EP1556487A2 - Cellule transplantable - Google Patents

Cellule transplantable

Info

Publication number
EP1556487A2
EP1556487A2 EP03747785A EP03747785A EP1556487A2 EP 1556487 A2 EP1556487 A2 EP 1556487A2 EP 03747785 A EP03747785 A EP 03747785A EP 03747785 A EP03747785 A EP 03747785A EP 1556487 A2 EP1556487 A2 EP 1556487A2
Authority
EP
European Patent Office
Prior art keywords
cell
cells
human
animal
gene
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Withdrawn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
EP03747785A
Other languages
German (de)
English (en)
Inventor
Mark Burcin
Sybille Esser
Manfred Ruediger
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.)
F Hoffmann La Roche AG
Original Assignee
F Hoffmann La Roche AG
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG filed Critical F Hoffmann La Roche AG
Priority to EP03747785A priority Critical patent/EP1556487A2/fr
Publication of EP1556487A2 publication Critical patent/EP1556487A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • 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
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular 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
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
    • A61P5/16—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4 for decreasing, blocking or antagonising the activity of the thyroid hormones
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N5/0676—Pancreatic cells
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A01K2267/025—Animal producing cells or organs for transplantation
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • 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
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • 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
    • 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
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/02—Cells from transgenic animals
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the invention relates to a human or animal non-totipotent cell which contains at least one nucleic acid which codes for at least one immunomodulator under the control of a gene switch molecule which can be regulated by adding an active substance, and to its production and use for transplantation, for inhibiting a
  • a large number of human diseases are based on the death or malfunction of specific cells, tissues or organs and often cannot be adequately treated with medication.
  • Conventional therapy has been to repair the damaged tissue or organ by transplanting healthy cells, tissues or organs, e.g. To replace the heart, lungs, kidneys, pancreas or cells or tissues obtained from healthy human donors.
  • the need for donor tissue can only be insufficiently met. This deficiency could be remedied by transplanting tissues or cells from specially bred non-human donor mammals.
  • replacement cells can also be obtained from cell lines. These cells can also be of human or non-human origin.
  • B cells, T cells, antigen presenting cells of the immune system and can ultimately lead to rejection of the transferred cells, tissue or organ .
  • immunomodulators which suppress the immune system or bring about a tolerance of the recipient to the transplant and thus prevent such a rejection reaction.
  • immunomodulators e.g. Steroids (prednisolone and derivatives), calcineurin inhibitors (cyclosporin A, tacrolimus), rapamycin (sirolimus), mycophenolate mofetil (MMF), azathioprine (imuran), lymphocyte antisera (ALG - anti-leukocyte globulin, ATG - anti thym monoMonal antibodies (anti-CD25: Zenapax, Simulect) were used.
  • Steroids prednisolone and derivatives
  • calcineurin inhibitors cyclosporin A, tacrolimus
  • rapamycin rapamycin
  • mycophenolate mofetil MMF
  • azathioprine imuran
  • lymphocyte antisera AG - anti-leukocyte globulin, ATG - anti thym monoMonal antibodies (anti-CD25: Zenapax, Simulect) were used.
  • the most significant side effects of conventional immunomodulator therapy include high blood pressure, kidney and liver damage, the increased incidence of opportunistic infections and an increased rate of various malignancies, e.g. Cancer and lymphoproliferative disorders.
  • various malignancies e.g. Cancer and lymphoproliferative disorders.
  • the infection with the cytomegalovirus which usually has only minor symptoms, leads to the formation of hepatitis, pneumonia and meningitis in immunosuppressed patients and is therefore a main cause of the increased mortality rate of transplant recipients (Transplantation Clinical Management, Vol. 5, 2000 Medscape, Inc., Web MD Health Network, NY, USA).
  • immunomodulators in particular immunosuppressants, can generally have toxic properties regardless of their effect on the immune system.
  • calcineurin inhibitors often cause renal insufficiency, high blood pressure, hyperlipidemia and the development of diabetes mellitus.
  • regular medication treatment severely impairs the quality of life and is therefore often not carried out by the patient to the extent that is medically necessary.
  • a clinically proven approach to reducing the side effects of immunomodulation is e.g. in combining newly developed immunoreactive drugs in low concentrations and thus replacing standard therapeutic agents (e.g. steroids, cyclosporin A).
  • the side effects of the individual immunomodulators of this combination such as however, the drop in the level of leukocytes, the appearance of oral abscesses and indigestion could not be avoided even with this treatment.
  • Another therapeutic approach could be to achieve immunological tolerance to the foreign tissue in the patient by administering novel immunomodulatory substances.
  • tolerance is characterized as the lack of an immune response or rejection reaction against the graft without continuous immunosuppression.
  • immunomodulatory substances are administered by the customary methods known to those skilled in the art (see, for example, WO 00/12138; WO 97/41232; WO 96/26274; WO 01/87330; Ferrari-Lacraz et al, The Journal of Immunology, 2001 , Vol. 167 pp.
  • a disadvantage of the conventional therapy approach is that tolerance-inducing immunomodulators are usually administered systemically (for example intravenously).
  • Another disadvantage is that the immunomodulators have to be isolated as therapeutic products from natural sources or biotechnologically produced as recombinant molecules in order to then administer them externally to the patient.
  • production-related aspects can mean that an immunomodulator in native form or as a recombinant molecule cannot be isolated or produced in sufficient quantity or activity.
  • the ex vivo production of the immunomodulator may require the addition of substances that may pose a further health risk to the patient (eg substances that have been isolated from animal organisms and can therefore potentially transmit zoonoses).
  • substances that may pose a further health risk to the patient eg substances that have been isolated from animal organisms and can therefore potentially transmit zoonoses.
  • the object was to develop an immunotherapy which avoids or reduces the disadvantages described above, in particular which has its immunomodulatory, in particular immunosuppressive, effect in the organism exactly where the immune response has been activated or can be activated, ie at the location of the transplanted cells or organs, and at the same time enables an immunomodulatory effect which can be regulated in terms of time and quantity
  • controllable expression of the immunomodulator in cells was achieved with the help of an adjustable gene expression system.
  • This regulatable expression system makes it possible that immunomodulators no longer have to be administered systemically or only at low concentrations continuously.
  • Another significant advantage of the invention is that even at the location of the transplant, there is no need for sustained suppression of the immune response if this is not necessary from a medical point of view, but is only activated when necessary. This reduces side effects and stress on the body, especially the transplant region. This also means a physical and psychological relief for the patient, since he is not continuously dependent on the administration of immunomodulators, in particular immunosuppressive agents.
  • Immunosuppressants in this sense are understood to mean substances which inhibit in whole or in part an immune response caused by the transplant, in particular cell (s), tissue and / or organ (s) in the organism.
  • this invention provides the advantage that the immunomodulator no longer has to be isolated from a native source or produced and purified recombinantly.
  • the immunomodulator is formed in therapeutically active amount and form in the recipient organism and / or at the site of action in vivo and thus retains its full native activity.
  • An object of the present invention therefore relates to a transplantable human or animal non-totipotent cell which contains at least one nucleic acid which codes for at least one immunomodulator under the control of a gene expression system which can be regulated by adding an active substance.
  • a nucleic acid in the sense of the present invention means an RNA or DNA, in particular genomic DNA, recombinantly produced DNA, cDNA or synthetic DNA, which was synthesized, for example, at the phosphoramidation level. Combinations and / or modifications of nucleotides of these nucleic acids are also included. This term also includes single and double-stranded nucleic acids.
  • nucleic acids which contain functionally linked components, for example one or more genes or active parts thereof coding for one or more immunomodulators and at least one regulatable gene expression system, the activation state of which is regulated by the addition of an active substance, and regulatable elements, for example promoters and regulators Nucleotide sequences and a polyadenylation signal, for example an SV40 polyadenylation signal.
  • the components are functionally linked if they are linked in such a way that under the influence of the transcription regulation the sequence (s) of the genes contained or the gene are or will be transcribed.
  • immunomodulator of the present invention essentially encompasses any type of molecule which has immunomodulatory, in particular immunosuppressive, activity, for example proteins, fusion proteins and soluble ligands, wherein a fusion protein is understood to mean an expression product of a fused gene which results from the linkage of two or more Genes or gene fragments are created.
  • An immunomodulating effect is present when the immune response of an organism, a cell and / or a tissue is essentially inhibited, for example by an altered or suppressed receptor binding.
  • An immunomodulating effect is also present when an immunological tolerance to a transplanted cell, tissue or organ is brought about.
  • the effect of the immunomodulator comprises, for example, one or more of the following activities: the inhibition of an antigen recognition mediated by T cells, • the inhibition of a signal mediated by a receptor on a T cell, the activation of a mediated by a receptor on a T cell Signal, the inhibition of the growth of T cells, • the inhibition of molecules which support the survival of T cells, the inhibition of effector molecules of T cells (such as TNF-alpha, IFN-gamma), the inhibition of the adhesion of T- Cells, the inhibition of a T-cell costimulatory interaction (the activation of a lymphocyte takes place via two signals: one is done
