EP1127109A2 - Empreinte realisee sur des cellules par techniques genetiques et utilisation dudit procede pour assurer la prophylaxie et le traitement d'affections - Google Patents

Empreinte realisee sur des cellules par techniques genetiques et utilisation dudit procede pour assurer la prophylaxie et le traitement d'affections

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Publication number
EP1127109A2
EP1127109A2 EP99953880A EP99953880A EP1127109A2 EP 1127109 A2 EP1127109 A2 EP 1127109A2 EP 99953880 A EP99953880 A EP 99953880A EP 99953880 A EP99953880 A EP 99953880A EP 1127109 A2 EP1127109 A2 EP 1127109A2
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EP
European Patent Office
Prior art keywords
receptor
factor
growth
interleukin
cells
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.)
Withdrawn
Application number
EP99953880A
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German (de)
English (en)
Inventor
Hans-Harald Sedlacek
Klaus Havemann
Rolf Müller
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sanofi Aventis Deutschland GmbH
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Aventis Pharma Deutschland GmbH
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Publication of EP1127109A2 publication Critical patent/EP1127109A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • transplantation of bone marrow cells is an established method for the treatment of leukaemias or tumor diseases after high-dose chemotherapy.
  • the transfer of cells into an organism is problem-free if the cells to be transferred can be transferred to a cell suspension without impairing their function and / or their survivability.
  • Such cell suspensions are relatively easy to administer into the circulation, into a body cavity, into an organ or locally.
  • the invention relates to a method in which cells are inserted at least one gene for a growth and / or differentiation factor and / or at least one gene for a receptor for a growth and / or differentiation factor and these cells are forced to move by the introduced gene to differentiate in vitro or in vivo to a stage in which they have the desired function.
  • this function is associated with an integration of the transfected cell into a tissue association.
  • the invention relates in particular to cells in which at least one gene for a growth and / or differentiation factor and / or at least one gene for the associated receptor for the growth and / or differentiation factor has been inserted and the use of these cells for the purpose of prophylaxis or Therapy of a disease.
  • the invention further relates to mononuclear cells obtained from the bone marrow, lymphatic organs, body cavities, body exudates, blood, blood vessels and connective tissue, into which at least one gene for a growth and differentiation factor and / or its receptor has been introduced for the prophylaxis or therapy of a disease .
  • Such cells express the gene or genes introduced into them and, due to the expressed growth factor and / or receptor in the cell culture, but especially after administration into an organism, experience a further development towards a desired differentiation stage.
  • the expression of the gene (s) introduced into the cell is placed under the control of promoters.
  • promoters can be non-specific, cell type-specific, metabolically and / or pharmacologically activatable and / or self-reinforcing.
  • the growth and differentiation of the transduced cell can be directed as necessary by the choice of the respective promoters.
  • the cells according to the invention can already be used as a therapeutic or prophylactic without further treatment.
  • nucleotide sequences are inserted which, under the control of different promoters (e.g. cell type-specific, cell cycle-specific, metabolic, pharmacologically activatable and / or self-reinforcing), code for prophylactically or therapeutically active proteins or for enzymes for the activation of the precursor of a drug in one Drug.
  • promoters e.g. cell type-specific, cell cycle-specific, metabolic, pharmacologically activatable and / or self-reinforcing
  • nucleotide sequences are for the prophylaxis and therapy of vascular diseases (EP A 0 777 739), diseases of the central nervous system (EP A 0 777 740), tumors (EP A 0 804 601) and diseases caused by the immune system conditional (EP A 0 807 183) have already been described in the cited patent applications as well as in the patent applications EP A 0 859 058, EP A 0 864 651 and DE19752299.8.
  • the method claimed in this invention and the cells made by this method are new.
  • the state of the art is to carry out the differentiation in vitro in cell culture by adding growth factors and to use the differentiated cells after mechanical or proteolytic detachment from the cell culture vessel.
  • the method of embossing cells towards differentiation claimed in this invention is to be distinguished from the method of dedifferentiating cells known from the literature. This dedifferentiation takes place by inserting a gene into a cell, which leads to immortalization of this cell. Examples of such genes are:
  • the selection of the starting cells, the promoters and the nucleotide sequences coding for receptors and / or growth factors and differentiation factors takes place according to the intended use for these cells, i.e. the state of differentiation of the cells sought in the organism and the therapeutic goal sought with these cells.
