPA and uPA
(Kalyan et al., Gene 36, 205 (1988), Devries et al., Biochem. 2Z, 2565 (1988))(Kalyan et al., Gene 36, 205 (1988), Devries et al., Biochem. 2Z, 2565 (1988))
- Protein CProtein C
(Foster et al., PNAS 62, 4673 (1985))(Foster et al., PNAS 62, 4673 (1985))
- Hirudin (Maerki et al., Semin. Thromb. Hemostas. H, 88 (1991), De Taxis du Poet et al., Blood Coag. Fibrin. 2, 113 (1991), Harvey et al., PNAS USA 63, 1084- Hirudin (Maerki et al., Semin. Thromb. Hemostas. H, 88 (1991), De Taxis du Poet et al., Blood Coag. Fibrin. 2, 113 (1991), Harvey et al., PNAS USA 63, 1084
(1986), Sachhieri et al., EP 0324 712 B1 , EP 0142 860 B1)(1986), Sachhieri et al., EP 0324 712 B1, EP 0142 860 B1)
Serin Proteinase Inhibitoren (Serpine), wie beispielsweiseSerine proteinase inhibitors (Serpine), such as
* C-1S-lnhibitor (Bock et al., Biochem. 25, 4292 (1986), Davis et al., PNAS USA 33, 3161 (1986), Que, BBRC 13Z, 620 (1986), Rauth et al., Proteine Sequences and Data Analysis 1 , 251 (1988), Carter et al., Eur. J. Biochem. 1Z3, 163 (1988), Tosi et al., Gene 42, 265 (1986), Carter et al., Eur. J. Biochem. 12Z, 301 /1991), Eidering et al., J. Biol. Chem. 26Z, 7013 (1993))* C-1S inhibitor (Bock et al., Biochem. 25, 4292 (1986), Davis et al., PNAS USA 33, 3161 (1986), Que, BBRC 13Z, 620 (1986), Rauth et al., Protein Sequences and Data Analysis 1, 251 (1988), Carter et al., Eur. J. Biochem. 1Z3, 163 (1988), Tosi et al., Gene 42, 265 (1986), Carter et al., Eur. J. Biochem. 12Z, 301/1991), Eidering et al., J. Biol. Chem. 26Z, 7013 (1993))
*α1-Antitrypsin (Tosi et al., Gene 42, 265 (1986), Graham et al., Hum. Genetics 35, 381 (1990), Hafeez et al., J. Clin. Invest. 32, 1214 (1992), Tikunova et al., Bioorganicheskaja Khimia 1Z, 1694 (1991), Kay et al., Human Gene Ther. 3, 641 (1992), Lemarchand et al., Molekulaiaruaia Biologica 2Z, 1014 (1993), Lambach et al., Human Mol. Gen. 2, 1001 (1993))* α1-Antitrypsin (Tosi et al., Gene 42, 265 (1986), Graham et al., Hum. Genetics 35, 381 (1990), Hafeez et al., J. Clin. Invest. 32, 1214 (1992) , Tikunova et al., Bioorganicheskaja Khimia 1Z, 1694 (1991), Kay et al., Human Gene Ther. 3, 641 (1992), Lemarchand et al., Molekulaiaruaia Biologica 2Z, 1014 (1993), Lambach et al., Human Mol. Gen. 2, 1001 (1993))
*Antithrombin III (Stackhouse et al., J. Biol. Chem. 253, 703 (1983), Olds et al., Biochem. 32, 4216 (1993), Laue et al., Nucl. Acids Res. 22, 3556 (1994))* Antithrombin III (Stackhouse et al., J. Biol. Chem. 253, 703 (1983), Olds et al., Biochem. 32, 4216 (1993), Laue et al., Nucl. Acids Res. 22, 3556 ( 1994))
Tissue Factor Pathway Inhibitor (TFPI)Tissue Factor Pathway Inhibitor (TFPI)
(Enjyoji et al., Genomics 1Z, 423 (1993), Wun et al., J. Biol. Chem. 263,(Enjyoji et al., Genomics 1Z, 423 (1993), Wun et al., J. Biol. Chem. 263,
6001 (1988), Girard et al., Thromb. Res. 55, 37 (1989))6001 (1988), Girard et al., Thromb. Res. 55, 37 (1989))
4.4. Auswahl des Liganden für glatte Muskelzellen (siehe S. 9, Zeilen 5-21 supra)4.4. Selection of the ligand for smooth muscle cells (see p. 9, lines 5-21 supra)
Als Ligand in kolloidalen Dispersionen, beispielsweise Polylysin-Ligand- Konjugaten, werden Substanzen bevorzugt, welche an die Oberfläche von glatten Muskelzellen binden. Hierzu gehören Antikörper oder Anti¬ körperfragmente, gerichtet gegen Membranstrukturen von glatten Muskel¬ zellen, wie beispielsweiseSubstances which bind to the surface of smooth muscle cells are preferred as ligands in colloidal dispersions, for example polylysine-ligand conjugates. These include antibodies or antibody fragments, directed against membrane structures of smooth muscle cells, such as, for example
- der Antikörper 10F3- the antibody 10F3
(Printseva et al., Exp. Cell Res. 132, 85 (1987),American J. Path. 134, 305 (1989)) oder - Antikörper gegen Actin(Printseva et al., Exp. Cell Res. 132, 85 (1987), American J. Path. 134, 305 (1989)) or - Antibodies against actin
(Desmonliere et al., Comptes Reudus des Seances de la Soc. de Biol et de ses Filiales 132, 391 (1988)) oder(Desmonliere et al., Comptes Reudus des Seances de la Soc. De Biol et de ses Filiales 132, 391 (1988)) or
- Antikörper gegen Angiotensin Il-Rezeptoren (Butcher et al., BBRA 126, 1280 (1993)) oder- Antibodies against angiotensin II receptors (Butcher et al., BBRA 126, 1280 (1993)) or
- Antikörper gegen Rezeptoren für Wachstumsfaktoren- Antibodies against receptors for growth factors
(Übersichten bei Mendelsohn, Prog. All. 45, 147 (1988), Sato et al., J. Nat. Canc. Inst. 61, 1600 (1989), Hynes et al., BBA 1198. 165 (1994))(Reviews by Mendelsohn, Prog. All. 45, 147 (1988), Sato et al., J. Nat. Canc. Inst. 61, 1600 (1989), Hynes et al., BBA 1198. 165 (1994))
oder Antikörper gerichtet beispielsweise gegenor antibodies directed against, for example
* EGF-Rezeptoren * EGF receptors
(Fan et al., Cancer Res. 56, 4322 (1993), Bender et al., Cancer Res. 52, 121 (1992), Aboud-Pirak et al., J. Nat. Cancer Inst. 62, 1605 (1988), Sato et al., Mol. Biol. Med. 1, 511 (1983), Kawamoto et al., PNAS 62, 1337 (1983))(Fan et al., Cancer Res. 56, 4322 (1993), Bender et al., Cancer Res. 52, 121 (1992), Aboud-Pirak et al., J. Nat. Cancer Inst. 62, 1605 (1988 ), Sato et al., Mol. Biol. Med. 1, 511 (1983), Kawamoto et al., PNAS 62, 1337 (1983))
* oder gegen PDGF-Rezeptoren (Yu et al., J. Biol. Chem. 262, 10668 (1994), Kelly et al., J. Biol. Chem. 266, 8987 (1991), Bowen-Pope et al., J. Biol. Chem. 25Z, 5161 (1982)) * or against PDGF receptors (Yu et al., J. Biol. Chem. 262, 10668 (1994), Kelly et al., J. Biol. Chem. 266, 8987 (1991), Bowen-Pope et al., J. Biol. Chem. 25Z, 5161 (1982))
* oder gegen FGF-Rezeptoren (Vanhalteswaran et al., J. Cell Biol. 115. 418 (1991), Zhan et al., J. Biol. Chem. 262, 20221 (1994)) * or against FGF receptors (Vanhalteswaran et al., J. Cell Biol. 115, 418 (1991), Zhan et al., J. Biol. Chem. 262, 20221 (1994))
* oder Antikörper gegen Endothelin A-Rezeptoren. * or antibodies against endothelin A receptors.
Die murinen monoklonalen Antikörper sind bevorzugt in humanisierter Form einzusetzen. Die Humanisierung erfolgt in der von Winter et al. (Nature 349. 293 (1991) und Hoogenbooms et al. (Rev. Tr. Transfus. Hemobiol. 35, 19 (1993) dargestellten Weise. Antikörperfragmente werden entsprechend dem Stand der Technik hergestellt, beispielsweise in der von Winter et al., Nature 349. 293 (1991), Hoogenboom et al., Rev. Tr. Transfus. Hemobiol. 36, 19 (1993), Girol, Mol. Immunol. 23, 1379 (1991) oder Huston et al., Int. Rev. Immunol. 12, 195 (1993) beschriebenen Weise.The murine monoclonal antibodies are preferably used in humanized form. Humanization takes place in the method described by Winter et al. (Nature 349, 293 (1991) and Hoogenbooms et al. (Rev. Tr. Transfus. Hemobiol. 35, 19 (1993). Antibody fragments are prepared according to the prior art, for example in the manner described by Winter et al., Nature 349, 293 (1991), Hoogenboom et al., Rev. Tr. Transfus. Hemobiol. 36, 19 (1993), Girol, Mol. Immunol. 23, 1379 (1991) or Huston et al., Int. Rev. Immunol 12, 195 (1993).
Zu den Liganden gehören des weiteren alle Wirksubstanzen, welche an Membranstrukturen oder Membranrezeptoren auf glatten Muskelzellen binden (Übersicht bei Pusztai et al., J. Pathol. 162, 191 (1993), Harris, Current Opin. Biotechnol. 2, 260 (1991)). Beispielsweise gehören hierzu Wachstumsfaktoren oder deren Fragmente bzw. Teilsequenzen von ihnen, die an Rezeptoren exprimiert durch glatte Muskelzellen binden wie beispielsweiseThe ligands also include all active substances which bind to membrane structures or membrane receptors on smooth muscle cells (review by Pusztai et al., J. Pathol. 162, 191 (1993), Harris, Current Opin. Biotechnol. 2, 260 (1991)). For example, this includes growth factors or their fragments or partial sequences of them, which bind to receptors expressed by smooth muscle cells, for example
- PDGF- PDGF
(Westermark et al., Cancer Res. 51, 5087 (1991), Ponten et al., J. Invest. Dermatol. 122, 304 (1994)(Westermark et al., Cancer Res. 51, 5087 (1991), Ponten et al., J. Invest. Dermatol. 122, 304 (1994)
- EGF- EGF
(Modjtahedi et al., Int. J. Oncol. 4, 277 (1994), Carpenter et al., J. Biol. Chem. 265, 7709 (1990))(Modjtahedi et al., Int. J. Oncol. 4, 277 (1994), Carpenter et al., J. Biol. Chem. 265, 7709 (1990))
- TGFß- TGFß
(Segarini, BBA 1155. 269 (1993))(Segarini, BBA 1155.269 (1993))
- TGFα- TGFα
(Salomon et al., Cancer Cells 2, 389 (1990)(Salomon et al., Cancer Cells 2, 389 (1990)
- FGF- FGF
(Burgess et al., Annu. Rev. Biochem. 53, 575 (1989)(Burgess et al., Annu. Rev. Biochem. 53, 575 (1989)
- Endothelin A- endothelin A
(Oreiily et al., J. Cardiovasc. Pharm. 22, 18 (1993)).(Oreiily et al., J. Cardiovasc. Pharm. 22, 18 (1993)).