  • Stimulation via the antigen receptor there is a further signal for clonal expansion and differentiation of an unembossed lymphocyte; these costimulatory interaction can be inhibited by an immunomodulator), • the JmhibiKING the activation, proliferation, survival, antigen presentation of signaling and / or effector functions of other cells involved in an immune response, such as general and specific antigen Cells, in particular, for example, dendritic cells and monocytes / macrophages B cells, neutrophils and NK cells inhibit the cellular interaction of different cells
  • Cells either via surface receptors or via secreted molecules, such as cytokines, chemokines or growth factors, which are involved in an immune response, such as general and specific ones antigen-presenting cells, in particular, for example, dendritic cells and monocytes / macrophages, T-cells, B-cells, neutrophilic granulocytes and NK cells, the inhibition of the migration of cells which are involved in an immune response, such as, for example, special antigen-presenting cells, in particular, for example, dendritic cells and monocytes / macrophages, T cells, B cells, neutrophilic granulocytes and NK cells, the inhibition of components of the complement system, the inhibition of phagocytotic activities in connection with the presentation of foreign or autoimmune antigens, or by the binding of antibodies to antigens, and / or the inhibition of inflammatory reactions.
  • antigen-presenting cells in particular, for example, dendritic cells and monocytes / macrophages, T-cells, B-cell
  • Antibodies are suitable as immunomodulators. It is particularly preferably an antibody against IL-15, IL-1, IL-2, IL-6, IL-7, IL-12, IL-17, IL-18, IL-21, interferon gamma, TNF- alpha, CD2, CD3, CD4, CD8, CD28, CD40, CD80, CD86 or CD 154 or against their receptors.
  • Immunomodulators are also preferably FasL, PD-Ll or PD-L2.
  • immunomodulators are IL-15, IL-10, IL-4, IL-2, interferon gamma or TGF-beta, in particular as a fusion protein.
  • the fusion protein particularly preferably comprises on the one hand wild-type IL-15, wild-type IL-10, wild-type IL-4, wild-type interferon gamma or wild-type TGF-beta and on the other hand an Fc fragment.
  • the fusion protein furthermore particularly preferably comprises mutated IL-15 on the one hand, preferably those in which “Q” has been replaced by “D” at positions 101 and 108 or mutated IL-2 and on the other hand an Fc fragment (see, for example, WO 97/41232 ; Kim et al, J Immunol. ( ⁇ 998), 160 (12): 5742-5748; WO 01/87330), which is fused, for example, to the C-terminus of the mutated IL-15 molel il via the hinge region.
  • immunomodulators are fusion proteins from TNF-alpha receptor (type 1 or 2), ICOS, CTLA-4, PSGL-1, ICAM-1 or VCAM-1 on the one hand and an Fc fragment on the other hand, as in the case of TNF- Receptor fusion proteins, those which are disclosed in EP 417,563 A.
  • immunomodulators are secreted variants of cytokine or growth factor receptors, such as e.g. IL-15Ralpha, IL-6, IL-7, IL-12, IL-17, IL-18 receptors, for example as variants without a transmembrane domain and cytoplasmic tail, preferably as a fusion protein with an Fc fragment.
  • cytokine or growth factor receptors such as e.g. IL-15Ralpha, IL-6, IL-7, IL-12, IL-17, IL-18 receptors, for example as variants without a transmembrane domain and cytoplasmic tail, preferably as a fusion protein with an Fc fragment.
  • the fusion protein is a chimeric fusion protein.
  • Suitable fusion proteins are, for example, IL-15 derivatives, comprising IL-15 or imitated IL-15 and a heterologous Fc fragment.
  • Fc (fragment crystallizable) fragment is to be understood as the fragment of an antibody which does not bind any antigens, for example one which comprises all constant domains or all constant domains except the first constant (partially or completely) domain, such as, for example, a one that includes the hinge region, the second (CH2) and third constant (CH3) heavy chain domains.
  • the Fc fragment can come from a natural source, can be produced recombinantly and / or can be synthesized. Appropriate
  • the Fc fragment can also have one or more mutations with respect to the natural sequence, for example those which have a suitable interface for the construction of a
  • the Fc fragment is one of an immunoglobulin (Ig) G, in particular a human IgGl, IgG2, IgG3, IgG4 and / or an analogous mammal IgG and / or an IgGM, in particular a human IgM or a analog mammal IgM and / or a murine IgG2a.
  • Ig immunoglobulin
  • the immunomodulators can have wild-type sequences or else mutated sequences.
  • the immunomodulators are preferably already in a functionally active form, for example in a functionally active soluble form or a functionally active viral form.
  • a soluble form is understood to mean a molecule that is not bound to a cell membrane, e.g. a soluble receptor molecule.
  • a viral form is to be understood as a protein isoform which is endogenously encoded by a virus genome, e.g. viral IL-10.
  • a mutated sequence according to the present invention is to be understood as a nucleotide or amino acid sequence which contains deviations from the wild-type sequence by, for example, deletion, addition, insertion or substitution of one or more nucleotides or amino acids, but without completely losing the immunomodulatory effect ,
  • Mutated immunomodulators for the purposes of the present invention are therefore molecules which preferably have a sequence homology of at least approximately 80%, preferably at least approximately 90%, particularly preferably at least approximately 95%, most preferably at least approximately 99% of the sequence to the wild-type sequence.
  • Sequence homology in the sense of the present invention is understood to mean the degree of similarity (% positive) of two sequences, which is determined for polynucleotides using BLASTN 2.0.14, for example, the filter being set ⁇ off and BLOSUM 62 being (Altschul et al 1997, Nucl. Acids Res, 25: 3389-3402). Sequence homology can be used with common sequence homology Programs z. B. can be checked online at http://www.hgsc.bcm.tmc.edu / SearchLauncher /.
  • regulatable gene expression system in the sense of the present invention means the combination of a sequence coding for a gene switch molecule and a gene switch binding sequence, the binding of the gene switch molecule to the gene switch binding sequence being regulated by the addition of an active substance, thereby controlling the expression of a target gene, here a target gene coding for an immunomodulator is carried out (see also Burcin et al. 1998, Frontiers in Bioscience 3: cl-7).
  • a gene switch molecule can activate or inhibit the transcription of the target gene by binding to a suitable gene switch binding sequence.
  • the activation can be based on the fact that the gene switch molecule e.g. Provides contact points for the RNA polymerase and / or involved transcription factors.
  • the inhibition can be brought about in that the gene switch molecule blocks the DNA binding sites required for the transcription complex by its binding to the DNA and thereby makes it inaccessible to, for example, the RNA polymerase and / or involved transcription factors.
  • an active substance can influence the transcription of the target gene positively (activation) or negatively (inhibition).
  • the target gene is not expressed in the absence of the active substance.
  • it binds to the gene switch molecule and thereby causes the activation and binding of the gene switch molecule to the gene switch binding sequence, which subsequently initiates the transcription of the target gene.
  • the gene switch molecule binds to the DNA in the absence of the active substance and activates the transcription. After addition and binding of the active substance, the gene switch molecule is inactivated and the transcription of the target gene is ended.
  • gene switch molecule means a molecule, preferably a protein, in particular a fusion protein, containing a binding site for an active substance and a transcription activation domain, which changes its activation state after binding of the active substance.
  • gene switch binding sequence in the sense of the present invention is preferably understood to mean a nucleic acid sequence located 5 'upstream from the translation start (+1) of the gene, or active part thereof, which codes for an immunomodulator, which sequence determines the transcription of the target gene, in particular with regard to the transcription rate and / or the tissue specificity, controlled or the translation controlled.
  • a regulatory nucleic acid sequence with promoter activity, preferably also with enhancer activity, is bound directly or indirectly to this gene switch binding sequence.
  • the functioning of the regulatable gene expression system according to the invention can be described, for example, as follows: If no gene switch molecule is bound to the gene switch binding sequence, the coupled target gene is not expressed and in this case no immunomodulator is produced.
  • an active substance When an active substance is added, it binds, for example, to the dimerization domain of the gene switch molecule. This binding results in a conformational change in the dimensioning domain, which causes two gene switch molecules to dimerize and subsequently bind to the gene switch binding sequence. This binding brings the activation domain of the gene switch molecule close to the minimal TATA promoter and thus initiates the transcription of the coupled target gene coding for an immunomodulator. After the addition of the active substance has ended, the binding of the gene switch molecule to the gene switch binding sequence is released and thus the target gene expression is ended.
  • the gene switch molecule of the present invention can accordingly be regulated, for example inhibited or activated, preferably activated by adding an active substance and then binds to the gene switch binding site.
  • Preferred active substance is to be understood as a pharmacologically acceptable substance which directly or indirectly effects the regulated expression of a gene or several genes via its gene switch (s), for example mifepristone, tetracycline, doxycycline or rapamycin.
  • s gene switch
  • the regulated gene expression system of the present invention is in particular a progesterone gene expression system which comprises a gene switch which represents an artificially composed transcription factor consisting of a GAL4 DNA binding domain (Gal4-DBD), a dimerization domain derived from a mutated progesterone receptor with a shortened ligand binding site (hPR-LBD), and an activation domain of the p65 subunit of the human NF- ⁇ B protein (p65-AD).
  • the gene switch binding sequence is a nucleotide sequence consisting, for example, of a 17-nucleotide GAL4 binding sequence with subsequent minimal TATA promoter to which the corresponding target gene is coupled.
  • Such a regulatable gene expression system with the gene switch components Gal4-DBD / hPR-LBD / p65-AD mentioned is described, for example, in Wang et al. 1994, PNAS 91: 8180-8184 or Wang et al. 1997, Gene Therapy 4: 432-441.
  • the active substance which is suitable in this system is, for example, mifepristone, an artificial hormone-like molecule which does not occur in the mammal and which specifically binds to the dimerization domain of the gene switch molecule and activates it through the dimerization which is thereby brought about.
  • Another controllable gene expression system of the present invention is a tetracycline gene expression system, which is one by tetracycline (Tet) or that Derivative doxycycline (dox) inducible system, in which the gene switch molecule consists of a Tet transactivator protein.
