EP1220900A2 - Gentransfervektoren zur therapie von autoimmunerkrankungen und erkrankungen mit immunpathogenese - Google Patents

Gentransfervektoren zur therapie von autoimmunerkrankungen und erkrankungen mit immunpathogenese

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Publication number
EP1220900A2
EP1220900A2 EP00984828A EP00984828A EP1220900A2 EP 1220900 A2 EP1220900 A2 EP 1220900A2 EP 00984828 A EP00984828 A EP 00984828A EP 00984828 A EP00984828 A EP 00984828A EP 1220900 A2 EP1220900 A2 EP 1220900A2
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Prior art keywords
cells
nucleic acid
apoptosis
vectors
acid sequence
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German (de)
English (en)
French (fr)
Inventor
Fritz Schwarzmann
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Lophius Biosciences GmbH
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Fritz Schwarzmann
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    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a gene transfer vector comprising at least one nucleic acid molecule, comprising a first nucleic acid sequence coding for one or more apoptosis-triggering ligands, a second nucleic acid sequence coding for one or more antigen (s) and optionally, a third nucleic acid sequence coding for a or more anti-apoptosis molecule (s), and optionally a fourth nucleic acid sequence, coding for one or more suicide enzyme (s).
  • autoimmune diseases such as multiple sclerosis (MS) or diabetes (type 1) is based on an uncontrolled activation of immune cells that are healthy
  • T-lymphocytes which are fragments of the body's own proteins in the body
  • autoimmune diseases In autoimmune diseases, this tolerance is disturbed or insufficient. Autoimmune diseases may be viral or bacterial Infections triggered. It is believed that due to the similarities between pathogen-specific and cell-type-specific proteins, uninfected cells are also attacked by pathogen-specific antibodies or T cells. In connection with chronic persistent viral infections, there are also inflammatory processes, some of which affect vital organs such as the liver (hepatitis), although the immune response is primarily directed against pathogen-specific structures. In such cases, one speaks of immunopathogenesis, since the damage and symptoms are primarily caused by the patient's own immune system and not by the pathogen. The same thing happens with the rejection of transplanted organs. A combination of foreign MHC molecules from the donor, complexed with processed cellular proteins, is recognized as foreign by the recipient's T cells.
  • autoimmune diseases and other diseases with immunopathogenesis are treated by strong immunosuppression and / or by administration of regulatory-effective cytokines such as interferon-ß.
  • regulatory-effective cytokines such as interferon-ß.
  • a large number of different preparations from different manufacturers are available for classic chemotherapy with immunosuppressants, which differ considerably in the area of application and the mode of action.
  • a particularly serious disadvantage of these substances is the occurrence of diverse and sometimes life-threatening side effects (including kidney damage, liver damage, pancreatitis, anemia, fever), which are observed in a large number of patients.
  • Such non-specific inhibition of the immune system e.g. by cyclosporin or FK506 not only leads to a greatly weakened immune system and thus to an increased susceptibility to infectious diseases, but also favors the development of tumors.
  • Fas-mediated apoptosis also plays a critical role in maintaining immune-privileged areas in the body.
  • the immune privileged condition of the testicles and anterior chamber require a strong expression of Fas ligand on the corresponding parenchymal cells of these organs. In these cases, it is believed that the expression of Fas ligand on the parenchymal cells protects these tissues from destruction by T cells by inducing apoptosis in the T cells.
  • a viral infection in the anterior chamber leads to systemic T cell tolerance to the virus. It is believed that APCs that present the Fas ligand on their cell surface along with privileged antigens derived from the privileged cells induce apoptosis of T cells in the periphery of the body, thereby causing systemic T cell tolerance.
  • mice treated with the APCs showed a significantly prolonged persistence of the virus in the liver on subsequent infection with an adenovirus, in accordance with a repressed T cell response against cells infected with adenovirus. Furthermore, the T cell tolerance was dependent on the function of the Fas ligand, since no tolerance could be induced in Fas-negative mice. A year later, experiments by the same group to induce T cell tolerance to alloantigens were also published in the mouse model (Zhang et al. (1999) J Immunol 162: 1423-1430).
  • FIG. 2 graphically shows the result of the inhibition of allogeneic stimulation of T cells by FasL-expressing antigen-presenting cells.
