EP1392836A2 - Gefässendothel-spezifischer promotor des humanen vascular endothelial cadherin-2 (hve-cad-2) gens und seine therapeutischen verwendungen - Google Patents

Gefässendothel-spezifischer promotor des humanen vascular endothelial cadherin-2 (hve-cad-2) gens und seine therapeutischen verwendungen

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Publication number
EP1392836A2
EP1392836A2 EP01996615A EP01996615A EP1392836A2 EP 1392836 A2 EP1392836 A2 EP 1392836A2 EP 01996615 A EP01996615 A EP 01996615A EP 01996615 A EP01996615 A EP 01996615A EP 1392836 A2 EP1392836 A2 EP 1392836A2
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EP
European Patent Office
Prior art keywords
nucleic acid
cell
gene
acid fragment
diseases
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP01996615A
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German (de)
English (en)
French (fr)
Inventor
Robert Zweigerdt
Manfred Rüdiger
Thomas G. Moll
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Cardion AG
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Cardion AG
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Publication date
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Publication of EP1392836A2 publication Critical patent/EP1392836A2/de
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination

Definitions

  • the invention relates to a nucleic acid fragment containing the regulatory sequence of the VE-Cad-2 gene, completely or a functionally active variant thereof; a nucleic acid construct containing the nucleic acid fragment and a functional nucleic acid sequence or a heterologous gene; a nector or vector system; a cell or cell line; a transgenic non-human animal; a drug; a diagnostic; an array; a method of identifying pharmacologically active substances; a method for identifying functionally active variants; a method for isolating endothelial cells from stem cells; a procedure for treatment; a method of administering a drug; the use of the nucleic acid fragment, the nucleic acid construct, the transformed cell or cell line of the test and the array.
  • the vascular endothelium is a single-layered association consisting of a large number of partially overlapping individual cells, which lines all arterial and venous blood vessels of higher vertebrates.
  • the cells of the vascular endothelium form a barrier that prevents the interaction of blood cells with the vascular wall.
  • the vascular endothelium plays an important role in the regulation of coagulation, the adhesion of leukocytes, the growth of smooth muscle cells in the vessels and the control of the transvascular diffusion of metabolites.
  • vascular endothelia in the body therefore offers the possibility, with specific expression of genes in vascular endothelial cells, of expressing a therapeutically active gene either systemically in the whole body, or, depending on the transfer system, locally in certain organs or specifically in partial sections of the vascular system ,
  • donor organs removed for transplantation can be perfused outside the body via the vascular system.
  • suitable vectors such as adenoviruses or liposomes
  • the vascular dothelium can be efficiently transfected and used to express genes that protect the transplanted organ and prevent rejection in the recipient.
  • promoters which control an efficient and specific expression of therapeutically relevant genes in the vascular endothelium.
  • vascular endothelial cells A number of DNA sequences that cause expression in vascular endothelial cells have been published. In some of these sequences, however, gene activation is not limited to the vascular endothelium, but also leads to expression in other cells and tissues. These include regulatory DNA elements from the following genes: von Willebrand factor (Guan J. et al. (1999). Blood. 94 (10): 3405-12.), platelet / endothelial cell adhesion molecule-1 (PECAM-1; Botella LM. et al (2000) J Immunol. 164 (3): 1372-1378), Preproendothelin-1 (Aversa CR. Et al. (1997).
  • von Willebrand factor Guan J. et al. (1999). Blood. 94 (10): 3405-12.
  • PECAM-1 platelet / endothelial cell adhesion molecule-1
  • Botella LM. et al (2000) J Immunol. 164 (3): 1372-1378 Preproendo
  • genes such as the receptor tyrosine kinase TIE-2 (Dube A. et al. (1999). Circ Res. 84 (10): 1177-1185), E-Selectin (Smith GM. Et al. (1993). Biochem Biophys Res Commun. 194 (1): 215-221) and the "Vascular Endothelial Growth Factor Receptor" -1 (Flt-1; Wakiya K. et al. (1996) J Biol Chem. 271 (48): 30823- 30828) and-2 (Flk-1; Kappel A. et al. (1999) Blood. 93 (12): 4284-4292) are specifically expressed in the vascular endothelium, but mainly after cell activation by inflammatory cytokines or only during the proliferation of vessels.
  • promoters show largely uniform vascular endothelial cell-specific gene expression in transgenic mice. These include the promoter of the vascular endothelial cadherin-1 gene (Gory S. et al. (1999). Blood. 93 (1): 184-192) and regulatory elements of the ICAM-2 gene (Cowan PJ. Et al. ( 1998) J Biol Chem. 273 (19): 11737-11744).
  • the uniform gene expression in the vascular endothelium of all organs of the body can certainly be an advantage or even a prerequisite for some applications.
  • a gene is introduced into the tumor, the protein product of which converts the systemically administered chemotherapeutic agent from the precursor form into the active form.
  • the expression of this transgene in the vascular endothelium of strongly vascularized cancerous ulcers leads to a high local concentration of the effective chemotherapeutic agent inside the tumor. At the same time, the systemic burden on the patient with the tissue-toxic agent is low.
  • Pro rz.g therapy examples include the transfection of tumors with the gene for thymidine kinase (TK) from the herpes simplex virus, which gancic lovir converts into the active, cell-killing form (Culver KW. Et al. ( 1992) Science 256: 1550-1552; Link CJ. Et al. (1995) Hybridoma 14: 143-147; Tanaka T. et al. (1996) Cancer Res.
  • TK thymidine kinase
  • the second strategy is based on the expression of genes, the products of which cause the initiation of programmed cell death, the so-called apoptosis, and thus can kill Kebs cells.
  • Examples of the "pro-apoptosis” therapy are the successful expression of the tumor necrosis factor-alpha (TNF- ⁇ ) in vascular endothelial cells of tumors (Jaggar RT. Et al. (1997) Hum Gene Ther. 8 (18): 2239- 2247) or the expression TRAIL (“TNF-related apoptosis-inducing ligand”), a member of the TNF- ⁇ superfamily.
  • FasL Fas ligand
  • CD95 CD95 ligand
  • the induction of apoptosis is the result of the specific interaction between the ligand and its receptor, Apol / Fas (CD95).
  • FasL In addition to the induction of cell death in cancer cells that carry the Apol / Fas receptor, the expression of FasL locally in the vessel wall also successfully prevented the formation of neointima after balloon dilation (Sata M. et al. (1998). Proc Natl Acad Be US A. 95 (3): 1213-1217).
  • FasL Another property of FasL is the maintenance of an immune privileged status in some organs like the brain, the anterior eye chamber and the testicle.
  • the expression of FasL in endothelial cells of transplanted organs prevented the rejection of the grafts (Swenson KM. Et al. (1998). Transplantation. 65 (2): 155-160; Tran TH. Et al. (1998). Transplantation 66 (9): 1126-1131).
  • adenoviral vectors are currently the most efficient and therefore most frequently used vehicles for introducing foreign genes into mammalian cells in vitro and in vivo.
  • the vectors are particularly suitable for the transduction of vascular endothelial cells, with high expression of the transgene being achieved (Crystal RG. (1999). Cancer Chemother Pharmacol. 43 Suppl: S90-99; O'Brien T, Simari RD. (2000) Mayo Clin Proc. Aug; 75 (8): 831-834).
  • the object of the invention to provide promoters which enable genes to be expressed efficiently in the endothelium of blood vessels, but preferably at the same time exclude any activation in the liver, including the vascular endothelium of the liver.
  • the object of the invention was therefore to provide nucleic acid fragments for targeted in vivo gene transfer which, especially in vivo, enable genes in venous and arterial endothelial cells of all organs and tissues to be switched on as specifically as possible, preferably with the exception of expression in the liver.
  • the object of the invention was surprisingly provided by providing a nucleic acid fragment containing a regulatory sequence of the human VE-Cad-2 gene extending upstream from the translation start to 5 'or a functionally active variant thereof, the nucleic acid fragment being vascular endothelium -specific expression enables.
  • the nucleic acid fragment according to the invention enables vascular endothelium-specific expression except expression in the liver.
  • the invention further relates to a nucleic acid fragment containing the sequence according to SEQ ID No. 1 or a functionally active variant thereof.
  • the nucleic acid fragment according to the invention enables vascular endothelium-specific expression, with the exception of expression in the liver.
  • the study provides no experimental evidence for the function or Activity of this potentially regulatory sequence, which would enable a person skilled in the art to design or obtain a regulatory sequence of the human VE-Cad-2 gene which would enable vascular endothelium-specific expression.
  • the expression data of the VE-Cad-2 gene show solid expression of the gene in the liver. The person skilled in the art would therefore see no signs or be motivated to assume that the putative regulatory sequences of the VE-Cad-2 gene according to Ludwig et al. (2000, supra) Vascular endothelium-specific expression, except expression in the liver.
  • this object was surprisingly achieved by providing a nucleic acid fragment which is characterized in that it is under the control of a 5 'regulatory sequence of the human manen VE-Cadherin-2 gene (hVE-CAD-2) comprises a functional nucleotide sequence; as well as by providing vector systems which comprise such a fragment, in particular as part of an expression cassette, and transgenic animals and cell lines which can be obtained by introducing such a nucleic acid fragment.