  • This transactivator (tTA) is a fusion protein from a VP16 activation domain and the Tet Repressor (TetR) from Escherichia coli. In the absence of tetracycline, the tTA has a high affinity for its gene switch binding site, the Tet-responsive element (TRE), and activates the expression of the target gene.
  • rtTA reverse transactivator
  • the wild-type TetR is replaced by a mutated TetR (rTetR), as a result of which the fusion protein binds the gene switch binding site of the DNA only in the presence of doxycycline and thus induces coupled target genes, as described, for example, in Gossen 1995, Science 268: 1766-1769; Gossen et al. 1992, PNAS 89: 5547-5551; Linstedt et al. 1997, Mol. Biol. Cell 8: 1073-1087; Mehlen et al. 1998, Nature 395: 801-804 or Joosse et al. 2000, Hum. Mol. Genet. 9: 3075-3082.
  • Another controllable gene expression system of the present invention is a rapamycin gene expression system. This is an inducible dimerization of the proteins FKBP12 and FRAP mediated by the active substance rapamycin. Dimerization of these two proteins causes a DNA binding of the activation domain bound to FRAP and thus the activation of a coupled target gene.
  • a regulatable gene expression system is described, for example, in Rivera et al. 1996, Nature Med 2: 1028-1032.
  • This active substance can be administered by methods familiar to the person skilled in the art, for example intravenously, intraperitoneally, intramuscularly, subcutaneously, intracranially, intraorbitally, intracapsularly, intraspinally, transmuscularly, topically, orally or via the mucous membrane, for example the nose or the oral cavity. Further methods of administration are, for example, systemic or local injection, perfusion or catheter-based administration. Tablets or capsules are suitable as oral dosage forms. Administration via the lungs takes place, for example, with the aid of sprays and via the skin in the form of disposers which are implanted under the skin. Transdermal therapeutic systems (TTS) are known, for example, from EP 0 944 398-A1, EP 0 916 336-A1, EP 0 889 723-A1 or EP 0 852 493-A1.
  • TTS Transdermal therapeutic systems
  • the cells are preferably transplantable.
  • Transplantable in the sense of the present invention means that the living cell can be transferred to another location of the same organism or to another (recipient) organism, which is preferably a non-tumorigenic cell, or if it is one of is a cell derived from a tumor cell, it is treated accordingly before the transplantation (for example by mitotic inactivation) in order to inhibit its proliferation (see, for example, WO 00/64459 and US 5,175,103; Pleasure et al. (1992), The Journal of Neuroscience, 12 (5): 1802-1815 ".
  • the transplantable human or animal non-totipotent cell according to the invention is in particular a mammalian cell, including a human cell, and originates, for example, from a human, a mouse, a rat, a guinea pig, a rabbit, a cow, a sheep, a goat, a horse , a pig, a dog, a cat or a monkey, preferably from a human.
  • Cells according to the invention are, for example, epithelial cells, endothelial cells, liver cells, heart cells, skin cells, muscle cells, nerve cells, bone marrow cells, bone cells, cartilage cells, blood cells,
  • a non-totipotent cell is a cell that cannot develop into a complete organism on its own.
  • the cell is a stem cell, a precursor cell and / or an immortalized cell. It is preferably a pluripotent or multipotent embryonic, fetal, neonatal or adult stem cell.
  • Particularly preferred stem cells derived from, but not limited to, adult tissue include neuronal stem cells, bone marrow stem cells, mesenchymal stem cells, hematopoietic stem cells, epithelial stem cells, and stem cells from the digestive tract, skin, adipose tissue, intestine, the placenta and duct of the pancreas.
  • a cell of the present invention also includes a cell which contains the above-described invention and which is introduced into a tissue or an organ of a human or animal organism before and / or after that tissue or organ in the same or another human or animal Organism is transplanted.
  • a preferred embodiment relates to a cell according to the invention in the form of a cell line.
  • a cell line according to the invention can be produced, for example, by transforming or infecting a cell line with the one described above Nucleic acid according to the invention with the aid of methods which are known to the person skilled in the art, for example transfection, transformation or infection.
  • the nucleic acid additionally codes a selection cassette, in particular a suitable transfection marker gene and / or differentiation marker gene.
  • a selection cassette in the sense of the present invention is a nucleic acid sequence which codes for at least one gene which effects a targeted selection of certain cells, for example transfected or differentiated cells.
  • Differentiation marker genes, transfection marker genes and reporter genes can be used for such a selection.
  • genes are predominantly used which impart resistance to certain toxic substances, for example antibiotics.
  • the most common antibiotics used in this context are neomycin, hygromycin (hph), zeocin (Sh ble) and puromycin (pacA).
  • Further examples of such genes, in particular for the selection of stem cells are, for example, genes which regulate the expression of fluorescent markers, for example GFP, with the aid of which the cells to be selected can be purified via fluorescence-mediated cell sorting (FACS).
  • FACS fluorescence-mediated cell sorting
  • selection markers are surface molecules, for example growth factor receptors, with the aid of which cells can be enriched via magnetic immunobeads (Bonini C, Science Vol 276, 1719-1724, 1997).
  • Further examples are genes which code for an enzyme activity (for example thymidine kinase) which convert a precursor of a toxic substance, so-called "prodrug” (for example ganciclovir) into a toxic substance. In this case, a negative selection can take place, ie only those cells that do not express the promoter upstream of the gene survive.
  • genes of the selection cassette according to the invention can be lacZ (coding for ⁇ -lactamase), ⁇ -lactamase, chloramphenicol acetyl transferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), and xanthinguanine phosphoribosyl transferase (XG.
  • CAT chloramphenicol acetyl transferase
  • ADA adenosine deaminase
  • DHFR dihydrofolate reductase
  • XG xanthinguanine phosphoribosyl transferase
  • the nucleic acid additionally encodes an NK cell-inhibiting and / or a killer cell-inhibiting molecule, preferably a human MHC class I molecule, a chimeric MHC class I molecule or a viral MHC class I homolog.
  • an NK cell-inhibiting and / or a killer cell-inhibiting molecule preferably a human MHC class I molecule, a chimeric MHC class I molecule or a viral MHC class I homolog.
  • killer cells as a heterogeneous population of mononuclear cells with spontaneous or acquired cytotoxic potential.
  • NK cells naturally killer cells
  • NK cells are to be understood as killer cells which are naturally present, i.e. which are not the result of an immune response and are therefore not induced by specific antigens.
  • the present invention further relates to a nucleic acid which codes for at least one immunomodulator and at least one gene expression system which can be regulated by adding an active substance, as has already been described in more detail above.
  • the nucleic acid preferably also codes for at least one element which controls gene expression.
  • elements are understood to mean, for example, promoters or regulatory nucleic acid sequences.
  • Suitable expression conditions for the expression of a nucleic acid can be created by these and by expression vectors (explained in more detail below).
  • expression vectors contain the promoters suitable for the respective cell or the gene to be transcribed in each case.
  • regulatable elements which enable consumer expression in eukaryotes are promoters which are recognized by RNA polymerase III.
  • Such promoters for constitutive expression in all cell and tissue types are, for example, the pGK (phosphoglycerate kinase) promoter, the CMV (cytomegalovirus) promoter, the TK (thymidine kinase) promoter, the EFl ⁇ (elongation factor-1-alpha) promoter, the SV40 (Simian Virus) promoter, the RSV (Rous Sarcoma Virus) promoter and the pUB (Ubiquitin) promoter.
  • pGK phosphoglycerate kinase
  • CMV cytomegalovirus
  • TK thymidine kinase
  • EFl ⁇ elongation factor-1-alpha
  • SV40 Seimian Virus
  • RSV Ra Sarcoma Virus
  • pUB Ubiquitin
  • regulatable elements which enable cell-specific or tissue-specific expression in eukaryotes are promoters or activator sequences from promoters or enhancers of those genes which code for proteins which are only expressed in certain cell types.
  • promoters are for example the insulin promoter for beta cells of the pancreas, the Sox-2 promoter for nerve cells, the myosin heavy chain promoter for muscle cells, the VE-cadherin promoter for endothelial cells and the keratin promoter for epithelial cells.
  • regulatable elements which enable regulatable expression in eukaryotes are the tetracycline operator in combination with a corresponding repressor (Gossen M. et al. (1994) Curr. Opin. Biotechnol. 5, 516-20).
  • Expression can also be controlled via regulatory nucleotide sequences which influence expression in terms of quantity and / or as a function of time. These include, for example, enhancer sequences, leader sequences, polyadenylation sequences, IRES sequences, introns, insulator sequences and repressor sequences.
  • the nucleic acid according to the invention can be localized on one or more nucleic acid molecules. According to the invention, however, in the case of several nucleic acid molecules, these must functionally interact.
  • the sequence for the gene switch molecule, (ii) the sequence for the immunomodulator under regulation of the gene switch binding site and (iii) the sequences for the selection markers can be on three different nucleic acid molecules.
  • Another object of the present invention is therefore also a vector which contains at least one nucleic acid according to the invention.
  • Vectors in the sense of the present invention can be plasmids, shuttle vectors, phagemids, cosmids, adenoviral vectors, retroviral vectors, expression vectors and vectors which are active in gene therapy.
  • Expression vectors in the sense of the present invention include at least one nucleic acid according to the invention, at least one translation initiation signal, a translation termination signal and / or a polyadenylation signal for expression in eukaryotes.
  • Gene-therapeutic vectors are, for example, plasmid vectors, virus vectors, for example adenovirus vectors, retroviral vectors or vectors which are based on replicons of RNA viruses (see, for example, 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).
  • 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 dioleoxylphosphatidylethanolamine
  • lipid formulations are DOTAP N- [l- (2,3-dioleoyloxy) propyl] -N, N, N-trimethyl-ammoniumethyl-sulfate or DOGS (TRANSFECTAM; dioetadecylamidoglycylspermin).
  • 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. 8, 213) or, for example, liposomes (Uhlmann and Peimann, 1990, Chem. Rev. 90, 544).
  • the Cells according to the invention can also be used to express a heterologous gene.
  • Gene therapy vectors can be introduced into cells by transfection (e.g. electroporation, lipofection, calcium phosphate precipitation) or infection.
  • the present invention furthermore relates to a medicament which contains at least one cell according to the invention and suitable auxiliaries and / or additives.