  • Macrophages were isolated from B6-lpr / lpr mice and infected with adenoviruses expressing either LacZ (AdLacZ) or FasL (AdFasL).
  • the infected macrophages were co-cultivated with either T cells from (A) Fas-expressing B6 - + / + mice or (B) Fas-deficient B6-lpr / lpr mice and stimulation of the T cells by incorporating 3 H-tymidine measured (Mixed Lymphocyte Response, MLR).
  • FIG. 6 graphically shows the result of a reduced production of autoantibodies in cytomegalovirus (MCMV) -infected mice which were treated before the infection with AdFasL-infected antigen presenting cells (APC).
  • MCMV cytomegalovirus
  • APC AdFasL-infected antigen presenting cells
  • FIG. 8 graphically shows the results of an experiment to demonstrate the modulating influence of IL-10 and tumor necrosis factor (TNF) on the function of dentritic cells (DC) as antigen-presenting cells.
  • Dentritic cells were generated from mononuclear cells of the peripheral blood by treatment with IL-4 and GM-CFS in vitro. The DCs were either treated with TNF or with IL-10 and then incubated with allogeneic T cells and the stimulation and proliferation of the T cells measured by the incorporation of 3 H-labeled thymidine.
  • APC antigen presenting cells
  • DC (TNF) Dentritic cells stimulated with tumor necrosis factor (TNF);
  • DC (IL-10) Dentritic cells stimulated with interleukin-10 (IL-10); allo T cells: T cells of a donor with an allogeneic MHC pattern, ie MHC pattern deviating from the DCs.
  • the X axis shows the amount of APCs that were used in the reaction.
  • the Y axis shows the radioactive decays per minute (CPM) as a measure of the incorporation of the radioactively labeled nucleotide or as a measure of the stimulation of the T cells.
  • CPM radioactive decays per minute
  • the invention further relates to the use of the gene transfer vectors for the ex vivo modification of eukaryotic cells, in particular animal or mammalian cells, in particular human cells.
  • Retroviral vectors for the ex vivo modification of eukaryotic cells, in particular animal or mammalian cells, in particular human cells.
  • compositions of liposomes and immunostimulatory reconstituted influenza virosomes are also suitable as vehicles for the transfer of the vectors according to the invention into mammalian and human cells (US Pat. No. 5,879,685).
  • foreign nucleic acid sequences can be integrated into a vector with suitable control sequences, bound to synthetic gene transfer molecules such as polymeric DNA-binding cations (e.g. polylysine, protamine and albumin) and coupled with cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose or transferrin.
  • the vectors according to the invention encode and express the following epitopes, individually or in combination: AS 52-71, AS 72-91, AS 82-101, AS 102-121, AS 132-151, AS 142-161, AS 182-201, AS 192 - 207.
  • Glutamic acid decarboxylase 65 is one of the target structures for autoimmune reactions in patients with type I diabetes. Mononuclear cells in the blood of patients react to the protein with cell division and over 70% of the patients also have antibodies against GAD65. A combination of antibodies against GAD65 and IA-2 in people who are genetically predisposed to diabetes due to their MHC type is a strong indicator of developing type I diabetes.
  • the vectors can be used as an antigenic target for T cells in diabetes encode and express the entire GAD65 protein or protein fragments.
  • the heat shock protein Hsp60 represents another target for activated immune cells in type I diabetes.
  • the vectors encode and express, for example, the area of AS 437-460
  • Cell death by apoptosis is characterized by a compression of the chromatin , a fragmentation of the chromosomal DNA, a kind of blistering of the membrane, a shrinkage of the cell, and finally a decay of the dead cell in the membrane-enclosed vesicle (apoptotic body).
  • the path relevant for the present invention leads to a stimulation of apoptosis receptors on the surface of the cells, such as CD95 / Fas / Apol, TRAIL or Apo3, and mediation by adapter molecules, such as FADD and TRADD, for the activation of the regulatory active caspase 8 ( FLICE, initiator caspase) and subsequent caspases such as Caspase 3 (effector caspase), which trigger apoptosis.
  • apoptosis receptors on the surface of the cells such as CD95 / Fas / Apol, TRAIL or Apo3, and mediation by adapter molecules, such as FADD and TRADD, for the activation of the regulatory active caspase 8 ( FLICE, initiator caspase) and subsequent caspases such as Caspase 3 (effector caspase), which trigger apoptosis.