  • Erythrocytes, granulocytes and other cell components are separated from these body fluids by density gradient centrifugation and thrombocytes by differential centrifugation according to the methods known to the person skilled in the art
  • Endothelial cells can be obtained using the methods known to the person skilled in the art, for example from adipose tissue, by scraping veins or by detaching from the umbilical cord endothelium.
  • VEGF vascular endothelial growth factor
  • KDR or Fit ligands such as VEGF-B, VEGF-C, VEGF-D, Neuropilin
  • IGF-1 Insulin-like growth factor
  • PDGF-AA, -AB, -BB platelet derived growth factor
  • PEGF platelet derived endothelial cell growth factor
  • SCF stem cell factor
  • TGF-ß transforming growth factor ß
  • Tie-2 ligands such as angiopoietin-1
  • BMP bone morphogenic proteins
  • BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7 and BMP-8 (Takahara et al. , Genomics 29, 9 (1995), Celeste et al., PNAS USA 87, 9843 (1990), Ozkaynak et al., EMBO J. 9, 2085 (1990), Ozkaynak et al., J. Biol. Chem. 267 , 1992, 25220, Hino et al., Bichem. Biophys. Res. Commun. 223, 304 (1996), Ruppert et al., Eur. J. Biochem. 237, 295 (1996)), pleiotrophin (PTN) and Midkine (Kurtz et al., Crit. Rev. Oncogenesis 6, 151 (1995), Int. Dev. Bio. 37, 183 (1993)).
  • BMP bone morphogenic proteins
  • promoters of the genes for the BMP can be metabolically activated, self-reinforcing and / or pharmacologically controllable and combinations thereof (see section 4)
  • All growth factors which contribute to the growth and differentiation of glial cells are suitable for the purposes of the invention. These include, for example:
  • GGF glia growth factor
  • NT-4 Neurotrophin-4
  • trk-C Neurotrophin-4
  • BdNF brain derived neurotrophic factor
  • CNF ciliary neurotrophic factor
  • GDNF glia cell line derived neurotrophic factor
  • NGF nerve growth factor
  • - can be activated without restriction, can be activated specifically for glia cells, can be activated metabolically, self-reinforcing and / or pharmacologically controllable and combinations thereof (see section 4).
  • growth factors include all those proteins such as Cytokines, cytokine inhibitors, enzyme inhibitors, anti-adhesion molecules, antagonists of oxygen radicals and growth factors for cartilage cells, which lead to an anti-inflammatory reaction and differentiation of synovial cells.
  • TGF Transforming growth factor
  • IL-10 interleukin 10
  • IGF-1 Insulin-like growth factor-1
  • interleukin-4 IL-4
  • FGF Fibroblast Growth Factor
  • Plasminogen Activator Inhibitor (PAI-1, -2)
  • PDGF Platelet derived growth factor
  • TGFß receptors These include, for example: TGFß receptors
  • lymphocyte and / or macrophage specific and / or synovial cell specific see section 4
  • Suitable for the purposes of the invention are all anti-allergic or cytokines and their receptors which inhibit the antibody reaction or the cellular immune reaction. These include, for example:
  • TNF ⁇ Tumor necrosis factor ⁇
  • lymphocytes and / or macrophages can be specifically activated, metabolically activated, self-reinforcing and / or pharmacologically controllable and combinations thereof (see section 4).
  • cytokines and / or their receptors which promote an antibody-mediated or a cellular immune reaction are suitable for the purposes of the invention.
  • - Can be activated without restriction, lymphocytes and / or macrophages specifically activated, metabolically activated, self-reinforcing and / or pharmacologically controllable and Kombinatine ⁇ thereof (see section 4).
  • nucleotide sequences are to be used as promoter sequences which, after binding transcription factors, activate the transcription of a transgene located at the 3 'end, such as a gene for a receptor of a growth factor or differentiation factor or a gene for a growth factor or differentiation factor.
  • a transgene located at the 3 'end such as a gene for a receptor of a growth factor or differentiation factor or a gene for a growth factor or differentiation factor.
  • at least one promoter sequence is inserted into the cell according to the invention. This promoter sequence can be combined with at least one further promoter sequence. The choice of which to combine with The promoter sequence depends on the disease to be treated.
  • the promoter sequence can be activated without restriction, endothelial cell-specific, under certain metabolic conditions, such as, for example, by hypoxia or inducible by a pharmacon, virus-specific and / or cell cycle-specific.
  • Such promoters have already been described in patent applications EP A 0 804 601; EP A 0 777 739; EP A 0 807 183; EP A 0 777 740; EP A 0 753 580; EP A 0 857 781, EP A 0 790 313; EP A 0 860 445; EP A 0 864 651 and EP A 0 805 209. Reference is made to these patent applications.
  • the promoter sequences to be selected include, for example:
  • binding sequences include, for example, binding sequences for c-myc proteins.
  • binding sequences include monomers or multimers of the nucleotide sequence designated as Myc E-Box (5'-GGAAGCAGACCACGTGGTCTGCTTCC-3 '(SEQ ID NO .: 1); Blackwood and Eisenmann, Science 251, 121 1 (1991)).
  • a promoter when combining the same or different promoters, can be inducible, for example in the form of a promoter which can be activated or deactivated by tetracycline in the form of the tetracycline operator in combination with a corresponding repressor.
  • the promoter can also be self-reinforcing with or without a pharmacologically controllable promoter unit.
  • Enhancers of those genes that code for proteins preferentially formed in endothelial cells.
