WO2004035787A2

WO2004035787A2 - Transplantable cell with immune modulator

Info

Publication number: WO2004035787A2
Application number: PCT/CH2003/000665
Authority: WO
Inventors: Mark Burcin; Sybille Esser; Manfred Ruediger
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2002-10-14
Filing date: 2003-10-13
Publication date: 2004-04-29
Also published as: AU2003266903A1; BR0315286A; CA2502312A1; WO2004035787A3; EP1556487A2; PL376397A1; JP2006502716A; MXPA05003912A; CN1705744A; KR20050071579A; RU2005114511A

Abstract

The invention relates to a human or animal non-totipotent cell which contains at least one nucleic acid which codes for at least one immune modulator by controlling a gene switch molecule which is adjusted by adding an active substance. The invention also relates to the production and use thereof in transplants in order to inhibit transplant rejection and also for the prophylaxis and/or therapy of diseases resulting from a transplant and/or auto immune diseases.

Description

Transplantable cell

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

Transplant rejection reaction and for the prophylaxis and / or therapy of secondary transplant and / or autoimmune diseases.

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. However, due to the shortage of organ 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. Alternatively, replacement cells can also be obtained from cell lines. These cells can also be of human or non-human origin.

However, with every transplantation of non-human cells, tissues or organs into a human organism (xenotransplantation) or of human donor cells, donor tissue or donor organs from a genetically non-identical person into a human recipient (allotransplantation) there is the problem of immune-mediated transplant rejection. The Rejection is based on the recognition of the histocompatibility antigens present on the donor cells as foreign proteins, which triggers an immune response directed against the graft. The immune response against allogeneic and xenogeneic cells, tissues and organs is based on numerous complex interactions between different cells (e.g. B cells, T cells, antigen presenting cells) of the immune system and can ultimately lead to rejection of the transferred cells, tissue or organ , To suppress the immune reactions, the recipient must therefore be treated with medications, so-called immunomodulators, which suppress the immune system or bring about a tolerance of the recipient to the transplant and thus prevent such a rejection reaction.

As 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.

While antibody therapies are given as supportive therapies during the first weeks and months after the transplant (induction therapy), the calcineurin inhibitors, steroids, and often MMF or imuran, are usually used from the time of the transplant over the entire period in which the patient wears the transplant , ie partially lifelong, administered. Regardless of the type, mode of action and combination of the immunomodulatory substances currently used, the medications are mostly administered intravenously or orally and thus distributed over the entire body of the patient. This means that they not only reach their place of action, ie, the transplanted organ or tissue or the place where the transplanted cells grow, but also all other tissues and organs of the organism. This leads to a general suppression of the immune system, which is not limited to the graft. In the non-target Tissues and organs impair the function of the immune system and thus prevent the physiological functioning of many organs, which can lead to numerous side effects.

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. For example, 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). Other studies show that immunosuppressed allograft receivers have a three to four times higher risk of cancer after conventional treatment, which can even increase by a factor of 20-500 in certain types of cancer (Penn L, Clin. Transpl. 147-158, 1998). In addition, immunomodulators, in particular immunosuppressants, can generally have toxic properties regardless of their effect on the immune system. For example, calcineurin inhibitors often cause renal insufficiency, high blood pressure, hyperlipidemia and the development of diabetes mellitus. Furthermore, 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.

Another disadvantage is that the medication must be administered in a relatively high dosage so that it can still reach the therapeutic concentration at the transplant site after distribution over the entire bloodstream. As a result, the occurrence of the side effects mentioned is further promoted or intensified. To counteract these facts, numerous novel immunotherapeutic and accompanying diagnostic approaches have been developed in recent years.

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). This was e.g. successfully performed in the transplantation of allogeneic islet cells for the treatment of diabetes mellitus, in which the diabetogenic effect of glucocorticoids was circumvented by using a combination of daclizumab, sirolimus and low-dose tacrolimus (Shapiro J., The New England Journal of Medicine, Vol 343, N. 4, 230-238, 2000). 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. In this context, tolerance is characterized as the lack of an immune response or rejection reaction against the graft without continuous immunosuppression. Such 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. 3478-3485; Kim et al, The Journal of Immunology, 1998, 160: 5742-5748; Penn, Transplant Proc, 1991, 23: 1101; Beveridge et al, Lancet, 1984, 1: 788) , 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. However, 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. In addition, 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). As a result, not all patients can be treated adequately with the immunomodulator or the treatment is associated with additional health risks.

Within the scope of the invention, 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

According to the invention, controllable expression of the immunomodulator in cells was achieved with the help of an adjustable gene expression system.

With the help of this regulatable gene expression system it is possible to produce an immunomodulator locally (eg in cell transplants) and in doses. Such a controllable production of immunomodulators has not been shown so far. It was all the more surprising to find that in transient cell culture experiments a controllable production of the immunomodulator MutlL-15 / mFc was obtained (see Example 1 and Kim et al, I. Immunology 1998, 160, 5742-5748).