  • FLICE regulatory active caspase 8
  • Caspase 3 effector caspase
  • the apoptosis-triggering ligands are membrane-bound or soluble proteins, for example CD95L / FasL / ApolL, Apo2L / TRAIL or Apo3L, which interact with specific receptor molecules, such as CD95 / Fas / Apol, TRAIL or Apo3, on another or the same cell, and thereby trigger apoptosis in the receptor-carrying cells.
  • the ligands that trigger apoptosis belong, for example, to the superfamily of tumor necrosis factors (TNF).
  • TNF tumor necrosis factors
  • Adapter proteins represent a connection between the effectors (caspases) and the regulators (receptors Bcl-2 family) of apoptosis.
  • the adapters interact physically via homotypic interactions with the three groups of factors via so-called death domains (DD), death ejfector domains ( DED) and caspase recruitment domains (CARD).
  • the vectors according to the invention comprise nucleic acid sequences which code for ligands which trigger apoptosis.
  • the vectors code, for example, for the ligand CD95L, which binds to the receptor molecule CD95 / Fas / Apo-1.
  • the vectors according to the invention can comprise nucleic acid sequences which code for the protein TRAIL (APO-2L) which binds to the specific receptor molecules TRAIL-Rl (DR4), TRAIL-R2 (KILLER, DR5, TRICK2), TRAIL-R3 (LIT, DcRl ) and TRAIL-R4 (TRUNDD, DcR2) binds.
  • TRAIL has been detected naturally on a number of cells, such as type II interferon-stimulated monocytes, cytomegalovirus-infected fibroblasts, type I interferon and antigen-stimulated T cells or NK cells.
  • the vectors according to the invention can comprise nucleic acid sequences which code for the APO-3 ligand (Apo3L / TWEAK).
  • Apo3L is a type II transmembrane protein with a length of 149 amino acids. The extracellular region of ApoL3 shows high homology to TNF. Apo3L mRNA was detected in a wide variety of lymphoid and non-lymphoid tissues. ApoL3 binds to the receptor molecule Apo3 (DR3, WSL-1, TRAMP, LARD) and induces apoptosis in the receptor-bearing cells.
  • Apo3L is a type II transmembrane protein with a length of 149 amino acids. The extracellular region of ApoL3 shows high homology to TNF. Apo3L mRNA was detected in a wide variety of lymphoid and non-lymphoid tissues. ApoL3 binds to the receptor molecule Apo3 (DR3, WSL-1, TRAMP,
  • the receptor for Apo3L is primarily expressed on the cell surface of lymphocytes, for example on unstimulated resting lymphocytes in peripheral blood (PBL), phytohaemagglutinin (PHA) -treated PBL, CD4 + T cells, CD8 + T cells and B cells.
  • PBL peripheral blood
  • PHA phytohaemagglutinin
  • the induction of apoptosis via Apo3L is mediated by FADD / MORT1.
  • ApoL3-mediated apoptosis is blocked by the viral caspase inhibitors CrmA of the cowpox virus and by the p35 protein of baculoviruses.
  • the induction of apoptosis can be inhibited at the various stages of the cascade of receptors, adapters and caspases.
  • Viruses in particular have invented numerous strategies to defend themselves against apoptosis as an immune mechanism.
  • the present invention uses these viral and cellular mechanisms to protect the cells altered by the vectors from autocrine induction of apoptosis.
  • the vectors according to the invention can comprise, for example, nucleic acid sequences which code for adenoviral proteins from the E3 region of the virus. These proteins inhibit membrane-bound biochemical processes that are induced when TNFR is activated.
  • the vectors according to the invention can comprise nucleic acid sequences which code for proteins which have high homology to the DED domains. These proteins are called FLEPs or the viral proteins are called vFLIPs.
  • FLEPs proteins which have high homology to the DED domains.
  • vFLIPs the viral proteins
  • FLIPs / vFLIPs the signal cascade between receptors and FADD on the one hand and Caspase 8 (FLICE) on the other hand is blocked and apoptosis is thereby prevented.
  • the LMP-1 protein from Epstein-Barr virus interacts with various TRAF molecules (TNFR- associated factors) and thereby blocks the signal transmission from the TNFR.