4.5. Herstellung des Wirkstoffes für glatte Muskelzellen4.5. Production of the active ingredient for smooth muscle cells
Die Herstellung des erfindungsgemäßen Wirkstoffes wird anhand folgender Beispiele näher beschrieben:The preparation of the active ingredient according to the invention is described in more detail using the following examples:
a) Konstruktion des Chimären Promotors Mvooenin-PromotorCDE-CHR-lnra) Construction of the chimeric promoter Mvooenin promoter CDE-CHR no
Der humane Myogenin-Promotor (Pos. < -210 bis > +54, der von Salmin et al., J. Cell Biol. 115. 905 (1991) publizierten DNA-Sequenz) wird an seinem 3' Ende mit dem 5'-Terminus des CDE-CHR-Inr Moduls des humanen cdc25C-Gens (Pos. < -20 bis > +121 , der von Lucibello et al., EMBO J. 14, 132 (1995) publizierten Sequenz) verknüpft (siehe Fig. 6). Die Verknüpfung erfolgt mit Hilfe von dem Fachmann bekannten und käuflichen Enzymen. Außerdem werden verschiedene Fragmente der Myogenin-Promotorse- quenz verwendet (siehe Fig. 6). So wird die TATA-Box enthaltende DNA- Sequenz des Myogenin-Promotors verwendet. Jedoch kann gleichermaßen auch die Promotorsequenz Position < -210 bis > -40 zur Anwendung kommen (siehe Fig. 6).The human myogenin promoter (pos. <-210 to> +54, the DNA sequence published by Salmin et al., J. Cell Biol. 115. 905 (1991)) is connected at its 3 'end with the 5'- Term of the CDE-CHR-Inr module of the human cdc25C gene (pos. <-20 to> +121, linked to the sequence published by Lucibello et al., EMBO J. 14, 132 (1995)) (see FIG. 6) . The linkage takes place with the aid of enzymes known and commercially available to the person skilled in the art. Various fragments of the myogenin promoter sequence are also used (see FIG. 6). The DNA sequence of the myogenin promoter containing the TATA box is used. However, the promoter sequence position <-210 to> -40 can also be used come (see Fig. 6).
b^ Konstruktion eines Plasmids enthaltend den zentralen Bestandteil des Wirkstoffesb ^ Construction of a plasmid containing the central component of the active ingredient
Die so hergestellte chimäre Myogenin-Promotormodul-Transkriptionskon- trolleinheit wird an ihrem 3' Ende mit dem 5'-Terminus einer DNA, die den kompletten kodierenden Bereich der humanen ß-Glucuronidase (DNA Pos. < 27- > 1982, der von Oshima et al., PNAS USA 64, 684 (1987) publizierten Sequenz) enthält, verknüpft (siehe Fig. 6).The chimeric myogenin promoter module transcription control unit thus produced is labeled at its 3 'end with the 5' terminus of a DNA which encompasses the complete coding region of the human β-glucuronidase (DNA pos. <27-> 1982, which was described by Oshima et al., PNAS USA 64, 684 (1987) published sequence), linked (see Fig. 6).
Diese DNA enthält auch die für eine Sekretion notwendige Signalsequenz (22 N-terminale Aminosäuren). Zur Erleichterung der zellulären Ausschleusung ist diese Signalsequenz bevorzugt auszutauschen gegen die Signalsequenz des Immunglobulins (Position < 63 bis > 107; Riechmann et al., Nature 332, 323 (1988) (Fig. 7). Die so hergestellten Transkriptions¬ kontrolleinheiten und die DNA für die humane ß-Glucuronidase werden in pUC18/19 oder Bluescript-abgeleiteten Plasmidvektoren einkloniert, die direkt oder in kolloidalen Dispersionssystemen für eine in vivo Applikation genutzt werden können. Alternativ können die Chimären Gene in virale Vektoren oder andere geeignete Vektoren transferiert und injuziert werden.This DNA also contains the signal sequence necessary for secretion (22 N-terminal amino acids). In order to facilitate cellular removal, this signal sequence should preferably be exchanged for the signal sequence of the immunoglobulin (position <63 to> 107; Riechmann et al., Nature 332, 323 (1988) (FIG. 7). The transcription control units thus produced and the DNA for human β-glucuronidase, clones are cloned into pUC18 / 19 or Bluescript-derived plasmid vectors which can be used directly or in colloidal dispersion systems for in vivo application.Alternatively, the chimeric genes can be transferred into viral vectors or other suitable vectors and injected.
c^ Konstruktion eines Plasmids enthaltend die Gene für ß-Glucuronidase wie auch für Tissue Plasminogen Activatorc ^ Construction of a plasmid containing the genes for β-glucuronidase as well as for tissue plasminogen activator
Das wie unter 2) hergestellte Myogenin-Repressormodul-ß-Glucuronidase- Element wird an seinem 3' Ende mit dem 5'-Terminus der cDNA der "internal ribosome entry site" des Poliovirus (Position < 140 bis > 630 des 5' UTR Elementes, Pelletier und Sonenberg, Nature 334, 320 (1988)) verknüpft. An dessen 3' Ende wird wiederum der 5'-Terminus der DNA des Tissue Plasminogen Activator (Position < 85 bis > 1753, Pennica et al., Nature 301. 214 (1983)) verknüpft (Fig. 7). Anschließend wird das gesamte Konstrukt in pUC17/19 oder Bluescript-abgeleiteten Plasmidvektoren einkloniert, die direkt oder in kolloidalen Dispersionssystemen für einen in vivo Transfer genutzt werden können. Alternativ können die Chimären Gene in virale Vektoren oder andere geeignete Vektoren transferiert werden (Fig. 8). 5. Wirkstoff zur Inhibition der GerinnungThe myogenin repressor module-β-glucuronidase element produced as under 2) becomes at its 3 'end with the 5' terminus of the cDNA the "internal ribosome entry site" of the poliovirus (position <140 to> 630 of the 5 'UTR element , Pelletier and Sonenberg, Nature 334, 320 (1988)). At its 3 'end the 5' terminus of the DNA of the tissue plasminogen activator (position <85 to> 1753, Pennica et al., Nature 301, 214 (1983)) is again linked (FIG. 7). The entire construct is then cloned into pUC17 / 19 or Bluescript-derived plasmid vectors, which can be used directly or in colloidal dispersion systems for in vivo transfer. Alternatively, the chimeric genes can be transferred to viral vectors or other suitable vectors (Fig. 8). 5. Active ingredient for inhibiting coagulation
5.1. Auswahl der Aktivatorsequenz zur Inhibition oder Gerinnung5.1. Selection of the activator sequence for inhibition or coagulation
Als Aktivatorsequenzen im Sinne der Erfindung sind bevorzugt genregulatorische Sequenzen bzw. Elemente aus Genen zu verwenden, welche in glatten Muskelzellen, in aktivierten Endothelzellen, in aktivierten Makrophagen oder in aktivierten Lymphozyten nachweisbare Proteine kodieren.As activator sequences in the sense of the invention, gene regulatory sequences or elements from genes are preferably used which encode detectable proteins in smooth muscle cells, in activated endothelial cells, in activated macrophages or in activated lymphocytes.
a) glatte Muskelzellena) smooth muscle cells
Beispiele für Aktivatorsequenzen für Gene in glatten Muskelzellen sind bereits unter 4.1. aufgeführt.Examples of activator sequences for genes in smooth muscle cells are already under 4.1. listed.
b^ aktivierte Endothelzellenb ^ activated endothelial cells
Beispiele für Proteine, welche besonders in aktivierten Endothelzellen gebildet werden, sind von Burrows et al. (Pharmac. Therp. 64, 155 (1994)) und Plate et al. (Brain Pathol. 4, 207 (1994)) beschrieben worden. Im besonderen zählen zu diesen in Endothelzellen verstärkt auftretenden Proteinen beispielsweise:Examples of proteins which are formed particularly 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 increasingly occur in endothelial cells, include:
- Hirn-spezifischer, endothelialer Glucose-1 -Transporter Endothelzellen des Hirns zeichnen sich durch eine sehr starke Expression dieses Transporters aus, um den transendothelialen Transport von D- Glucose in das Hirn zu bewerkstelligen (Gerhart et al., J. Neurosci. Res. 22, 464 (1989)). Die Promotorsequenz wurde von Murakami et al. (J. Biol. Chem. 267. 9300 (1992)) beschrieben.- Brain-specific, endothelial glucose-1 transporter Brain endothelial cells are characterized by a very strong expression of this transporter to accomplish the transendothelial transport of D-glucose into the brain (Gerhart et al., J. Neurosci. Res. 22, 464 (1989)). The promoter sequence was developed by Murakami et al. (J. Biol. Chem. 267, 9300 (1992)).
- Endoglin- Endoglin
Endoglin scheint ein nicht signalübertragender Rezeptor des TGFß zu sein (Gougos et al., J. Biol. Chem. 265, 8361 (1990), Cheifetz, J. Biol. Chem. 26Z, 19027 (1992), Moren et al., BBRC 132, 356 (1992)). In geringen Mengen kommt er auf normalem Endothel vor, ist jedoch verstärkt exprimiert auf proliferierendem Endothel (Westphal et al., J. Invest. Derm. 122, 27 (1993), Burrows et al., Pharmac. Ther. 64, 155 (1994)). Die Promotorsequenz wurde von Bellon et al. (Eur. J. Immunol. 23, 2340 (1993)) und Ge et al. (Gene 133. 201 (1994)) beschrieben.Endoglin appears to be a non-signal-transmitting receptor for TGFß (Gougos et al., J. Biol. Chem. 265, 8361 (1990), Cheifetz, J. Biol. Chem. 26Z, 19027 (1992), Moren et al., BBRC 132, 356 (1992)). It occurs in small amounts on normal endothelium, but is increasingly expressed on proliferating endothelium (Westphal et al., J. Invest. Derm. 122, 27 (1993), Burrows et al., Pharmac. Ther. 64, 155 (1994 )). The promoter sequence was developed by Bellon et al. (Eur. J. Immunol. 23, 2340 (1993)) and Ge et al. (Gene 133, 201 (1994)).
VEGF-RezeptorenVEGF receptors
Zwei Rezeptoren werden unterschieden (Plate et al., Int. J. Cancer 52, 520 (1994)):Two receptors are distinguished (Plate et al., Int. J. Cancer 52, 520 (1994)):
* VEGF-Rezeptor-1 (flt-1) * VEGF receptor-1 (flt-1)
(de Vries et al., Science 255, 989 (1992))(de Vries et al., Science 255, 989 (1992))
(enthält im zytoplasmischen Teil eine fms-ähnliche Tyrosinkinase) und der(contains an fms-like tyrosine kinase in the cytoplasmic part) and the
* VEGF-Rezeptor-2 (flk-1 , KDR) (Terman et al., BBRC 1SZ, 1579 (1992)) * VEGF receptor-2 (flk-1, KDR) (Terman et al., BBRC 1SZ, 1579 (1992))
(enthält im zytoplasmischen Teil eine Tyrosinkinase).(contains a tyrosine kinase in the cytoplasmic part).
Beide Rezeptoren sind fast ausschließlich auf endothelialen Zellen (Senger et al., Cancer Metast. Rev. 12, 303 (1993)) anzutreffen.Both receptors are found almost exclusively on endothelial cells (Senger et al., Cancer Metast. Rev. 12, 303 (1993)).
andere endothelspezifische Rezeptortyrosinkinasenother endothelium-specific receptor tyrosine kinases
* til-1 oder til-2 * til-1 or til-2
(Partanen et al., Mol. Cell Biol. 12, 1698 (1992), Schnürch and Risau, Development 119. 957 (1993), Dumont et al., Oncogene Z, 1471 (1992))(Partanen et al., Mol. Cell Biol. 12, 1698 (1992), Schnürch and Risau, Development 119, 957 (1993), Dumont et al., Oncogene Z, 1471 (1992))
* B61 -Rezeptor (Eck-Rezeptor) * B61 receptor (corner receptor)
(Bartley et al., Nature 363, 558 (1994), Pandey et al., Science 233, 567 (1995), van der Geer et al., Ann. Rev. Cell Biol. 12, 251 (1994))(Bartley et al., Nature 363, 558 (1994), Pandey et al., Science 233, 567 (1995), van der Geer et al., Ann. Rev. Cell Biol. 12, 251 (1994))
B61B61
Das B61-Molekül stellt den Liganden dar für den B61-Rezeptor.The B61 molecule represents the ligand for the B61 receptor.
(Holzman et al., J. Am. Soc. Nephrol. 4, 466 (1993), Bartley et al., Nature 363, 558 (1994))(Holzman et al., J. Am. Soc. Nephrol. 4, 466 (1993), Bartley et al., Nature 363, 558 (1994))
- Endothelin, im speziellen- endothelin, in particular
* Endothelin B* Endothelin B
(Oreilly et al., J. Cardiovasc. Pharm. 22, 18 (1993), Benatti et al., J. Clin. Invest. 21, 1149 (1993), O'Reilly et al., BBRC 123, 834 (1993). Die Promotorsequenz wurde von Benatti et al., J. Clin. Invest. 21. 1149 (1993) beschrieben.(Oreilly et al., J. Cardiovasc. Pharm. 22, 18 (1993), Benatti et al., J. Clin. Invest. 21, 1149 (1993), O'Reilly et al., BBRC 123, 834 (1993 The promoter sequence was described by Benatti et al., J. Clin. Invest. 21, 1149 (1993).
* Endothelin-1 * Endothelin-1
(Yanasigawa et al., Nature 332, 411 (1988).(Yanasigawa et al., Nature 332, 411 (1988).