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. In addition, 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.

Also included are 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.

The term 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, on the other hand there is a further signal for clonal expansion and differentiation of an unembossed lymphocyte; these costimulatory interaction can be inhibited by an immunomodulator), ^• the Jmhibierung 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.

Antibodies, for example, 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.

Further preferred 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. (Ϊ998), 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.

Further preferred 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.

Further preferred 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.

Preferably 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.

An ^• 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

Methods are known to the person skilled in the art. 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

Include fusion protein (see Kim et al. Supra). In a further embodiment of the invention, 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.

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 /.

The term 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). In general, it is possible that 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.

The addition of an active substance can influence the transcription of the target gene positively (activation) or negatively (inhibition). For example, the target gene is not expressed in the absence of the active substance. After adding 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. Another example is that 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. The term 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. The term 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.

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.

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-κB 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. When the active substance tetracycline is added, the DNA binding and thus the target gene activation is inhibited. This regulable gene expression system is described, for example, in Gossen 1995, Science 268: 1766-1769; Early 1995, Nature 375: 415-418; Chao et al. 1998, Mol. Cell. Biol. 18 (8): 4883-4898; Halappnavar et al. 1999, J. Biol. Chem. 274 (52): 37097-37104 or van der Viag et al. 2000, J. Biol. Chem. 275 (1): 697-704.

A modification of this regulatory Tet system is the reverse transactivator (rtTA). In this system, 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. Such 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.

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,

Connective tissue cells and cells from the pancreas, the kidney, the eye or the lungs.

A non-totipotent cell is a cell that cannot develop into a complete organism on its own.

In a further embodiment, 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.

In a further embodiment of the invention, 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. As such, 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). Further examples of 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. Other genes of the selection cassette according to the invention can be lacZ (coding for β-lactamase), β-lactamase, chloramphenicol acetyl transferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), and xanthinguanine phosphoribosyl transferase (XG. Reagents are known to the person skilled in the art, which can optionally be used to secure or increase the function of these genes, for example additional nucleic acid sequences.

In a preferred embodiment, 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.

The skilled worker understands killer cells as a heterogeneous population of mononuclear cells with spontaneous or acquired cytotoxic potential. NK cells (natural killer 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. These 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). In general, expression vectors contain the promoters suitable for the respective cell or the gene to be transcribed in each case. Examples of 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 EFlα (elongation factor-1-alpha) promoter, the SV40 (Simian Virus) promoter, the RSV (Rous Sarcoma Virus) promoter and the pUB (Ubiquitin) promoter.

Examples of 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. Such 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.

Further examples of 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. For the purposes of the present invention, for example, (i) 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. By transcription of the gene switch molecule in the cell nucleus, the gene switch protein formed in the cytoplasm after translation can in turn bind in the cell nucleus to the gene switch binding site of the immunomodulator sequence and regulate its expression.

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.

Such expression vectors, in particular for expression in mammalian cells, are commercially available, for example, pIRES (from Clontech, Heidelberg, DE), pCI-neo vector (from Promega, Mannheim, DE), pCMV-Script (from Stratagene, La Jolla) , USA) and pcDNA3 vector (Invitrogen, Karlsruhe, DE) or can be put together individually from individual elements. Gene-therapeutic vectors according to the invention 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. In 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. In addition to the lipid mixtures DOTMA (1, 2-dioleyloxypropyl-3-trimethylammonium bromide) and DPOE (dioleoxylphosphatidylethanolamine), numerous new lipid formulations have now been synthesized and tested for their transfection efficiency in various cell lines. (Behr et al. 1989, Proc. Natl. Acad. Sci. USA 86: 6982-6986; Gao and Huang, 1991, Biochem. Biophys. Acta 1189, 195-203; Feigner et al. 1994, J. Biol. Chem . 269, 2550-2561). Examples of the new 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

(a) rheumatic diseases, for example rheumatic arthritis, Sjogren's syndrome, scleroderma, dermatomyositis, polymyositis, Reiter's syndrome or

Behcef's disease,

(b) Type I or LADA diabetes

(c) autoimmune diseases of the thyroid gland, for example Graves' I rankheit, (d) autoimmune diseases of the central nervous system, for example multiple sclerosis,

(f) skin diseases, for example psoriasis or neurodermatitis,

(g) inflammatory bowel diseases, for example ulcerative colitis or Crohn's disease

(h) Immune Disorders (i) Vascular Cranes and

(j) post-transplant disorders, for example graft rejection reactions.

Suitable 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.

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

Administration and - for ß-cells subcutaneously, intravenously, intraperitoneally, encapsulated or intramuscularly.

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 DΝA 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.

It is known that 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.

The terms 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.