  • LMP-1 also binds TRADD. Probably due to a modified binding site for TRADD, however, the apoptosis signal cascade is not triggered.
  • LMP-1 additionally induces the expression of the antiapoptotic proteins A20, Bcl-2 and Mcl-1.
  • the vectors according to the invention also express, for example, the LT protein of SV40, which mediates resistance to Fas-induced apoptosis via a protein kinase C-mediated route.
  • the vectors according to the invention code, for example, for the polyoma proteins ST and MT, both of which mediate resistance to CD95- and TNFR-mediated apoptosis.
  • the ST protein achieves this by binding and inhibiting the PP2A protein.
  • the MT protein directly activates signaling pathways that promote cell survival. This includes the PI3 kinase, which subsequently phosphorylates the proapopic protein Bad and thus inactivates it.
  • Inhibitors of caspases can also be encoded on the vectors according to the invention in order to prevent autocrine induction of apoptosis in the changed cells.
  • the p35 protein of the Baculoviruses Autographica californica nuclear polyhedrosis Virus (AcNPV) and Bombyx mori NHV (BmNPV) is synthesized in the early phase of virus multiplication and prevents apoptosis by a number of different stimuli.
  • p35 blocks the induction of apoptosis by TNF and CD95 ligands.
  • p35 is cleaved by a series of caspases (caspase 1, 3, 6, 7, 8 and 10).
  • proteins Serp-1 and Serp-2 of the myxomavirus and SPI-4 of the rabbit pox virus have corresponding antiapoptotic properties.
  • homologous proteins can be encoded into cellular cLAPs, such as, for example, vIAPs, or cellular proteins, such as, for example, FLAME-1 or I-FLICE.
  • Cydia pompnella granulosis virus (CpGP), Orgyiapse audotsugata polyherdosis virus (OpMNPV) and AcNPV code for viral IAPs (vIAPs) that are in the signaling cascade above of p35 act.
  • vIAPs bind and inhibit inactive procaspases and caspase 8, but cannot inhibit caspases that have already been activated like p35.
  • cIAPs interact with TRAF molecules, but can also prevent apoptosis through non-TNFR-associated processes.
  • a conserved RTNG finger motif and at least one so-called BIR motif (baculovirus IAP repeat) are necessary for the antiapoptotic activity.
  • FLAME-1 is a cellular protein that inhibits apoptosis by the CD95 / TNF receptor (Srinivasula et al. (1997) J Biol Chem 272: 18542-18545).
  • FLAME-1 has high homology to Caspase 10 and Caspase 8 (FLICE).
  • Two neighboring regions are located in the amino terminal area and show homology to the DED domains of FADD, which enable homotypic interactions with other DED proteins.
  • a third neighboring region shows homology to the functional caspase domain of caspases 8 and 10.
  • FLAME-1 interacts directly with FADD, caspase 8 and caspase 9, but has no caspase activity.
  • the vectors of the invention either synthesize a single antisense RNA or a combination of different ones which are specifically directed, for example, against individual apoptosis receptors, caspases or adapter molecules.
  • the vectors express a single antisense RNA, which contains several regions, each of which is specific for individual apoptosis receptors, caspases or adapter molecules, and in combination prevents the expression of several proteins involved in apoptosis.
  • the vectors according to the invention can code, for example, for CD95-specific antisense RNAs and block the expression of CD95 / Fas. Blocking CD95 prevents induction of apoptosis via CD95L / FasL.
  • the gene therapy vectors encode and express, for example, TNFR-specific antisense RNAs which block expression of TNFR and protect the cell from TNF-mediated apoptosis.
  • the vectors encode and express, for example, TRAIL-R1 or TRALL-R2-specific antisense RNAs which prevent expression of the receptors TRAIL-R1 or TRAIL-R2. This makes the cells resistant to TRAIL-mediated apoptosis.
  • the vectors according to the invention either synthesize a single antisense RNA or a combination of different antisense RNAs which are specifically directed against individual adapter proteins.
  • the vectors express an antisense RNA which contains individual regions which are in each case specific for an adapter protein and, in combination, prevents the expression of several adapter proteins.
  • the vectors according to the invention are characterized in that they optionally comprise nucleic acid sequences which code for suicide enzymes by means of which genetically modified cells can be used at any time, e.g. in vivo, can be eliminated.
