WO1996006938A1 - Therapie genetique de maladies vasculaires avec une substance active specifique de la cellule et dependant du cycle cellulaire - Google Patents

Therapie genetique de maladies vasculaires avec une substance active specifique de la cellule et dependant du cycle cellulaire 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)
English (en)
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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 DK95930524T priority Critical patent/DK0777739T3/da
Priority to EP95930524A priority patent/EP0777739B1/fr
Priority to AT95930524T priority patent/ATE191506T1/de
Priority to PCT/EP1995/003368 priority patent/WO1996006938A1/fr
Priority to AU33874/95A priority patent/AU690733B2/en
Priority to JP8508477A priority patent/JPH10507626A/ja
Priority to DE59508144T priority patent/DE59508144D1/de
Publication of WO1996006938A1 publication Critical patent/WO1996006938A1/fr
Priority to GR20000401227T priority patent/GR3033535T3/el

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    • 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
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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
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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
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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).

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Abstract

L'invention concerne une séquence d'ADN pour la thérapie génétique de maladies vasculaires. Les éléments essentiels de la séquence d'ADN sont la séquence activatrice, le module promoteur et le gène de codage de la substance active. La séquence activatrice est activée spécifiquement dans les cellules des muscles lisses, dans les cellules endothéliales activées, dans des macrophages activés ou dans des lymphocytes activés. L'activation est régulée en fonction du cycle cellulaire par le module promoteur. La substance active représente un inhibiteur de la croissance des cellules des muscles lisses et/ou de la coagulation. La séquence d'ADN décrite est insérée dans un vecteur viral ou non viral complété par un ligand ayant une affinité pour les cellules cibles.
PCT/EP1995/003368 1994-08-26 1995-08-25 Therapie genetique de maladies vasculaires avec une substance active specifique de la cellule et dependant du cycle cellulaire WO1996006938A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
DK95930524T DK0777739T3 (da) 1994-08-26 1995-08-25 Genterapeutisk behandling af karsygdomme med et cellespecifikt cellecyklusafhængigt aktivt stof
EP95930524A EP0777739B1 (fr) 1994-08-26 1995-08-25 Therapie genetique de maladies vasculaires avec une substance active specifique de la cellule et dependant du cycle cellulaire
AT95930524T ATE191506T1 (de) 1994-08-26 1995-08-25 Gentherapeutische behandlung von gefässerkrankungen durch einen zellspezifischen, zellzyklusabhängigen wirkstoff
PCT/EP1995/003368 WO1996006938A1 (fr) 1994-08-26 1995-08-25 Therapie genetique de maladies vasculaires avec une substance active specifique de la cellule et dependant du cycle cellulaire
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
JP8508477A JPH10507626A (ja) 1994-08-26 1995-08-25 細胞サイクルによって変化する細胞に特異的な活性化合物を用いる血管系の疾病の遺伝子療法
DE59508144T DE59508144D1 (de) 1994-08-26 1995-08-25 Gentherapeutische behandlung von gefässerkrankungen durch einen zellspezifischen, zellzyklusabhängigen wirkstoff
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

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GB9417366A GB9417366D0 (en) 1994-08-26 1994-08-26 Cell cycle regulated repressor and DNA element
GB9417366.3 1994-08-26
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 (fr) 1994-08-26 1995-08-25 Therapie genetique de maladies vasculaires avec une substance active specifique de la cellule et dependant du cycle cellulaire

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0805209A2 (fr) * 1996-05-03 1997-11-05 Hoechst Aktiengesellschaft Construction d'acides nucléiques contenant des gènes codant pour des signaux de transport
EP0848061A2 (fr) * 1996-12-11 1998-06-17 Hoechst Aktiengesellschaft Auto-augmentation de l'expression, par des systèmes controlâble pharmacologiquement
EP0859008A2 (fr) * 1997-02-18 1998-08-19 Hoechst Aktiengesellschaft Vecteur en ADN pour l'expression des gènes structuraux réglé par le cycle cellulaire
EP0860445A1 (fr) * 1997-02-18 1998-08-26 Hoechst Aktiengesellschaft Nouvelles sequences nucleotidiques pour l'expression des gènes structuraux régulé par le cycle cellulaire
EP0790313A3 (fr) * 1996-02-13 1998-12-02 Hoechst Aktiengesellschaft Produit d'acide nucléique pour l'expression de gènes qui est régulée par le cycle cellulaire, cellules le contenant et son utilisation pour la production de médicaments
WO2000004178A1 (fr) * 1998-07-14 2000-01-27 Aventis Pharma Deutschland Gmbh Systemes d'expression contenant des agents promoteurs chimeres dotes de points de liaison destines a des facteurs de transcription recombinants
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
US6576758B1 (en) 1996-09-24 2003-06-10 Aventis Pharma Deutschland Gmbh Nucleic acid constructs containing hybrid promoters
US6733753B2 (en) 1997-02-10 2004-05-11 Amgen Inc. Composition and method for treating inflammatory diseases