The person skilled in the art understands transplantation to mean the transmission or transplantation of living material, for example cells, tissue and

Organs, to another place in the same organism (autotransplantation) or from one organism (donor) to another organism (recipient). A distinction is made between transplantation into another organism

Synotransplantation, in which donors and recipients belong to the same species and are genetically completely or largely identical,

Allotransplantation, in which donors and recipients belong to the same species, but are immunogenetically different and

Xenotransplantation, in which the donor and recipient do not belong to the same species and are therefore immunologically completely different.

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 person skilled in the art understands 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.

Treatment based on the use of cells can be achieved in that 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. Preferred 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.

The standard methods of transfection, transformation, electroporation or injection familiar to the person skilled in the art are used to introduce a nucleic acid, a vector, a differentiation marker gene or a transfection marker gene or a cell according to the present invention into a cell.

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.

In a further embodiment, 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. .

Numerous methods for the selection of cells are known. Methods for selecting cells from differentiated embryonic stem cells are described, for example, in Klug et al. (J. Clin. Invest. 1996 Jul 1; 98 (l): 216-24) and Soria et al. (Diabetes. 2000 Feb .; 49 (2): 157-62).

In a preferred method of selection, 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.

In a preferred embodiment, it is a pluripotent or multipotent embryonic, fetal, neonatal or adult stem cell. Another object of the present invention is a method for

Generation of a transgenic non-human mammal according to the invention, 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.

It is preferred that the non-human mammalian foster mother be mock pregnant by mating with a male with a severed spermatic duct.

Methods for introducing 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. In a subsequent PCR (polymerase chain reaction) analysis, primers are used which specifically recognize the transgene for the nucleic acid according to the invention. Integration of the transgene into the genome can be demonstrated in this way. Another way of identification can be done using Southern blot. Here, 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.

Methods for producing a transgenic non-human mammal according to the invention 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. 4,736,866; US 5,625,122; US 5,698,765; US 5,583,278 and US 5,750,825 are known and include 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).

Numerous processes for the production of transgenic animals, in particular transgenic mice, 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).

In further embodiments, 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.

In the case of cell transplantation, 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.

For the purposes of the present invention, 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.

For example, 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.

The terms pharmacologically active substance and toxic substance in the sense of the invention include all those molecules, compounds and or

To understand compositions and mixtures of substances that are suitable

Conditions have a pharmacological or toxic influence on individual

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, anti-sense sequences of nucleic acids, peptides, proteins or

Be complex and antibodies. Examples are organic molecules made up of

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,

Dopamine of nerve cells, the muscle cell contraction and / or the migration behavior of cells, the metabolic activity of cells, the electrophysiological activity of cells, the enzymatic activity of cell products, the cell differentiation, the cell organization to tissues or organs.Application to the entire organism of an animal or human mammal this includes an influence on, for example, the cardiovascular system, the endocrine system, the gastrointestinal system, ^• the nervous system and metabolic activities.

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:

(i) Human or animal non-totipotent cell containing at least one nucleic acid coding for at least one immunomodulator under the control of a gene expression system which can be regulated by adding an active substance.

(ii) Cell according to (i), characterized in that the cell is a stem cell, a precursor cell and / or an immortalized cell.

(iii) Cell according to (i) or (ii), characterized in that it is a pluripotent or multipotent embryonic, fetal, neonatal or adult stem cell.

(iv) Cell according to at least one of (i) to (iii) in the form of a cell line.

(v) Cell according to at least one of (i) - (iv), characterized in that the regulatable gene expression system is a progesterone gene expression system, a tetracycline expression system and / or a rapamycin gene expression system.

(vi) 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. the inhibition of a 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. 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, k. the inhibition of components of the complement system

1. 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 m. the inhibition of inflammatory reactions.

(vii) cell according to at least one of (i) to (vi), characterized in that the immunomodulator is an antibody.

(viii) cell according to at least one of (i) to (vi), characterized in that the immunomodulator a. a receptor is b. a soluble secreted receptor is c. is a secreted protein or peptide

(ix) cell according to (viii), wherein the immunomodulator is a fusion protein of a mutated IL 15 and an Fc fragment, the Fc fragment fusing to the C-terminus of the mutated IL 15 molecule, preferably via the hinge region is.

(x) cell according to (ix), characterized in that 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

(xiv) cell according to (xiii), characterized in that said inhibiting molecule is a human MHC class I molecule, a chimeric MHC class I molecule or a viral MHC class I homolog.

(xv) nucleic acid coding for at least one immunomodulator and at least one gene expression system which can be regulated by adding an active substance.

(xvi) vector containing at least one nucleic acid according to (xv). (xvii) Medicament containing at least one cell according to one of (i) to (xiv) and suitable auxiliaries and / or additives.

(xviii) Human or animal organ-specific tissue and / or human or animal mammalian organ, containing at least one cell according to one of (i) to (xiv).

(xix) Transgenic non-human mammal, containing at least one cell according to one of (i) to (xiv).