  • the suicide genes encode and express, for example, enzymes which convert biological substrates (prodrugs) which are supplied to the body from the outside into toxic substances or modify the substances in such a way that the enzymes of the cell use them as substrates.
  • suicide enzymes code for substances that are toxic per se, but the expression of these genes in the genetically modified cell is strictly controlled.
  • the genes for substances which are toxic per se are strongly repressed in the cell and are not synthesized. By adding biological or chemical substances, the genes that code for the toxic proteins are activated and the cells die.
  • the vectors described in connection with this invention preferably code for suicide genes which convert non-toxic or little toxic prodrugs into toxic substances.
  • Antigen-presenting cells can be treated with one of the vectors according to the invention for the treatment of diseases such as, for example, autoimmune diseases or diseases which are based on immunopathogenesis or diseases which are based on rejection of transplanted tissues or organs.
  • APCs can be treated, for example, with a gene transfer vector comprising nucleic acid sequences coding for the antigens, ligands which trigger apoptosis, antiapoptosis molecules and for suicide enzymes.
  • the APCs can be treated with any type of combination of vectors, for example with several of the vectors according to the invention, which code for different antigens.
  • promoters of non-viral genes are also suitable for the efficient expression of gene sequences in mammals.
  • the expression can be carried out by a consumable or regulatable (inducible) promoter.
  • a glucocorticoid-inducible promoter can be used in certain cell types, such as hormone-stimulable cells.
  • the expression rates can usually be increased by combining the above-mentioned promoter elements with so-called E / z / z ⁇ «cer elements.
  • viral EM ⁇ Hcer elements often have a particular efficiency, since they usually have a broader host spectrum than enhancers from mammalian cells.
  • Very efficient representatives of viral enhancers include the SV40 early gene enhancer and the promoter / enhancer combinations from the LTR of Rous sarcoma virus and the human cytomegalovirus.
  • adjustable enhancer elements can be used, e.g. are only active in the presence of inductors, such as hormones or metal ions.
  • the expression efficiency of cDNA gene sequences which do not contain any introns can be increased significantly by 10-20 times in some cases by fusion of an intron in the 5 'region of the ORF.
  • a particular effectiveness in different cells has, for example for a large number of different introns, a synthetic intron SIS, which was generated by the fusion of an adenovirus splice donor with an immunoglobulin gene splice acceptor, or a SV40 19S late mRNA intron ,
  • the expression efficiency of the desired nucleic acid sequence can be increased by selecting and using suitable, host-specific codons (codon usage).
  • apoptosis receptors on the surface, including the packaging cell lines that can be used to produce viral gene transfer vectors and the cells to be transduced, antigen-presenting cells, transduction of these cells with the gene for an apoptosis-triggering ligand would lead to paracrine and autocrine induction of apoptosis. Switching off the expression of the ligand in the packaging lines or in the transduced cells in culture prevents an unspecific interaction between these cells and enables cultivation.
  • the expression of the ligands is preferably switched off using the methods described below. 1. Using the RevTet system from ClonTech, USA. 2. Another possibility, which is based on the principle of double infection with an expression vector and a regulation vector, is based on the use of bacterial regulation systems in eukaryotes. The gene for the apoptosis-inducing ligand is under the control of the strong prokaryotic T7 promoter. Since neither in the packaging line If T7 polymerase is still present in the cells to be transduced later, the ligand is not expressed. The co-transduction of the gene for the T7 polymerase by means of a second regulatory vector leads to the expression of the ligand in the double-transduced cells. Another suitable system the Cre-loxP system of the bacteriophage Pl
  • vectors which, for example, enable the vectors to multiply in bacteria or eukaryotic cells
  • vectors comprise regulatory nucleic acid sequences which enable expression of the coding regions, or nucleic acid sequences which enable the vector to be packaged or the nucleic acid to be packaged in a vector
  • nucleic acids which code for one or more suicide enzymes and for one or more anti-apoptosis molecules.
  • the expression of the antiapoptosis molecules is always linked to the expression of the suicide enzymes and is dependent on them.