Die Promotorsequenz wurde von Wilson et al., Mol. Cell. Biol. 12, 4654The promoter sequence was described by Wilson et al., Mol. Cell. Biol. 12, 4654
(1990) beschrieben.(1990).
- Endothelin-Rezeptoren, insbesondere der Endothelin-B-Rezeptor (Webb et al., Mol. Pharmacol. 4Z, 730 (1995), Haendler et al., J. Cardiovasc. Pharm. 22, 1 (1992)).- Endothelin receptors, in particular the endothelin B receptor (Webb et al., Mol. Pharmacol. 4Z, 730 (1995), Haendler et al., J. Cardiovasc. Pharm. 22, 1 (1992)).
- Mannose-6-Phosphat-Rezeptoren- Mannose-6-phosphate receptors
(Perales et al., Eur. J. Biochem. 225, 225 (1994).(Perales et al., Eur. J. Biochem. 225, 225 (1994).
Die Promotorsequenzen sind von Ludwig et al. (Gene 142, 311 (1994)), Oshima et al. (J. Biol. Chem. 263, 2553 (1988)) und Pohlmann et al. (PNAS USA 64. 5575 (1987)) beschrieben worden.The promoter sequences are from Ludwig et al. (Gene 142, 311 (1994)), Oshima et al. (J. Biol. Chem. 263, 2553 (1988)) and Pohlmann et al. (PNAS USA 64, 5575 (1987)).
- von Willebrand Faktor- von Willebrand factor
Die Promotorsequenz wurde von Jahroudi und Lynch (Mol. Cell. Biol. 14, 999 (1994), Ferreira et al., Biochem. J. 223, 641 (1993) und Aird et al., PNAS USA 22, 4567 (1995)) beschrieben.The promoter sequence was described by Jahroudi and Lynch (Mol. Cell. Biol. 14, 999 (1994), Ferreira et al., Biochem. J. 223, 641 (1993) and Aird et al., PNAS USA 22, 4567 (1995) ) described.
- IL-1α, IL-1ß.- IL-1α, IL-1ß.
IL-1 wird von aktivierten Endothelzellen produziert (Warner et al., J. Immunol. 132, 1911 (1987)).IL-1 is produced by activated endothelial cells (Warner et al., J. Immunol. 132, 1911 (1987)).
Die Promotorsequenzen wurden von Hangen et al., Mol. Carcinog. 2, 68 (1986), Turner et al., J. Immunol. 143, 3556 (1989), Fenton et al., J. Immunol. 133, 3972 (1987), Bensi et al., Cell Growth Diff. 1, 491 (1990), Hiscott et al.. Mol. Cell. Biol. 13, 6231 (1993) und Mori et al., Blood 34, 1688 (1994) beschrieben - IL-1 -RezeptorThe promoter sequences were described by Hangen et al., Mol. Carcinog. 1986, 2: 68, Turner et al., J. Immunol. 143: 3556 (1989), Fenton et al., J. Immunol. 133, 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 34, 1688 (1994) - IL-1 receptor
Die Promotorsequenz wurde von Ye et al., PNAS USA 22, 2295 (1993) beschrieben.The promoter sequence was described by Ye et al., PNAS USA 22, 2295 (1993).
- Vascular Cell Adhesion Molecule (VCAM-1)- Vascular Cell Adhesion Molecule (VCAM-1)
Die Expression von VCAM-1 in Endothelzellen wird aktiviert durch Lipopolysaccharide, TNF-α (Neish et al., Mol. Cell. Biol. 3, 2558 (1995)), IL-4 (lademarko et al., J. Clin. Invest. 25, 264 (1995)) und IL-1 (Marni et al., J. Clin. Invest. 22, 1866 (1993)).Expression of VCAM-1 in endothelial cells is activated by lipopolysaccharides, TNF-α (Neish et al., Mol. Cell. Biol. 3, 2558 (1995)), IL-4 (lademarko et al., J. Clin. Invest 25, 264 (1995)) and IL-1 (Marni et al., J. Clin. Invest. 22, 1866 (1993)).
Die Promotorsequenz von VCAM-1 wurde beschrieben von Neish et al., Mol. Cell. Biol. 15, 2558 (1995), Ahmad et al., J. Biol. Chem. 2Z2, 8976 (1995), Neish et al., J. Exp. Med. 1Z6, 1583 (1992), lademarco et al., J. Biol. Chem. 26Z, 16323 (1992) und Cybulsky et al., PNAS USA 66, 7859 (1991).The promoter sequence of VCAM-1 was described by Neish et al., Mol. Cell. Biol. 15, 2558 (1995), Ahmad et al., J. Biol. Chem. 2Z2, 8976 (1995), Neish et al., J. Exp. Med. 1Z6, 1583 (1992), lademarco et al., J. Biol. Chem. 26Z, 16323 (1992) and Cybulsky et al., PNAS USA 66, 7859 (1991).
- synthetische Aktivatorsequenz- synthetic activator sequence
Als Alternative zu natürlichen endothelspezifischen Promotoren lassen sich auch synthetische Aktivatorsequenzen verwenden, die aus oligomerisierten Bindestellen für Transkriptionsfaktoren, die preferentiell oder selektiv in Endothelzellen aktiv sind, bestehen.As an alternative to natural endothelium-specific promoters, synthetic activator sequences can also be used which consist of oligomerized binding sites for transcription factors which are preferentially or selectively active in endothelial cells.
Ein Beispiel hierfür ist der Transkriptionsfaktor GATA-2, dessen Bindestelle im Endothelin-1 Gen 5'-TTATCT-3' ist (Lee et al., Biol. Chem. 266. 16188 (1991), Dorfmann et al., J. Biol. Chem. 26Z, 1279 (1992) und Wilson et al., Mol. Cell. Biol. 12, 4854 (1990)).An example of this is the transcription factor GATA-2, whose binding site in the endothelin-1 gene is 5'-TTATCT-3 '(Lee et al., Biol. Chem. 266, 16188 (1991), Dorfmann et al., J. Biol Chem. 26Z, 1279 (1992) and Wilson et al., Mol. Cell. Biol. 12, 4854 (1990)).
aktivierte Makrophagen und/oder aktivierte Lymphozytenactivated macrophages and / or activated lymphocytes
Als Aktivatorsequenz im Sinne dieser Erfindung sind außerdem Promo¬ torsequenzen der Gene für Proteine zu verstehen, welche bei der Immunreaktion in Makrophagen und/oder in Lymphozyten verstärkt gebildet werden. Hierzu gehören beispielsweise:In the sense of this invention, the activator sequence is also to be understood as meaning promoter sequences of the genes for proteins which are formed to an increased extent in the immune reaction in macrophages and / or in lymphocytes. These include, for example:
* IL-1 (Bensi et al., Gene 52, 95 (1987), Fibbe et al., Blut 52, 147 (1989)) * IL-1 (Bensi et al., Gene 52, 95 (1987), Fibbe et al., Blut 52, 147 (1989))
* IL-1 -Rezeptor (Colotta et al., Immunol. Today 13., 562 (1994), Sims et al., Clin. Immunol. Immunopath. Z2, 9 1994), Ye et al., PNAS USA 22, 2295 (1993)) * IL-1 receptor (Colotta et al., Immunol. Today 13, 562 (1994), Sims et al., Clin. Immunol. Immunopath. Z2, 9 1994), Ye et al., PNAS USA 22, 2295 (1993))
* IL-2 (Jansen et al. Cll 32, 207 (1994), Ohbo et al., J. Biol. Chem. 270. 7479 (1995))* IL-2 (Jansen et al. Cll 32, 207 (1994), Ohbo et al., J. Biol. Chem. 270, 7479 (1995))
* IL-2-Rezeptor (Semenzato et al., Int. J. Clin Lab. Res. 22, 133 (1992))* IL-2 receptor (Semenzato et al., Int. J. Clin Lab. Res. 22, 133 (1992))
* IFN γ (Kirchner, DMW Hl, 64 (1986), Lehmann et al., J. Immunol. 153. 165 (1994))* IFN γ (Kirchner, DMW Hl, 64 (1986), Lehmann et al., J. Immunol. 153, 165 (1994))
* IL-4 (Paul, Blood ZZ, 1859 (1991), te Velde et al., Blood Z6, 1392 (1990)) * IL-4 (Paul, Blood ZZ, 1859 (1991), Te Velde et al., Blood Z6, 1392 (1990))
* IL-4-Rezeptor (Vallenga et al., Leukemia 7, 1131 (1993), Galizzi et al., Int. Immunol. 2, 669 (1990)) * IL-4 receptor (Vallenga et al., Leukemia 7, 1131 (1993), Galizzi et al., Int. Immunol. 2, 669 (1990))
* IL-3 (Frendl, Int. J. Immunopharm. 14, 421 (1992)) * IL-3 (Frendl, Int. J. Immunopharm. 14, 421 (1992))
* IL-5 (Azuma et al., Nucl. Acid Res. 14, 9149 (1986), Yokota et al., PNAS 64, 7388 (1987)) * IL-5 (Azuma et al., Nucl. Acid Res. 14, 9149 (1986), Yokota et al., PNAS 64, 7388 (1987))
* IL-6 (Brack et al., Int. J. Clin. Lab. Res. 22, 143 (1992)) or * IL-6 (Brack et al., Int. J. Clin. Lab. Res. 22, 143 (1992)) or
* LIF (Metcalf, Int. J. Cell Clon. 2, 95 (1991), Samal, BBA 1260. 27 (1995)) * LIF (Metcalf, Int. J. Cell Clon. 2, 95 (1991), Samal, BBA 1260. 27 (1995))
* IL-7 (Joshi et all, 21, 681 (1991)) * IL-7 (Joshi et all, 21, 681 (1991))
* IL-10 (Benjamin et al., Leuk. Lymph. 12, 205 (1994), Fluchiger et al., J. Exp. Med. lZ2, 91 (1994)) * IL-10 (Benjamin et al., Leuk. Lymph. 12, 205 (1994), Fluchiger et al., J. Exp. Med. IZ2, 91 (1994))
* IL-11 (Yang et al., Biofactors 4, 15 (1992)) * IL-11 (Yang et al., Biofactors 4, 15 (1992))
* IL-12 (Kiniwa et al., J. Clin. Invest. 22, 262 (1992), Gatelay, Cancer Invest. 11, 500 (1993)) * IL-12 (Kiniwa et al., J. Clin. Invest. 22, 262 (1992), Gatelay, Cancer Invest. 11, 500 (1993))
* IL-13 (Punnonen et al., PNAS 22, 3730 (1993), Muzio et al., Blood 63, 1738 (1994)) * IL-13 (Punnonen et al., PNAS 22, 3730 (1993), Muzio et al., Blood 63, 1738 (1994))
* GM-CSF (Metcalf, Cancer 15, 2185 (1990)) * GM-CSF (Metcalf, Cancer 15, 2185 (1990))
* GM-CSF Rezeptor (Nakagawa et al., J. Biol. Chem. 262, 10905 (1994)) * GM-CSF receptor (Nakagawa et al., J. Biol. Chem. 262, 10905 (1994))
* Adhäsionsproteine wie Integrin beta2 protein (Nueda et al., J. Biol. Chem. 268. 19305 (1993)) * Adhesion proteins such as integrin beta2 protein (Nueda et al., J. Biol. Chem. 268. 19305 (1993))
Promotorsequenzen für diese Proteine sind durch folgende Veröffent¬ lichungen zugänglich:Promoter sequences for these proteins are accessible through the following publications:
* IL-1 -Rezeptor * IL-1 receptor
(Ye et al., PNAS USA 22, 2295 (1993))(Ye et al., PNAS USA 22, 2295 (1993))
* IL-1α * IL-1α
(Hangen et al., Mol. Carcinog. 2, 68 (1986), Turner et al., J. Immunol. 143. 3556 (1989), Mori et al., Blood 64, 1688 (1994))(Hangen et al., Mol. Carcinog. 2, 68 (1986), Turner et al., J. Immunol. 143. 3556 (1989), Mori et al., Blood 64, 1688 (1994))
IL-1ßIL-1ß
(Fenton et al., J. Immunol. 133, 3972 (1987), Bensi et al., Cell Growth Diff.(Fenton et al., J. Immunol. 133, 3972 (1987), Bensi et al., Cell Growth Diff.
1, 491 (1990), Turner et al., J. Immunol. 143, 3556 (1989), Hiscott et al.,1: 491 (1990), Turner et al., J. Immunol. 143: 3556 (1989), Hiscott et al.