  • the medicament according to the invention can be used for the prophylaxis and / or therapy of diseases, for example of
  • rheumatic diseases for example rheumatic arthritis, Sjogren's syndrome, scleroderma, dermatomyositis, polymyositis, Reiter's syndrome or
  • autoimmune diseases of the thyroid gland for example Graves' I rankheit
  • autoimmune diseases of the central nervous system for example multiple sclerosis
  • auxiliaries and additives which serve, for example, to stabilize and / or preserve the medicament, are generally familiar to the person skilled in the art. These include, for example, physiological saline solutions, Ringer's dextrose, Ringer's lactate, University of Wisconsin solution / NiaSpan® (Beizer UW), EuroCollins solution, DMSO, ethylene glycol, sucrose, trehalose, Ficoll, perfluorocarbons, demineralized water, stabilizers, antioxidants, complexing agents , antimicrobial compounds, proteinase inhibitors and / or inert gases.
  • physiological saline solutions include, for example, physiological saline solutions, Ringer's dextrose, Ringer's lactate, University of Wisconsin solution / NiaSpan® (Beizer UW), EuroCollins solution, DMSO, ethylene glycol, sucrose, trehalose, Ficoll, perfluorocarbons, demineralized water, stabilizers, antioxidants, complexing agents
  • the medicament according to the invention is administered by methods which are suitable for the particular cell, tissue or organ type to which it is to be administered. Such methods are familiar to the person skilled in the art.
  • the drug can then be administered intravenously, for example, for liver cells, intramuscularly for heart muscle cells or also by catheter-based
  • the drug can be introduced into the organism either using an ex vivo approach in which the cells are removed from the patient, genetically modified, for example by D ⁇ A transfection, and then reintroduced into the patient or using an in vivo approach, in which gene therapy-effective vectors according to the invention are introduced into the patient's body as naked DNA or using viral or non-viral vectors according to the invention or cells according to the invention.
  • the dosage of drugs depends on several factors, for example on the body weight, the general state of health, the extent of the body surface, the age of the patient and the interaction with other drugs. Dosage also depends on the type of administration. The dosage must therefore be determined by the specialist in each individual case for each patient. The drug can be administered once or several times a day and for several days; this can also be determined by a person skilled in the art.
  • the present invention further provides a human or animal organ-specific tissue and / or a human or animal mammalian organ which contains at least one cell according to the invention.
  • organ-specific tissue and mammalian organ in the sense of the present invention include, for example, the mammalian organs heart, skin, pancreas, kidneys, liver, muscles, nerves, eyes, lungs, bone marrow, cartilage, bones, vessels, connective tissue or tissues of these organs.
  • the present invention furthermore relates to a transgenic non-human mammal which contains at least one cell according to the invention.
  • Transgenic animals generally show a tissue-specific increased expression of nucleic acids and are therefore very suitable for the analysis of, for example, immune reactions.
  • Transgenic mice are preferably used.
  • a non-human mammal according to the invention is, for example, a mouse, a rat, a guinea pig, a rabbit, a cow, a sheep, a goat, a horse, a pig, a dog, a cat or a monkey.
  • Another object of the present invention is the use of a cell according to the invention, a human or animal organ-specific tissue according to the invention and / or a human or animal mammalian organ according to the invention for transplantation into a human or animal Mammal.
  • the transplantation according to the invention is preferably an auto, allo- or xenotransplantation.
  • transplantation to mean the transmission or transplantation of living material, for example cells, tissue and
  • Another object of the present invention is the use of a cell according to the invention, a human or animal organ-specific tissue according to the invention and / or a human or animal mammalian organ according to the invention for inhibiting a graft rejection reaction in an animal mammal or in humans.
  • An animal mammal within the meaning of the present invention is understood to mean, for example, a mouse, a rat, a guinea pig, a rabbit, a cow, a sheep, a goat, a horse, a pig, a dog, a cat or a monkey.
  • a graft rejection reaction as a process by which transplanted material, for example cells, tissue or an organ, are rejected by the recipient organism.
  • This rejection reaction is caused by cellular and humoral immunity.
  • the cause of this rejection reaction is a difference in Protein structure between transplanted material and recipient.
  • the protein structure of the transferred tissue is recognized as immunogenic by the recipient's immune system, which triggers an immune response.
  • Another object of the present invention is the use of a cell according to the invention, a human or animal organ-specific tissue according to the invention and / or a human or animal mammalian organ according to the invention for the prophylaxis and / or therapy of secondary transplant diseases and / or autoimmune diseases. Examples of such diseases have already been described above in the areas of application of the medicament according to the invention.
  • the uses according to the invention for inhibiting a graft rejection reaction and for the prophylaxis and / or therapy of secondary transplant diseases and / or autoimmune diseases can take place, for example, by introducing the cells, tissues and / or organs according to the invention into a human or animal mammal.
  • the expression of the immunomodulator according to the invention is controlled via the regulatable gene expression system according to the invention. This control takes place through the administration or withdrawal of an active substance according to the invention, whereby the gene switch molecule is activated or deactivated. When activated, the gene switch activates the transcription of the target gene which codes for the immunomodulator.
  • the immunomodulator expressed thereby essentially inhibits a defense reaction of the immune system to the transplanted cell, tissue or organ, and in particular in the transplant region of the organism of the human or animal mammal.
  • cells according to the invention consist of epithelial cells, endothelial cells,
  • Liver cells, derivatives of non-totipotent embryonic stem cells or non-totipotent embryonic germ cells, or stem cells derived from adult tissue include neuronal stem cells, bone marrow stem cells, mesenchymal stem cells, hematopoietic stem cells, epithelial stem cells, stem cells from the digestive tract, skin, adipose tissue, intestine, placenta and duct of the pancreas all in one human or animal mammal are introduced.
  • the present invention furthermore relates to a method for producing a cell according to the invention, the method comprising the following steps: a. Introducing at least one nucleic acid according to the invention and / or at least one vector according to the invention into a transplantable human or animal non-totipotent cell, and b. Expression of the nucleic acid with the addition of at least one suitable active substance for regulating the gene switch molecule.
  • Suitable conditions which cause or enhance expression of the nucleic acid have already been described above. These include, for example, expression vectors, promoters and regulatable nucleic acid sequences, e.g. Enhancers, polyadenylation sequences.
  • the present invention furthermore relates to an in vitro method for producing a human or animal organ-specific one according to the invention Tissue and / or a human or animal mammalian organ according to the invention, the method comprising the following steps: a. Introduction of at least one nucleic acid according to the invention and / or at least one vector according to the invention as well as at least one differentiation gene into at least one non-totipotent
  • Stem cell, a non-totipotent progenitor cell and / or a non-totipotent immortalized cell b. Differentiation of the cell from step a, c. Selection of the differentiated cell from step b. and d. Introducing the selected cell from step c. in a human or animal organ-specific tissue and / or in a human or animal mammalian organ.
  • step a preference is given to the in vitro method according to the invention mentioned after, before or simultaneously with step a.
  • at least one suitable transfection marker gene is introduced into a non-totipotent stem cell, a non-totipotent progenitor cell and / or a non-totipotent immortalized cell and after step a. preferably the transfected cell from step a. selected.
  • the differentiation of the cells containing the nucleic acid according to the invention can e.g. by "embroid body formation", preferably by culturing the cells in solutions, by culturing the cells in high density, by cell aggregation, by withdrawing the cultivation on feeder cells, withdrawing substances which inhibit differentiation (for example LIF or medium conditioned by feeder cells), by adding Cytokines, growth factors, hormones, vitamins (e.g. nicotinamide), retinoic acid, sodium butyrate or DMSO to the cultured cells or by adding other substances known to initiate differentiation. .
  • the selection cassette according to the invention contains a marker gene, specifically an antibiotic resistance gene.
  • the cells are selected or isolated by enriching the differentiated cells after adding a suitable antibiotic during or after the differentiation step. Only the differentiated cells that express the marker gene are resistant to the antibiotic. Undifferentiated cells die.
  • the transfected cells can also be selected using the same method.
  • An antibiotic according to the invention means an antibiotic against which the antibiotic resistance gene (s) used as the selection cassette according to the invention produces resistance (s). After the antibiotic has been added to the cultured stem cells, only those stem cells which contain the reporter gene expression vector essentially survive and differentiate.
  • a selection method can also be used, the gene or genes of the selection cassette according to the invention coding for luciferase, green fluorescent protein, red fluorescent protein and / or yellow fluorescent protein.
  • the cells to be selected are isolated by means of fluorescence-activated cell sorting (FACS) or selected by affinity purification. Furthermore, cells can be concentrated with the help of surface molecules, for example growth factor receptors, using magnetic immunobeads (Bonini C, Science Vol 276, 1719-1724, 1997).
  • a second marker gene can preferably be introduced into the cells, as a result of which a selection of the cells in which the introduction of the nucleic acid and / or the vector according to step a. of the in vitro method according to the invention was successful. This double selection makes it possible to obtain an approximately 90%, preferably approximately 95-100% pure cell population of the desired cells.
  • the present invention further provides a method for producing a transgenic non-human mammal according to the invention, the method comprising the following steps: a. Introducing at least one nucleic acid according to the invention and / or at least one vector according to the invention as well as at least one suitable transfection marker gene into at least one oocyte, stem cell, a precursor cell and / or an immortalized cell of a non-human mammal, b. Selection of the transfected cell from step a, c. Introduce the after step b. selected cell into at least one non-human mammalian blastocyst, d. Introduce the blastocyst from step c. or the embryo from step d. into a non-human mammal foster mother and e. Identification of the transgenic non-human mammal developed from said blastocyte.
