WO2001027254A2

WO2001027254A2 - Gene transfer vectors for treating autoimmune diseases and diseases with immunopathogenesis by therapy

Info

Publication number: WO2001027254A2
Application number: PCT/DE2000/003608
Authority: WO
Inventors: Fritz Schwarzmann
Original assignee: Fritz Schwarzmann
Priority date: 1999-10-12
Filing date: 2000-10-12
Publication date: 2001-04-19
Also published as: CA2387146A1; DE10083095D2; EP1220900A2; WO2001027254A3; AU782255B2; AU2147901A; IL148805A0; JP2003513619A

Abstract

The invention relates to a gene transfer vector, comprising at least one nucleic acid molecule comprising a first nucleic acid sequence which codes for one or more ligands that trigger apoptosis, a second nucleic acid sequence which codes for one or more antigens, optionally, a third nucleic acid sequence which codes for one or more anti-apoptosis molecules, and optionally, a fourth nucleic acid sequence which codes for one or more suicide enzymes.

Description

Gene transfer vectors for the therapy of autoimmune diseases and diseases with immunopathogenesis

The invention relates to a gene transfer vector comprising at least one nucleic acid molecule, comprising a first nucleic acid sequence coding for one or more apoptosis-triggering ligands, a second nucleic acid sequence coding for one or more antigen (s) and optionally, a third nucleic acid sequence coding for a or more anti-apoptosis molecule (s), and optionally a fourth nucleic acid sequence, coding for one or more suicide enzyme (s).

The development of autoimmune diseases such as multiple sclerosis (MS) or diabetes (type 1) is based on an uncontrolled activation of immune cells that are healthy

Attack and destroy cells in the body. Crucially involved in the pathogenesis of

Autoimmune diseases are T-lymphocytes, which are fragments of the body's own proteins in the body

Recognize the connection with the body's own MHC (major histocompatibility com /? / Ex) molecules. T helper cells (CD4 "/ CD8 ^" ), which recognize a combination of peptide and MHC class II, have a key function in immunopathogenesis, since they stimulate the induction of autoreactive antibodies and immune cells by secretion of different cytokines. Proteins of cellular or viral origin, which are synthesized in cells, are degraded intracellularly in proteasomes and, together with MHC class I molecules, are presented to the T lymphocytes on the cell surface. These cells are specifically recognized and eliminated by cytolytic T cells (CD4 ^" / CD8 ⁺ ).

Usually, T cells that recognize the body's own proteins are either eliminated

(clonal deletion) or inactivated (anergy). The result is a tolerance to cells and

Structures of your own body. In autoimmune diseases, this tolerance is disturbed or insufficient. Autoimmune diseases may be viral or bacterial Infections triggered. It is believed that due to the similarities between pathogen-specific and cell-type-specific proteins, uninfected cells are also attacked by pathogen-specific antibodies or T cells. In connection with chronic persistent viral infections, there are also inflammatory processes, some of which affect vital organs such as the liver (hepatitis), although the immune response is primarily directed against pathogen-specific structures. In such cases, one speaks of immunopathogenesis, since the damage and symptoms are primarily caused by the patient's own immune system and not by the pathogen. The same thing happens with the rejection of transplanted organs. A combination of foreign MHC molecules from the donor, complexed with processed cellular proteins, is recognized as foreign by the recipient's T cells.

According to current knowledge, autoimmune diseases and other diseases with immunopathogenesis are treated by strong immunosuppression and / or by administration of regulatory-effective cytokines such as interferon-ß. A large number of different preparations from different manufacturers are available for classic chemotherapy with immunosuppressants, which differ considerably in the area of application and the mode of action. A particularly serious disadvantage of these substances is the occurrence of diverse and sometimes life-threatening side effects (including kidney damage, liver damage, pancreatitis, anemia, fever), which are observed in a large number of patients. Such non-specific inhibition of the immune system (e.g. by cyclosporin or FK506) not only leads to a greatly weakened immune system and thus to an increased susceptibility to infectious diseases, but also favors the development of tumors. For example, after organ transplants, where lifelong immunosuppression is required, the risk of developing tumors associated with Epstein-Barr virus is increased many times over. The use of immunotherapeutics (e.g. interferons) is a significant improvement over classic chemotherapy because it has fewer side effects. Nevertheless, a cure for autoimmune diseases is also not possible with these novel immunotherapeutics, since they do not have a specific effect.