Mol. Cell. Biol. 13, 6231 (1993))Mol. Cell. Biol. 13, 6231 (1993))
IL-2IL-2
(Fujita et al., Cell 46, 401 (1986), Hama et al., J. Exp. Med. 131, 1217(Fujita et al., Cell 46, 401 (1986), Hama et al., J. Exp. Med. 131, 1217
(1995), Kant et al., Lymph. Rec. Interact. 1Z2 (1989), Kamps et al., Mol.(1995), Kant et al., Lymph. Rec. Interact. 1989, 1Z2, Kamps et al., Mol.
Cell. Biol. 12, 5464 (1990), Williams et al., J. Immunol. 141, 662 (1988),Cell. Biol. 12, 5464 (1990), Williams et al., J. Immunol. 141, 662 (1988),
Brunvand, FASEB J. 6, A998 (1992))Brunvand, FASEB J. 6, A998 (1992))
IL-2-RezeptorIL-2 receptor
(Ohbo et al., J. Biol. Chem. 2Z2, 7479 (1995), Shibuya et al., Nucl. Acids(Ohbo et al., J. Biol. Chem. 2Z2, 7479 (1995), Shibuya et al., Nucl. Acids
Res. 13, 3697 (1990), Lin et al., Mol. Cell. Biol. 13, 6201 (1993), Williams et al., J. Immunol. 141, 662 (1988))Res. 13, 3697 (1990), Lin et al., Mol. Cell. Biol. 13, 6201 (1993), Williams et al., J. Immunol. 141, 662 (1988))
IFN γIFN γ
(Ye et al., J. Biol. Chem. 262, 25728 (1994))(Ye et al., J. Biol. Chem. 262, 25728 (1994))
IL-4IL-4
(Rooney et al., EMBO J. 13, 625 (1994), Hama et al., J. Exp. Med. 131,(Rooney et al., EMBO J. 13, 625 (1994), Hama et al., J. Exp. Med. 131,
1217 (1995), Li-Weber et al., J. Immunol. 153, 4122 (1994), 143, 19131217 (1995), Li-Weber et al., J. Immunol. 153, 4122 (1994), 143, 1913
(1992), Min et al., J. Immunol. 143, 1913 (1992), Abe et al., PNAS 32,(1992), Min et al., J. Immunol. 143, 1913 (1992), Abe et al., PNAS 32,
2864 (1992))2864 (1992))
IL-4-RezeptorIL-4 receptor
(Beckmann et al., Chem. immunol. 51, 107 (1992), Ohara et al., PNAS(Beckmann et al., Chem. Immunol. 51, 107 (1992), Ohara et al., PNAS
35, 8221 (1988))35, 8221 (1988))
IL-3IL-3
(Mathey-Prevot et al., PNAS USA SZ, 5046 (1990), Cameron et al., Blood(Mathey-Prevot et al., PNAS USA SZ, 5046 (1990), Cameron et al., Blood
33, 2851 (1994), Arai et al., Lymphokine Res. 2, 551 (1990))33, 2851 (1994), Arai et al., Lymphokine Res. 2, 551 (1990))
IL-3-Rezeptor (α-subunit)IL-3 receptor (α-subunit)
(Miyajima et al., Blood 35, 1246 (1995), Rapaport et al., Gene 13Z, 333(Miyajima et al., Blood 35, 1246 (1995), Rapaport et al., Gene 13Z, 333
(1993), Kosugi et al., BBRC 223, 360 (1995))(1993), Kosugi et al., BBRC 223, 360 (1995))
IL-3-Rezeptor (ß-subunit)IL-3 receptor (ß-subunit)
(Gorman et al., J. Biol. Chem. 26Z, 15842 (1992), Kitamura et al., Cell 66,(Gorman et al., J. Biol. Chem. 26Z, 15842 (1992), Kitamura et al., Cell 66,
1165 (1991), Hayashida et al., PNAS USA SZ, 9655 (1990))1165 (1991), Hayashida et al., PNAS USA SZ, 9655 (1990))
IL-5IL-5
(Lee et al., J. Allerg. Clin. Immunol. 24, 594 (1994), Kauhansky et al., J.(Lee et al., J. Allerg. Clin. Immunol. 24, 594 (1994), Kauhansky et al., J.
Immunol. 152, 1812 (1994), Staynov et al., PNAS USA 22, 3606 (1995)) * IL-6Immunol. 152, 1812 (1994), Staynov et al., PNAS USA 22, 3606 (1995)) * IL-6
(Lu et al., J. Biol. Chem. 2Z2, 9748 (1995), Gruss et al., Blood 62, 2563 (1992), Ray et al., PNAS S5, 6701 (1988), Droogmans et al., DNA- Sequence 3, 115 (1992), Mori et al., Blood 34, 2904 (1994), Liberman et al., Mol Cell. Biol. IQ, 2327 (1990), Ishiki et al., Mol. Cell. Biol. Q, 2757 (1990))(Lu et al., J. Biol. Chem. 2Z2, 9748 (1995), Gruss et al., Blood 62, 2563 (1992), Ray et al., PNAS S5, 6701 (1988), Droogmans et al., DNA Sequence 3, 115 (1992), Mori et al., Blood 34, 2904 (1994), Liberman et al., Mol Cell. Biol. IQ, 2327 (1990), Ishiki et al., Mol. Cell. Biol Q, 2757 (1990))
* IL-7* IL-7
(Pleiman et al., Mol. Cell. Biol. 11, 3052 (1991), Lapton et al., J. Immunol. 144, 3592 (1990))(Pleiman et al., Mol. Cell. Biol. 11, 3052 (1991), Lapton et al., J. Immunol. 144, 3592 (1990))
* IL-8 * IL-8
(Chang et al., J. Biol. Chem. 252, 25277 (1994), Sprenger et al., J. Immunol. 153, 2524 (1994))(Chang et al., J. Biol. Chem. 252, 25277 (1994), Sprenger et al., J. Immunol. 153, 2524 (1994))
* IL-10 * IL-10
(Kim et al., J. Immunol. 1 Q, 3618 (1992), Platzer et al., DNA Sequence 4, 399 (1994), Kube et al., Cytokine 7, 1 (1995))(Kim et al., J. Immunol. 1 Q, 3618 (1992), Platzer et al., DNA Sequence 4, 399 (1994), Kube et al., Cytokine 7, 1 (1995))
* IL-11 * IL-11
(Yang et al., J. Biol. Chem. 262, 32732 (1994))(Yang et al., J. Biol. Chem. 262, 32732 (1994))
* GM-CSF * GM-CSF
(Nimer et al., Mol. Cell. Biol. 12, 6084 (1990), Staynov et al., PNAS USA 22, 3606 (1995), Koyano-Nakayawa et al., Int. Immunol. 5, 345 (1993), Ye et al., Nucl. Acids Res. 22, 5672 (1994))(Nimer et al., Mol. Cell. Biol. 12, 6084 (1990), Staynov et al., PNAS USA 22, 3606 (1995), Koyano-Nakayawa et al., Int. Immunol. 5, 345 (1993) , Ye et al., Nucl. Acids Res. 22, 5672 (1994))
* GM-CSF-Rezeptor (α-Kette) * GM-CSF receptor (α chain)
(Nakagawa et al., J. Biol. Chem. 262, 10905 (1994))(Nakagawa et al., J. Biol. Chem. 262, 10905 (1994))
* Makrophagen-Colony Stimulating Factor (M-CSF)-Rezeptor * Macrophage Colony Stimulating Factor (M-CSF) receptor
(Yue et al., Mol. Cell. Biol. 13, 3191 (1993), Zhang et al., Mol. Cell. Biol. 14, 373 (1994))(Yue et al., Mol. Cell. Biol. 13, 3191 (1993), Zhang et al., Mol. Cell. Biol. 14, 373 (1994))
* Typ I und II Makrophagen Scavenger Rezeptoren (Moulton et al., Mol. Cell. Biol. 14, 4408 (1994))* Type I and II macrophage scavenger receptors (Moulton et al., Mol. Cell. Biol. 14, 4408 (1994))
* IL-13 * IL-13
(Staynov et al., PNAS USA 22, 3606 (1995))(Staynov et al., PNAS USA 22, 3606 (1995))
* LIF* LIF
(Gough et al., Ciba Found. Symp. 15Z, 24 (1992), Stahl et al., Cytokine 5, 386 (1993))(Gough et al., Ciba Found. Symp. 15Z, 24 (1992), Stahl et al., Cytokine 5, 386 (1993))
* Interferon Regulator/ Factor 1 , dessen Promotor durch IL-6 wie auch durch IFNγ oder beta stimuliert wird* Interferon regulator / factor 1, whose promoter is stimulated by IL-6 as well as by IFNγ or beta
(Harrock et al., EMBO J. 13, 1942 (1994)) * IFNγ Responsive Promotor (Lamb et al., Blood 33, 2063 (1994))(Harrock et al., EMBO J. 13, 1942 (1994)) * IFNγ Responsive Promoter (Lamb et al., Blood 33, 2063 (1994))
* IFNγ* IFNγ
(Hardy et al., PNAS 32, 8173 (1985))(Hardy et al., PNAS 32, 8173 (1985))
* MAC-1 * MAC-1
(Dziennis et al., Blood 35, 319 (1995), Bauer et al., Hum. Gene Ther. 5, 709 (1994), Hickstein et al., PNAS USA 32, 2105 (1992))(Dziennis et al., Blood 35, 319 (1995), Bauer et al., Hum. Gene Ther. 5, 709 (1994), Hickstein et al., PNAS USA 32, 2105 (1992))
* LFA-1α * LFA-1α
(Nueda et al., J. Biol. Chem. 266, 19305 (1993), Agura et al., Blood Z2, 602 (1992), Comwell et al., PNAS USA 22, 4221 (1993))(Nueda et al., J. Biol. Chem. 266, 19305 (1993), Agura et al., Blood Z2, 602 (1992), Comwell et al., PNAS USA 22, 4221 (1993))
* p150,95 * p150.95
(Noti et al., DNA and Cell Biol. H, 123 (1992), Lopezcabrera et al., J. Biol. Chem. 266, 1187 (1993))(Noti et al., DNA and Cell Biol. H, 123 (1992), Lopezcabrera et al., J. Biol. Chem. 266, 1187 (1993))
5.2. Auswahl der Wirksubstanz zur Inhibition der Gerinnung5.2. Selection of the active substance to inhibit coagulation
Als Wirksubstanz im Sinne dieser Erfindung ist eine DNA-Sequenz zu verwenden, welche ein Protein kodiert, welches direkt oder indirekt die Thrombozytenaggregation oder einen Blutgerinnungsfaktor inhibiert oder die Fibrinolyse stimuliert.A DNA sequence which encodes a protein which directly or indirectly inhibits platelet aggregation or a blood coagulation factor or stimulates fibrinolysis is to be used as active substance in the sense of this invention.
Eine derartige Wirksubstanz wird als Gerinnungshemmer bezeichnet. Als Gerinnungshemmer sind Gene für beispielsweise Plasminogenaktivatoren (PA), so der Gewebe-PA (tPA) oder der Urokinase-ähnliche PA (uPA) oder Protein C, Antithrombin-Ill, C-1 S-lnhibitor, α1-Antitrypsin, der Tissue Factor Pathway Inhibitor (TFPI) oder Hirudin einzusetzen. Die DNA-Sequenzen für diese Gerinnungshemmer wurden bereits im Abschnitt 4.3. beschrieben:Such an active substance is called an anticoagulant. Genes for, for example, plasminogen activators (PA), such as tissue PA (tPA) or urokinase-like PA (uPA) or protein C, antithrombin III, C-1S inhibitor, α1-antitrypsin, the tissue factor, are anticoagulants Use pathway inhibitor (TFPI) or hirudin. The DNA sequences for these anticoagulants have already been described in section 4.3. described:
5.3. Kombination zweier gleicher oder unterschiedlicher Wirksubstanzen zur Inhibition der Gerinnung5.3. Combination of two identical or different active substances to inhibit coagulation
Gegenstand der Erfindung ist des weiteren ein Wirkstoff, in welchem eine Kombination der DNA-Sequenzen von zwei gleichen gerinnungshemmenden Substanzen (A,A) oder zwei unterschiedlichen gerinnungshemmenden Substanzen (A,B) vorliegt. Zur Expression beider DNA-Sequenzen ist vorzugsweise die cDNA einer "internal ribosomal entry site" (IRES) als regulatorisches Element zwischengeschaltet.The invention further relates to an active ingredient in which there is a combination of the DNA sequences of two identical anticoagulant substances (A, A) or two different anticoagulant substances (A, B). To express both DNA sequences preferably the cDNA of an "internal ribosomal entry site" (IRES) interposed as a regulatory element.