(xx) Use of a cell according to one of (i) to (xiv) and / or a human or animal organ-specific tissue and / or a human or animal mammalian organ according to (xviii) for transplantation into a human or animal mammal.

(xxi) Use according to (xx), characterized in that it is an allo-, auto- or xenotransplantation.

(xxii) Use of a cell according to one of (i) to (xiv), a nucleic acid according to (xv), a human or animal organ-specific tissue and / or a human or animal mammalian organ according to (xviii) for the manufacture of a medicament for inhibiting a transplant rejection reaction in a human or animal mammal, optionally in the presence of at least one immunomodulator.

(xxiii) Use of a cell according to one of (i) to (xiv), one

Nucleic acid according to (xv), a human or animal organ-specific tissue and / or a human or animal isphatic mammalian organ according to (xviii) for the manufacture of a medicament for Prophylaxis and / or therapy of secondary transplant diseases and / or autoimmune diseases.

(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.

(xxv) In 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.

(xxviii) Method for producing a transgenic non-human mammal according to (xviii), comprising the following steps: f. Introduction of at least one nucleic acid according to (xv) and / or at least one vector according to (xvi) as well as at least one suitable transfection marker gene into at least one oocyte, 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.

(xxix) Method according to (xxviii), characterized in that it is a pluripotent or multipotent embryonic, fetal, neonatal or adult stem cell.

(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

Mammal foster mother and f. Identification of the transgenic non-human mammal developed from said mammalian oocyte.

(xxxi) Transgenic non-human mammal, characterized in that it was generated by the method according to one of (xxviii) or (xxix).

(xxxii) Transgenic non-human mammal, characterized in that it is a descendant of the mammal according to (xxx).

(xxxiii) Use of a transgenic non-human mammal according to one of the (xix), (xxx) or (xxxi) for obtaining a non-human cell, a non-human organ-specific tissue and or a non-human mammalian organ for the allo- and / or xenotransplantation.

(xxxiv) Use of a transgenic non-human mammal according to one of the (xix), (xxx) or (xxxi), a human or animal organ-specific tissue and / or a human or animal

Mammalian organ according to (xviii) for the detection of pharmacologically active substances and / or for the identification of toxic substances.

The following figures and examples are intended to illustrate the present invention without, however, restricting it:

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

Gene switch binding domain UAS recognizes. 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. ii) 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

Genenschalteφroteins in an active conformation caused by dimerization. iii) A p65 activation domain (P65-AD) which activates the transcription of the target gene by the gene switch. In the absence of mifepristone, the gene switch is transcribed in small amounts by the upstream ubiquitous weak basal minimal thymidine kinase promoter (pTK). However, 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 marker genes controlled by a cell type-specific promoter, such as the rat insulin promoter RIP), are used to select transgenic cells of a specific cell type.

To generate the transgenic animals of the invention, the cells must contain at least elements 1 and 2. To produce the transgenic cells from cell lines, the cells can additionally contain element 3.

Examples

Two experimental models were developed to investigate and demonstrate the mode of action of the regulated expression of the immunomodulating protein MutIL-15 / mFc: a) Transgenic cell line for transplantation

In this model, 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.

b Transgenic mouse model. 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.

Example 1: Vector cloning for a transgenic mouse model

In the vector 17x4 / pGL3 Basic (modified pGL3Basic vector from Promega with TATA box and 4 copies of the 17-oligomer as a gene switch binding site) 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). This completes the CD5-MutIL-l 5 / mFc gene by means of an SV40polyA sequence (vector name 17x4 / IL15). An oligonucleotide with the interfaces Sbfl and Pmel was then introduced before the 17-oligomers (vector name 17x4 / ILl 5 / oligo). These interfaces were used to insert the gene for the gene switch molecule (GS = Gal4-DBD / hPR-LBD / p65-AD) including the upstream regulatory region (Gal4UAS-PTK-IVS8), which were isolated from the vector pswitch (Jfnvitrogen, Karlsruhe) ( Vector name 17x4 / IL15 / Oligo / GS). In addition, 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. In addition, however, 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). These elements were inserted 5 'below the CD5-MutIL-15 / mFc gene and alternatively cloned in two directions of transcription. (Vector names 17x4 / IL15 / Oligo / RIPhn / GS and 17x4 / IL15 / Oligo / RIPnh / GS). In addition, 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