  • it is a pluripotent or multipotent embryonic, fetal, neonatal or adult stem cell.
  • Another object of the present invention is a method for
  • transgenic non-human mammal the method comprising the following steps: a. Introducing both at least one nucleic acid according to the invention and / or at least one vector according to the invention and at least one suitable transfection marker gene into one of the two precursors of a fertilized non-human mammalian oocyte, b. Introduce the mammalian oocyte from step a. into a non-human mammal foster mother and c. Identification of the transgenic non-human mammal developed from said mammalian oocyte.
  • non-human mammalian foster mother be mock pregnant by mating with a male with a severed spermatic duct.
  • blastocytes and / or oocytes into the foster mother are known to the person skilled in the art. It can be done, for example, by injection into the fallopian tube or uterus (see e.g. Hogan, B, Beddington, R, Constantini, F. and Lacy, E, A laboratory Manual (1994), Cold Spring Harbor Laboratory Press, page 173-181).
  • a transgenic non-human mammal can be identified, for example, by extracting genomic DNA from the transgenic non-human mammal, for example from the tail of a mouse.
  • genomic DNA is transferred to a membrane and detected by means of DNA probes, for example radioactively labeled DNA probes, which are specific for the transgene sought.
  • transgenic non-human mammal by regenerating a non-human stem cell, oocyte, precursor cell or immortalized cell to form a transgenic non-human animal, in particular transgenic mice, are known to the person skilled in the art, for example, 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 (see, for example, Polites and Pihkert: DNA micromjection and transgenic animal production, Page 15-68 in Pinkert, 1994: Transgenic Animal Technology: A Laboratory Handbook, Academic Press, London, UK; Houdebine 1997, Harwood Academic Publishers, Amsterdam, The Netherlands; Doetschman: Gene Transfer in Embryonic Stern Cells, page 115-146 in Pinkert, 1994, supra; Wood: Retrovirus-Mediated Gene Transfer, pages 147-176 in Pinkert, 1994, supra; Monastersky: Gene Transfer Technology: Alternative Techniques and Applications, pages 177-220 in Pinkert, 1994, supra).
  • transgenic animals in particular transgenic mice
  • processes for the production of transgenic animals are also known to the person skilled in the art, inter alia, from WO 98/36052, WO 01/32855, DE 196 25 049, US 4,736,866, US 5,625,122, US 5,698,765, US 5,583,278 and US 5,750,825 and include transgenic animals that can be generated, for example, by direct injection of vectors according to the invention into embryos or spermatocytes or via the transfection of vectors or nucleic acids into embryonic stem cells (see also Polites and Pinkert, in Pinkert, (1994) Transgenic animal technology, A Laboratory Handbook, Academic Press, London, UK, pages 15 to 68; Doetschman, in Pinkert, 1994, supra, pages 115 to 146).
  • the stem cell is used in the in vitro methods according to the invention for producing a human or animal organ-specific tissue according to the invention and / or a human or animal mammalian organ according to the invention and in the methods for producing a transgenic non-human mammal according to the invention is used to generate a pluripotent or multipotent embryonic, fetal, neonatal or adult stem cell.
  • the present invention also relates to a transgenic non-human mammal, which was produced by the method according to the invention described above, and the descendant (s) of this mammal.
  • Another object of the present invention is the use of a transgenic non-human mammal according to the invention for obtaining a human or animal cell, a human or animal organ-specific tissue and / or a human or animal mammalian organ for allo- and / or xenotransplantation.
  • this can take place, for example, by means of an implantation method or by means of a catheter injection method through the blood vessel wall.
  • extraction means the removal of the cell, tissue and / or organ mentioned from the organism of a transgenic non-human mammal according to the invention. Methods for such removal are common.
  • Another object of the present invention is the use of a transgenic non-human mammal according to the invention, a cell according to the invention, a human or animal organ-specific tissue according to the invention and / or a human or animal mammalian organ according to the invention for finding pharmacologically active substances and / or for identifying toxic substances substances.
  • one such method could be to target cells of the present invention to e.g. sow a 96-well microtiter plate, then add a pharmacologically active or toxic substance to be investigated and then use cell counting to analyze whether the substance has caused the death of an increased number of cells.
  • pharmacologically active substance and toxic substance in the sense of the invention include all those molecules, compounds and or
  • Exercise cells individual tissues, individual organs or the entire organism of an animal or human mammal.
  • Possible pharmacologically active substances and toxic substances can be simple chemical (organic or inorganic) molecules or compounds, nucleic acids or analogues of
  • nucleic acids nucleic acids, anti-sense sequences of nucleic acids, peptides, proteins or
  • Substance libraries originate and are examined for their pharmacological or toxic activity.
  • Pharmacologically active substances are, for example, active substances which have an influence on: the ability of cells to divide and / or survive, the secretion of proteins, eg insulin from beta cells of the pancreas,
  • Toxic substances are, for example, active substances that • stimulate cells to apoptosis after certain signals, for example stress, influence the cardiovascular system, influence the nervous system and / or influence metabolic activities.
  • the identified pharmacologically active substances and toxic 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 prophylaxis and / or therapy of Transplantation complications and / or autoimmune diseases, as exemplified above, can be used.
  • the present invention also relates to:
  • the regulatable gene expression system is a progesterone gene expression system, a tetracycline expression system and / or a rapamycin gene expression system.
  • cell according to at least one of (i) - (v), characterized in that the immunomodulator has at least one of the following functional properties: a. inhibiting antigen recognition mediated by T cells b. inhibiting a signal mediated via a receptor on a T cell, c. the activation of a signal mediated via a receptor on a T cell, d. inhibiting the growth of T cells, e. inhibiting molecules that support the survival of T cells f. inhibition of T cell effector molecules (such as TNF-alpha, IFN-gamma), g. inhibition of T cell adhesion, h.
  • T cell effector molecules such as TNF-alpha, IFN-gamma
  • T-cell costimulatory interaction the activation of a lymphocyte takes place via two signals: on the one hand there is stimulation via the antigen receptor, on the other hand there is another signal for the clonal expansion and differentiation of an unimpressed lymphocyte; this costimulatory interaction can be done by one Immunomodulator are inhibited) i. inhibition of activation, proliferation, survival,
  • Antigen presentation, signaling, and / or the effector functions of other cells that are involved in an immune response such as, for example, general and special antigen-presenting cells, in particular, for example, dendritic cells and monocytes / macrophages B cells, neutrophilic granulocytes and NK cells inhibiting the cellular Interaction of different cells, either via surface receptors or via secreted molecules such as cytokines, chemokines or growth factors that are involved in an immune response, such as general and special antigen-presenting cells, in particular, for example, dendritic cells and monocytes / macrophages, T cells, B cells, neutrophilic granulocytes and JNK cells, j.
  • general and special antigen-presenting cells in particular, for example, dendritic cells and monocytes / macrophages, T cells, B cells, neutrophilic granulocytes and JNK cells, j.
  • a receptor is b.
  • a soluble secreted receptor is c. is a secreted protein or peptide
  • the Fc fragment of the antibody is one of an IgG, in particular a human IgGl, IgG2, IgG3, IgG4 or an analog mammal IgG or an IgM, in particular a human IgM or an analog mammal IgM.
  • (xi) cell according to at least one of (i) l to (x), characterized in that the nucleic acid additionally codes a selection cassette, in particular a suitable transfection marker gene and or differentiation marker gene.
  • (xii) cell according to at least one of (i) to (xi), characterized in that the nucleic acid additionally encodes a molecule which inhibits NK cells and / or killer cells.
  • (xiii) cell according to at least one of the (i) - (xi), characterized in that the nucleic acid additionally encodes a molecule that a. Inhibits dendritic cells b. Monocytes and or macrophages inhibited c. B cells inhibit d. Polymer nuclear cells, e.g. Neutrophil granulocytes inhibited
  • (xxiv) Method for producing a cell according to one of (i) to (xiv), comprising the following steps: c. Introducing at least one nucleic acid according to (xv) and / or at least one vector according to (xvi) into a transplantable human or animal non-totipotent cell, and d. Expression of the nucleic acid with the addition of at least one suitable active substance to regulate the gene switch.
  • v / tro process for producing a human or animal organ-specific tissue and / or human or animal mammalian organ according to (xviii), comprising the following steps: e. Introducing at least one nucleic acid according to (xv) and / or at least one Velrtor according to (xvi) and also at least one differentiation marker gene into at least one non-totipotent stem cell, a non-totipotent precursor cell and / or a non-totipotent immortalized cell, f. Differentiation of the cell from step a, g. Selection of the differentiated cell from step b. and h. Introducing the selected cell from step c. in a human or animal organ-specific tissue and / or in a human or animal mammalian organ.
  • (xxvi) Method according to (xxv), characterized in that after, before or simultaneously with step a. at least one suitable transfection marker gene is introduced into at least one non-totipotent stem cell, a non-totipotent precursor cell and / or a non-totipotent immortalized cell and after step a. preferably the transfected cell from step a. is selected.
  • (xxvii) Method according to one of (xxv) or (xxvi), characterized in that it is a pluripotent or multipotent embryonic, fetal, neonatal or adult stem cell.
  • Progenitor cell and / or an immortalized cell of a non-human mammal g. Selection of the transfected cell from step a, h. Introduce the after step b. selected cell in at least one non-human mammalian blastocyst, i. Introduce the blastocyst from step c. into a non-human mammal foster mother and j. Identification of the transgenic non-human mammal developed from the blastocyst.