  • promoters of the genes for the following proteins are to be used, for example:
  • VCAM-1 Vascular Cell Adhesion Molecule
  • LCAM-3 Interstitial Cell Adhesion Molecule
  • PECAM platelet endothelial cell adhesion molecule
  • 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.
  • An example of this is the transcription factor GATA-2, whose binding site in the endothelin-1 gene is 5'-TTATCT-3 '(Lee et al., Biol. Chem. 16188 (1991), Dormann et al., J. Biol. Chem 1279 (1992) and Wilson et al., Mol. Cell Biol. 4854 (1990)).
  • Promoter and activator sequences that are activated in synovial cells are, for example, the promoter sequences of genes coding for matrix Metalloproteinases (MMP), such as MMP-1 (interstitial collagenase), MMP-3 (Stromelysin / Transi ⁇ ) or tissue inhibitors of metal proteinases (TIMP), such as TIMP-1, -2, -3.
  • MMP matrix Metalloproteinases
  • MMP-1 interstitial collagenase
  • MMP-3 Stromelysin / Transi ⁇
  • TIMP-1, -2, -3 tissue inhibitors of metal proteinases
  • Promoters and activator sequences that are activated in lymphocytes and / or macrophages are, for example, the promoter and activator sequences of the genes coding for cytokines, cytokine receptors and adhesion molecules and receptors for the Fc fragment of antibodies such as e.g.
  • IRF-1 interferon regulatory factor 1
  • the promoter of IRF-1 being activated by IL-6 as well as by IFN ⁇ or IFNß, IFN ⁇ Responsive promoter, IL-7, IL-8, IL-10, IL-11, IFN ⁇ , GM-CSF, GM-CSF receptor ( ⁇ chain), IL-13, LIF, macrophage-colony stimulating factor (M- CSF) - receptor, type I and II macrophage scavenger receptors, MAC-1 (leukocyte functional antigen) LFA-1 ⁇ (leukocyte functional antigen) or p150.95 (leukocyte functional antigen).
  • a chimeric promoter represents the combination of an upstream cell-specific, metabolically or virus-specific activator sequence with a downstream promoter module which contains the nucleotide sequence CDE-CHR or E2FBS-CHR, to which suppressive proteins bind, which hereby activates the upstream activator sequence in Go and can inhibit the d phase of the cell cycle (WO 96/06943; Lucibello et al., EMBO J., 12 (1994)).
  • Such transcription factors include, for example, Sp1 and NF-Y.
  • hybrid promoters have already been described in patent application EP A 0 848 063.
  • a gene construct is selected which contains the following components in total: The nucleotide sequence of the endothelial cell-specific promoter in a form in which at least one binding site is mutated for a transcription factor. This mutation blocks the initiation of the transcription of the effector gene.
  • a transgene that codes as an effector gene for an active ingredient is provided.
  • At least one further unspecific, cell-specific, virus-specific, promoter or enhancer sequence which can be activated by tetracycline and / or cell cycle-specific and which activates the transcription of at least one gene for at least one transcription factor which is mutated in such a way that it binds to the mutated binding site (s). can bind in the endothelial cell-specific promoter and activate it.
  • the mutation in the promoter sequence can e.g. represent a mutation of the TATA box of the cdc25B promoter.
  • the mutation of the TATA can be, for example, TGTATAA.
  • TBP normal TATA box-binding protein
  • the nucleic acid sequence which codes for the TBP must have a commutation.
  • the TBP binds to the mutated TATA box (e.g. to TGTATAA) and thus leads to the efficient transcription of the effector gene.
  • Such commutations of the TBP gene are described, for example, by Strubin and Struhl (Cell, 721 (1992)) and by Heard et al. (EMBO J., 3519 (1993)).
  • transgene that codes as an effector gene for an active ingredient.
  • NSS nuclear retention signal
  • the transcription product of the nuclear retention signal preferably has a binding structure for a nuclear export factor.
  • NEF nuclear export factor
  • the gene coding for the nuclear retention signal is preferably selected from the group comprising the Rev-responsive element (RRE) of HIV-1 or HIV-2, the RRE-equivalent retention signal of retroviruses or the RRE-equivalent retention signal of the HBV.
  • RRE Rev-responsive element
  • the nuclear export factor is preferably a gene selected from the group comprising the Rev gene of the viruses HIV-1, HIV-2, Visna-Maidi virus, Caprine arthritis encephaiitis virus, the virus of the infectious anemia of the horse, the immunodeficiency virus of the cat , of retroviruses, of HTLV or the gene of the hnRNP-A1 protein or the gene of the transcription factor TFIII-A.
  • An activator-responsive promoter unit consists of the following components:
  • promoter or enhancer sequence which can be activated, for example, cell cycle-specific, cell proliferation-dependent, metabolically, endothelial cell-specific or virus-specific or both cell cycle-specific and metabolically, endothelial cell-specific or virus-specific (so-called chimeric promoters)
  • one or more identical or different activator units which are located downstream of the promoter or enhancer sequences and are activated by their basal transcription
  • an activator-responsive promoter which is activated by the expression products of one or more activator subunit (s).
  • activator-responsive promoter units according to the invention can represent binding sequences for chimeric transcription factors from DNA-binding domains, protein-protein interaction domains and transactivation domains. All of the transcription factor binding sites mentioned in the application can be single (monomers) or in multiple copies (multimers, for example up to 10 copies).