Transient transfections of cos-7 cells (DSMZ, Braunschweig) and A293 cells (Quantum, Montreal, Canada) were carried out. For this purpose 5-7.5xl0 ⁵ cells / hole were sown on 6-hole plates the day before the transfection. For the transfection, which was carried out with 1-2 μg DNA hole, 10-20 μl DNA with a concentration of 10 ng / ml were mixed with 250 μl OptiMEM-I medium (Life Technologies, Karlsruhe). In parallel, 2 μl lipofectamine 2000 (Life Technologies, Karlsruhe # 11668-019) / μg DNA were mixed with 250 μl OptiMEM-I medium (Life Technologies, Karlsruhe # 31985-062). Equal volumes of both batches were mixed and left at room temperature for 20 minutes. In the meantime, the 6-well plates with the 50-70% confluent cells were washed once with PBS and then 1.5 ml of growth medium / well (DMEM + 10% FCS) were added. 500 μl of the DNA / lipofectamine mixture were added dropwise to these cells per hole. Cells stimulated with mifepristone received an additional 2 μl of a 10 ^" M solution of mifepristone (Sigma, Deisenhofen; final concentration 10 ^" M) in 80% ethanol (Invitrogen, Karlsruhe). The next day, the medium was removed and 2 ml of fresh medium (DMEM + 10% FCS) were added. The cells stimulated with mifepristone received an additional 2 μl of mifepristone. 3 days after the transfection, the medium was removed from the cells, centrifuged to remove cell residues and then frozen away at -80 ° C. The cells were washed once with PBS, scraped into PBS, transferred into a 1.5 ml Eppendorf tube and for 30-60 min at 4 ° C. with 50 μl RffA buffer (PBS with 1% IGEPAL / Sigma, Deisenhofen 1 -3021, 0.5% sodium deoxycholate, 0.1% SDS, 4mM EDTA and Complete EDTA-free Protease Inhibitor Cocktail Röche, Mannheim # 1873580) lysed. The centrifuged lysates were snap frozen in liquid nitrogen and stored at -80 ° C.

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 μl 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. 100 μl of undiluted medium supernatants were added to the coated plates, these were incubated for 1 h at room temperature and then washed 5 times with 200 μl of PBS / 0.1% Tween20 and once with 200 μl of PBS. The enzyme-coupled antibody HRP antiMaus IgG 2a clone R19-15 (BD PharMingen, Heidelberg # 02017E-553391) was also incubated again for 1 h at room temperature with the plates to detect the mouse Fc part and the plates were then washed again as described above. 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 μl H2O2 30%) made visible, stopped by adding 3 M HC1 and then measured in an ELISA reader (μ Quant, BIO-TEK Instruments Inc.) at 490 nm.

In addition, undiluted media supernatants were analyzed in the immunoblot. For this purpose, 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. with a 1: 500 dilution of mouse anti-human IL15 antibody (Becton-Dickinson, Heidelberg # 554712), washed extensively with PBS / 0.1% Tween20 and then for one hour treated at room temperature with a 1: 3000 dilution of a peroxidase-coupled sheep anti-mouse antibody (Amersham, Freiburg # NA9310). After extensive washing, the membrane was washed for 5 minutes at room temperature with detection reagent (NEN; bath Homburg # NE103E) treated and the color reaction detected by placing an X-ray film.

The analysis of media supernatants from transfected cells with or without mifepristone addition showed the following result: The following transfections were carried out:

1. mCD5.6 (positive control for MutIL-15 / mFc secretion, vector with CD5-MutIL-15 / mFc under the control of a CMV promoter)

2.17x4 / ILl 5 / Oligo +/- mifepristone (negative control without gene switch)

3. 17x4 / IL15 / Oligo + pcDNA3switch +/- Mifepriston 4. 17x4 / IL15 / Oligo / GS +/- Mifepriston

5. 17x4 / IL15 / Oligo / GS + pcDNA3switch +/- mifepristone

6. 17x4 / ILl 5 / Oligo / IVS8 / GS +/- mifepristone

7. 17x4 / ILl 5 / Oligo / IVS8 / GS + pcDNA3switch +/- Mifepriston

No significant expression of MutIL-15 / mFc was observed in cells which had been transfected with the control vector without a gene switch molecule (2.) and in cells which had not been stimulated with mifepristone. After transfection with constructs that only contained the autoregulated gene switch molecule (4th + 6th), only a slight increase in the secretion of MutIL-15 / mFc after mifepristone stimulation could be detected using the ELISA. This was probably due to insufficient formation of active gene switch molecules by autoregulatory gene activation in the course of the transient transfection. Therefore, the gene switch quantity was increased externally by cotransfection with the vector pcDNA3 switch (gene switch under the control of the CMV promoter), which causes a constant high production of gene switches. In these co-transfections, 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.

Table 1: Measurement of MutIL-15 / mFc in media supernatants from transiently transfected A293 cells (mean of duplicate values)

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).

These results demonstrate that the transcription and secretion of MutIL-15 / mFc fusion protein can be induced by adding mifepristone in cells which had been transfected with the vectors presented above (eg 17x4 / IL15 / Oligo / IVS8 / GS). This proved the functionality of the regulated expression of MutIL-15 / mFc. Example 4: Examination of the regulated expression and functionality of the gene switch protein fGal4UAS-PTK-IVS8-Gal4-DBD / hPR-LBD / p65-AD ^' ) in the vectors mentioned in Examples 1 and 2. As mentioned above, 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).