  • hVE-CAD-2 human manen VE-Cadherin-2 gene
  • nucleic acid fragment to mean a nucleic acid, in particular a DNA or RNA sequence, preferably a single or double-stranded, especially a double-stranded, DNA sequence.
  • regulatory sequence in the sense of the present invention is generally understood to mean a nucleic acid sequence located upstream from the translation start (+1) of the human VE-Cad-2 gene, which transcription of a sequence with this sequence towards the 3 'end connected, downstream nucleic acid sequence in particular with respect to the correct transcription start, the transcription rate, kinetics and / or the tissue specificity for the vascular endothelium or modulates the control of translation.
  • the regulatory sequence has enhancer or promoter activity.
  • a heterologous gene in relation to the regulatory sequence of the VE-Cad-2 gene is understood by the person skilled in the art to mean all genes which are not naturally linked to this sequence in the direction of the 3 'end.
  • the genes include naturally occurring genes, mutated genes or genes that encode fusion proteins.
  • the genes can come from humans, animals, plants, algae or bacteria.
  • a heterologous gene can also comprise two or more genes arranged in series; for example, the genes can be separated by an "internal ribo some entry site” (IRES) (Vagner et al.
  • nucleic acid fragment according to the invention Be transcribed fragment, wherein the nucleic acid fragment according to the invention is preferably 5 'upstream of the expression-regulated genes.
  • a “functionally active variant” in the sense of the present invention is understood to mean a nucleic acid sequence which was obtained from the sequence according to SEQ ID No. 1 by single or multiple nucleotide addition, insertion, substitution or deletion and a sequence homology of at least 25% of the sequence according to SEQ ID No. 1 and vascular endothelium-specific expression, preferably excluding expression in the liver.
  • a functionally active variant is understood to mean all those DNA sequences that are complementary to a DNA sequence that hybridize with the reference sequence under stringent conditions and a similar activity to the nucleic acid fragment according to the invention of the sequence according to SEQ ID No. 1 to have.
  • a functionally active variant can also contain sequences located upstream from position 105932 of BAC clone 50g21 (accession number AC005740), which is the first nucleotide of the nucleic acid fragment according to SEQ ID No. 1.
  • “Stringent hybridization conditions” are to be understood as those conditions in which hybridization takes place at 60 ° C. in 2.5 ⁇ SSC buffer, followed by several washing steps at 37 ° C. in a lower buffer concentration and remains stable.
  • Functionally active variants within the meaning of the invention are also nucleic acid fragments which preferably have a sequence homology of at least approximately 30%, 50% or 65%) or 80%, preferably at least approximately 90%, particularly preferably at least approximately 95% of the sequence after the Have SEQ ID No. 1.
  • Examples of these functionally active variants are therefore homologues of the Nucleic acid fragments according to the invention which originate from organisms other than human or mouse, preferably from non-human mammals such as monkeys, pigs and rats.
  • the activity of the nucleic acid fragment to be tested can be compared, for example, with the activity of the nucleic acid fragment according to SEQ ID No. 1. Assuming that the nucleic acid fragment to be tested fulfills the conditions of the functionally active variant at the level of the percent sequence homology, the nucleic acid fragment to be tested is a functionally active variant if the activity in the functional test is similar or is identical to the activity which the nucleic acid fragment according to SEQ ID No. 1 shows.
  • Such a functional test includes, for example, the tests described in Examples 2 and 3, in which the activity of the nucleic acid fragments according to the invention is examined for the expression of a reporter gene in various cells transfected with luciferase reporter gene constructs and which make it possible to measure the strength to evaluate the controlled expression and cell specificity of the regulatory activity of the nucleic acid fragments according to the invention.
  • nucleic acid fragment according to the invention contains a functional part of the sequence according to SEQ ID No. 1.
  • a functional part of 1-3804 bp is particularly preferred, a functional part of 2724-3804 bp according to the sequence is particularly preferred of SEQ ID No. 1.
  • a “functional part” in the sense of the present invention is understood to mean a nucleic acid sequence which was obtained from the sequence according to SEQ ID No. 1 nucleotide deletion at the 5 'or 3' end and has the same function as the unmodified one comprises nucleic acid fragment according to the invention.
  • a further embodiment of the invention relates to nucleic acid fragments according to the invention which have a sequence homology of at least approximately 80%, preferably of at least approximately 90%, particularly preferably of at least approximately 95% with the sequence according to SEQ ID No. 1 or with a functional part have of it.
  • Vascular endothelium-specific expression in the context of the invention is to be understood to mean that the expression of a gene or a functional part thereof or a gene which codes for a fusion protein on cells of the vascular endothelium is limited.
  • the vascular endothelium includes, for example, vascular cells that line the vessels (arteries and veins) and capillaries of the body of an adult or an embryo, including the precursors of these cells, as well as endothelial cells that cover the cardiovascular system including the endocardium, the heart valves and venous valves line.
  • vascular endothelial cells such as, for example, bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVEC).
  • BAEC bovine aortic endothelial cells
  • HUVEC human umbilical vein endothelial cells
  • Vascular endothelium-specific expression, except expression in the liver in the sense of the invention means that the expression of a gene or a functional part thereof or a gene which codes for a fusion protein on such cells of the vascular endothelium or cell lines as just stated is limited. Excepted from expression, i.e. the essentially lack of expression or the essentially impossible to detect expression are cells of the liver including endothelial cells of the liver. Preferably comprising the endothelial cells in the liver, vascular endothelial cells, mesenchymal cells and smooth muscle cells of the vessels and capillaries in the liver or cell lines derived from liver cells. Preference is also given to an essentially absent expression in all non-endothelial cells of the liver, including, for example, hepatocytes, copper star cells and epithelial cells.
  • the invention further relates to a nucleic acid construct containing a nucleic acid fragment according to the invention and a heterologous gene.
  • the heterologous gene codes for a therapeutically active gene product.
  • a nucleic acid construct in the sense of the present invention is understood to mean a nucleic acid fragment which contains a nucleic acid Fragment as well as functionally linked to it contains one or more heterologous genes.
  • this gene can be a marker gene or a gene that codes for a therapeutically active gene product.
  • a marker gene could be a fluorescent protein such as green fluorescent protein (GFP), beta-galactosidase, luciferase red fluorescent protein, yellow fluorescent protein, or His, Myc, or Flag tag linked to a heterologous gene.
  • GFP green fluorescent protein
  • beta-galactosidase beta-galactosidase
  • luciferase red fluorescent protein luciferase red fluorescent protein
  • yellow fluorescent protein or His, Myc, or Flag tag linked to a heterologous gene.
  • the "heterologous gene” is understood to mean a heterologous gene as defined above.
  • a therapeutically effective gene product could, for example, the isoforms of the hemoxygenase, the isoforms of the MCP (Monocyte Chemoattractant Protein) z.
  • MCP-1, GM-CSF the isoforms of nitrogen monoxide synthase (e.g. iNOS: inducible nitrogen monoxide synthase; eNOS: endothelial nitrogen monoxide synthase; nNOS: neuronal nitrogen monoxide synthase) or the Fas ligand.
  • nitrogen monoxide synthase e.g. iNOS: inducible nitrogen monoxide synthase
  • eNOS endothelial nitrogen monoxide synthase
  • nNOS neuronal nitrogen monoxide synthase
  • Hemoxygenase 1, MCP-1, iNOS are particularly preferred.
  • genes of interest for use as heterologous genes in gene therapy include tumor suppressor genes such as p53 (Takahashi et al, 1992, Cancer Res. 52, 2340-2343) and retinoblastoma or RB; Cell cycle blockers such as GATA-6 (Suzuki et al., Genomics, 1996, 38, 283-290); Anti-angiogenesis genes such as Endostatin and angistatin (Folkman K., Nature Med. 1, 27-31, 1995); Antisense gene sequences (Wang & Becker, Nature Med. 3, 887-893, 1997) and genes coding for viral subunits vaccine (Viral Subunit Vaccines, Donelly et al, Nature Med. 1, 583-587, 1995).
  • tumor suppressor genes such as p53 (Takahashi et al, 1992, Cancer Res. 52, 2340-2343) and retinoblastoma or RB
  • Cell cycle blockers such as GATA-6 (Suzuki et
  • beta-galactosidase reporter gene under the control of the 5'-hVE-CAD-2 promoter was expressed in many if not all vascular endothelial cells of transgenic animals. A significant exception to this were the vessels of the liver, in which there was essentially no activation of the reporter gene, i.e. essentially no expression of the reporter gene was detectable.
  • this provides a regulatory sequence, i.e. a nucleic acid fragment according to the invention which enables the connection of therapeutic genes in the vascular endothelium, but at the same time excludes the expression of toxic gene products in the liver. For the first time, this enables the gene therapy application of many genes with cytotoxic properties, such as those used to combat tumors, for the first time.
  • the invention relates to an isolated nucleic acid fragment which consists of the promoter of the human vascular endothelial cadherin-2 (hVE-CAD2) gene or functional parts thereof and is used for example for the expression of a transgene or a heterologous gene in vascular endothelial cells.