Antigen-presenting cells (APCs) play an important role both in triggering a T cell response and in inducing a T cell tolerance. The induction of extensive activation and proliferation of T cells is dependent on two signals that the T cell must receive. The first signal is through the T cell receptor, which contains an antigen in the Detects association with MHC on the surface of APCs, directed to the T cell. In the absence of the second signal, the so-called costimulatory signal, the T cell becomes anergic. Anergy describes a state in which the T cells are neither reactive nor multiply. APCs are also able to influence the function of the T helper cells and to direct their properties either towards the Th (T helper cell) I or Th2 phenotype, and thereby, for example, to promote the development of autoimmune diseases. The combination of the different functions of the antigen-presenting properties of the APCs determines the profile of the response of the T helper cell. Therefore, APCs can control whether an immune response is immunogenic or tolerogenic.

Fas is expressed on the surface of cells and mediates apoptosis after interaction with the specific ligand FasL or after binding of an agonistically active anti-Fas antibody. The so-called "ictivation-induced cell death" (AICD) of T cells, ie their programmed cell death after activation of the cells, is triggered by an autocrine feedback after the interaction of Fas and FasL on one and the same activated T cell, which underscores the importance of Fas-associated apoptosis for maintaining T cell tolerance.

APC's Fas-mediated apoptosis plays a role in down-regulating immune responses. Activated T cells express increased amounts of Fas ligand and thus induce apoptosis in the APCs. On the other hand, activated macrophages also express Fas ligand and can in turn induce apoptosis in the T cells. For example, a large amount of Fas ligand on HIV-infected macrophages is associated with the depletion of HIV-specific CD4 + T cells.

The importance of Fas-dependent apoptosis for maintaining T-cell tolerance and avoiding autoimmune diseases has been shown, among other things, by the fact that mutations that affect the genes for Fas or the ligand of Fas, FasL, lead to the development of autoimmune diseases in Iprllpr or gld / gld mice lead, gldlgld or Iprllpr denotes mice that are homozygous for inactivating mutations for FasL or for Fas. Both the clonal deletion of T cells in the periphery and the maintenance of T cell tolerance to the body's own antigens (autoantigens) and superantigens is disturbed in / p / V pr mice. A correction of the Fas-associated apoptosis defect in the T cells by an artificially in The Fas gene inserted and expressed in the cells can prevent the development of autoimmune diseases in pr // pr mice.

Fas-mediated apoptosis also plays a critical role in maintaining immune-privileged areas in the body. The immune privileged condition of the testicles and anterior chamber require a strong expression of Fas ligand on the corresponding parenchymal cells of these organs. In these cases, it is believed that the expression of Fas ligand on the parenchymal cells protects these tissues from destruction by T cells by inducing apoptosis in the T cells. A viral infection in the anterior chamber leads to systemic T cell tolerance to the virus. It is believed that APCs that present the Fas ligand on their cell surface along with privileged antigens derived from the privileged cells induce apoptosis of T cells in the periphery of the body, thereby causing systemic T cell tolerance.

In 1998, Zhang et al. (Zhang et al. (1998) Nat Biotechnol 16: 1045-1049) showed the induction of an antigen-specific T cell tolerance using Fas ligand (FasL) -producing antigen-presenting cells (APC) in the mouse model. Antigen-presenting cells, which were infected by an adenoviral vector which, in addition to FasL, also encoded adenoviral proteins, were able to generate T cell tolerance in mice. T cell tolerance was specific for adenovirus proteins and showed no influence on infection with mouse cytomegalovirus. Mice treated with the APCs showed a significantly prolonged persistence of the virus in the liver on subsequent infection with an adenovirus, in accordance with a repressed T cell response against cells infected with adenovirus. Furthermore, the T cell tolerance was dependent on the function of the Fas ligand, since no tolerance could be induced in Fas-negative mice. A year later, experiments by the same group to induce T cell tolerance to alloantigens were also published in the mouse model (Zhang et al. (1999) J Immunol 162: 1423-1430). Another year later, the working group published experiments that showed that inflammatory diseases such as those that occur after an infection of Fas-deficient mice with mouse cytomegalovirus, for example, can be prevented or treated using FasL-expressing antigen-presenting cells (Zhang et al. (2000) J Clin Invest 105: 813-821). In this experimental approach, too, the therapy was based on the induction of one antigen-specific, here mouse cytomegalovirus-specific, T-cell tolerance. Zhang et al. used adenoviral vectors for the experiments described, which ensure the advantage of a high infection rate and good gene expression.

However, adenoviral vectors have decisive disadvantages for use in humans in particular, first and foremost the expression of antigenic viral proteins, which have so far failed gene therapy approaches with adenoviral vectors. Since adenoviral vectors express regulatory and structural proteins, the transduced cells are recognized and eliminated by the immune system regardless of the targeted expression of the antigens. In order to avoid this reaction, the mice described in the experiment were previously made tolerant to adenoviral proteins with corresponding FasL-positive APCs which expressed adenoviral proteins. An appropriate approach to make the immune system tolerant of a potential viral pathogen is unsuitable for humans.