Since the ELISA is not sensitive enough to detect expression of MutIL-15 / mFc at the single cell level, co-transfections with the plasmid pGene / N5-His / lacZ (Invitrogen, Karlsruhe) were carried out. This vector contains a lacZ gene, the transcription of which is also regulated by gene switch binding sites. This means that ß-galactosidase is only produced in cells that contain active gene switches by mifepristone stimulation. The gene switch was provided by a second vector (in this case the constructs with an auto-regulated gene switch). β-galactosidase could be detected as a blue precipitate in individual cells by a substrate reaction. As a result, a function of the vector constructs could be demonstrated with much higher sensitivity. A293 cells were transfected as described in Example 3 with the following constructs and then partially stimulated with mifepristone:

1. pCMVß (positive control for lacZ, vector with lacZ gene under the control of a CMV promoter)

2.17x4 / ILl 5 / Oligo + pGene / V5-His / lacZ +/- mifepristone (negative control without gene switch)

3. 17x4 / ILl 5 / Oligo / GS + pGene / V5-His / lacZ +/- mifepristone

4. 17x4 / ILl 5 / Oligo / IVS8 / GS + pGene / V5-His / lacZ +/- mifepristone 5. pcDNA3switch + pGene / V5-His / lacZ +/- mifepristone (positive control with constitutively high gene switch)

6. pGene / N5-His / lacZ mifepristone (negative control without gene switch)

On the third day after the transfection, 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 μl of 400 mM potassium ferricyanide, 60 μl of 400 mM potassium ferrocyanide, 60 μl of 200 mM MgC12, 300 μl 20 mg / ml X-Gal, 5.52 ml PBS).

The light microscopic evaluation of the transfected cells gave the following result (Tab. 2):

Table 2: Intensity of the β-galactosidase staining of transiently transfected A293 cells

No significant blue staining of cells was observed without a gene switch (2.). In cells that had been transfected with an autoregulated gene switch (3rd + 4th), mifepristone stimulation caused a clear blue staining of 3-5% of the cells. Since this low number of cells produces a mutIL-15 / mFc amount that is only very small, it is below the detection limit of the ELISA. However, lacZ staining can also detect and detect gene switch activity in individual cells.

These experiments demonstrate that the activated gene switch provided by constructs 17x4 / IL15 / Oligo / GS and 17x4 / IL15 / Oligo / IVS8 / GS can stimulate lacZ transcription after stimulation with mifepristone. This data proves the functionality of the auto-regulated gene switch of the above-mentioned constructs.

Example 5: Production and characterization of transgenic mice 50 μg 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

GGC TTC TGA GGC GGA AAG AAC CAG C - 3 ') and GS-L15 RV.3 (5'- G

CAG AGA CCC CAT GGG CAT GGT GGC TAG -3 '). The PCR products obtained accordingly have a length of 211 bp (without intron) or 335 bp (with intron IVS8).

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 μg / 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. 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. 1 μg 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.

Primer CD5.6-FW: 5'-CCTGCTGGGGATGCTGGTC

Primer CD5.6-RV: 5'-TTTTCCTCCAGTTCCTCACATTC

MgCl ₂ : 3 mM

The PCR conditions for the detection of the gene switch are as follows: Denaturation: 95 ° C, 600 sec

Cycles: 95 ° C 15 sec, 53 ° C 5 sec, 72 ° C 10 sec. Primer GS-FW: 5'-GACTTAAAAAGCTCAAGTGCTCCAAAG Primer GS-RV: 5'-TATATCCTGTAAAGAATCCAT MgCl ₂ : 3 mM In addition, blood is drawn from the animals at regular intervals before or during the mifepristone treatment and the amount of MutlL-15 / mFc contained therein is determined by ELISA.

For comparison, mice of the same age are used in the same transgenic line, who had not previously been treated with mifepristone.

Alternatively, the transgenic mice are produced in a two-step process. First, 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"). 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. 50 μg 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 In islet transplantation, 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 μg / kg) so that they demonstrably produce MutIL-15 / mFc on the day the organ is removed. To prepare the islands, donor pancreata are perfused in situ with type IV collagenase (2 mg / ml; Worthington Biochemical Coφ.). After 30 minutes of digestion at 37 ° C., the islands are purified using a discontinuous Ficoll gradient and then cultured in RPMI1640 medium (Gibco, Karlsruhe) 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). As a control, syngeneic islands (i.e., islands from B6AF1 mice) are transplanted into the diabetic 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 islands are then injected into this cavity using a sterile pipette tip. Then the kidney is put back into the abdomen and the abdomen is closed. The recipient animals are treated with mifepristone 250 μg / 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). 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.

As a control, 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.