Figure imgf000034_0001
Figure imgf000034_0001
Derartige IRES wurden beispielsweise von Montford und Smith (TIG H, 179 (1995), Kaufman et al., Nucl. Acids Res. 12, 4485 (1991), Morgan et al., Nucl. Acids Res. 22, 1293 (1992, Dirks et al., Gene 126, 247 (1993), Pelletier und Sonenberg, Nature 334, 320 (1988) und Sugitomo et al., BioTechn. 12, 694 (1994) beschrieben.Such IRES have been described, for example, by Montford and Smith (TIG H, 179 (1995), Kaufman et al., Nucl. Acids Res. 12, 4485 (1991), Morgan et al., Nucl. Acids Res. 22, 1293 (1992, Dirks et al., Gene 126, 247 (1993), Pelletier and Sonenberg, Nature 334, 320 (1988) and Sugitomo et al., BioTechn. 12, 694 (1994).
So kann die cDNA der IRES-Sequenz des Poliovirus (Position < 140 bis > 630 des 5' UTR (Pelletier und Sonenberg, Nature 334, 320 (1988)) zur Verknüpfung der DNA der antithrombotischen Substanz A (am 3' Ende) und der DNA der antithrombotischen Substanz B (am 5' Terminus) verwendet werden.Thus, the cDNA of the IRES sequence of the poliovirus (position <140 to> 630 of the 5 'UTR (Pelletier and Sonenberg, Nature 334, 320 (1988)) for linking the DNA of the antithrombotic substance A (at the 3' end) and the DNA of the antithrombotic substance B (at the 5 'terminus) can be used.
Eine derartige Kombination zweier gleicher oder unterschiedlicher Gene bewirkt eine additive Wirkung (bei gleichen Genen) oder eine synergistische Wirkung der gewählten antithrombotischen Substanzen.Such a combination of two identical or different genes produces an additive effect (with the same genes) or a synergistic effect of the selected antithrombotic substances.
5.4. Auswahl des Liganden zur Inhibition der Gerinnung5.4. Selection of ligand to inhibit coagulation
Als Ligand für virale oder nicht-virale Vektoren, beispielsweise in kolloidalen Dispersionen, enthaltend Polylysin-Ligand-Konjugaten, werden Substanzen bevorzugt, welche an die Zelloberfläche von glatten Muskelzellen oder von proliferierenden Endothelzellen oder von aktivierten Makrophagen und/oder Lymphozyten binden.Substances which bind to the cell surface of smooth muscle cells or of proliferating endothelial cells or of activated macrophages and / or lymphocytes are preferred as ligands for viral or non-viral vectors, for example in colloidal dispersions containing polylysine-ligand conjugates.
a) Liganden für glatte Muskelzellena) Ligands for smooth muscle cells
Beispiele für Liganden, die an glatte Muskelzellen binden, wurden schon im Abschnitt 4.4. aufgeführt. b) Liganden für aktivierte EndothelzellenExamples of ligands that bind to smooth muscle cells have already been described in section 4.4. listed. b) Ligands for activated endothelial cells
Hierzu gehören im Sinne der Erfindung Antikörper oder Antikörperfragmente, gerichtet gegen Membranstrukturen von Endothelzellen wie sie bei¬ spielsweise von Burrows et al. (Pharmac. Ther. 64, 55 (1994)), Hughes et al. (Cancer Res. 42, 6214 (1989) und Maruyama et al. (PNAS-USA 6Z, 5744 (1990) beschrieben wurden. Insbesondere zählen hierzu Antikörper gegen die VEGF-Rezeptoren.For the purposes of the invention, this includes antibodies or antibody fragments, directed against membrane structures of endothelial cells, as described, for example, by Burrows et al. (Pharmac. Ther. 64, 55 (1994)), Hughes et al. (Cancer Res. 42, 6214 (1989) and Maruyama et al. (PNAS-USA 6Z, 5744 (1990). In particular, these include antibodies against the VEGF receptors.
Die murinen monoklonalen Antikörper sind bevorzugt in humanisierter Form einzusetzen. Die Humanisierung erfolgt in der im Abschnitt 4.4. dargestellten Weise. Antikörperfragmente werden entsprechend dem Stand der Technik hergestellt, beispielsweise in der in Abschnitt 4.4. beschriebenen Weise.The murine monoclonal antibodies are preferably used in humanized form. Humanization takes place in the section 4.4. illustrated way. Antibody fragments are produced according to the state of the art, for example in section 4.4. described way.
Zu den Liganden gehören des weiteren alle Wirkstoffe, welche an Membranstrukturen oder Membranrezeptoren auf Endothelzellen binden. Beispielsweise gehören hierzu Substanzen, die endständig Mannose enthalten des weiteren IL-1 oder Wachstumsfaktoren oder deren Fragmente bzw. Teilsequenzen von ihnen, die an Rezeptoren exprimiert durch Endo¬ thelzellen binden, wie beispielweise PDGF, bFGF, VEGF, TGFß (Pusztain et al., J. Pathol. 162, 191 (1993)).The ligands also include all active substances which bind to membrane structures or membrane receptors on endothelial cells. For example, these include substances that contain mannose, IL-1 or growth factors or their fragments or partial sequences thereof that bind to receptors expressed by endothelial cells, such as PDGF, bFGF, VEGF, TGFß (Pusztain et al., J. Pathol. 162, 191 (1993)).
Des weiteren gehören hierzu Adhäsionsmoleküle, welche an aktivierte und/oder proliferierende Endothelzellen binden. Derartige Adhäsionsmole¬ küle, wie beispielsweise SLex, LFA-1 , MAC-1 , LECAM-1 oder VLA-4, wur¬ den bereits beschrieben (Übersichten bei Augustin-Voss et al., J. Cell Biol. 119. 483 (1992), Pauli et al., Cancer Metast. Rev. 2, 175 (1990), Honn et al., Cancer Metast. Rev. H, 353 (1992)).This also includes adhesion molecules that bind to activated and / or proliferating endothelial cells. Adhesion molecules of this type, 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. 2, 175 (1990), Honn et al., Cancer Metast. Rev. H, 353 (1992)).
c) Liganden für aktivierte Makrophagen und/oder aktivierte Lymphozytenc) ligands for activated macrophages and / or activated lymphocytes
Zu den Liganden im Sinne der Erfindung gehören des weiteren Substanzen, welche an die Oberfläche von Immunzellen spezifisch binden. Hierzu gehören Antikörper oder Antikörperfragmente gerichtet gegen Membranstrukturen von Immunzellen, wie sie beispielsweise von Powelson et al., Biotech. Adv. H, 725 (1993) beschrieben wurden. Desweiteren gehören zu den Liganden auch monoklonale oder polyklonale Antikörper oder Antikörperfragmente, die mit ihren konstanten Domänen an Fcγ- oder μ-Rezeptoren von Immunzellen binden (Rojanasakul et al., Pharm. Res. 11, 1731 (1994)).For the purposes of the invention, the ligands also include substances which specifically bind to the surface of immune cells. These include antibodies or antibody fragments directed against membrane structures of immune cells, as described, for example, by Powelson et al., Biotech. Adv. H, 725 (1993). Furthermore, the ligands also include monoclonal or polyclonal antibodies or antibody fragments which bind with their constant domains to Fcγ or μ receptors of immune cells (Rojanasakul et al., Pharm. Res. 11, 1731 (1994)).
Auch hier werden die murinen monoklonalen Antikörper bevorzugt in humanisierter Form eingesetzt (siehe Abschnitt 4.4.) und Fragmente, beispielsweise mit der in Abschnitt 4.4. zitierten Methodik, hergestellt.Here too, the murine monoclonal antibodies are preferably used in humanized form (see section 4.4.) And fragments, for example with the one in section 4.4. cited methodology.
Zu den Liganden gehören des weiteren alle Substanzen, welche an Membranrezeptoren auf der Oberfläche von Immunzellen binden. Beispiels¬ weise gehören hierzu Wachstumsfaktoren, wie Zytokine, EGF, TGF, FGF oder PDGF, oder deren Fragmente bzw. Teilsequenzen von ihnen, die an Rezeptoren exprimiert durch derartige Zellen binden.The ligands also include all substances that bind to membrane receptors on the surface of immune cells. Examples include growth factors such as cytokines, EGF, TGF, FGF or PDGF, or their fragments or partial sequences thereof, which bind to receptors expressed by such cells.
5.5. Herstellung des Wirkstoffes zur Inhibition der Gerinnung5.5. Production of the active substance for inhibiting coagulation
Die Herstellung des erfindungsgemäßen Wirkstoffes wird anhand folgender Beispiele näher beschrieben:The preparation of the active ingredient according to the invention is described in more detail using the following examples:
a^ Konstruktion des Chimären Promotors Endothelin 1 CDE-CHR-Inra ^ Construction of the chimeric promoter endothelin 1 CDE-CHR-Inr
Der humane Endothelin-1 Promotor (Position < -170 bis > -10, Wilson et al., Mol. Cell. Biol. 12, 4854 (1990)) oder eine um die TATA-Box verkürzte Variante (Position < -170 bis > -40) werden an ihrem 3' Ende mit dem 5'- Terminus des CDE-CHR-Inr Moduls (Position < -20 bis > +121) des humanen cdc25C-Gens (Lucibello et al., EMBO J., 14, 132 (1995)) verknüpft (Fig. 9). Die Verknüpfung erfolgt mit Hilfe von dem Fachmann bekannten und käuflichen Enzymen.The human endothelin-1 promoter (position <-170 to> -10, Wilson et al., Mol. Cell. Biol. 12, 4854 (1990)) or a variant shortened by the TATA box (position <-170 to> -40) are at their 3 'end with the 5' terminus of the CDE-CHR-Inr module (position <-20 to> +121) of the human cdc25C gene (Lucibello et al., EMBO J., 14, 132 (1995)) linked (Fig. 9). The linkage takes place with the aid of enzymes known and commercially available to the person skilled in the art.
b) Konstruktion eines Plasmids enthaltend den Chimären Promotor Endothelin- 1 -CDE-CHR-Inr im zentralen Bestandteil des Wirkstoffesb) Construction of a plasmid containing the chimeric promoter endothelin-1-CDE-CHR-Inr in the central component of the active ingredient
Die beschriebene chimäre Endothelin-1 Promotormodul-Transkrip¬ tionseinheit wird an ihren 3' Enden mit dem 5'-Terminus einer DNA, die den kompletten kodierenden Bereich des Tissue Plasminogen Activators (Position < 85 bis > 1753, Pennica et al., Nature 221, 214 (1983)) enthält, verknüpft (Fig. 9). Diese PNA enthält auch die für eine Sekretion notwendige Signalsequenz. Transkriptionskontrolleinheiten und die DNA für Tissue Plasminogen Activator werden in pUC19/19 oder Bluescript-abgeleiteten Plasmidvektoren einkloniert, die direkt oder in kolloidalen Disper¬ sionssystemen für eine in vivo Applikation genutzt werden können. Alternativ können die Chimären Gene in virale Vektoren oder andere geeignete Vektoren transferiert und injiziert werden.The chimeric endothelin-1 promoter module transcription unit described is at its 3 'ends with the 5' terminus of a DNA which encompasses the entire coding region of the tissue plasminogen activator (Position <85 to> 1753, Pennica et al., Nature 221, 214 (1983)), linked (Fig. 9). This PNA also contains the signal sequence necessary for secretion. Transcription control units and the DNA for tissue plasminogen activator are cloned into pUC19 / 19 or Bluescript-derived plasmid vectors which can be used directly or in colloidal dispersion systems for in vivo application. Alternatively, the chimeric genes can be transferred into viral vectors or other suitable vectors and injected.
c) Konstruktion des Chimären Promotors Myogenin-CDE-CHR-Inrc) Construction of the chimeric promoter myogenin-CDE-CHR-Inr
Der humane Myogenin-Promotor (Pos. < -210 bis > +54, der von Salmin et al., J. Cell Biol. 115. 905 (1991 publizierten DNA-Sequenz) wird an seinem 3'-Ende mit dem 5'-Terminus des CDE-CHR-Inr Moduls des humanen cdc25C-Gens (Pos. < -20 bis > +121 , der von Lucibello et al., EMBO J. 14, 132 (1995) publizierten Sequenz) verknüpft (siehe Fig. 10). Die Verknüpfung erfolgt mit Hilfe von dem Fachmann bekannten und käuflichen Enzymen. Außerdem werden verschiedene Fragmente der Myogenin-Promotorse- quenz verwendet (siehe Fig. 10). So wird die TATA-Box enthaltende DNA- Sequenz des Myogenin-Promotors verwendet. Jedoch kann gleichermaßen auch die Promotorsequenz Position < -210 bis > -40 zur Anwendung kom¬ men.The human myogenin promoter (pos. <-210 to> +54, that of Salmin et al., J. Cell Biol. 115. 905 (published in 1991) is labeled with the 5'- at its 3 'end. Term of the CDE-CHR-Inr module of the human cdc25C gene (pos. <-20 to> +121, linked to the sequence published by Lucibello et al., EMBO J. 14, 132 (1995)) (see FIG. 10) The linkage takes place with the aid of enzymes known and commercially available. In addition, various fragments of the myogenin promoter sequence are used (see FIG. 10). Thus, the DNA sequence of the myogenin promoter containing the TATA box is used. However, equally the promoter sequence position <-210 to> -40 are used.