  • the hVE-Cad-2 promoter or the regulatory sequence preferably comprises the nucleic acid sequence according to the invention shown in FIG. 1 according to SEQ ID No. 1 of 5015 bp, which is immediately 5'-above the translation start of the hVE-CAD2 gene or a functional one Equivalent to this sequence.
  • Another object of the invention is to provide recombinant nucleic acid molecules which are suitable for gene expression in vascular endothelial cells and which contain the following functionally linked components:
  • genes i.e. heterologous genes or active parts thereof
  • heterologous genes can include any gene for which vascular endothelium-specific expression is required.
  • the polyadenylation signal can be of any suitable type.
  • a preferred embodiment in the 3 'position relative to the functional sequence preferably comprises an SV40 polyadenylation signal.
  • the recombinant nucleic acid molecules can comprise further conventional regulatory nucleotide sequences, e.g. Leader sequences, IRES sequences, enhancer sequences, polyadenylation signals and the expression rate in terms of quantity or sequences which control their chronological course.
  • regulatory nucleotide sequences e.g. Leader sequences, IRES sequences, enhancer sequences, polyadenylation signals and the expression rate in terms of quantity or sequences which control their chronological course.
  • Another object of the invention is to provide vector systems which contain the aforementioned recombinant nucleic acid molecules.
  • Vector systems in the sense of the invention are understood to mean vectors which contain a nucleic acid fragment according to the invention or a nucleic acid construct according to the invention.
  • a particularly preferred vector is selected from the group consisting of plasmids, shuttle vectors, phagemids, Cosmids, first or third generation of adenoviral vectors, expression vectors and gene therapy vectors.
  • the nucleic acid construct according to the invention or the vector according to the invention can also contain other regulatory sequences which are functionally linked to the regulatory sequences according to the invention.
  • the regulatory sequences are "functionally linked” if they are linked in a manner, preferably covalently linked, that the genes are placed under the influence of the transcription regulation of all functionally linked regulatory sequences.
  • the additional regulatory sequences include, for example, tetracycline-inducible sequences or other regulatory sequences that can be controlled by adding or removing a transcription-inducing agent.
  • cell cycle-dependent regulatory sequences can be used, such as the promoter of the E2F-1 gene, which enables expression control during the S phase and allows the expression of transgenes to be specifically controlled in actively dividing cells such as tumor cells (Parr et al. Nature Med., 1997, 3: 1145-1149).
  • heterologous genes By functionally linking different regulatory sequences, it is possible to control the expression of heterologous genes on the basis of a combination of parameters, such as, for example, tissue or cell specificity (ie vascular endothelium-specific expression, except expression in the liver), cell - cycle status-specific and expression based on the presence or absence of a transcription-inducing agent such as, for example, tetracycline, and thereby the expression of gene therapy genes are restricted to a certain group of cells.
  • tissue or cell specificity ie vascular endothelium-specific expression, except expression in the liver
  • cell - cycle status-specific based on the presence or absence of a transcription-inducing agent such as, for example, tetracycline
  • “Expression vectors” in the sense of the invention comprise at least one nucleic acid fragment according to the invention, at least one translation initiation signal, at least one heterologous gene, a translation termination signal and or a polyadenylation signal for expression in prokaryotes and or eukaryotes.
  • Suitable expression vectors can be prokaryotic or eukaryotic expression vectors.
  • prokaryotic expression vectors are e.g. for expression in E. coli the vectors pGEM or pUC derivatives and for eukaryotic expression vectors for expression in Saccharomyces cerevisiae e.g. the vectors p426MET25 or p426GALl (Mumberg et al. 1994, Nucl. Acids Res., 22, 5767-5768), for expression in insect cells e.g.
  • Bakulovirus vectors as described in EP-B1-0 127 839 or EP-B1-0 549 721, and for expression in mammalian cells for example the vectors Rc / CMV and Rc / RSV or SV40 vectors, all of which are general are available.
  • the invention further relates to the use of a nucleic acid fragment according to the invention or of a vector according to the invention for the expression of at least one heterologous gene.
  • the invention also relates to a knock-out gene construct which contains a nucleic acid fragment according to the invention.
  • Knock-out gene constructs are e.g. from U.S. Patents 5,625,122; US 5,698,765; US 5,583,278 and US 5,750,825 known.
  • the invention relates to a cell which contains a nucleic acid construct according to the invention, a vector according to the invention or a contains the appropriate knock-out gene construct.
  • the cell according to the invention can be used to express a heterologous gene.
  • Another preferred cell according to the invention is a cell selected from the group consisting of embryonic stem cells, embryonic germ cells and stem cells derived from adult tissue. Particularly preferred stem cells derived from adult tissue include, but are not limited to, neural stem cells, bone marrow stem cells, mesenchymal stem cells, hematopoietic stem cells, epithelial stem cells, gastrointestinal stem cells, and duct stem cells. "Duct" in the sense of the invention is understood to mean all ducts including the ductus arteriosus botalli.
  • the cells according to the invention can be produced, for example, by transfection of at least one cell with a nucleic acid fragment according to the invention, with a nucleic acid construct according to the invention, with a vector according to the invention or with a knock-out gene construct according to the invention.
  • the nucleic acid fragment can be in the form of a plasmid, as part of a viral or not -viral vector.
  • viral vectors baculoviruses, vaccinia viruses, adenoviruses, adeno-associated viruses and herpes viruses.
  • non-viral vectors virosomes, liposomes, cationic lipids, or poly-lysine-conjugated DNA.
  • vectors which are active in gene therapy are virus vectors, for example adenovirus vectors, retroviral vectors or vectors which are based on replicons of RNA viruses (Lindemann et al., 1997, Mol. Med. 3: 466-76; Springer et al., 1998, Mol. Cell. 2: 549-58; Khromykh, 2000, Curr. Opin. Mol. Ther .; 2: 555-69).
  • Eukaryotic expression vectors are suitable in isolated Form for gene therapy use, since naked DNA can penetrate skin cells, for example, when applied topically. (Hengge et al., 1996, J.Clin. Invest. 97: 2911-6; Yu et al., 1999, J. Invest. Dermatol. 112: 370-5).
  • Vectors with gene therapy effects can also be obtained by complexing the nucleic acid fragments according to the invention with liposomes.
  • lipofection small unilamellar vesicles are made from cationic lipids by ultrasound treatment of the liposome suspension.
  • the DNA is bound ionically on the surface of the liposomes in such a ratio that a positive net charge remains and the plasmid DNA is 100% complexed by the liposomes.
  • DOTMA 1,2-dioleyloxypropyl-3-trimethylammonium bromide
  • DPOE diioloxylphosphatidylethanolamine
  • lipid formulations are DOTAP N- [l- (2,3-dioleoyloxy) propyl] -N, N, N-trimethyl-ammoniumethyl-sulfate or DOGS (TRANSFECTAM; dioeta-decylamidoglycylspermin).
  • Auxiliaries that increase the transport of nucleic acids into the cells can be, for example, proteins or peptides that are bound to DNA or synthetic peptide-DNA molecules that enable the transport of the nucleic acid into the nucleus of the cell (Schwartz et al., 1999, Gene Therapy 6: 282; Branden et al. 1999, Nature Biotechs. 17: 784).
  • Auxiliaries also include molecules that enable the release of nucleic acids into the cytoplasm of the cell (Planck et al., 1994, J. Biol. Chem. 269, 12918; Kichler et al., 1997, Bioconj. Chem.
  • the cells of the invention can be used to express a heterologous gene.
  • the invention further relates to a cell, the cell being in particular a mammalian cell, including human cells.
  • the invention further relates to a cell according to the invention, the cell originating from a cell line.
  • the cell line is preferably selected from the group consisting of embryonic stem cells, embryonic germ cells and stem cells derived from adult tissue.
  • Particularly preferred stem cells derived from adult tissue include, but are not limited to, neural stem cells, bone marrow stem cells, mesenchymal stem cells, hematopoietic stem cells, epithelial stem cells, digestive stem cells, and duct stem cells.
  • a cell line according to the invention can be produced by transfection, transformation or infection of a cell line with a nucleic acid fragment according to the invention or a vector according to the invention with the aid of methods which have been explained above.
  • the invention relates to a cell, the cell being a transgenic non-human stem cell.
  • the stem cell contains a nucleic acid construct according to the invention, a vector according to the invention or a knock-out gene construct according to the invention.
  • Transgenic non-human stem cells can be produced by transfecting the stem cell with a nucleic acid fragment according to the invention or with a vector according to the invention or with a knock-out gene construct according to the invention.
  • Methods for transforming stem cells are known to the person skilled in the art and include, for example. Electroporation or microinjection. Another object of the invention is to provide a method for expressing one or more genes in the vascular endothelium of animals.
  • the method comprises the transfer of one or more genes, ie heterologous genes, into the animal mentioned, each nucleic acid construct containing the nucleic acid fragment according to the invention and also functionally linked to it, ie functionally linked, containing one or more heterologous genes.
  • nucleic acid construct mentioned can be transferred, for example, by means of viral vectors or liposomal administration systems, as described, for example, by Evans R. et al. (1994) Ann N Y Acad Sei. 716: 257-264.