Example 7: Heterotopic heart transplant

In heterotopic heart transplantation, the donor heart is connected to the large vessels in the recipient's abdomen (Ono et al, J. Cardiovasc. Surg. 1969, Corry et al, Transplantation 1973). 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 μg / kg) so that they demonstrably produce MutlL-15 / mFc on the day the organ is removed. In the anesthetized mice, 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. 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 μg / 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. As a control, 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 μg DNA of constructs 17x4 / IL15 / Oligo / RIPnh / GS,

17x4 / IL15 / Oligo / RIPhn / GS, 17x4 / IL15 / Oligo / RIPhn / IVS8 / GS and

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 μl 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. Approximately 3 × 10 ⁷ cells were washed twice with 5-10 ml of cold PBS and then the cell pellet was taken up in cold PBS, so that the final volume including DNA is 800 μl. The digested DNA was then added to the cells, the solution was mixed and incubated on ice for at least 10 minutes. Under the sterile bench, the Zeil / DNA mixture was filled into pre-cooled electroporation cuvettes with an electrode gap of 0.4 cm (BioRad, Munich). The electroporation was carried out with a Genepulser-2 device (BioRad, Munich) at 0.8 kV and 3 μF. The cells were then treated with ES cell medium (DMEM with 20 mM Hepes, 15% heat-inactivated FCS, 50 U / ml penicillin, 50 μg / ml streptomycin, 0.1 mM non-essential amino acids, 0.1 mM mercaptoethanol, 10 ³ U / ml Leukemia Inhibitory Factor) mixed and evenly distributed over ten 10 cm cell culture dishes coated with 0.1% gelatin. The next day the selection was started by adding 200 μg / ml hygromycin (Sigma, Deisenhofen H 3274) in ES cell medium. 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.

After addition of 10 nM mifepristone, 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. Alternatively, 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. The double transgenic obtained Lines are examined by RT-PCR for simultaneous expression of the two transgenes as described in Example 5. In this way, double-transgenic ES cells are obtained which express the immune modulator regulated by gene switch molecules. 100 μg 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):

157-62.

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 μg / 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.

As a control, 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. Alternatively, 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.

Studies of the blood sugar level in animals that have received insulin-producing cells made from stem cells show that Cell grafts in animals treated with mifepristone are functionally active for a long time.

Claims

Patent claims

1. Human or animal non-totipotent cell containing at least one nucleic acid encoding at least one immunomodulator among the

Control of a gene expression system that can be regulated by adding an active substance.

2. Cell according to claim 1, characterized in that the cell is a stem cell, a precursor cell and / or an immortalized one

cell acts.

3. Cell according to claim 1 or 2, characterized in that it is a pluripotent or multipotent embryonic, fetal, neonatal or adult stem cell.

4. Cell according to at least one of claims 1 to 3 in the form of a cell line.

5. Cell according to at least one of claims 1-4, characterized in that the regulatable gene expression system is a progesterone gene expression system, a tetracycline expression system and / or a rapamycin gene expression system.

6. Cell according to at least one of claims 1-5, characterized in that the immunomodulator has at least one of the following functional properties:

a. the inhibition of antigen recognition mediated by T cells b. the inhibition of 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. the inhibition of the growth of T cells, e. the inhibition of molecules that support the survival of T cells f. the inhibition of effector molecules of T cells (such as TNF-alpha, IFN-gamma), g. the inhibition of the adhesion of T cells, h. the inhibition of a T cell costimulatory interaction (the activation of a lymphocyte occurs 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 unimprinted lymphocyte; this costimulatory interaction can be activated by an immunomodulator be inhibited) i. the inhibition of the activation, proliferation, survival, antigen presentation, signaling, and/or effector functions of other cells involved in an immune response, such as general and specific antigen presenting ones

Cells, in particular e.g. dendritic cells and monocytes/macrophages B cells, neutrophil granulocytes and NK cells, inhibit 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 dendritic cells and monocytes/macrophages, T cells, B cells, neutrophil granulocytes and NK cells, j. the inhibition of the migration of cells that are involved in an immune response, such as special antigen-presenting cells, in particular dendritic cells and monocytes/macrophages, T cells, B cells, neutrophil granulocytes and NK cells, k. the inhibition of components of the complement system

1. the inhibition of phagocytotic activities in connection with the presentation of foreign or autoimmune antigens, or through the binding of antibodies to antigens, and/or m. the inhibition of inflammatory reactions.

7. Cell according to at least one of claims 1 to 6, characterized in that the immunomodulator is an antibody.

8. Cell according to at least one of claims 1 to 6, characterized in that the immunomodulator d. a receptor is e. is a soluble secreted receptor ^• f. is a secreted protein or peptide

9. Cell according to claim 8, wherein the immunomodulator is a fusion protein of a mutated IL 15 and an Fc fragment, the Fc fragment being fused to the C terminus of the mutated IL 15 molecule, preferably via the hinge region.

10. Cell according to claim 9, characterized in that the Fc fragment of

Antibody one of an IgG, in particular a human IgGl, IgG2, IgG3, IgG4 or an analogue mammalian IgG or an IgM, in particular a human IgM or an analogue mammalian IgM.