  • the LexA operator in conjunction with the SV40 promoter is an example of an activator-responsive promoter activated by two activator subunits.
  • the first activator subunit comprises the cDNA for the LexA DNA binding protein coding for amino acids 1-81 or 1 -202, the 3 'end of which is linked to the 5' end of the cDNA for the Gal80 protein (amino acids 1-435).
  • the second activator subunit comprises the cDNA of the Gal80 binding domain of the Gal4 protein coding for amino acids 851-881, the 3 'end of which is linked to the 5' end of the cDNA of the SV40 large T antigen coding for amino acids 126-132 , the 3 'end of which is linked to the 5' end of the cDNA for the transactivation domain of the VP16 of HSV-1 coding for amino acids 406-488.
  • Another example of an activator-responsive promoter activated by two activator subunits is the binding sequence for the Gal4 protein in connection with the SV40 promoter.
  • the first activation unit comprises the cDNA for the DNA binding domain of the Gal4 protein (amino acids 1-147), the 3 'end of which is linked to the 5' end of the cDNA for the Gal ⁇ O protein (amino acids 1-435).
  • the second activation subunit comprises the cDNA for the Gal80 binding domain of Gal4 (amino acids 851 -881), the 3 'end of which is linked to the 5' end of the cDNA of the nuclear localization signal of SV40 (SV40 large T; amino acids 126-132) , the 3 'end of which is linked to the 5' end of the cDNA for the transactivation domain of the VP16 of HSV-1 coding for amino acids 406-488.
  • activator subunits that activate the activator-responsive promoter, consisting of the binding sequence for the Gal4 protein and the SV40 promoter, is shown
  • a first activation unit which comprises the cDNA for the cytoplasmic domain of the CD4-T-cell cell gene (amino acids 397-435), the 5 'end of which is linked to the 3'-end of the cDNA for the transactivation domain of the VP16 of HSV-1 (Amino acids 406-488), the 5 'end of which is in turn linked to the 3' end of the cDNA of the nuclear localization signal of SV40 (SV40 large T; amino acids 16-132) and
  • the second activation unit comprising the cDNA of the nuclear localization signal of SV40 (SV40 large T; amino acids 126-132), the cDNA for the DNA binding domain of the Gal4 protein (amino acids 1-147), the 3 'end of which is linked to the 5th 'End of the cDNA for the CD4 binding sequence of the p56 Ick protein (amino acids 1-71). 5) Examples to illustrate the subject of the invention
  • Cells might look like endothelial cells.
  • PBMC peripheral, mononuclear cells of the blood
  • the PBMCs obtained are washed twice in cold PBS and magnet beads (CD14 micro beads, Miltenyi Biotec) coupled with anti-CD14 antibody for 15 min. Incubated at 4 ° C, then washed with cold PBS and suspended in PBS containing 0.002% EDTA and 1% human serum albumin.
  • the CD14 + cells are detached from the column using the Vario MACS Separation kit (Mite ⁇ yi Biotec) in accordance with the manufacturer's instructions.
  • the purity of the CD14 + cells thus produced is in the range between 70% and 95%.
  • FIG. 1 This nucleotide sequence is shown schematically in FIG. 1:
  • FIG. 1 This nucleic acid sequence (FIG. 1) is cloned into a pxP2 plasmid vector (Norden, BioTechniques 6, 454 (1988)).
  • the respective components of the nucleic acid construct are connected to one another via suitable restriction sites which are introduced at the ends of the different components with the aid of PCR amplification.
  • the components are connected with the help of restriction-specific enzymes and with the help of DNA ligases.
  • the CD14 + cells are adjusted to a concentration of 1x10 7 / ml culture medium [medium 199 with 20% fetal calf serum and penicillin / streptomycin / amphotericin (from Gibco-BRL)], sown in 60 mm culture dishes and with a complex from the in b) plasmid shown (coding for VEGF receptor) and Superfect (from Quiagen, Düsseldorf) for 10 min. incubated at 37 ° C.
  • the complex is produced according to the manufacturer of Superfect (Quiagen).
  • the success of the transaction is determined by detecting the VEGF Receptor II (DR) m-RNA using RT-PCR.
  • the RT-PCR is carried out as described by Sewing et al., J. Cell Sei. 104, 545 (1993).
  • VEGF receptor II (KDR) expressing CD14 cells are adjusted to 1x10 6 / ml and in the culture medium "EGM-2 (from Biowhittaker), which contains 50 ng / ml VEGF (Pepro Tech, London, England) and 10% fetal calf serum, 5th % Horse serum and 0.8 ⁇ g / ml hydrocortisone had been added, incubated for 7 days.
  • ECM-2 from Biowhittaker
  • the cells are examined immunocytochemically with the method known to those skilled in the art of the alkaline-phosphatase-anti-aicaline phosphatase (APAAP) method using specific antibodies (see Table 1).
  • APAAP alkaline-phosphatase-anti-aicaline phosphatase
  • m-RNAs specifically for CD14, CD31, CD 34, CD36, CD 144 and vWF are detected with the aid of RT-PCR (Sewing et al., J. Cell Sei. 104, 545 (1993)).
  • CD54 intercellular adhesion molecule PharMingen (ICAM-1)
  • CD51 / 61 ⁇ v / ß3 integrin complex (vitronectin PharMingen receptor)
  • CD62E E-selectin / ELAM-1 endothelial PharMingen leukocyte adhesion molecule
  • VCAM-1 CD106 vascular cell adhesion molecule PharMingen
  • VE vascular endothelial
  • cadherin 5 CD144 vascular endothelial (VE) -cadherin PharMingen (cadherin 5) cells of the CD14 receptor for LPS-LPS-BP Becton Dickinson monocytic lineage HLA-DR MHC class VI molecule Immunotech
  • DC marker CD1a (lgSF) - -