  • the said construct can be transferred into an animal in such a way that it is injected into the fertilized egg cell by microinjection from which the said animal originates. This is a standard technique that can be carried out by a trained specialist.
  • the invention thus furthermore relates to transgenic non-human animals, preferably mammals, which contain at least one nucleic acid construct according to the invention or at least one cell according to the invention, ie at least one non-human stem cell.
  • Transgenic non-human animals according to the invention can be produced, for example, by regenerating a cell according to the invention, ie a transgenic non-human stem cell, into a transgenic non-human animal.
  • the transgenic non-human animal according to the invention can e.g. B. for the expression of a heterologous gene in the sense of the invention or for the analysis of gene therapy-effective nucleic acids or vectors according to the invention.
  • the invention further relates to a method for producing a transgenic non-human animal according to the invention, characterized in that a cell according to the invention, ie a transgenic non-human stem cell, is regenerated into a transgenic non-human animal.
  • a cell according to the invention ie a transgenic non-human stem cell
  • Processes for producing transgenic animals, in particular transgenic mice, are known to the person skilled in the art from DE 196 25 049 and US Pat. No.
  • transgenic animals which can be generated, for example, by direct injection of expression vectors according to the invention into embryos or spermatocytes or via the transfection of expression vectors into embryonic stem cells
  • knock-out mice can be generated, which are generally heterozygous mice show reduced expression of the nucleic acid, while homozygous mice no longer have expression of the nucleic acid.
  • the animals produced in this way can also be used for analysis, for example for screening, and for the identification of pharmacologically active substances which interact with the nucleic acid fragments according to the invention.
  • the invention further relates to a test for the identification of pharmacologically active substances which modulate the function of the nucleic acid fragments according to the invention, the test containing at least one nucleic acid fragment according to the invention, at least one vector according to the invention and / or at least one cell according to the invention , optionally combined or together with suitable additives and / or auxiliaries.
  • the test according to the invention can be used to identify pharmacologically active substances which modulate the function of the nucleic acid fragments according to the invention.
  • pharmacologically active substance within the meaning of the invention is understood to mean all those molecules, compounds and / or compositions and substance mixtures which can interact under suitable conditions with the nucleic acid fragments according to the invention, if appropriate together or in combination with suitable auxiliaries and / or additives.
  • Possible pharmacologically active substances are simple chemical (organic or inorganic) molecules or compounds; peptides, proteins or complexes thereof can also be included.
  • Examples of pharmacologically active substances are organic molecules which come from compound libraries and which have been examined for their pharmacological activity.
  • the pharmacologically active substances can influence the function (s) of the nucleic acid fragments according to the invention in vivo or in vitro or alternatively only bind to the nucleic acid fragments according to the invention or enter into other interactions with them in a covalent or non-covalent manner ,
  • the nucleic acid sequences, the transcription of which is controlled or modulated by the nucleic acid fragments according to the invention are preferably located 3 'downstream of the nucleic acid fragments according to the invention.
  • the function of the nucleic acid fragment according to the invention comprises initiation of the transcription, modulation of the transcription, ie activation or inhibition of the transcription of the transcription-controlled nucleic acid sequences.
  • the nucleic acid fragments according to the invention can increase the transcription rate, which leads to a faster production of the transcripts or an increase in the transcription duration, as a result of which a larger number of transcripts is generated compared to control values.
  • the nucleic acid fragments according to the invention can bring about a reduction in the transcription rate, which causes a slower synthesis of the transcripts or a shortening of the duration of the transcription, as a result of which a reduced number of transcripts is produced in comparison with control values.
  • the pharmacologically active substances influence the function of a nucleic acid fragment according to the invention in an activating or inhibiting manner.
  • the transcription controlled or modulated by the nucleic acids according to the invention should be increased, whereby an increase in the amount of the gene expression product compared to the expression determined in the absence of the pharmacologically active substance is achieved.
  • the transcription controlled or modulated by nucleic acid fragments according to the invention should be inhibited, as a result of which a decrease in the amount of the gene expression product compared to the strength of the expression, determined in the absence of the pharmacologically active substance, is brought about.
  • a particularly preferred embodiment of the test is provided by modifying the test described in Example 2 or 3.
  • the hVE-2-5.0 vector for example, it is possible to use the hVE-2-5.0 vector.
  • the cloned PCR fragment hVE-PCR1 which is used in Example 2, can be replaced by any nucleic acid fragment according to the invention.
  • the pharmacologically active substance to be tested can be added to a group of the transfected cells and the cells are cultivated, for example, for 48 hours.
  • the pharmacologically active substance to be tested is not supplied to a control group of transfected cells and the cells are cultivated in the same way as the other group of cells.
  • the expression of the reporter gene in the various cells is then measured and the effect of the pharmacologically active substance to be tested on the transcription control of the nucleic acid fragments according to the invention is determined.
  • Pharmacologically active substances in the sense of the invention are those substances which have a modulatory influence, ie inhibiting or activating, on transcription which is controlled and / or modulated by nucleic acid fragments according to the invention. This is determined by the rate and the kinetics of the expression of the reporter gene.
  • the test is not limited to the use of the vectors and reporter genes used in Examples 2 and 3. Other expression vectors and other reporter genes mentioned above can also be used.
  • Such tests according to the invention can be produced by combining at least one nucleic acid fragment according to the invention, at least one vector according to the invention and / or at least one cell according to the invention with suitable additives and / or auxiliaries.
  • the identified pharmacologically active substances can optionally be combined or together with suitable additives and / or auxiliary substances for the production of a diagnostic agent or a medicament for the diagnosis, prevention and / or treatment of diseases selected from the group consisting of vascular diseases, genetically determined diseases, diseases which associated with pathological vasodilation or vasoconstriction, atherosclerosis, diabetes, cancerous diseases, inflammatory diseases and / or immunogenic diseases.
  • the pharmacologically active substances can be, for example, inorganic or organic molecules, for example nucleic acids or analogs of nucleic acids, anti-sense sequences of nucleic acid fragments according to the invention, peptides, proteins or antibodies.
  • examples of pharmacologically active substances are furthermore organic molecules contained in substance libraries which have been examined for their pharmacological activity.
  • the invention further relates to an array bound to a carrier material which comprises at least one nucleic acid fragment according to the invention and / or at least one cell according to the invention.
  • a carrier material which comprises at least one nucleic acid fragment according to the invention and / or at least one cell according to the invention.
  • Such arrays can be used, for example, for the identification of substances that bind to nucleic acid fragments according to the invention, which in turn can be used again as potential pharmacologically active substances in the test described above.
  • the array can also be used for analysis in connection with diseases selected from the group consisting of vascular diseases, genetic diseases, diseases associated with pathological vasodelation or vasoconstriction, atherosclerosis, diabetes, cancerous diseases, inflammatory diseases and / or immunogenic diseases.
  • An array according to the invention can be produced by fixing at least one nucleic acid fragment according to the invention and / or at least one cell according to the invention on a carrier material.
  • Carrier material in the sense of the invention is understood to mean, for example, porous materials such as nitrocellulose, but also non-porous materials such as glass, chemically sensitized glass, as described, for example, in WO 98/18961
  • Arrays according to the invention can be brought into contact with substances or substance libraries and can respond to interactions, e.g. for binding or confirmation changes. It is also possible that substances to be tested contain a detectable marker, e.g. the substance can be radioactively labeled, fluorescence-labeled or luminescence-labeled or contain a marker that allows indirect detection, e.g. Biotin.
  • a diagnostic agent in a further embodiment of the invention, is provided, the diagnostic agent having at least one nucleic acid fragment according to the invention, at least one nucleic acid construct according to the invention, at least one vector and / or at least one cell according to the invention, optionally together or in combination with suitable auxiliaries or additives , contains.
  • a diagnostic agent can be used, for example, for the diagnosis of diseases selected from the group consisting of vascular diseases, genetically determined diseases, diseases associated with pathological vasodelation or vasoconstriction, atherosclerosis, diabetes, cancerous diseases, inflammatory diseases and / or immunogenic diseases be used.
  • a diagnostic agent according to the invention can be obtained by combining at least one nucleic acid fragment according to the invention, at least one nucleic acid construct according to the invention, at least one vector according to the invention and / or at least one cell according to the invention can be produced with suitable additives and / or auxiliary substances.
  • a diagnostic agent according to the invention preferably contains a nucleic acid fragment according to the invention, the nucleic acid fragment being a DNA probe.
  • Suitable probes are e.g. DNA or RNA fragments with a length of approximately 100-1000 nucleotides, preferably with a length of approximately 200-500 nucleotides, particularly preferably with a length of approximately 300-400 nucleotides, the sequence following the nucleic acid fragment according to SEQ ID NO 1 can originate or a functionally active variant thereof.
  • nucleic acid fragments according to the invention it is possible to use the nucleic acid fragments according to the invention to synthesize oligonucleotides which are suitable as primers for the polymerase chain reaction.
  • oligonucleotides which are suitable as primers for the polymerase chain reaction.
  • the nucleic acid fragments described above or parts thereof can be duplicated or isolated from genomic DNA.