d) Konstruktion eines Plasmids enthaltend den Chimären Promotor Myoαenin- CDE-CHR-Inr im zentralen Bestandteil des Wirkstoffesd) Construction of a plasmid containing the chimeric promoter Myoαenin- CDE-CHR-Inr in the central component of the active ingredient
Die so hergestellte Chimäre Myogenin-Promotormodul-Transkriptionskontroll- einheit wird an ihrem 3' Ende mit dem 5'-Terminus einer DNA, die den kompletten kodierenden Bereich des Tissue Plasminogen Activators enthält, verknüpft (siehe Fig. 10). Diese DNA enthält auch die für eine Sekretion notwendige Signalsequenz. Transkriptionskontrolleinheiten und die DNA für den Tissue Plasminogen Activator werden in pUC18/19 oder Bluescript- abgeleiteten Plasmidvektoren einkloniert, die direkt oder in kolloidalen Dispersionssystemen für eine in vivo Applikation genutzt werden können. Alternativ können die Chimären Gene in virale Vektoren oder andere geeignete Vektoren transferiert und injiziert werden. e) Konstruktion eines Plasmids enthaltend zwei Gene für WirksubstanzenThe chimeric myogenin promoter module transcription control unit produced in this way is linked at its 3 'end to the 5' terminus of a DNA which contains the complete coding region of the tissue plasminogen activator (see FIG. 10). This DNA also contains the signal sequence necessary for secretion. Transcription control units and the DNA for the tissue plasminogen activator are cloned into pUC18 / 19 or Bluescript-derived plasmid vectors which can be used directly or in colloidal dispersion systems for in vivo application. Alternatively, the chimeric genes can be transferred into viral vectors or other suitable vectors and injected. e) Construction of a plasmid containing two genes for active substances
Die, wie unter c) beschriebene, Myogenin-CDE-CHR-Inr-Transkriptionsein- heit wird an ihrem 3' Ende mit dem 5' Ende der DNA für den Tissue Factor Pathway Inhibitor (TFPI, Position < 133 bis > 957; Wun et al., J. Biol. Chem. 263, 6001 (1988) oder Position < 382 bis > 1297; Girard et al., Thromb. Res. 55, 37 (1989)) verknüpft. Die Verknüpfung erfolgt mit Hilfe von dem Fach¬ mann bekannten und käuflichen Enzymen.The myogenin-CDE-CHR-Inr transcription unit, as described under c), is at its 3 'end with the 5' end of the DNA for the tissue factor pathway inhibitor (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 linkage takes place with the aid of enzymes known and commercially available to the person skilled in the art.
Das 3' Ende der DNA für TFPI wird nunmehr verknüpft mit dem 5* Ende der cDNA der internal ribosome entry site (Position < 140 bis > 630; Pelletier und Sonnenberg, Nature 334. 320 (1988)) und ausschließend deren 3' Ende mit dem 5' Ende der DNA für den Tissue Plasminogen Activator verknüpft (siehe Fig. 11). Dieser so hergestellte Wirkstoff wird ausschließlich in puc18/19 oder in Bluescript-abgeleiteten Plasmidvektoren einkloniert, die direkt oder in kolloidalen Dispersionssystemen für eine in vivo Applikation genutzt werden können. Alternativ können die Chimären Gene in virale Vektoren oder andere geeignete Vektoren transferiert und injiziert werden.The 3 'end of the DNA for TFPI is now linked to the 5 * end of the cDNA of the internal ribosome entry site (position <140 to>630; Pelletier and Sonnenberg, Nature 334, 320 (1988)) and, exclusively, its 3' end linked to the 5 'end of the DNA for the tissue plasminogen activator (see FIG. 11). This active ingredient thus produced is cloned exclusively into puc18 / 19 or into Bluescript-derived plasmid vectors which can be used directly or in colloidal dispersion systems for in vivo application. Alternatively, the chimeric genes can be transferred into viral vectors or other suitable vectors and injected.
6. Wirkung des Wirkstoffes auf glatte Muskelzellen und/oder auf die Gerinnung6. Effect of the active ingredient on smooth muscle cells and / or on coagulation
Ein Wirkstoff gemäß der vorliegenden Erfindung ermöglicht nach lokaler oder systemischer, bevorzugt intravenöser oder intraarterieller Gabe eine vorwiegende, wenn nicht ausschließliche Wirkung an solchen glatten Muskelzellen, welche durch Schäden oder Verletzungen des Gefäßes (insbesondere der Endothelschicht) und gegebenenfalls nach Wanderung in die Intima vom Gefäßvolumen her direkt zugänglich sind.An active substance according to the present invention, after local or systemic, preferably intravenous or intra-arterial, administration has a predominant, if not exclusive, effect on such smooth muscle cells, which are caused by damage or injuries to the vessel (in particular the endothelial layer) and possibly after migration into the intima of the vessel volume are directly accessible from here.
Durch die Kombination aus gewebespezifischer Aktivatorsequenz und zellzyklusreguliertem Repressormodul ist gewährleistet, daß der Zelizyklusinhibitor überwiegend oder ausschließlich in sich teilenden glatten Muskelzellen aktiviert wird.The combination of tissue-specific activator sequence and cell cycle-regulated repressor module ensures that the cell cycle inhibitor is activated predominantly or exclusively in dividing smooth muscle cells.
Durch die erfindungsgemäße Verwendung von mutierten Zelizyklusinhibitoren ist deren längerfristige proliferationshemmende Wirkung gewährleistet. Ein Wirkstoff gemäß der vorliegenden Erfindung ermöglicht des weiteren, daß nach lokaler (z.B. in Gewebe, Körperhöhlen oder Gewebezwischenräumen) oder nach systemischer, bevorzugt intravenöser oder intraarterieller Gabe, vorwiegend, wenn nicht ausschließlich, glatte Muskelzellen, aktivierte prolife¬ rierende Endothelzellen, aktivierte Lymphozyten oder aktivierte Makrophagen die antithrombotische Substanz exprimieren und diese somit am Ort der Entstehung der Thrombose freigesetzt wird.The use of mutant cell cycle inhibitors according to the invention ensures their longer-term proliferation-inhibiting effect. An active ingredient according to the present invention further enables that after local (for example in tissue, body cavities or tissue spaces) or after systemic, preferably intravenous or intraarterial administration, predominantly, if not exclusively, smooth muscle cells, activated proliferating endothelial cells, activated lymphocytes or activated macrophages express the antithrombotic substance and this is thus released at the site of thrombosis.
Da der Wirkstoff sowohl durch seine Zeil- als auch Zellzyklusspezifität ein hohes Maß an Sicherheit verspricht, kann er auch in hohen Dosierungen und, falls notwendig, mehrmals in Abständen von Tagen oder Wochen zur Prophylaxe oder zur Therapie von Gefäßverschlüssen, verursacht durch proliferierende glatte Muskelzellen und/oder zur Prophylaxe und/oder Therapie von Thrombosen angewandt werden. Since the active ingredient promises a high degree of safety due to its cell and cell cycle specificity, it can also be used in high doses and, if necessary, several times in intervals of days or weeks for the prophylaxis or therapy of vascular occlusions, caused by proliferating smooth muscle cells and / or used for the prophylaxis and / or therapy of thromboses.
Legende zu Fig. 1 - 11:Legend for Fig. 1 - 11:
Fig. 1:Fig. 1:
Nukleotidsequenz des cdc25C - Promotorbereichs mit den in vivo gefundenen Proteinbindungsstellen (genomisches DMS Footprinting; ■ (gefüllte Kreise): vollständiger konstitutiver Schutz; o (offene Kreise: partieller konstitutiver Schutz; * (Sternchen): zellzyklusregulierter, G1- spezifischer Schutz). CBS: constitutive binding site; CDE: cell cycle- dependent element. Grau unterlegte Bereiche zeigen die Yc-Boxen (NF-Y Bindestellen) an. Startstellen sind durch gefüllte Quadrate markiert.Nucleotide sequence of the cdc25C promoter region with the protein binding sites found in vivo (genomic DMS footprinting; ■ (filled circles): complete constitutive protection; o (open circles: partial constitutive protection; * (asterisk): cell cycle-regulated, G1-specific protection). CBS: constitutive binding site; CDE: cell cycle-dependent element. Gray areas indicate the Y c boxes (NF-Y binding points). Starting points are marked by filled squares.
Fig. 2:Fig. 2:
Derepression des cdc25C-Promotors spezifisch in Gn durch Mutation des cdc.Depression of the cdc25C promoter specifically in Gn by mutation of the cdc.
Fig. 3:Fig. 3:
Schematische Darstellung der cdc25C Enhancer-Regulation durch das CDE.Schematic representation of the cdc25C enhancer regulation by the CDE.
Fig. 4:Fig. 4:
Gn / G-] - spezifische Repression des SV40-Enhancers durch das CDE.Gn / G-] - specific repression of the SV40 enhancer by the CDE.
Fig. 5:Fig. 5:
Homologien im CDE-CHR-Bereich und den 5' gelegenen Yc-Boxen in den cdc25C, cyclin A und cdc2-Promotoren Fig. 6:Homologies in the CDE-CHR area and the 5 'Yc boxes in the cdc25C, cyclin A and cdc2 promoters Fig. 6:
Chimäre Konstrukte bestehend aus verschiedenen Anteilen des humanen Myogenin (Myf-4) Promotors, dem 3' fusionierten Promotorrnodul mit den CDE und CHR Repressorelementen sowie einer DNA für humane ß-glucuronidase (kompletter kodierender Bereich, Position < 27 bis > 1982) als Effektor (Oshima et al., PNAS USA 34. 685 (1987)). Positionsangaben beziehen sich auf die Angaben von Salminen et al., J. Cell Biol. 115, 905 (1991) für das Myogenin Gen bzw. auf das von Lucibello et al., EMBO J. 14, 132 (1995) verwendete System für cdc25C.Chimeric constructs consisting of different portions of the human myogenin (Myf-4) promoter, the 3 'fused promoter module with the CDE and CHR repressor elements and a DNA for human ß-glucuronidase (complete coding area, position <27 to> 1982) as effector ( Oshima et al., PNAS USA 34, 685 (1987)). Position information refers to the information from Salminen et al., J. Cell Biol. 115, 905 (1991) for the myogenin gene or to the system used by Lucibello et al., EMBO J. 14, 132 (1995) for cdc25C .
Fig. 7:Fig. 7:
Ersatz der homologen Signalsequenz der ß-glucoronidase durch eine heterologe Signalsequenz (des humanen Immunglobulins). Positionsangabe der Signalsequenz (MGWSCIILFLVATAT) des Immunglobulins (HuVHcAMP) beziehen sich auf Riechmann et al., Nature 332, 323 (1988).Replacement of the homologous signal sequence of the ß-glucoronidase with a heterologous signal sequence (human immunoglobulin). Position of the signal sequence (MGWSCIILFLVATAT) of the immunoglobulin (HuVHcAMP) refer to Riechmann et al., Nature 332, 323 (1988).
Alternative: Einbau des Signalpeptids des Ig zur besseren extrazellulären Ausschleusung der ß-glucuronidase, siehe (B).Alternative: Incorporation of the Ig signal peptide for better extracellular removal of the ß-glucuronidase, see (B).