  • (xxx) A method for producing a transgenic non-human mammal according to (xix), comprising the following steps: d. Introducing both at least one nucleic acid according to (xv) and / or at least one vector according to (xvi) and at least one suitable transfection marker gene into one of the two pre-nuclei of a fertilized non-human mammalian oocyte, e. Introduce the mammalian oocyte from step a. into a non-human
  • Mammalian organ according to (xviii) for the detection of pharmacologically active substances and / or for the identification of toxic substances are provided.
  • Fig. 1 shows an immunoblot analysis of media supernatants of the 17x4 / IL15 / Oligo + pcDNA3switch +/- mifepristone (Example 3);
  • Fig. 2 shows a schematic illustration of the mechanism of action of the gene expression system according to the invention which can be regulated by adding an active substance.
  • the gene switch of the present invention is a chimeric protein consisting of three functional units: i) a Gal4 DNA binding domain (Gal-DBD), which
  • the UAS sequence of the invention contains four copies of a sequence motif of 17 nucleotides, each motif of which can serve as a binding site for two Gal4-DBD molecules.
  • a truncated ligand-binding domain of the human progesterone receptor (PR-LBD) which mediates the binding of the active substance mifepristone to the gene switch and the conversion of the
  • a p65 activation domain (P65-AD) which activates the transcription of the target gene by the gene switch.
  • P65-AD A p65 activation domain which activates the transcription of the target gene by the gene switch.
  • pTK ubiquitous weak basal minimal thymidine kinase promoter
  • these gene switch molecules are present as monomers and cannot yet bind any DNA regions or initiate transcription. After adding the active substance mifepristone Activation of the gene switch system.
  • the ligand-bound Genschalter homodimer binds to all regions of the DNA which contain UAS sequences (such as, for example, the regulatory region in front of the gene for MutIL15-mFc regulated by the TATA promoter) and thus activates the transcription of the immunomodulatory protein. Since the TK promoter of the gene switch red gene is additionally preceded by DNA regions with UAS sequences, the transcription of the gene switch protein itself is activated in an autoregulatory feedback mechanism and thus the amount of the active gene switch is increased. Furthermore, the transplantable cells of the invention produced from cell lines can contain marker genes, for example for resistance to the antibiotics neomycin (Neo-R) or hygromycin (Hygro-R).
  • the marker genes which are regulated by a ubiquitous promoter such as, for example, the phosphoglycerate inase promoter (pGK), are used to select the cell for the uptake of the DNA construct.
  • the cells must contain at least elements 1 and 2.
  • the cells can additionally contain element 3.
  • stem cells were transfected with vector constructs which, in addition to the elements for the regulatable expression of the immunomodulator MutIL-15 / mFc, contained a selection cassette which made it possible to produce a specific cell type (eg insulin-producing cell) (see US Pat. No. 5,733,727) , In this way, differentiated cells of a specific cell type can be produced and isolated from undifferentiated transgenic stem cells.
  • These transgenic differentiated cells can be transplanted into a suitable recipient mouse (e.g. diabetic mouse). If the transplanted animals are treated with mifepristone, the transplanted cells themselves form MutIL-15 / mFc and thus prevent their own rejection.
  • Transgenic mice were generated which contain a construct in the genome which effects the regulated expression of MutIL-15 / mFc by adding mifepristone. Since the expression of MutIL-15 / mFc is regulated by a ubiquitous promoter, the mice produced MutIL-15 / mFc when stimulated by adding mifepristone. Can be removed organs (eg heart and kidney) cells (islet cells, neural cells) from the transgenic animals and transplanted into an ⁇ other non-transgenic mouse. If the transplanted animals are treated with mifepristone, the transplanted organ / cells themselves form MutlL-15 / mFc and thus prevent their own rejection.
  • organs eg heart and kidney
  • transplanted organ / cells themselves form MutlL-15 / mFc and thus prevent their own rejection.
  • Example 1 Vector cloning for a transgenic mouse model
  • the luciferase gene was identified by the gene for a fusion protein from mutated IL-15 protein and mouse Fc part (hereinafter referred to as as MutIL-15 / mFc: Kim et al, The Journal of Immunology, 1998, 160: 5742-5748), which additionally contains a CD5 Leader sequence contains (Jones H, Nature 323 (6086), 346-349, 1986).
  • GS Gal4-DBD / hPR-LBD / p65-AD
  • Gal4UAS-PTK-IVS8 the upstream regulatory region
  • a vector was alternatively produced in which the intron IVS8 (also from pswitch) was inserted between the TATA box and the start codon of the CD5-MutIL-15 / mFc gene (vector name 17x4 / IL15 / Oligo / IVS8 / GS). The transitions between the segments of all cloning products were checked by sequencing.
  • Example 2 Vector cloning for transgenic in vitro transplants from insulin-producing cells
  • these vectors were produced analogously to the vectors for the transgenic mouse model.
  • these plasmids also contain a fragment which contains a neomycin resistance gene (neo) regulated by the rat insulin promoter (RIP) and a hygromycin resistance gene (hygro) regulated by the mouse phosphoglycerate kinase promoter (pGK).
  • neo neomycin resistance gene
  • hygro hygromycin resistance gene
  • pGK mouse phosphoglycerate kinase promoter
  • vectors were alternatively produced in which the intron IVS8 (also from pswitch) was used between the TATA box and the start codon of the CD5-mutIL15-mFc gene (vector names 17x4 / IL15 / Oligo / RIPhn / IVS8 / GS and 17x4 / IL15 / Oligo / RIPnh / IVS8 / GS). The transitions between the segments of all cloning products were checked by sequencing.
  • Example 3 Examination of the regulated expression and secretion of CD5-mutIL15-mFc in the vectors mentioned in Examples 1 and 2
  • CD5-mutIL15-mFc The secretion of CD5-mutIL15-mFc was analyzed using an ELISA that recognizes the mouse Fc part of the fusion protein. For this were 96 holes Plates (Nunc, Wiesbaden # 439454) were coated with 100 ⁇ l each of an anti-mouse IgG2a antibody (clone R1-89, BD PharMingen, Heidelberg # 02251D-553446) for one hour at 37 ° C. The plates were then washed three times with PBS and incubated for one hour at 37 ° C. with DMEM + 10% FCS in order to saturate non-specific bindings.
  • an anti-mouse IgG2a antibody clone R1-89, BD PharMingen, Heidelberg # 02251D-553446
  • the bound amount of the fusion protein was determined by a color reaction after adding an OPD-containing substrate solution (25 ml 0.1 M citric acid, 25 ml 0.1 M dipotassium hydrogen phosphate, ad 100 ml with H2O + 1 tablet OPD (Sigma Deisenhofen # P8412) + 40 ⁇ l H2O2 30%) made visible, stopped by adding 3 M HC1 and then measured in an ELISA reader ( ⁇ Quant, BIO-TEK Instruments Inc.) at 490 nm.
  • OPD-containing substrate solution 25 ml 0.1 M citric acid, 25 ml 0.1 M dipotassium hydrogen phosphate, ad 100 ml with H2O + 1 tablet OPD (Sigma Deisenhofen # P8412) + 40 ⁇ l H2O2 30%
  • media supernatants were analyzed in the immunoblot.
  • media supernatants were mixed with Lämmli sample buffer, heated at 92 ° C. for 5 minutes, then applied to a 12.5% polyacrylamide gel and separated electrophoretically at 150 V.
  • the proteins were then transferred to a nitrocellulose membrane (Schleicher and Schuell, Dassel CD0564-1).
  • the membrane was treated with 5% milk powder / PBS / 0.1% Tween20 to saturate non-specific bonds. Then it was incubated for 16 hours at 4 ° C.
  • mCD5.6 positive control for MutIL-15 / mFc secretion, vector with CD5-MutIL-15 / mFc under the control of a CMV promoter
  • the media supernatant contained cells that were both gene-free (3rd) and containing Constructs (5th + 7th) had been transfected after mifepristone treatment with an increased amount of MutIL-15 / mFc, which was detected by ELISA.
  • Immunoblot analyzes of media supernatants from transfection 3 (Fig. 1) also confirm that an increased amount of MutIL-15 / mFc (50 kDa band) is produced after mifepristone stimulation (lane 1) compared to unstimulated cells (lane 2).
  • the number of active gene switch molecules was that in transiently transfected cells that only produced autoregulated gene switches (transfection with 17x4 / IL15 / Oligo / GS and 17x4 / IL15 / Oligo / IVS8 / GS).
  • pCMVß positive control for lacZ, vector with lacZ gene under the control of a CMV promoter
  • the cells were fixed for 10 min at -20 ° C. with ice-cold methanol, washed 3 ⁇ with PBS and then for 2.5 h at 37 ° C. with lacZ staining solution (60 ⁇ l of 400 mM potassium ferricyanide, 60 ⁇ l of 400 mM potassium ferrocyanide, 60 ⁇ l of 200 mM MgC12, 300 ⁇ l 20 mg / ml X-Gal, 5.52 ml PBS).
  • lacZ staining solution 60 ⁇ l of 400 mM potassium ferricyanide, 60 ⁇ l of 400 mM potassium ferrocyanide, 60 ⁇ l of 200 mM MgC12, 300 ⁇ l 20 mg / ml X-Gal, 5.52 ml PBS.
  • Example 5 Production and characterization of transgenic mice 50 ⁇ g DNA of the constructs 17x4 / IL15 / Oligo / GS and 17x4 / IL15 / Oligo / IVS8 / GS were cut with the restriction enzymes Eco47III and Notl and the desired fragments of 4800 bp and 4924 bp in length purified.
  • the fragments were then injected into the pronuclei of C3HeB / FeJ mice and the embryos then implanted in sham pregnant women.
  • the genomic DNA was extracted from the mice obtained and examined for the integration of the transgene with the aid of PCR analysis.
  • the following primers were used for the PCR analysis: GS-IL15FW.2 (5'-TAT
  • the PCR products obtained accordingly have a length of 211 bp (without intron) or 335 bp (with intron IVS8).
  • mice Of 44 mice obtained from the oocyte injection of the construct 17x4 / ILl 5 / oligo / GS, 16 animals were transgenic (36%). The founder animals (F0 generation) were tested with wild-type DBA 2 mice and the FI offspring obtained were in turn examined for the genomic presence of the transgene. The transgenic FI animals were then examined for regulated expression of MutIL-15 / mFc. At the age of approx. 8-16 weeks, the animals were injected with 250 ⁇ g / kg mifepristone (Sigma, Deisenhofen M 8046) dissolved in sesame oil a total of three times intraperitoneally every other day. The animals were sacrificed one day after the last injection and RNA or protein was extracted from the tissues.
  • mifepristone Sigma, Deisenhofen M 8046
  • RNA The expression of gene switching protein or immunomodulator in the tissues was then analyzed using ELISA and Western blot.
  • the amount of RNA is determined by means of a quantitative reverse transcription PCR (RT-PCR) in order to determine the amount of transcribed gene switch or immunomodulator.
  • RT-PCR quantitative reverse transcription PCR
  • 1 ⁇ g RNA each are transcribed into cDNA using the Expand Reverse Transcriptase (Röche, Mannheim) according to the manufacturer's instructions.
  • the expression of the gene switch or the mutILl 5 / mFc immunomodulator is then examined quantitatively using the Light Cycler Fast Start DNA Master SYBR Green Kit (Röche, Mannheim).
  • the PCR conditions for the detection of the immunomodulator are as follows: Denaturation: 95 ° C, 600 sec cycles: 95 ° C 15 sec, 60 ° C 5 sec, 72 ° C 10 sec.
  • the PCR conditions for the detection of the gene switch are as follows: Denaturation: 95 ° C, 600 sec
  • mice of the same age are used in the same transgenic line, who had not previously been treated with mifepristone.
  • the transgenic mice are produced in a two-step process.
  • two different lines of transgenic mice are generated, which either express only the gene switch molecule (lines “A") or only the immunomodulator regulated by the gene switch binding site (lines “B”).