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993010135A1 (fr) * 1991-11-12 1993-05-27 The United States Of America As Represented By The Secretary Department Of Health And Human Services Caracterisation du promoteur de lactoferrine de souris sensible a l'×strogene
WO1993013807A1 (fr) * 1992-01-10 1993-07-22 Georgetown University Procede permettant d'administrer des cellules endotheliales produites par genie genetique au niveau de sites d'angiogenese afin d'effectuer une therapie genetique
WO1994029469A2 (fr) * 1993-06-07 1994-12-22 Vical Incorporated Plasmides adequats pour une therapie genique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993010135A1 (fr) * 1991-11-12 1993-05-27 The United States Of America As Represented By The Secretary Department Of Health And Human Services Caracterisation du promoteur de lactoferrine de souris sensible a l'×strogene
WO1993013807A1 (fr) * 1992-01-10 1993-07-22 Georgetown University Procede permettant d'administrer des cellules endotheliales produites par genie genetique au niveau de sites d'angiogenese afin d'effectuer une therapie genetique
WO1994029469A2 (fr) * 1993-06-07 1994-12-22 Vical Incorporated Plasmides adequats pour une therapie genique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
F.EHLERT ET AL.: "Cell cycle-regulated transcription of the human cdc25C gene is controlled by a novel regulatory element", BRITISH JOURNAL OF HAEMATOLOGY, vol. 87, no. Supplement 1, 2 June 1994 (1994-06-02) - 5 June 1994 (1994-06-05), pages 124 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0790313A3 (fr) * 1996-02-13 1998-12-02 Hoechst Aktiengesellschaft Produit d'acide nucléique pour l'expression de gènes qui est régulée par le cycle cellulaire, cellules le contenant et son utilisation pour la production de médicaments
US6235526B1 (en) 1996-05-03 2001-05-22 Aventis Pharma Deutschland Gmbh Nucleic acid constructs containing genes encoding transport signals
EP0805209A3 (fr) * 1996-05-03 1998-04-22 Hoechst Aktiengesellschaft Construction d'acides nucléiques contenant des gènes codant pour des signaux de transport
EP0805209A2 (fr) * 1996-05-03 1997-11-05 Hoechst Aktiengesellschaft Construction d'acides nucléiques contenant des gènes codant pour des signaux de transport
US6576758B1 (en) 1996-09-24 2003-06-10 Aventis Pharma Deutschland Gmbh Nucleic acid constructs containing hybrid promoters
EP0848061A2 (fr) * 1996-12-11 1998-06-17 Hoechst Aktiengesellschaft Auto-augmentation de l'expression, par des systèmes controlâble pharmacologiquement
EP0848061A3 (fr) * 1996-12-11 2004-02-18 Aventis Pharma Deutschland GmbH Auto-augmentation de l'expression, par des systèmes controlâble pharmacologiquement
KR19980064289A (ko) * 1996-12-11 1998-10-07 야코비, 피셔 약리학적으로 통제가능한 자가 증진 발현 시스템
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 (fr) * 1997-02-18 2000-04-05 Hoechst Aktiengesellschaft Vecteur en ADN pour l'expression des gènes structuraux réglé par le cycle cellulaire
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 (fr) * 1997-02-18 1998-08-26 Hoechst Aktiengesellschaft Nouvelles sequences nucleotidiques pour l'expression des gènes structuraux régulé par le cycle cellulaire
EP0859008A2 (fr) * 1997-02-18 1998-08-19 Hoechst Aktiengesellschaft Vecteur en ADN pour l'expression des gènes structuraux réglé par le cycle cellulaire
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 (fr) * 1998-07-14 2000-01-27 Aventis Pharma Deutschland Gmbh Systemes d'expression contenant des agents promoteurs chimeres dotes de points de liaison destines a des facteurs de transcription recombinants

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