Fig. 8:Fig. 8:
Chimäre Konstrukte bestehend aus verschiedenen Anteilen des humanen Myogenin (Myf-4) Promotors, dem 3' fusionierten Promotorrnodul mit den CDE und CHR Repressorelementen sowie einer DNA für humane ß-glucuronidase, der internal ribosome entry site als regulierende Nukleotidsequenz und der DNA für Tissue Plasminogen Activator. Die Positionsangaben beziehen sich auf Salminen et al., J. Cell Biol. 115. 905 (1991) für das Myogenin, Lucibello et al. EMBO J. 14, 132 (1995) für das CDE/CHR-Inr Element, Oshima et al., PNAS USA 84, 685 (1987) für ß-glucuronidase, Riechmann et al., Nature 332, 323 (1988) für die Signalsequenz des Immunoglobulins, Pelletier und Sonenberg, Nature 334, 320 (1988) für die Internal ribosome entry site des Poliovirus und auf Pennica et al., Nature 301 , 214 (1983) für den humanen Tissue Plasminogen Activator. Fig. 9:Chimeric constructs consisting of different parts of the human myogenin (Myf-4) promoter, the 3 'fused promoter module with the CDE and CHR repressor elements as well as a DNA for human ß-glucuronidase, the internal ribosome entry site as a regulating nucleotide sequence and the DNA for tissue plasminogen Activator. The position information relates to Salminen et al., J. Cell Biol. 115. 905 (1991) for the myogenin, Lucibello et al. EMBO J. 14, 132 (1995) for the CDE / CHR-Inr element, Oshima et al., PNAS USA 84, 685 (1987) for β-glucuronidase, Riechmann et al., Nature 332, 323 (1988) for the Signal sequence of the immunoglobulin, Pelletier and Sonenberg, Nature 334, 320 (1988) for the internal ribosome entry site of the poliovirus and on Pennica et al., Nature 301, 214 (1983) for the human tissue plasminogen activator. Fig. 9:
Chimäre Konstrukte bestehend aus verschiedenen Anteilen des humanen Endothelin-1 Promotors, dem 3' fusionierten Promotorrnodul mit den CDE und CHR Repressorelementen sowie einer DNA für den humanen Tissue Plasminogen Activator (kompletter kodierender Bereich, Pennica et al., Nature 301. 214 (1983)) als Effektor. Positionsangaben beziehen sich auf die Angaben von Wilson et al., Mol. Cell. Biol. IQ, 4854 (1990) für das Endothelin- 1 Gen bzw. auf das von Lucibello et al., EMBO J. 14, 132 (1995) verwendete System für cdc25C.Chimeric constructs consisting of different portions of the human endothelin-1 promoter, the 3 'fused promoter module with the CDE and CHR repressor elements and a DNA for the human tissue plasminogen activator (complete coding area, Pennica et al., Nature 301, 214 (1983)) ) as an effector. Positions refer to the information provided by Wilson et al., Mol. Cell. Biol. IQ, 4854 (1990) for the endothelin-1 gene or on the system used by Lucibello et al., EMBO J. 14, 132 (1995) for cdc25C.
Fig. 10:Fig. 10:
Chimäre Konstrukte bestehend aus verschiedenen Anteilen des humanen Myogenin (Myf-4) Promotors, dem 3' fusionierten Promotorrnodul mit den CDE und CHR Repressorelementen sowie einer DNA für den humanen Tissue Plasminogen Activator (kompletter kodierender Bereich) als Effektor. Positionangaben beziehen sich auf die Angaben von Salminen et al., J. Cell Biol. 115. 905 (1991) für das Myogenin Gen bzw. auf das von Lucibello et al., EMBO J. 14, 132 (1995) verwendete System für cdc25C und auf die Angaben von Pennica et al., Nature 301. 214 (1983) für den Tissue Plasminogen Activator.Chimeric constructs consisting of different portions of the human myogenin (Myf-4) promoter, the 3 'fused promoter module with the CDE and CHR repressor elements and a DNA for the human tissue plasminogen activator (complete coding area) as effector. Positions refer to the information from Salminen et al., J. Cell Biol. 115, 905 (1991) for the myogenin gene and to the system used by Lucibello et al., EMBO J. 14, 132 (1995) for cdc25C and on the specifications of Pennica et al., Nature 301, 214 (1983) for the tissue plasminogen activator.
Fig. 11 :Fig. 11:
Chimäre Konstrukte mit zwei EffektorgenenChimeric constructs with two effector genes
Positionsangabe für den Tissue Factor Pathway Inhibitor beziehen sich aufPositions for the tissue factor pathway inhibitor refer to
*) Wun et al., J. Biol. Chem. 263, 6001 (1988)*) Wun et al., J. Biol. Chem. 263, 6001 (1988)
**) Girard et al., Thromb. Res. 55, 37 (1989) und für die internal ribosome entry site (IRES) cDNA basierend auf Pelletier und Sonenberg, Nature 334, 320 (1988) Tabelle 1: Rolle von CDE und CHR bei der zellzyklusregulierten Transkription von cdc25C, cyclin A und cdc2 * *) Girard et al., Thromb. Res. 55, 37 (1989) and for the internal ribosome entry site (IRES) cDNA based on Pelletier and Sonenberg, Nature 334, 320 (1988) Table 1: Role of CDE and CHR in the cell cycle-regulated transcription of cdc25C, cyclin A and cdc2
Tab. 1Tab. 1
Gθ Growing FactorGθ Growing Factor
wt cdc25C 0.8 13.1 17.5wt cdc25C 0.8 13.1 17.5
cyclin A 0.7 27.1 41.7 cdc2 1.0 41.2 41.2cyclin A 0.7 27.1 41.7 cdc2 1.0 41.2 41.2
mCDE(-13) cdc25C 7.6 11.6 1.5mCDE (-13) cdc25C 7.6 11.6 1.5
cyclin A 13.4 23.9 1.8 cdc2 11.3 33.9 3.0cyclin A 13.4 23.9 1.8 cdc2 11.3 33.9 3.0
mCHR(-6/-3) cdc25C 14.4 21.0 1.5mCHR (-6 / -3) cdc25C 14.4 21.0 1.5
cyclin A 15.5 28.3 1.8 cdc2 18.6 38.6 2.1cyclin A 15.5 28.3 1.8 cdc2 18.6 38.6 2.1
Ergebnisse transienter Transfektionen in HIH3T3-Zellen sind als RLUs/1000 dargestellt. mCDE: mutiertes CDE (Pos. -13: G → T); mCHR: mutiertes CHR (Pos. -6 bis -3). Results of transient transfections in HIH3T3 cells are shown as RLUs / 1000. mCDE: mutated CDE (pos. -13: G → T); mCHR: mutated CHR (pos. -6 to -3).

Claims

Patentansprüche claims
1. Wirkstoff zur Prophylaxe oder Therapie von Tumorerkrankungen, dadurch charakterisiert, daß er ein DNA-Konstrukt enthält, welches aus einer Aktivatorsequenz, einem Promotorrnodul und einer DNA-Sequenz für eine antitumorale Substanz besteht.1. Active ingredient for the prophylaxis or therapy of tumor diseases, characterized in that it contains a DNA construct which consists of an activator sequence, a promoter module and a DNA sequence for an antitumor substance.
2. Wirkstoff nach Anspruch 1), bei welchem das Promotorrnodul die Elemente CDE-CHR-Inr besitzt und die Positionen < -20 bis > +30 des cdc25C Promotorbereiches enthält (Nukleotidsequenz GGCTGGCGGAAGGTTTGAATGGTCAACGCCTGCGG- CTGTTGATATTCTTG), wobei CDE das "cell cycle dependent element" (Nukleotid- sequenz TGGCGG), CHR die "cell cycle gene homology region" (Nukleotidsequenz GTTTGAA) und Inr die Initiationsstelle (Position +1) sowie die für die Inititation wichtigen benachbarten Sequenzen darstellen.2. Active ingredient according to claim 1), in which the promoter module has the elements CDE-CHR-Inr and contains the positions <-20 to> +30 of the cdc25C promoter region (nucleotide sequence GGCTGGCGGAAGGTTTGAATGGTCAACGCCTGCGG-CTGTTGATATTCTTG cell cycle, dependent element "the CDE das" cycle) (Nucleotide sequence TGGCGG), CHR represent the "cell cycle gene homology region" (nucleotide sequence GTTTGAA) and Inr the initiation site (position +1) and the neighboring sequences important for initiation.
3. Wirkstoff nach Anspruch 1) enthaltend eine Aktivatorsequenz (Promotor¬ oder Enhancersequenz), welche durch Transkriptionsfaktoren gebildet in Endothelzellen oder in Zellen in unmittelbarer Nachbarschaft zu proliferierenden Endothelzellen, reguliert wird.3. Active ingredient according to claim 1) containing an activator sequence (promoter or enhancer sequence) which is regulated by transcription factors formed in endothelial cells or in cells in the immediate vicinity of proliferating endothelial cells.
4. Wirkstoff nach Anspruch 3) enthaltend4. Active ingredient according to claim 3) containing
- den CMV-Promotor, den CMV-Enhancer oder den SV40 Promotor oder- the CMV promoter, the CMV enhancer or the SV40 promoter or
- die Promotorsequenz für den endothelialen Glucose-1 -Transporter, Endo¬ glin, VEGF-Rezeptor-1 oder -2, Rezeptortyrosinkinase til-1 oder til-2, B61 Rezeptor, B61 Ligand, Endothelin, insbesondere Endothelin-B, -1 , Mannose-6-Phosphat-Rezeptor, IL-1α oder IL-1 ß, IL-1 -Rezeptor, VCAM- 1 , von Willebrand Faktor oderthe promoter sequence for the endothelial glucose-1 transporter, endoglin, VEGF receptor 1 or -2, receptor tyrosine kinase til-1 or til-2, B61 receptor, B61 ligand, endothelin, in particular endothelin B, -1, Mannose-6-phosphate receptor, IL-1α or IL-1β, IL-1 receptor, VCAM-1, from Willebrand factor or
- eine synthetische Aktivatorsequenz aus oligomerisierten Bindestellen für Transkriptionsfaktoren, die preferentiell oder selektiv in Endothelzellen aktiv sind wie GATA-2, dessen Bindungsstelle 5'-TTATCT-3' ist oder - die Promotorsequenz für VEGF oder die Enhancersequenz für VEGF oder die cDNA-Sequenz für c-SRC oder für v-SRC, welche das VEGF- Gen regulieren odera synthetic activator sequence from oligomerized binding sites for transcription factors which are preferentially or selectively active in endothelial cells such as GATA-2, the binding site of which is 5'-TTATCT-3 'or the promoter sequence for VEGF or the enhancer sequence for VEGF or the cDNA sequence for c-SRC or for v-SRC which regulate the VEGF gene or
- die Promotorsequenz des Maus-Mammatumor-Virus oder die Promotorsequenz eines Steroidrezeptors.the promoter sequence of the mouse mammary tumor virus or the promoter sequence of a steroid receptor.
5. Wirkstoff nach Anspruch 1-4), bei welchem die DNA-Sequenz für den anti- tumoralen Wirkstoff5. Active ingredient according to claims 1-4), in which the DNA sequence for the anti-tumor active ingredient
- das Retinoblastomprotein p110 oder p107 und p130 Proteine oder- the retinoblastoma protein p110 or p107 and p130 proteins or
- das p53 Protein ist oder- the p53 protein is or
- das p21 Protein, p16 Protein oder einen anderen "Cyclin- dependent Kinase (cdK)"- Inhibitor oderthe p21 protein, p16 protein or another "cyclin-dependent kinase (cdK)" inhibitor or
- das GADD45 Protein oder- the GADD45 protein or
- das bak Protein oder- the bak protein or
- ein Protein, welches die Angiogenese inhibiert oder- a protein that inhibits angiogenesis or
- ein Protein, welches eine zytostatische Wirkung aufweist oder- a protein which has a cytostatic effect or
- ein Protein, welches Entzündungen stimuliert oder- a protein that stimulates inflammation or
- ein Enzym für die Spaltung von Vorstufen von Zytostatika in Zytostatika kodiert.- encodes an enzyme for the cleavage of precursors from cytostatics into cytostatics.
6. Wirkstoff nach Anspruch 5),6. Active ingredient according to claim 5),
- bei welchem das Retinoblastomprotein (pRb/p110) durch Austausch der Aminosäuren in den Positionen 246, 350, 601 , 605, 780, 786, 787, 800 und 804 nicht mehr phosphorylierbar wird, ohne jedoch seine Bindungsaktivität mit dem großen T-Antigen einzubüßen, beispielsweise, daß die Aminosäuren Thr-246, Ser-601 , Ser-605, Ser-780, Ser-786, Ser- 787 und Ser-800 mit Ala, der Aminosäure Thr-350 mit Arg und der Aminosäure Ser-804 mit Glu ausgetauscht werden oder- in which the retinoblastoma protein (pRb / p110) can no longer be phosphorylated by replacing the amino acids in positions 246, 350, 601, 605, 780, 786, 787, 800 and 804, but without losing its binding activity with the large T antigen , for example, that the amino acids Thr-246, Ser-601, Ser-605, Ser-780, Ser-786, Ser-787 and Ser-800 with Ala, the amino acid Thr-350 with Arg and the amino acid Ser-804 with Glu be exchanged or
- bei welchem das p107 Protein in analoger Weise wie das pRb/110 mutiert ist oder- in which the p107 protein is mutated in an analogous manner to the pRb / 110 or
- bei welchem das p130 Protein in analoger Weise wie das pRb/110 mutiert ist.- in which the p130 protein is mutated in an analogous manner to the pRb / 110.