  • Suitable primers are e.g. DNA fragments with a length of approximately 10-100, preferably with a length of approximately 15-50 nucleotides, particularly preferably with a length of approximately 20-30 nucleotides, the sequence of which is obtained from the nucleic acid fragment according to SEQ ID No. 1 can be. This opens another way to identify mutations in the regulatory sequence of the VE-Cad-2 gene that could cause disease, especially genetic disorders of the vascular epithelium. Such methods are generally known to the person skilled in the art.
  • the invention further relates to a medicament, the medicament comprising at least one nucleic acid fragment according to the invention, at least one nucleic acid construct according to the invention, at least one vector according to the invention, and / or at least one cell according to the invention, optionally together or in combination with suitable additives and / or auxiliary substances.
  • a medicament comprising at least one nucleic acid fragment according to the invention, at least one nucleic acid construct according to the invention, at least one vector according to the invention, and / or at least one cell according to the invention, optionally together or in combination with suitable additives and / or auxiliary substances.
  • the use of the medicament for somatic gene therapy is particularly preferred.
  • a medicament according to the invention can be produced by combining at least one nucleic acid fragment according to the invention, at least one nucleic acid construct according to the invention, at least one vector according to the invention and / or at least one cell according to the invention with suitable additives and / or auxiliaries.
  • the invention further relates to a method for the treatment of mammals or humans, wherein a pharmaceutically effective amount of the medicament according to the invention is administered to the mammal or human.
  • the drug is preferably administered by a method selected from the group consisting of systemic injection, local injection, perfusion or catheter-based administration.
  • the drug can be introduced into the organism either using the ex vivo approach, in which the cells are removed from the patient, genetically modified by DNA transfection and then reintroduced into the patient, or using the in vivo approach, in which the gene therapeutic effective vectors are introduced into the patient's body, as naked DNA or using viral or non-viral vectors or cells according to the invention.
  • a suitable medicament is, for example, one which contains a nucleic acid fragment according to the invention in naked form or in the form of a gene-therapeutic vector as stated above or in complexed form with liposomes or gold particles.
  • Suitable additives or auxiliaries include, for example, a physiological buffer solution, preferably with a pH of approximately 6.0-8.0, preferably approximately 6.8-7.8, particularly preferably approximately 7.4 and / or an osmolarity of approximately 200-400 milliosmol / l, preferably from approximately 290-310 milliosmol I.
  • suitable stabilizers such as nuclease inhibitors, preferably complexing agents such as EDTA and / or other auxiliaries known to the person skilled in the art can be contained in the additives.
  • the nucleic acid fragment described can optionally be administered in the form of a viral vector as described above or as a liposome complex or gold particle complex with the aid of perfusion, systemic injection or local injection or catheter-based administration.
  • the pharmaceutical composition of the invention can also be administered in oral dosage forms such as e.g. Tablets or capsules over the mucous membrane e.g. the nose or oral cavity is implanted in the form of sprays in the lungs or in the form of disposers under the skin.
  • Transdermal therapeutic systems are e.g. known from EP 0 944 398-A1, EP 0 916 336-A1, EP 0 889 723-A1 or EP 0 852 493-A1.
  • a treatment based on the use of cells according to the invention which express at least one heterologous gene as described above can be achieved in that cells according to the invention selected from the group consisting of epithelial cells, vascular cells, liver cells, embryonic stem cells, embryonic germ cells, from adult Tissue-derived stem cells.
  • Preferred stem cells derived from adult tissue include, but are not limited to, neuronal stem cells, bone marrow stem cells, mesenchymal stem cells, hematopoietic stem cells, epithelial cells Stem cells, stem cells from the digestive tract and duct stem cells are used.
  • the medicament according to the invention for the prevention and / or treatment of diseases can be selected from the group consisting of vascular diseases, genetically caused diseases, diseases associated with pathological vasodilation or vasoconstriction, atherosclerosis, diabetes, cancerous diseases, inflammatory diseases and / or immunogenic diseases.
  • the invention relates to a method for identifying nucleic acid fragments according to the invention, comprising the steps:
  • nucleic acid fragments that enable vascular endothelial-specific expression, preferably excluding expression in the liver.
  • a “variant” is understood to mean a nucleic acid sequence which is obtained from the sequence according to SEQ ID No. 1 by adding, inserting, substituting or deleting one or more nucleotides.
  • the reporter gene used in the method for identifying nucleic acid fragments according to the invention can be selected from the group consisting of beta-gallactosidase, luciferase, green fluorescent protein (GFP), red fluorescent protein, yellow fluorescent protein , and His, Myc, or Flag tag linked to a heterologous gene.
  • GFP green fluorescent protein
  • red fluorescent protein red fluorescent protein
  • yellow fluorescent protein yellow fluorescent protein
  • His, Myc His, Myc
  • Flag tag linked to a heterologous gene can be selected from the group consisting of beta-gallactosidase, luciferase, green fluorescent protein (GFP), red fluorescent protein, yellow fluorescent protein , and His, Myc, or Flag tag linked to a heterologous gene.
  • GFP green fluorescent protein
  • His, Myc His, Myc, or Flag tag linked to a heterologous gene.
  • a “heterologous gene” is understood to mean a heterologous gene as defined above.
  • a reporter gene expression vector into cells or cell lines is not limited to transfection, but can also be carried out by transformation, injection, by "gene gun” bombardment or by other methods for introducing nucleic acids into cells which are generally known to the person skilled in the art , can be achieved.
  • the cells and cell lines according to the invention described above can be used for the purpose of the method for identifying nucleic acid fragments.
  • the reporter gene is luciferase
  • the expression strength of the reporter gene can be measured, for example, by using the luciferase assay system or the BCA protein assay system as described in Example 3 , be achieved.
  • other assays may be required to quantify the reporter gene expression products, which are generally known to those skilled in the art are known.
  • the cell specificity of the transcription control properties of the nucleic acid fragments to be tested can be compared with the transcription control properties of the nucleic acid fragments according to the invention, preferably nucleic acid fragments of the sequence according to SEQ ID No. 1 and lead to the identification of nucleic acid fragments which show similar or different transcription control properties as the nucleic acid fragments according to the invention and which enable vascular endothelial-specific expression, preferably excluding expression in the liver.
  • the identified nucleic acid fragments could, for example, induce an even more sustained and / or stronger expression of the reporter gene.
  • Such identified nucleic acid fragments could be even more suitable for use in gene therapy, since the gene therapy-active gene products could thus be produced in larger quantities over a longer period of time.
  • the invention further relates to a method for producing a vector, drug or diagnostic agent effective for gene therapy, wherein a nucleic acid fragment identified with the aid of the method just described is introduced into a vector containing at least one heterologous gene and vascular endothelium-specific expression, preferably with the exception of expression in the liver.
  • Methods for the production of genetically therapeutically active vectors, drugs or diagnostics have been described above.
  • the invention further relates to the use of a gene therapy-active vector, pharmaceutical or diagnostic agent for the diagnosis, prevention and / or therapy of diseases selected from the group consisting of vascular diseases, produced by the method just described.
  • diseases selected from the group consisting of vascular diseases, produced by the method just described.
  • the invention also relates to a method for selecting and / or immortalizing vascular endothelial cells from stem cells into which a construct according to the invention has been transferred using standard methods or which can be isolated by a person skilled in the art from transgenic animals or their embryos using well-characterized methods, which contain a construct according to the Invention.
  • the invention further relates to a method for selecting endothelial cells from stem cells, comprising the following steps: (1) combining at least one nucleic acid according to the invention
  • the stem cell (s) used in the selection process include embryonic stem cells, embryonic germ cells, and stem cells derived from adult tissue.
  • the stem cells derived from adult tissue include, but are not limited to, neuronal stem cells. cells, bone marrow stem cells, mesenchymal stem cells, hematopoietic stem cells, epithelial stem cells, digestive stem cells and duct stem cells.
  • the differentiation of the cultivated stem cells which are used for the selection in the method according to the invention can e.g. by "embroid body formation", preferably by culturing the stem cells in solutions, by culturing the stem cells in high density, by adding cytokines, growth factors, retinoic acid or DMSO to the cultivated cells or by adding other substances which are known to be they initiate differentiation, are initiated.
  • embroid body formation preferably by culturing the stem cells in solutions, by culturing the stem cells in high density, by adding cytokines, growth factors, retinoic acid or DMSO to the cultivated cells or by adding other substances which are known to be they initiate differentiation, are initiated.
  • a preferred embodiment of the invention relates to a method for selection, wherein the reporter gene is an antibiotic resistance gene and the endothelial cell (s) is (are) isolated by adding the differentiated endothelial cell (s) suitable antibiotic is (are) collected in step (3) or (4).
  • An antibiotic according to the invention means an antibiotic against which the antibiotic resistance gene used in the reporter gene expression vector generates resistance. After the antibiotic has been added to the cultivated stem cells, only those stem cells which contain the reporter gene expression vector essentially survive and differentiate.
  • a method for selection is provided, the antibiotic resistance gene being selected from the group consisting of hygromycin resistance gene (hph), zeocin Resistance gene (Sh ble), puromycin resistance gene (pacA) and gentamycin or G418 resistance gene (aph).