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  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Psychiatry (AREA)
  • Vascular Medicine (AREA)
  • Communicable Diseases (AREA)
  • Pain & Pain Management (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé permettant de préparer des cellules adaptées pour traiter le corps humain, selon lequel (a) des cellules sont prélevées sur le corps humain; (b) les cellules obtenues à l'étape (a) sont transfectées in vitro avec au moins un gène pour un facteur de croissance et/ou de différenciation, qui se trouve sous le contrôle d'un promoteur, de sorte que les cellules de l'étape (b) soient en mesure après avoir été replacées dans le corps humain d'effectuer des différenciations de manière voulue. L'invention concerne également des cellules pouvant être obtenues à l'aide dudit procédé et leur utilisation pour préparer un médicament pour traiter des affections.
EP99953880A 1998-11-05 1999-10-19 Empreinte realisee sur des cellules par techniques genetiques et utilisation dudit procede pour assurer la prophylaxie et le traitement d'affections Withdrawn EP1127109A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19850986 1998-11-05
DE19850986A DE19850986A1 (de) 1998-11-05 1998-11-05 Die gentechnische Prägung von Zellen und ihre Verwendung zur Prophylaxe und Therapie von Erkrankungen
PCT/EP1999/007902 WO2000028010A2 (fr) 1998-11-05 1999-10-19 Empreinte realisee sur des cellules par techniques genetiques et utilisation dudit procede pour assurer la prophylaxie et le traitement d'affections