  • lines "A" express only the gene switch molecule
  • lines "B” only the immunomodulator regulated by the gene switch binding site
  • From the transgenic mouse strains of the A lines obtained those animals are selected which express suitable amounts of the gene switch molecule and, in a second step, variate with transgenic animals of the B lines.
  • the progeny obtained are then examined for simultaneous expression of the two transgenes. In this way, double transgenic lines "A / B" are obtained which express immune modulators regulated by gene switch molecules.
  • DNA of the constructs pswitch (for lines “A") or 17x4 / IL15 / Oligo and 17x4 / IL15 / Oligo / IVS8 (for lines “B") are cut with suitable restriction enzymes and the desired fragments are purified. Then the fragments are injected into the precuclear nucleus of C3HeB / FeJ mice and the embryos are then implanted in apparently pregnant females. The genomic DNA is extracted from the mice obtained and included Using PCR analysis to examine the integration of the transgene.
  • Example 6 Transplantation of Isles from Donor Organs of Transgenic Mice
  • islets isolated from mouse pancreata are injected under the kidney capsule of diabetic mice (Ferrari-Lacraz et al, The Journal of Immunology, 2001, Vol.167 p. 3478-3485).
  • Donor Islands are isolated from transgenic mice of the strain DBA / 2J, which express MutIL-15 / mFc under the control of the gene switch.
  • the donor mice are pretreated with mifepristone (250 ⁇ g / kg) so that they demonstrably produce MutIL-15 / mFc on the day the organ is removed.
  • donor pancreata are perfused in situ with type IV collagenase (2 mg / ml; Worthington Biochemical Co ⁇ .). After 30 minutes of digestion at 37 ° C., the islands are purified using a discontinuous Ficoll gradient and then cultured in RPMI1640 medium (Gibco, Düsseldorf) with 5.6 mM glucose. Then 300-400 islets are transplanted under the recipient's kidney capsule.
  • the recipient is a 6-10 week old, non-transgenic B6AF1 mouse, in which diabetes was induced by intraperitoneal injection of the beta cell toxin streptozotocin (225 mg / kg; Sigma, Deisenhofen).
  • syngeneic islands i.e., islands from B6AF1 mice
  • Pancreatic islets are grafted aseptically under the left kidney capsule.
  • the mouse is anesthetized and an incision is made under the left costal arch.
  • the left kidney is mobilized out of the abdomen and the lower pole of the kidney capsule is cut briefly.
  • a pocket is formed under the kidney capsule with a blunt sterile cannula.
  • the function of the allograft is monitored via regular blood glucose measurements (Accu-Check III; Boehringer Mannheim, Mannheim). Before and after the diabetes induction and after the island transplant, blood is regularly taken from the tail vein or the retrobulbar venous plexus from the animals and the blood sugar level is determined. Additional controls urine test strips are used between blood tests.
  • Primary graft function is defined as blood sugar levels below 11mmol / l (200 mg / dl) on day 3-5 after the transplant. The graft is considered rejected if the blood sugar level has risen to over 500 mg / dl for at least two consecutive days after observing primary graft function.
  • island transplantations are carried out in which the animals either do not receive mifepristone and thus do not express gene switch-regulated MutIL-15 / mFc or in which the animals are treated with external addition of MutIL-15 / mFc.
  • the animals treated in the manner described can better regulate their blood sugar levels after the transplantation of islet cells which are obtained from transgenic donor mice and show a reduced rejection of the cell transplants when treated with mifepristone.
  • Donor hearts are isolated from transgenic DBA / 2J strain mice that express MutIL-15 / mFc under the control of the gene switch.
  • the donor mice are pretreated with mifepristone (250 ⁇ g / kg) so that they demonstrably produce MutlL-15 / mFc on the day the organ is removed.
  • mifepristone 250 ⁇ g / kg
  • the vena cava is isolated and heparin (400 U / kg) is injected for distribution over the entire circulation.
  • the animals are then bled by dividing the abdominal vessels and the heart exposed using a median sternotomy.
  • the aorta and pulmonary vessels are isolated and divided and the pulmonary veins and the vena cava en masse are ligated with 4-10 Tevdek (Deknatal, Queens Village).
  • the heart is stored at 4 ° C in physiological saline (Physiolosol, Abbot Laboratories, Illinois, USA) immediately after its removal.
  • physiological saline Physiolosol, Abbot Laboratories, Illinois, USA
  • the inferior vena cava and abdominal aorta of the recipient (6-8 Week-old, non-transgenic B6AF1 mouse) are dissected and the vessels are provided with loosely located vascular clamps.
  • the end of the donor aorta is connected to the side of the recipient abdominal aorta with an 8-0 prole thread.
  • the donor's pulmonary artery is fused to the recipient's vena cava in the same way.
  • the vascular clamps are removed and the donor heart is warmed with 37 ° C warm Ringer's lactate solution, so that the heart contraction starts again spontaneously.
  • the recipient animals are treated with mifepristone 250 ⁇ g / kg every other day from the day of transplantation, so that they continuously produce MutIL-15 / mFc. Survival of the graft is checked every other day by palpating the beating heart through the abdominal wall and assessed on a scale of 1+ to 4+ based on the strength and rate of the impulses.
  • the heart is said to be rejected when there are no more heart muscle contractions. Rejection is prevented if the donor hearts beat for a longer period than hearts in untreated control animals.
  • heart transplantations are carried out, in which the animals either do not receive mifepristone and thus do not express a gene switch-regulated MutIL-15 / mFc or in which the animals are treated with external addition of MutIL-15 / mFc.
  • This example shows that the heterotopic heart transplants in the animals treated with mifepristone remain functional longer than in the untreated animals.
  • Example 8 Preparation of transgenic ES cell lines 100 ⁇ g DNA of constructs 17x4 / IL15 / Oligo / RIPnh / GS,
  • 17x4 / IL15 / Oligo / RIPnh / IVS8 / GS were cut with the restriction enzymes Eco47III and Notl, the fragments were purified and resuspended in at least 100 ⁇ l sterile PBS.
  • the constructs 17x4 / IL15 / Oligo / RIPhn / GS and 17x4 / IL15 / Oligo / RIPhn / IVS8 / GS were then introduced by electroporation into mouse ES cells of the SVJ129 or RI line. To Exponentially growing mouse ES cells were trypsinized, separated and counted.
  • ES cell medium DMEM with 20 mM Hepes, 15% heat-inactivated FCS, 50 U / ml penicillin, 50 ⁇ g / ml streptomycin, 0.1 mM non-essential amino acids, 0.1 mM mercaptoethanol, 10 3 U / ml Leukemia Inhibitory Factor
  • the cells were selected for up to 5-9 days for uptake of the construct and individual transgenic clones were isolated by hand under the sterile bench and transferred to 96-well plates. The cells were passaged before reaching confluence and successively transferred to 48-well plates, 24-well plates, 6-well plates and 10 cm dishes.
  • the cell clones were checked for controllable expression of the gene switch and MutIL-15 / mFc using quantitative RT-PCR or MutIL-15 / mFc using ELISA and Western blot assays (as described above in Examples 3 and 4) ) examined.
  • the transgenic ES cells are produced in a 2-step process. First, transgenic ES cells are generated, which only express the gene switch molecule in a regulated manner. From the transgenic cell lines obtained, those clones are selected which express suitable amounts of the gene switch molecule. In a second step, these clones are supertransfected with DNA constructs that encode the immune modulator regulated by a gene binding site.
  • double-transgenic ES cells which express the immune modulator regulated by gene switch molecules.
  • 100 ⁇ g DNA of the gene switch construct are cut with suitable restriction enzymes, the desired fragment is purified, resuspended in sterile PBS and introduced into ES cells by electroporation as described above. Since the construct confers resistance to the antibiotic Zeocin, the cells can be selected for successful transfection by treatment with this antibiotic.
  • Transgenic clones are isolated from the cells obtained and the expression of the gene switch molecule as a function of mifepristone addition is examined by quantitative RT-PCR.
  • Those clones which express suitable amounts of the gene switch molecule are super-transfected in a second step by renewed electroporation with purified Eco47III / NotI fragments of the constructs 17x4 / IL15 / Oligo / RIPhn and 17x4 / IL15 / Oligo / RIPhn IVS8 and by hygromycin. Treatment selected for uptake of the second transgene. The simultaneous integration of both transgenes is checked by means of PCR analysis of the genomic DNA.
  • the double transgenic cell clones obtained are after addition of 10 nM mifepristone for controllable expression of the gene switch and MutIL-15 / mFc by means of quantitative RT-PCR or MutIL-15 / mFc protein with the aid of ELISA and Western blot assays as above examined in Example 5 examined. Subsequently, those clones are selected from the cell lines obtained which produce and secrete the immune modulator MutlL-15 / mFc in sufficient quantities only after adding mifepristone.
  • Example 9 Transplantation of insulin-producing cells which are produced from transgenic ES cells. Before the transplantation, undifferentiated hygromycin-resistant ES cell clones are used to produce insulin-producing cells which are under the control of the Gene switch MutIL-15 / mFc express Soria et al. (Diabetes. 2000 Feb .; 49 (2):
  • the insulin-producing cells are treated with mifepristone before the transplant so that they produce sufficient MutIL-15 / mFc. Then 1 million insulin-producing cells in streptozotocin-treated diabetic C57BL / 6 mice either under the kidney capsule (as described in Example 7) or in the spleen (Soria et al. (Diabetes. 2000 Feb .; 49 (2): 157-62) The recipient animals are treated with mifepristone (250 ⁇ g / kg) every other day from the day of transplantation so that they continuously produce MutIL-15 / mFc.
  • the function of the allograft is monitored via regular blood glucose measurements (Accu-Check III; Boehringer Mannheim, Mannheim).
  • Primary graft function is defined as blood sugar levels below 11mmol / l (200 mg / dl) on day 3-5 after the transplant. The graft is considered rejected if the blood sugar level rises to over 500 mg / dl for at least two consecutive days if primary graft function is previously observed.
  • insulin-producing cells are transplanted into recipient animals which have been produced from the same cell clone but are not pretreated with mifepristone and therefore do not express any gene switch-regulated MutIL-15 / mFc.
  • insulin-producing cells can also be used which contain the MutIL-15 / mFc construct but not the gene switch
  • Animals also serve as controls, which are treated with external addition of MutlL-15 / mFc after the receipt of insulin-producing cells derived from stem cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Diabetes (AREA)
  • Environmental Sciences (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Rheumatology (AREA)
  • Plant Pathology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Neurology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Husbandry (AREA)
  • Endocrinology (AREA)
  • Dermatology (AREA)
  • Emergency Medicine (AREA)