7. Wirkstoff nach Anspruch 5), bei welchem das Protein p53 C-terminal verkürzt ist um das Serin 392. 7. Active ingredient according to claim 5), in which the protein p53 is shortened C-terminal by the serine 392.
8. Wirkstoff nach Anspruch 1-4), bei welchem die antitumorale Substanz Plasminogenaktivatorinhibitor-1 (PAI-1), PAI-2, PAI-3, Angiostatin, Platelet Factor-4, TIMP-1 , TIMP-2 oder TIMP-3 ist.8. Active ingredient according to claim 1-4), wherein the antitumor substance plasminogen activator inhibitor-1 (PAI-1), PAI-2, PAI-3, angiostatin, platelet factor-4, TIMP-1, TIMP-2 or TIMP-3 is.
9. Wirkstoff nach Anspruch 1-4), bei welchem die antitumorale Substanz Perforin, Granzym, IL-2, IL-4, IL-12, Interferon wie IFNα, IFNß, IFN γ, TNFα, TNFß, Oncostatin M, RANTES, MCAF, IL-8, MIP-1α, MIP-1ß, NAP-2, IL-3, IL-5, LIF, IL-11 oder IL-13 ist.9. Active ingredient according to claims 1-4), in which the antitumor substance perforin, granzyme, IL-2, IL-4, IL-12, interferon such as IFNα, IFNß, IFN γ, TNFα, TNFß, Oncostatin M, RANTES, MCAF , IL-8, MIP-1α, MIP-1ß, NAP-2, IL-3, IL-5, LIF, IL-11 or IL-13.
10. Wirkstoff nach Anspruch 9), bei welchem die antitumorale Substanz ein Fusionsprotein mit dem Fc-Fragment des Immunglobulins darstellt.10. Active ingredient according to claim 9), in which the antitumor substance is a fusion protein with the Fc fragment of the immunoglobulin.
11. Wirkstoff nach Anspruch 1-4), bei welchem die antitumorale Substanz ein Enzym darstellt und dieses Enzym eine Herpes Simplex Virus Thymidin- Kinase, Cytosindeaminase, Varizella Zoster Virus Thymidin-Kinase, Nitroreduktase, ß-Glucuronidase (im besonderen eine humane, pflanzliche oder bakterielle ß-Glucuronidase), Carboxypeptidase (vorzugsweise von Pseudomonas), Lactamase (vorzugsweise von Bazillus cereus), Pyro- glutamat-Aminopeptidase, D-Aminopeptidase, Oxidase, Peroxidase, Phosphatase, Hydroxynitrillyase, Protease, Esterase oder eine Glycosidase ist.11. Active ingredient according to claims 1-4), in which the antitumor substance is an enzyme and this enzyme is a herpes simplex virus thymidine kinase, cytosine deaminase, varicella zoster virus thymidine kinase, nitroreductase, β-glucuronidase (in particular a human, vegetable or bacterial β-glucuronidase), carboxypeptidase (preferably from Pseudomonas), lactamase (preferably from Bacillus cereus), pyroglutamate aminopeptidase, D-aminopeptidase, oxidase, peroxidase, phosphatase, hydroxynitrile lyase, protease, esterase or a glycosidase.
12. Wirkstoff nach Anspruch 11), bei welchem durch Punktmutationen der DNA- Sequenz des Enzyms die lysosomale Speicherung verringert und die extrazelluläre Ausschleusung erhöht ist.12. Active ingredient according to claim 11), in which the lysosomal storage is reduced and the extracellular discharge is increased by point mutations of the DNA sequence of the enzyme.
13. Wirkstoff nach Anspruch 11), bei welchem die Signalsequenz des Enzyms ausgetauscht ist durch eine heterologe Signalsequenz zur Verbesserung der extrazellulären Ausschleusung.13. Active ingredient according to claim 11), in which the signal sequence of the enzyme is replaced by a heterologous signal sequence to improve the extracellular discharge.
14. Wirkstoff nach Anspruch 1-13), welcher die DNA-Sequenzen von mehreren gleichen oder unterschiedlichen antitumoralen Substanzen enthält, wobei jeweils zwei DNA-Sequenzen durch eine DNA-Sequenz für die internal ribosome entry site miteinander verbunden sind.14. Active ingredient according to claims 1-13), which contains the DNA sequences of several identical or different antitumor substances, wherein two DNA sequences are connected to each other by a DNA sequence for the internal ribosome entry site.
15. Wirkstoff nach Anspruch 1-13), eingefügt in einen Vektor.15. Active ingredient according to claim 1-13), inserted into a vector.
16. Wirkstoff nach Anspruch 15), bei welchem der Vektor ein Virus ist. 16. Active ingredient according to claim 15), in which the vector is a virus.
17. Wirkstoff nach Anspruch 16), bei welchem das Virus ein Retrovirus, Adenovirus, Adeno-assoziiertes Virus, Herpes Simplex Virus oder Vaccinia Virus darstellt.17. Active ingredient according to claim 16), in which the virus is a retrovirus, adenovirus, adeno-associated virus, herpes simplex virus or vaccinia virus.
18. Wirkstoff nach Anspruch 1-14) eingefügt in ein Plasmid.18. Active ingredient according to claim 1-14) inserted into a plasmid.
19. Wirkstoff nach Anspruch 15-18), zubereitet in einem kolloidalen Dispersionssystem.19. Active ingredient according to claim 15-18), prepared in a colloidal dispersion system.
20. Wirkstoff nach Anspruch 19), bei welchem das kolloidale Dispersionssystem Liposomen sind.20. Active ingredient according to claim 19), in which the colloidal dispersion system are liposomes.
21. Wirkstoff nach Anspruch 20), bei welchem das kolloidale Dispersionssystem Polylysin-Liganden sind.21. Active ingredient according to claim 20), in which the colloidal dispersion system are polylysine ligands.
22. Wirkstoff nach Anspruch 15-21), ergänzt um einen Liganden, welcher an Membranstrukturen von Endothelzellen bindet.22. Active ingredient according to claim 15-21), supplemented by a ligand which binds to membrane structures of endothelial cells.
23. Wirkstoff nach Anspruch 22), bei welchem der Ligand23. Active ingredient according to claim 22), in which the ligand
- ein polyklonaler oder monoklonaler Antikörper oder ein Antikörperfrag¬ ment hiervon ist, der mit seinen variablen Domänen an Membranstruktu¬ ren von Endothelzellen bindet,is a polyclonal or monoclonal antibody or an antibody fragment thereof which binds with its variable domains to membrane structures of endothelial cells,
- ein Zytokin oder Wachstumsfaktor oder ein Fragment bzw. eine Teilsequenz hiervon ist, der an Rezeptoren auf glatten Muskelzellen bindet-is a cytokine or growth factor or a fragment or a partial sequence thereof which binds to receptors on smooth muscle cells
- ein Adhäsionsmolekül darstellt, wie SLeX, LFA-1 , MAC-1 , LECAM-1 oder VLA-4.represents an adhesion molecule such as SLeX, LFA-1, MAC-1, LECAM-1 or VLA-4.
24. Wirkstoff nach Anspruch 23), bei welchem die Membranstruktur von Endothelzellen ein Rezeptor darstellt für Mannose, IL-1 oder einen Wachstumsfaktor, wie PDGF, FGF, VEGF, TGFß.24. Active ingredient according to claim 23), in which the membrane structure of endothelial cells is a receptor for mannose, IL-1 or a growth factor, such as PDGF, FGF, VEGF, TGFß.
25. Wirkstoff nach Anspruch 1-24) in einer Arzneimittelzubereitung für die intravenöse, intraarterielle, intracavitäre Injektion, für die Injektion in Gewebe, in Gewebespalten oder zur lokalen Verabreichung. 25. Active ingredient according to claims 1-24) in a pharmaceutical preparation for intravenous, intraarterial, intracavitary injection, for injection into tissue, in tissue columns or for local administration.
PCT/EP1995/003368 1994-08-26 1995-08-25 Genetic therapy of vascular diseases with a cell-specific active substance which is dependent on the cell cycle WO1996006938A1 (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
JP8508477A JPH10507626A (en) 1994-08-26 1995-08-25 Gene Therapy for Vascular Disease Using Cell-Specific Active Compounds That Change with the Cell Cycle
AU33874/95A AU690733B2 (en) 1994-08-26 1995-08-25 Genetic therapy of vascular diseases with a cell-specific active substance which is dependent on the cell cycle
DK95930524T DK0777739T3 (en) 1994-08-26 1995-08-25 Gene therapeutic treatment of vascular disease with a cell-specific cell cycle-dependent active substance
PCT/EP1995/003368 WO1996006938A1 (en) 1994-08-26 1995-08-25 Genetic therapy of vascular diseases with a cell-specific active substance which is dependent on the cell cycle
DE59508144T DE59508144D1 (en) 1994-08-26 1995-08-25 GENTHERAPEUTIC TREATMENT OF VASCULAR DISEASES BY A CELL-SPECIFIC, CELL-CYCLE-DEPENDENT ACTIVE SUBSTANCE
AT95930524T ATE191506T1 (en) 1994-08-26 1995-08-25 GENETHERAPEUTIC TREATMENT OF VASCULAR DISEASES USING A CELL-SPECIFIC, CELL CYCLE-DEPENDENT ACTIVE SUBSTANCE
EP95930524A EP0777739B1 (en) 1994-08-26 1995-08-25 Genetic therapy of vascular diseases with a cell-specific active substance which is dependent on the cell cycle
GR20000401227T GR3033535T3 (en) 1994-08-26 2000-05-31 Genetic therapy of vascular diseases with a cell-specific active substance which is dependent on the cell cycle

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GB9417366A GB9417366D0 (en) 1994-08-26 1994-08-26 Cell cycle regulated repressor and DNA element
GB9417366.3 1995-03-29
GBGB9506466.3A GB9506466D0 (en) 1994-08-26 1995-03-29 Cell cycle regulated repressor and dna element
GB9506466.3 1995-03-29
PCT/EP1995/003368 WO1996006938A1 (en) 1994-08-26 1995-08-25 Genetic therapy of vascular diseases with a cell-specific active substance which is dependent on the cell cycle

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EP0805209A3 (en) * 1996-05-03 1998-04-22 Hoechst Aktiengesellschaft Nucleic acid construct with genes coding for transportsignals
EP0805209A2 (en) * 1996-05-03 1997-11-05 Hoechst Aktiengesellschaft Nucleic acid construct with genes coding for transportsignals
US6576758B1 (en) 1996-09-24 2003-06-10 Aventis Pharma Deutschland Gmbh Nucleic acid constructs containing hybrid promoters
EP0848061A2 (en) * 1996-12-11 1998-06-17 Hoechst Aktiengesellschaft Self-enhancing, pharmacologically controllable expression systems
EP0848061A3 (en) * 1996-12-11 2004-02-18 Aventis Pharma Deutschland GmbH Self-enhancing, pharmacologically controllable expression systems
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US6733753B2 (en) 1997-02-10 2004-05-11 Amgen Inc. Composition and method for treating inflammatory diseases
US6380170B1 (en) 1997-02-18 2002-04-30 Aventis Pharma Deutschland Gmbh Nucleic acid construct for the cell cycle regulated expression of structural genes
EP0859008A3 (en) * 1997-02-18 2000-04-05 Hoechst Aktiengesellschaft Nucleic acid construct for the cell cycle regulated expression of structural genes
AU735514B2 (en) * 1997-02-18 2001-07-12 Aventis Pharma Deutschland Gmbh Nucleic acid construct for the cell cycle regulated expression of structural genes
EP0860445A1 (en) * 1997-02-18 1998-08-26 Hoechst Aktiengesellschaft New nucleotide sequences for the cell cycle regulated expression of structural genes
EP0859008A2 (en) * 1997-02-18 1998-08-19 Hoechst Aktiengesellschaft Nucleic acid construct for the cell cycle regulated expression of structural genes
US6033856A (en) * 1997-03-14 2000-03-07 Hoechst Aktiengesellschaft Promoter of the cdc25B gene, its preparation and use
US6465246B1 (en) 1997-11-21 2002-10-15 Aventis Pharma Deutschland Gmbh Oncogene- or virus-controlled expression systems
WO2000004178A1 (en) * 1998-07-14 2000-01-27 Aventis Pharma Deutschland Gmbh Expression system containing chimeric promoters with binding sites for recombinant transcription factors

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