Publications (1)

Publication Number Publication Date
EP1127109A2 true EP1127109A2 (fr) 2001-08-29

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EP99953880A Withdrawn EP1127109A2 (fr) 1998-11-05 1999-10-19 Empreinte realisee sur des cellules par techniques genetiques et utilisation dudit procede pour assurer la prophylaxie et le traitement d'affections

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EP (1) EP1127109A2 (fr)
JP (1) JP2002529080A (fr)
AU (1) AU1040700A (fr)
CA (1) CA2349497A1 (fr)
DE (1) DE19850986A1 (fr)
WO (1) WO2000028010A2 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0015761D0 (en) * 2000-06-27 2000-08-16 Univ Bristol Polypeptide
US6962982B2 (en) 2001-06-22 2005-11-08 Roche Diagnostics Corporation Soluble complexes of target proteins and peptidyl prolyl isomerase chaperones and methods of making and using them
DE10158680B4 (de) * 2001-11-30 2004-04-08 Universitätsklinikum Hamburg-Eppendorf Verfahren zur ex vivo-Expansion und -Differenzierung von multipotenten Stammzellen
FR3018819A1 (fr) * 2014-03-19 2015-09-25 Univ Bourgogne Traitement de la reponse inflammatoire et dysimmunitaire

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5980887A (en) * 1996-11-08 1999-11-09 St. Elizabeth's Medical Center Of Boston Methods for enhancing angiogenesis with endothelial progenitor cells
WO1998039427A2 (fr) * 1997-03-06 1998-09-11 University Of Massachusetts Therapie genique utilisant des greffes de moelle osseuse transfectees avec des genes therapeutiques sous la surveillance de promoteurs specifiques de tissus

Non-Patent Citations (1)

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Title
See references of WO0028010A2 *

Also Published As

Publication number Publication date
DE19850986A1 (de) 2000-05-25
WO2000028010A3 (fr) 2000-07-27
WO2000028010A2 (fr) 2000-05-18
CA2349497A1 (fr) 2000-05-18
AU1040700A (en) 2000-05-29
JP2002529080A (ja) 2002-09-10

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