  • Lymphocytes accessory cells and effector cells are the most prominent representatives of the acquired immune system. Lymphocytes are able to specifically recognize foreign antigens and to stimulate a specific humoral and cell-mediated immune response. Different subpopulations of lymphocytes are known which differ in the type of antigen recognition and the specific effector functions. B lymphocytes are the producers of antibodies. They recognize extracellular and antigens presented on the surface of cells and differentiate into antibody-secreting plasma cells after contact with an antigen. T lymphocytes, the mediators of the cell-mediated immune response, can be divided into several subtypes, of which the CD4 + T helper cells and the CD8 ⁇ cytotoxic T cells are most significant. Helpers and cytotoxic T cells have a restricted specificity for antigens.
  • T helper cells secrete cytokines which stimulate T cells and other immune cells, such as B cells and macrophages, to proliferate and differentiate.
  • Cytotoxic T cells CTL
  • suppressor T cells are a subtype of T helper cells that produce cytokines that suppress certain immune functions.
  • a third class of lymphocytes, the natural killer (NK) cells are part of the innate immune response to fight viruses and intracellular pathogens.
  • Mononuclear cells can be purified from peripheral blood, for example, using Ficoll-Hypaque density gradient centrifugation.
  • other methods based on the antibody-mediated recognition of immune cells can be used for the positive and negative selection of cell populations. Examples of such methods are immunomagnetic selection, "panning" for immobilized monoclonal antibodies, antibody / complement-mediated cell lysis and Cell sorting of fluorescence-labeled cells.
  • a suitable surface marker for the selection of T cells is CD3. Specific T helper cells are selected using the CD4 and cytotoxic T cells using the CD8 marker.
  • Suitable methods for separating T cells from mononuclear cell populations are based e.g. on the rosetting of T cells with red blood cells from sheep (SRBC). This method also allows the extraction of non-rosetting cell populations (B-lymphocytes, monocytes, macrophages). SRBCs treated with neuraminidase or 2-aminoethylisothiouronium bromide (AET) are preferred because they have an increased binding of T cells.
  • the different T cell populations can be positively or separated using the previously described methods based on the recognition of specific surface markers by antibodies.
  • Pluripotent stem cells then differentiate into lymphoid stem cells, bone marrow stromal cells, precursor T cells, precursor B cells, thymocytes, T helper cells, cytotoxic T cells and B cells.
  • This differentiation is modulated by growth factors such as IL-3, IL-4, IL-6, IL-7, GM-CSF, M-CSF, G-CSF, IL-2, and IL-5.
  • Granulocyte-monocyte precursors differentiate into monocytes, macrophages, and neutrophils. This differentiation is modulated by the growth factors GM-CSF, M-CSF, and IL-8.
  • Eosinophilic precursors differentiate into eosinophils. This process is modulated by GM-CSF and IL-5.
  • the differentiation of basophilic precursors in mast cells and basophils is stimulated by GM-CSF, IL-4, and IL-9. Megakaryocytes produce blood platelets after stimulation with GM-CSF, EPO and IL-6. Differentiate erythroid progenitor cells, stimulated by EPO, into red blood cells. The purity of the cell populations obtained is checked by FACS analysis using specific antibodies.
  • T lymphocytes This cross-linking induces the proliferation and activation of CD3 + cells, such as T-lymphocytes.
  • the activation of T lymphocytes by CD3-binding agents can also be increased by varying certain parameters, such as the concentration of the binding agent, the incubation time, the number of cells, the incubation temperature, and the binding affinity of the agent, the affinity, and the efficiency of the cell activation become.
  • T cells in peripheral blood monocytes can also be stimulated by phytohemaglutinin (PHA).
  • PHA phytohemaglutinin
  • Other lymphocyte stimulators include Tetanus Toxoid, Concanavalin A (Con A), Ionomycin and PMA.
  • the mammalian or human cells can be cultivated in a suitable nutrient medium to which at least one defined growth factor has been added.
  • growth factors have been described that promote the growth of different cell types. Typical representatives of such growth factors are cytokine mitogens such as IL-2, IL-10, IL-12, and IL-15, which e.g. promote the growth and activation of lymphocytes.
  • cytokine mitogens such as IL-2, IL-10, IL-12, and IL-15, which e.g. promote the growth and activation of lymphocytes.
  • Other cell types are particularly affected by a different class of growth factors, such as hormones, including the human pregnancy hormone chorionic gonadotropin (hCG) and human growth hormone.