  • hph hygromycin resistance gene
  • Sh ble zeocin Resistance gene
  • pacA puromycin resistance gene
  • aph gentamycin or G418 resistance gene
  • the method for selection according to the invention relates to a selection method, the reporter gene being selected from the group consisting of luciferase, green fluorescent protein, red fluorescent protein, and yellow fluorescent protein, and the endothelial cell ( n) is (are) isolated from the cultivated cell (s) by means of fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • the reporter gene being selected from the group consisting of beta-galactosidase, luciferase, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, or His, Myc, bound to a heterologous gene. , or Flag-Tag, and the endothelial cell (s) is (are) isolated from the cultured cells by means of affinity purification.
  • a “heterologous gene” is understood to mean a heterologous gene as defined above.
  • the selected and / or immortalized vascular endothelial cells can be used for cell-mediated transplantation, for generating artificial vessels in vitro for the production of artificial heart or venous valves and for somatic gene transfer in vivo.
  • the invention further relates to a method for producing an artificial tissue or organ containing at least one endothelial cell, wherein at least one endothelial cell obtained by the inventive method for selection described above is combined and cultivated with at least one suitable cell and / or a carrier to produce the artificial tissue or organ.
  • the artificial tissue is preferably selected from the group consisting of vessels, cardiac valves and venous valves.
  • a "suitable cell” in the sense of the invention means a nutrient cell or a cell other than the endothelial cell obtained by the selection process, which is required for the formation of the artificial organ or tissue or the part of the artificial tissue or Organ is.
  • suitable cells are known to the person skilled in the art.
  • a “carrier” in the sense of the invention is understood to mean a substance, molecule or material or matrix which serves as chemical, physiological or mechanical support for the tissue or organ to be produced. Such carriers are known to the person skilled in the art.
  • the invention further relates to a method for testing the pharmacological activity of a pharmacological substance, wherein at least one endothelial cell obtained by the method according to the invention is exposed to the pharmacological substance and the pharmacological activity of the pharmacological substance is determined.
  • a "pharmacological substance” in the sense of the invention means any substances, compounds, mixtures or compositions. Possible pharmacological substances are simple chemical (organic or inorganic) molecules or compounds, but they can also contain peptides, proteins or complexes thereof. Examples of pharmacological substances are toxic substances, compounds, mixtures or compositions; or organic molecules that come from compound libraries and that have been examined for their pharmacological activity.
  • “Pharmacological activity” in the sense of the invention means any response to a cell exposed to a pharmacological substance the level of morphology, metabolism, physiology or genetic activity.
  • the pharmacological activity of the cell tested can be examined at the level of vitality or apoptosis, i.e. the selected endothelial cell is exposed to the toxic substance, compound, mixture or composition and the vitality of the cell is determined.
  • This enables e.g. Identify endothelial cells that are resistant to a tested toxic substance or that enable the identification of toxic substances, compounds, mixtures or compositions that are useful for the induction of apoptosis.
  • Vitality and apoptosis tests are generally known to the person skilled in the art.
  • Methods according to the invention for testing the pharmacological activity can also be used for "high-throughput" screening (screening) of pharmacological substances which show interesting diagnostic or therapeutic properties in a selected cell type.
  • screening screening of pharmacological substances which show interesting diagnostic or therapeutic properties in a selected cell type.
  • the susceptibility or sensitivity of a selected endothelial cell exposed to a pharmacological substance to the pharmacological substance can be determined, as a result of which pharmacological substances with interesting susceptibility or sensitivity properties can be identified.
  • these cells can be used to establish an in vitro vascular endothelial cell model.
  • an in vitro model is suitable for the investigation of potentially therapeutically active substances, in particular for pharmacological examinations.
  • 1 shows the nucleic acid sequence of a 5015 base pair long 5 'upstream from the translation start point (ATGATG; position 5016-
  • the transcription start is at position 3805 (+1).
  • the recognition sequence for the restriction endonuclease EcoRV is at position 513-518, for Sacl at position 2725-2730, for Hindlll at position 3704-3709 and for Smal at position 4385-4390.
  • the top scheme (A) shows the genomic structure of the human VE-CAD2 promoter.
  • the first exon was identified by a box from the start of translation.
  • the arrow inside the box indicates the reading direction of the VE-CAD2 gene.
  • Interfaces for restriction endonucleases that were used for the cloning were identified (EcoRV, Sacl, Hindill, Smal).
  • hVE-PCRl contains at the 5 'end ne interface for the restriction enzyme Kpnl and at the 3 'end an interface for the restriction enzyme Seal.
  • C-G show the structure of reporter gene constructs which were obtained by cloning promoter fragments into the plasmid pGL3-basic (Promega).
  • pGL3-basic contains the coding sequence of the marker luciferase.
  • the luciferase (Luc) gene was identified as a box (Luc).
  • the bacterial components of the circular plasmids were not shown.
  • FIG. 3 shows the luciferase activity in different cells after transfection with the different reporter gene constructs from FIG. 2 in cell culture.
  • the mean values and the standard deviation (SEM) were given for the measured light units relative to the promoterless plasmid pGL3-basic, which was used as the control plasmid.
  • the following cells were transfected: primary endothelial cells from the human umbilical vein (human umbilical vein endothelial cells, HUVEC), primary endothelial cells from the bovine aorta (bovine aortic endothelial cells, BAEC), human tumor cell line (HeLa), fibroblast cell line from rodents (NIH 3T3). Each transfection was repeated 6 times.
  • FIG. 4 shows the schematic structure of constructs for generating transgenic mice.
  • (A) is the genomic structure of the hVE-CAD2 Promoter shown, which is already described under Figure 1 (A).
  • the construct pRZ-hVE-Zl (B) contains 5015 bp of the VE-CAD2 promoter coupled to the reporter gene beta-galactosidase (LacZ gene; shown as a white box).
  • the graphic shows the proportion of the construct obtained by cutting with the enzymes Sall and Notl and for which
  • Notl were used for the cloning of the constructs and for the analysis of transgenic animals.
  • Fig. 5 shows the X-Gal staining in transgenic pRZ-hVE-Zl mice of the two established transgenic strains (the strains were called pRZ-hVE-Zl- # 10 and pRZ-hVE-Zl- # 54). The pattern and intensity of the staining were the same in all strains examined in both strains.
  • (A) shows an embryo on day 10.5 of development, while the embryo under (B) was 12.5 days old (embryos of the transgenic strain pRZ-hVE-Zl- # 10 are shown).
  • the lacZ reporter gene is developed in developing vascular structures, such as, for example, capillaries in the head region, vessels lying between the somites, the head systems and the developing systems of the limbs (limb buds) (embryonic day 12.5; FIG. 5B). Sections of these embryos confirmed that the beta-galactosidase protein was restricted to the vascular endothelium (section data not shown).
  • the embryo under (C) was stained 15.5 on embryonic day. For this purpose, the skin was first removed and the embryo was then split lengthways to allow the dye to penetrate. At this stage, the reporter gene was intense in all of the developing blood vessels Skeletal muscles of the whole embryo expressed.
  • Kidney, heart and pancreas reporter gene expression was limited to the monocellular vascular endothelium of the blood vessels and capillaries.
  • An example shows the ventral view under (D) and the dorsal view of a heart under (E).
  • (F) shows an enlarged partial view of the illustration (E), with the X-Gal staining in all
  • Fig. 6 Northern blot analysis of VE-Cad-2 expression in adult mouse and human tissue.
  • Murine VE-Cad-2 mRNA is at the position of approximately 7 kb. Highly vascularized organs such as the lungs, heart, spleen, liver and kidney show a pronounced expression of VE-Cad-2.
  • mRNA of human VE-Cad-2 is approximately at position 4.4 kb. With the exception of the brain, the expression of human VE-Cad-2 is detectable in all organs examined, with pronouncedly high levels of expression in the heart, placenta, lungs and liver.
  • Fig. 7 RT-PCR analysis of VE-Cad-2 and beta-galactosidase expression in tissues of adult transgenic pRZ-hVE-Zl # 10 (left column), pRZ-hVE-Zl (middle column) and wild-type CD 1 mice (right column).
  • the middle and right column PCR products (divided by a broken line) are located on the same gel and therefore there is only one molecular weight marker (M) for these two columns, while the left column PCR products on a separate one Gel were run.
  • Ubiquitously expressed genes - "housekeeping" gene GAPDH and the ribosomal gene L7 - were detected in comparable strengths in the cDNA from all examined tissues in both transgenic and wild-type mice.
  • VE-Cad-2 expression could also be detected in slightly different strengths in all mice and all examined tissues including lungs, liver, heart, spleen and kidney. Beta-galactosidase expression was only in transgenic animals of the established strain pRZ-hVE-Zl # 10 and pRZ-hVE-
  • the published cDNA for the VE-CAD2 gene of the mouse (P. Telo et al., JBC 1998; accession number Y08715) was used for a genebank search (NCBI, basic BLAST). Sequence sections were found on a human BAC (bacterial artificial chromosome) which have a sequence homology of 81-96% to the cDNA of the mouse.