11. Cell according to at least one of claims 1 to 10, characterized in that the nucleic acid additionally encodes a selection cassette, in particular a suitable transfection marker gene and/or differentiation marker gene.

12. Cell according to at least one of claims 1 to 11, characterized in that the nucleic acid additionally encodes a molecule that inhibits NK cells and / or killer cells.

13. Cell according to at least one of claims 1-11, characterized in that the nucleic acid additionally encodes a molecule that a. Dendritic cells inhibited b. Monocytes and/or macrophages inhibited c. B cells inhibited d. Polmonuclear cells, e.g. neutrophil granulocytes, are inhibited

14. Cell according to claim 13, characterized in that said inhibitory molecule is a human MHC class I molecule, a chimeric MHC class I molecule or a viral MHC class I homologue.

15. Nucleic acid encoding at least one immunomodulator and at least one gene expression system that can be regulated by adding an active substance.

16. Vector containing at least one nucleic acid according to claim 15.

17. Medicaments containing at least one cell according to one of claims 1 to 14 and suitable auxiliaries and/or additives.

18. Human or animal organ-specific tissue and/or human or animal mammalian organ, containing at least one cell according to one of claims 1 to 14.

19. Transgenic non-human mammal containing at least one cell according to one of claims 1 to 14.

20. Use of a cell according to one of claims 1 to 14 and / or a human or animal organ-specific tissue and / or a human or animal mammalian organ according to claim 18 for transplantation into a human or animal mammal.

21. Use according to claim 20, characterized in that it is an allotransplantation, autotransplantation or xenotransplantation.

22. Use of a cell according to one of claims 1 to 14, a nucleic acid according to claim 15, a human or animal organ-specific tissue and / or a human or animal mammalian organ according to claim 18 for producing a medicament for inhibiting a transplant rejection reaction in a human or animal mammal , optionally in the presence of at least one immunomodulator.

23. Use of a cell according to one of claims 1 to 14, a nucleic acid according to claim 15, a human or animal organ-specific tissue and / or a human or animal mammalian organ according to claim 18 for the production of a medicament for the prophylaxis and/or therapy of

Post-transplant diseases and/or autoimmune diseases.

24. A method for producing a cell according to one of claims 1 to 14 comprising the following steps: c. Introducing at least one nucleic acid according to claim 15 and/or at least one vector according to claim 16 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.

25. In v/tro process for producing a human or animal organ-specific tissue and/or human or animal mammalian organ according to claim 18, comprising the following steps: e. Introducing at least one nucleic acid according to claim

15 and / or at least one cell according to claim 16 and as well as at least one differentiation marker gene into at least one non-totipotent stem cell, a non-totipotent progenitor cell and / or a non-totipotent immortalized cell, f. Differentiation of the cell from step a, G. Selecting the differentiated cell from step b. and h. Introduce the selected cell from step c. into a human or animal organ-specific tissue and/or into a human or animal mammalian organ.

26. The method according to claim 25, 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 progenitor cell and/or a non-totipotent immortalized cell and after step a. preferably the transferred one

Cell from step a. is selected.

27. Method according to one of claims 25 or 26, characterized in that it is a pluripotent or multipotent embryonic, fetal, neonatal or adult stem cell.

28. A method for producing a transgenic non-human mammal according to claim 18, comprising the following steps: f. introducing both at least one nucleic acid according to claim 15 and / or at least one vector according to claim 16 and at least one suitable transfection marker gene into at least one

Oocyte, stem cell, a progenitor cell and/or an immortalized cell of a non-human mammal, g. Selecting the transfected cell from step a, h. Introduce the following step b. selected cell into at least one non-human mammalian blastocyst, i. Insert the blastocyst from step c. into a non-human one

mammal foster mother and j. Identification of the transgenic non-human mammal developed from said blastocyst.

29. The method according to claim 28, characterized in that it is a pluripotent or multipotent embryonic, fetal, neonatal or adult stem cell.

30. Method for producing a transgenic non-human mammal according to

Address 19, containing the following steps: d. Introducing at least one nucleic acid according to claim

15 and/or at least one vector according to claim 16 as well as 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 foster mother and f. Identification of the transgenic non-human mammal developed from said mammalian oocyte.

31. Transgenic non-human mammal, characterized in that it was produced by the method according to one of claims 28 or 29.

32. Transgenic non-human mammal, characterized in that it is a descendant of the mammal according to claim 30.

33. Use of a transgenic non-human mammal according to one of claims 19, 30 or 31 for obtaining a non-human cell, a non-human organ-specific tissue and/or a non-human mammalian organ for allo- and/or xenotransplantation.

34. Use of a transgenic non-human mammal according to one of claims 19, 30 or 31, a human or animal organ-specific tissue and / or a human or animal mammalian organ according to Anspmch 18 for finding pharmacologically active active ingredients and / or for identifying toxic substances .