Abstract

L'invention concerne une cellule humaine ou animale non totipotente, contenant au moins un acide nucléique codant au moins un immunomodulateur sous contrôle d'une molécule de commutation génétique pouvant être régulée par addition d'une substance active. L'invention concerne également la fabrication et l'utilisation de ladite cellule pour la transplantation, l'inhibition d'une réaction de rejet de transplantation, et la prophylaxie et/ou la thérapie de maladies auto-immunes et/ou de maladies faisant suite à une transplantation.
EP03747785A 2002-10-14 2003-10-13 Cellule transplantable Withdrawn EP1556487A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP03747785A EP1556487A2 (fr) 2002-10-14 2003-10-13 Cellule transplantable

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP02022868 2002-10-14
EP02022868 2002-10-14
EP03747785A EP1556487A2 (fr) 2002-10-14 2003-10-13 Cellule transplantable
PCT/CH2003/000665 WO2004035787A2 (fr) 2002-10-14 2003-10-13 Cellule transplantable

Publications (1)

Publication Number Publication Date
EP1556487A2 true EP1556487A2 (fr) 2005-07-27

Family

ID=32103881

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03747785A Withdrawn EP1556487A2 (fr) 2002-10-14 2003-10-13 Cellule transplantable

Country Status (11)

Country Link
EP (1) EP1556487A2 (fr)
JP (1) JP2006502716A (fr)
KR (1) KR20050071579A (fr)
CN (1) CN1705744A (fr)
AU (1) AU2003266903A1 (fr)
BR (1) BR0315286A (fr)
CA (1) CA2502312A1 (fr)
MX (1) MXPA05003912A (fr)
PL (1) PL376397A1 (fr)
RU (1) RU2005114511A (fr)
WO (1) WO2004035787A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BRPI0920679A2 (pt) 2008-10-08 2022-05-17 Intrexon Corp Células construídas expressando múltiplos imunomoduladores e usos das mesmas

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5912411A (en) * 1993-06-14 1999-06-15 University Of Heidelberg Mice transgenic for a tetracycline-inducible transcriptional activator
US5602301A (en) * 1993-11-16 1997-02-11 Indiana University Foundation Non-human mammal having a graft and methods of delivering protein to myocardial tissue
FR2782732A1 (fr) * 1998-08-28 2000-03-03 Transgene Sa Systeme d'expression inductible
AU1685201A (en) * 1999-12-01 2001-06-12 Centre For Translational Research In Cancer Drug inducible system and use thereof
DE10014690A1 (de) * 2000-03-24 2001-10-18 Franz Wolfgang M Verfahren zur Isolierung in vito differenzierter Körperzellen
AU2001261585B2 (en) * 2000-05-12 2006-08-31 Beth Israel Deaconess Medical Center, Inc. Compositions and methods for achieving immune suppression

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of WO2004035787A3 *
STROM T B; FIELD L J; RUEDIGER M: "Allogeneic stem cells, clinical transplantation and the origins of regenerative medicine", CURRENT OPINION IN IMMUNOLOGY, CURRENT BIOLOGY LTD, vol. 14, 1 October 2002 (2002-10-01), England, pages 601 - 605, XP004377413 *

Also Published As

Publication number Publication date
MXPA05003912A (es) 2005-09-30
AU2003266903A1 (en) 2004-05-04
CN1705744A (zh) 2005-12-07
WO2004035787A3 (fr) 2004-08-26
WO2004035787A2 (fr) 2004-04-29
PL376397A1 (en) 2005-12-27
BR0315286A (pt) 2005-08-30
RU2005114511A (ru) 2005-11-20
KR20050071579A (ko) 2005-07-07
JP2006502716A (ja) 2006-01-26
CA2502312A1 (fr) 2004-04-29

Similar Documents

Publication Publication Date Title
DE69434168T2 (de) Liganden für die FLT3 Rezeptoren
DE69832402T2 (de) Erzeugung von hämatopoietischen zellen aus multipotenten neuronalen stammzellen
DE69534227T2 (de) Stoffe und verfahren zur beherrschung der hyperakuten abstossung von menschlichen transplantaten
KR102122267B1 (ko) 당뇨병 치료를 위한 복수 형질전환 돼지
DE69434986T2 (de) Homologe rekombination für universelle donorzellen und chimerische säugetierzellen
JPH04501510A (ja) 普遍的なドナー細胞及びキメラ性哺乳類宿主のための相同性組換え
KR102377189B1 (ko) C9orf72 유전자좌에서 파괴를 갖는 비-인간 동물
EP1554307A2 (fr) Antagonistes il-15
DE69632967T2 (de) Verfahren zur Verhinderung der Transplantabstoßung bei einer Transplantation und zur Herstellung einer universalen Gentherapie-Wirtszelle unter Anwendung von Lymphocyten-Aktivierungsgen (LAG-3)
DE69832798T2 (de) Regulatorische sequenzen für die in-vivo-expression einer heterologen dns-sequenz in endothelzellen und ihre verwendungen.
DE69432856T2 (de) Zufuhr von genprodukten mittels mesangium-zellen
WO2004003156A2 (fr) Le l-21, regulateur de la production d'immunoglobine
EP1556487A2 (fr) Cellule transplantable
JP2022512674A (ja) 人工トランス活性化因子による選択
US6677311B1 (en) Inducible HSV-TK in transformed cell populations
AU5268800A (en) Genetic modification of male germ cells for generation of transgenic species and genetic therapies
DE10128980B4 (de) Gen-modifizierte alloreaktive T-Zellen, Verfahren zu ihrer Herstellung und ihre Verwendung
US7294755B1 (en) Genetic modification of male germ cells for generation of transgenic species and genetic therapies
WO1994016065A1 (fr) Production de cellules destinees a la transplantation et reduisant le rejet du greffon par l'hote, et cellules ainsi obtenues
DE69938335T2 (de) Methoden zur kontrolle der zelldifferenzierung und des zellwachstums durch verwendung eines fusionsproteins und eines wirkstoffes
DE69734725T2 (de) Neues verfahren zur überprüfung des differenzierungszustands pankreatischer zellen eines säugetiers
DE69728263T2 (de) H2-M modifizierte transgene Tiere
Tortola et al. KappaBle fluorescent reporter mice enable low-background single-cell detection of NF-κB transcriptional activity in vivo
DE69731950T2 (de) Tumorvakzinierung mittels Verwendung autologer oder HLA-verwandter Antigen präsentierender Zellen (APC), die mit einem Tumorantigen sowie einem eine Immunantwort hervorrufenden Fremdantigen transduziert sind
RU2778405C2 (ru) Способы защиты плодов свиней от инфицирования вирусом

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050511

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20060516

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20070521