  • the BAC is described as follows: Homo sapiens chromosome 5p, BAC clone 50g21 (LBNL H154), complete sequence. The reference number is AC005740. The published sequence spans 186780 base pairs.
  • the BAC was obtained as an E.
  • the bacterial clone was cultivated and the BAC-DNA was isolated using a DNA purification kit (Qiagen, Hilden, Germany). Due to the homology to the cDNA of the murine VE-CAD2 gene, the translation start of the human gene was identified, which is located at position 110948 of the published BAC sequence.
  • the promoter was isolated using the PCR technique (polymerase chain reaction). The primers were defined based on the BAC sequence.
  • the 5 "primer hVE2-chrF2 (forward primer) lies between positions 105933 and 105956 of the BAC sequence.
  • the primer contains a recognition site for the restriction enzyme Kpnl and 6 random bases (n) PCR product obtained.
  • the primer sequence is: 5'- nnn nnn ggt acc cag aag tag tgc cct tcc tct cga - 3 '(SEQ ID No. 2).
  • the orientation of the S' primer hVE2-UP-ATG (reverse The primer lies between positions 110947 and 110924 of the BAC sequence and is set in such a way that the translation start of the human VE-CAD2 gene (adenosine at position 110948). Is not included.
  • hVE2-UP-ATG contains a recognition site for the restriction enzyme Seal and 6 randomly chosen nucleotides and has the following sequence: 5 ⁇ - nnn nnn agt act get tac ege aac gtg ggc tag att - 3' (SEQ ID no. 3).
  • 5 ⁇ - nnn nnn agt act get tac ege aac gtg ggc tag att - 3' (SEQ ID no. 3).
  • 50ng on purified BAC-DNA are used in a 50 ⁇ l PCR mixture.
  • the fragment obtained, which is referred to as hVE-PCR1 contains 5015 bp of the hVE-CAD2 promoter and is cut with Kpnl and Seal for cloning reporter gene constructs (cf. Example 2 and Example 4). The fragment was checked by sequencing.
  • hVE-PCR1 The PCR fragment hVE-PCR1 is used to clone the constructs (cf. Example 1).
  • hVE-PCRl contains an interface for the restriction enzyme Kpnl at the 5 'end and an interface for the restriction enzyme Seal at the 3' end.
  • the construct hVE2-5.0 kb (cf. FIG. 1C) is produced by cutting hVE-PCRl with Kpnl and Seal and cloning into the Kpnl and Smal interfaces of the plasmid pGL3-Basic (Seal and Smal interfaces are compatible). All other constructs are truncations of the hVE-2 5.0 kb construct at the 5 'end of the promoter.
  • hVE2-4.5 kb (cf. FIG. 1 D)
  • hVE-PCRl is cut with EcoRV and Seal and the 4.5 kb fragment is cloned into the Smal site of pGL3-basic. All interfaces are compatible.
  • hVE2-2.3 kb (cf. FIG. 1E) is obtained by cutting hVE-PCRl with Sacl and Seal. The 2.3 kb fragment is cloned into the SacI and Smal cut plamide pGL3-Basic.
  • hVE2-1.3 kb (cf. FIG.
  • hVE-PCRl is cut with Hindill and Seal and cloned into the Kpnl and Smal cut vector pGL3-basic. The ends are smoothed beforehand by treatment with T4-DNA polymerase.
  • the construct hVE2-0.6 (see FIG. 1 G) is created in the same way, with hVE-PCRl with Smal and Scal cut and the 0.6 kb fragment obtained is cloned into the Smal cut pGL3 basic.
  • Luciferase reporter gene constructs were used to transfect the following cells: primary endothelial cells from the human umbilical vein (human umbilical vein endothelial cells, HUVEC), primary endothelial cells from the bovine aorta (bovine aortic endothelial cells, BAEC), human Tumor cell line (HeLa), fibroblast rodent cell line (NIH-3T3). 2-3x10 5 cells are sown in DMEM with 10% FCS on a 6-well cell culture plate. The transfection is carried out 12 hours after the cells have been sown.
  • 1.5 ⁇ g of the respective plasmid DNA are mixed with 3.25 ⁇ l ExGen solution (MBI Fermentas) and a 150 mM sodium chloride solution in a 100 ⁇ l mixture.
  • the transfection solution is mixed with 1 ml of serum-free medium and applied to the cells for one hour.
  • the cells are then cultivated in medium containing serum for 48 hours.
  • the luciferase determination (Luciferase Assay System, Promega, Cat. No. E4030) and the protein measurement (BCA-Protein Assay, Pierce, Cat. No. 23223)
  • the cells are washed with PBS buffer and in 200 ⁇ l lysis buffer solved. 20 ⁇ l of the lysate are used for each assay.
  • the implementation takes place according to the manufacturer's instructions.
  • the luciferase measurement was carried out in a Packard luminometer. Each sample is measured twice. The luciferase values were compared via the protein determination.
  • the promoter-free plasmid pGL3-basic was transfected. The values of 6 independent transfection experiments were evaluated and averaged. These values were divided by the mean values after transfection with pGL3-basic and thus related to this control plasmid. The results are shown in the figure. 1. Expression of Luciferase Reporter Gene Constructs in Cell Culture
  • FIG. 3 show that the hVE-CAD-2 promoter is specifically switched on in cultured endothelial cells.
  • the construct hVE2-1, 3 brings about a clear activation of the luciferase gene only in arterial endothelial cells, whereas only background activity is observed in venous cells that does not differ from expression in control cell lines.
  • the c-D ⁇ A for the hVE-CAD-2 gene is published in the ⁇ CBI database under the access number AF240635.
  • the start of transcription of the 1211 bp gene is above the start of translation (see FIG. 1).
  • the construct hVE2-1,3 contains 1310 bp above the start of translation and thus obviously only 99 bp of the promoter 5 'above the start of transcription. This short promoter fragment nevertheless leads to a limited endothelial cell-specific expression.
  • the construct hVE2-0.6 which contains only transcribed sequence, shows, as expected, no promoter activity regardless of the cell line used.
  • the data show that the hVE-CAD-2 promoter mediates specific gene expression in vascular endothelial cells in vitro.
  • constructs containing 5.0 kb, 4.5 kb or 2.3 kb promoter fragments There is no clear difference between constructs containing 5.0 kb, 4.5 kb or 2.3 kb promoter fragments.
  • a sequence of just 2.3 kb is sufficient to effect high and vascular endothelial cell-specific gene expression in cell culture.
  • this observation does not allow a reliable statement about possible differences of the promoter fragments mentioned for gene expression in vivo.
  • Example 4 Production of transgenic mice by microiniection of beta-galactosidase constructs.
  • the PCR product hVE-PCRl (cf. Example 1 and Figure 1), which contains the human VE-CAD2 promoter, is cut with Kpnl and Seal and into the plasmid cut with Sall and Smal pPD 46.21 (A. Fire et al., Gene, 1990).
  • the ends of the DNA fragments are smoothed by treatment with T4 DNA polymerase.
  • the plasmid pPD 46.21 contains the coding sequence for the reporter gene beta-galactosidase (lacZ) from E. coli coupled to a nuclear localization signal which effects the transport of the protein into the cell nucleus.
  • the construct pRZ-hVE-Zl has a total size of 11.1 kb.
  • the construct is cut with Sall and Notl and the 8.5 kb fragment shown in FIG. 4 (B) is obtained, which contains the 5015 bp VE-CAD2 promoter and the LacZ gene (with a SV- 40 polyadenylation signal; not shown).
  • transgenic mice are generated as described by Hogan B. (Hogan B. et al. Manipulating the Mouse Embryo; a laboratory manual; Second Edition; Cold Spring Harbor Laboratory Press).
  • the DNA solution is injected into the preliminary nucleus of fertilized oocytes from the mouse strain CDl.
  • DNA is isolated from docked tail pieces from founder animals. For this, the pieces are overnight in 500 ⁇ l lysis buffer (50 ⁇ M tris / HCl, pH 8.0, 100 mM EDTA 100 mM NaCl, 1%. SDS addition of 35 ⁇ M Proteinase K (10 mg / ml)) at 55 ° C.
  • the mouse DNA is digested with EcoRI.
  • the 1.9 kb EcoV-EcoRI fragment (Z2) is used as a probe for Southern blot hybridization (cf. FIG. 4).
  • positive animals are crossed with female or male CDI wild-type partners.
  • the offspring showed a Mendelian distribution of the transgene. This confirmed the integration of the construct into the germline.
  • Embryos were prepared on the desired embryonic day and incubated in fixative solution (2% paraformaldehyde; 0.1 M PIPES pH 6.9; 2 mM MgCl 2 ; 2 Mm EGTA) at room temperature for 10-20 min. Embryos that were older than 14.5 embryonic days were halved median and fixed for a further 20 min. After washing three times in PBS / 0.01% sodium deoxycholate / 0.02% NP-40, the embryos were incubated for 12-16 h at 30 ° C.