  • hCG human pregnancy hormone chorionic gonadotropin
  • the selection of suitable growth factors for defined cell populations is described in detail and is state of the art.
  • 5xl0 6 macrophages were cultivated per well of a 6-well plate in complete DMEM medium for 24 hours and then with an adenoviral vector which led to the expression of the Fas ligand (FasL, CD95L) gene (AdFasL), or a control vector (AdLacZ, expression of the ⁇ -galactosidase) transfected After 48 hours, the transfected macrophages were tested for the expression and functionality of FasL.
  • FasL, CD95L adenoviral vector which led to the expression of the Fas ligand (FasL, CD95L) gene
  • AdLacZ expression of the ⁇ -galactosidase
  • AdFasL-APC AdFasL-APC
  • the mouse model used was tested intraperitoneal infection of Fas-deficient B6-lpr / lpr mice or FasL-deficient B ⁇ -gld / gld mice with lxl 0 6 PFU of the mouse cytomegalovirus (MCMV) induces a chronic inflammatory reaction in various organs, while B6- + / + Mice do not show any relevant organ changes after the elimination of virus-infected cells Hessen.
  • MCMV mouse cytomegalovirus
  • the spleens were also removed from the B6-gld / gld mice and the spleen cells were cocultivated with MCMV-pulsed APC from B6 mice for 48 hours in a mixed lymphocyte reaction (MLR).
  • MLR mixed lymphocyte reaction
  • the T cells were obtained 5 days after the start of the MLR and used in a second MLR against APC from a third donor ("third party") (FIG. 9) Allogeneic-specific suppression of the T cells could also be demonstrated for the IL-10 matured DC, since the response of the T cells, which had initially been co-cultivated with IL-10 matured DC, to the APC of the third donor was unaffected.
  • pcDNA3-FasL-IRES-PLP comprises nucleic acid sequences which code, for example, for the apoptosis-triggering ligand FasL and, for example, for the antigen proteolipid protein (PLP). Both areas are linked by an LRES sequence in such a way that the antigen is translated by the same mRNA as the ligand that triggers apoptosis.
  • the vector is based on the cloning vector pcDNA3.
  • the nucleic acid regions coding for FasL and for PLP were transcribed from RNA into cDNA by polymerase chain reaction (PCR).
  • RNA from cells and the use of PCR to rewrite specific RNA molecules in cDNA are state of the art.
  • the oligonucleotides which were used as primers for the PCR comprise, at their 5 'ends, interfaces for endonucleases which were subsequently used for the cloning of the cDNAs into the vector pcDNA3.
  • the area coding for FasL was first cloned into the vector pcDNA3 via the interfaces for Hind ⁇ ll and BamHI. Subsequently, the fragment coding for PLP was used via them interfaces of BamHI and Ec ⁇ RI.
  • nucleic acid fragment comprising the IR ⁇ S was cloned between FasL and PLP via the recognition sequence for BamHI.
  • the techniques for isolating nucleic acids, the cleavage of nucleic acids and the purification of nucleic acid cleavage products, as well as the ligation of individual nucleic acid fragments and the multiplication of the artificially generated ones Nucleic acids in bacteria are state of the art.
  • pcDNA3-TK-IRES-crmA (Fig. 10B) comprises nucleic acid sequences encoding a suicide enzyme such as thymidine kinase and an anti-apoptosis molecule such as crmA.
  • the expression of crmA is coupled to the expression of TK by an ERES sequence in such a way that crmA can only be expressed together with the TK.
  • the vector is based on the cloning vector pcDNA3 and the cloning strategy and the production of the vector according to the invention is comparable to that of pcDNA3-FasL-ERES-PLP.
  • the region coding for the thymidine kinase was first cloned into the vector pcDNA3 via the interfaces for Hz «dIII and BamKl. Then the fragment coding for crmA was used via them interfaces of Bam ⁇ l and Xhol. Finally, the nucleic acid fragment comprising the ERES was cloned between FasL and PLP via the recognition sequence for Bam ⁇ l.
  • the nucleic acid sequences of the two vectors pcDNA3-FasL-ERES-crmA and pcDNA3-TK-ERES-PLP are listed as SEQ ED NO: 1 and SEQ ID NO: 2, respectively.

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