  • fixative solution 2% paraformaldehyde; 0.1 M PIPES pH 6.9; 2 mM MgCl 2 ; 2 Mm EGTA
  • Vascular endothelium-specific activity of the 5.0 kb hVE-Cad-2 promoter in transgenic mice Analysis of the beta-galactosidase staining in transgenic animals shows promoter activity during the early embryonic development on day 11.5, in most if not all Vessels of transgenic animals. A detailed analysis of whole embryos and the corresponding sections showed the reporter gene activation in the vascular endothelium of the umbilical cord, the brain, the muscles and the organs that formed. Expression in fine capillaries was also observed. The promoter activity was absolutely specific, so that no ectopic reporter gene expression was detectable in non-endothelial cells. The expression pattern in the two lines obtained was largely identical. The promoter fragment used obviously contains all regulatory elements in order to bring about an gene expression independent of the insertion site in transgenic mice.
  • the hVE-CAD-2 promoter obviously consists of numerous regulatory elements, some of which specifically activate the gene in the vascular endothelium of the liver vessels. Accordingly, the promoter sequence comprising human 5015 bp used here contains all regulatory elements for gene expression in the entire vascular endothelium with the exception of the vascular endothelial cells in the liver. Alternatively, these liver-specific elements could be included but do not result in gene expression in this organ due to specific inhibitory elements present in the promoter fragment.
  • the 5.015 kb promoter of the VE-CAD-2 gene from humans which is preferably used according to the invention surprisingly contains all regulatory elements for a vascular endothelial cell-specific expression in the blood vessels, but at the same time contains a gene connection in the blood vessels of the liver excludes.
  • the promoter is therefore suitable for numerous gene therapy approaches that require gene expression in the vascular endothelium, but in which the risk of systemic side effects from liver damage is to be expected due to the therapeutic gene used.
  • Mouse VE-Cad-2 probes Using the published sequence of the mouse VE-Cad-2 cDNA (Telo et al. 1998, J. Jol. Chem. 273, 17565-17572), primers for the amplification of a 437 bp fragment were prepared. The fragment was named mVE-Northern.
  • the primer sequences are: mVE-2F2 (Forward Primer): TAG TTC TGC CAT TCC TGC TAG G (SEQ ID No. 4) and mVE-2R2 (Revers Primer): GAC CTT TAG AGT CTC TCA CGG A (SEQ ID No. 5).
  • mouse Cardiac tissue generated cDNA was used as a template to amplify the probe.
  • the following PCR reaction conditions were used: 2.0 ⁇ l (50 ng) cDNA, 0.5 ⁇ l (5 pM) primer mVE-2F2, 0.5 ⁇ l (5 pM) primer mVE-2R2, 1.5 mM MgCl, and 9.5 ul H 2 O were mixed to form a 12.5 ul master mix.
  • the master mix was supplemented with Taq polymerase and H 2 O to a total volume of 25 ⁇ l.
  • the reaction vessel was transferred to a "thermal cycler" and the following PCR program was used: 15 min at 95 ° C, followed by 30 cycles of: 30 sec at 95 ° C, 30 sec at 55 ° C, 60 sec at 72 ° C followed by 7 min at 72 ° C and 5 min at 95 ° C.
  • Human VE-Cad-2 probes Using the murine VE-Cad-2 gene sequence (Telo et al., 1998, supra), the putative open reading frame of the human VE-Cad-2 gene was identified in a Genbank database analysis. Based on the matched mouse VE-Cad-2 gene sequence and the BAC clone 50G21 sequence (accession number AC005740), primers were designed which recognize the translation start of the human VE-CAd-2 gene and also designed another primer, which recognizes a sequence located 3 'downstream of the translation start, both primers covering a 432 bp human VE-Cad-2 fragment.
  • hVE-2S2 forward primer
  • GTA AGC ATG ATG CAA CTT CTG SEQ ID ⁇ r. 6
  • hVE-2Al reverse Primer
  • AAG GGT TTC TGC TCG TCC TTG SEQ ID ⁇ r. 7
  • a cD ⁇ A derived from HUVEC cells was used as the template for the PCR.
  • the PCR reaction conditions used were the same as those used to generate the mouse VE-Cad-2 probes.
  • the isolated human fragment was designated hVE-Northern.
  • Northern blot hybridization Multiple tissue Northern blots (Multiple Tissue Northern Blots (MTN), Clone-tech were used. Mouse: (MTN) Blot Cat. No .: 7762-1 Lot. No: 9030523 and human: (MTN) Blot Cat. No .: 7760-1 Lot. No: 9010567: All buffers and solutions were provided by the manufacturer and used according to the manufacturer's instructions. 50 ng of the Northern probes described above were treated with alpha-P 32 dCTP (ICN) using the "Prime IT kit "(Stratagene) according to the manufacturer's instructions. After a final washing step, a Kodak X-ray film was exposed using the hybridized blots for 96 h.
  • ICN alpha-P 32 dCTP
  • the murine VE-Cad-2 mRNA is in a position of approximately 7 kb. Highly vascularized organs such as the lungs, heart, spleen, liver and kidney show a pronounced expression of VE-Cad-2 in the mouse (FIG. 6 (A)).
  • the human VE-Cad-2 mRNA is located at a position of approximately 4.4 kb. With the exception of the brain, the expression of human VE-Cad-2 is detectable in all organs examined with a pronounced high strength in the heart, placenta, lungs and liver.
  • RNA was isolated from adult transgenic mice produced according to Examples 1 and 2. Organs of a year-old pRZ-hVE-Zl # 10 female, a year-old pRZ-hVE-Zl # 54 female and a year-old CDl wild-type female (control) were carefully isolated and used for RNA preparation Using the Trizol method (Live Technologies, Cat. No. 15596-026 Lot. No: 1011120) used according to the manufacturer's instructions. Tissue was homogenized with an ultra-Turrax. The following organs were tested: lungs, liver, heart, spleen and kidney.
  • RNA from each organ was used as a template for reverse transcription (RT) using the "Expand Reverse Transcriptase Kit” which contains a DNase digestion step (Röche Cat. No: 1785-834 Lot. No: 92035623).
  • the resulting cDNA was diluted in a total volume of 20 ul. 2 ⁇ l of the cDNA preparation were used for the PCR, using the “Hot Star Tag TM Master Mix Kit” (Qiagen 1000 units Cat. No: 203445 Lot. No: 10921919).
  • the following primer pairs were used:
  • GAPDH R3 (Forward Primer): CAC CAC CTT CTT GAT GTC ATC A (SEQ ID No. 8) and F2 (Reverse Primer): GCC ATC AAT GAC CCC TTC ATT G (SEQ ID No. 9).
  • the amplified PCR fragment is 693 bp long.
  • L7 up (Forward Primer): GGT AGT GGT CAA ATG GCG ATT (SEQ ID No. 10) and L7 down (Reverse Primer): GCC ACC AAT CCC CAT ATG GAA (SEQ ID No. 11).
  • the amplified PCR fragment is 206 bp long.
  • mVE FI Forward Primer: GTC CGG TCC TCA TCA GAT TCT (SEQ ID No. 12) and R2 (Reverse Primer): GTG TGC TGC CCC AAC AAC ATT
  • the amplified PCR fragment is 612 bp long.
  • LacZ FI Forward Primer: GGT AGT GGT CAA ATG GCG ATT (SEQ ID No. 14) and R3 (Reverse Primer): GCC ACC AAT CCC CAT ATG GAA (SEQ ID No. 15).
  • the amplified PCR fragment is 400 bp long.
  • PCR reaction conditions were used: Mixing was carried out in separate reaction vessels: 2.0 ⁇ l cDNA, 0.5 ⁇ l (5 pm) primer (reverse primer), 0.5 ⁇ l (5 pm) primer (forward primer), 1.5 mM MgCl and 9.5 ul H 2 O to form a 12.5 ul master mix. Each reaction vessel was then filled with Taq polymerase and H 2 O to a total volume of 25 ⁇ l. The reaction tubes were transferred to a "thermal cycler" and the following PCR program was used: 15 min at 95 ° C, followed by 30 cycles of: 30 sec at 95 ° C, 30 sec at 55 ° C and 60 sec at 72 ° C followed by 7 min at 72 ° C and 5 min at 95 ° C. 15 ⁇ l of each PCR reaction were then separated electrophoretically on a 1.5% agarose gel with EtBr and then analyzed.
  • the isolated cDNAs were successfully amplified and the ubiquitously expressed genes GAPDH gene ("housekeeping gene”) and L7 gene (ribosomal gene) were observed in all tissues tested. All cDNAs used were therefore of a suitable quality.
  • the expression of the mouse VE-Cad-2 gene was also observed in all organs tested. This observation is consistent with Northern blot analysis of the adult mice and human tissue as described in Example 6 and shown in Figure 6.
  • the RT-PCR analysis of beta-galactosidase gene expression showed no detectable PCR product in the transgenic CD1 wild-type mouse (negative control, FIG. 7, right column, picture below). In contrast, in both transgenic strains pRZ-hVE-Zl # 10 (Fig.

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EP01996615A 2000-11-16 2001-11-16 Gefässendothel-spezifischer promotor des humanen vascular endothelial cadherin-2 (hve-cad-2) gens und seine therapeutischen verwendungen Withdrawn EP1392836A2 (de)

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