The problem of autocrine induction of apoptosis due to the interaction of the artificially expressed FasL molecules with the corresponding apoptosis receptors on the surface of the APCs was not taken into account in the experiments described. In the experiments by Zhang et al. (Zhang et al. (1998) Nαt Biotechnol 16: 1045-1049; Zhang et al. (1999) J Immuno l 162: 1423-1430; Zhang et al. (2000) J Clin Invest 105: 813-821) defective APCs infected with the Fas ligand-expressing vectors. For an efficient therapeutic application, it is imperative to protect the modified antigen-presenting cells against autocrine stimulation of apoptosis and at the same time to be able to avoid an uncontrollable immortalization or even a transformation of the cells in the direction of a tumor cell.

In all previous experiments, the problem of unintentional stimulation of the immune system by the expression of the antigen, for which a T cell tolerance is to be induced, without simultaneous expression of apoptosis-inducing ligands, has also not been taken into account.

It is therefore an object of the present invention to provide means for the prevention or therapy of auto-brain diseases and diseases with immunopathogenesis. The object is achieved by the subject-matter defined in the patent claims.

The invention is illustrated by the following figures.

FIG. 1 graphically shows the results of infection of mouse macrophages with an adenovirus that expresses FasL. Macrophages from CD95-deficient B6 mice were purified and infected with adenoviruses that expressed either LacZ (AdLacZ, Ad / CV; FasL control) or FasL (AdFasL, Ad / FL). (A) The expression of FasL on AdFasL-infected and non-infected macrophages was examined by means of FACS. The histogram shows the number of infected cells and the strength of the FasL expression (Y axis, number of fluorescent cells; X axis, strength of the fluorescence, determined by means of a fluorescence-coupled anti-FasL antibody). (B) ^{5 l} Cr release test to determine the ability of the infected FasL-expressing cells to induce apoptosis in target cells. The macrophages or non-infected control macrophages infected with the two different adenoviruses were incubated with chromium-labeled, Fas ⁺ target cells (A20 cells) and the lysis of the target cells was quantified by the release of ⁵¹ chromium into the culture supernatant. Counts: number of FasL-expressing cells; FL2-H / PE: strength of FasL expression; Specific lysis (%): release of ⁵¹ chromium, relative to a positive control with a maximum of ⁵¹ chromium release by lysis of the cells using SDS; E / T ratio: ratio of effector cells (infected macrophages) to target cells (A20 cells); M0-Ad / CV: macrophages infected with AdLacZ (adenovirus expressing LacZ); M0 -Ad / FL: macrophages infected with Ad-FasL (adenovirus expressing FasL); M0-FL: macrophages transfected with a FasL-expressing plasmid;

FIG. 2 graphically shows the result of the inhibition of allogeneic stimulation of T cells by FasL-expressing antigen-presenting cells. Macrophages were isolated from B6-lpr / lpr mice and infected with adenoviruses expressing either LacZ (AdLacZ) or FasL (AdFasL). The infected macrophages were co-cultivated with either T cells from (A) Fas-expressing B6 - + / + mice or (B) Fas-deficient B6-lpr / lpr mice and stimulation of the T cells by incorporating ³ H-tymidine measured (Mixed Lymphocyte Response, MLR). M0-CV: macrophages infected with AdLacZ (adenovirus expressing LacZ); M0-FL: Macrophages infected with Ad-FasL (adenovirus that expresses FasL). Figure 3 graphically shows the result of a quantitative analysis of the inflammatory response in the lungs, kidneys and liver. B6 ^{+ +} and B6-gld / gld mice were infected intraperitoneally with mouse cytomegalovirus (1 x 10 ³ pfu) and then the degree of inflammation and tissue damage in the lungs (upper panel), the kidney (middle panel) and the Liver (lower panel) classified according to a relative scale from 0 (no inflammation or damage) to 4 (most severe inflammation or damage). The bars in the illustration represent the mean ± standard deviation of the results of at least 5 mice at each time of the examination.

FIG. 4 graphically shows the result of the reduction in inflammation in the lungs, kidneys and liver in mouse cytomegalovirus-infected mice which were treated before infection with AdFasL-infected antigen presenting cells (APC). B6-gld / gld- and B6- lprllpr-MM.se. were infected with mouse cytomegalovirus and treated 28 days after infection with APCs, either with Ad-CMVLacZ (FasL negative control), with mouse cytomegalovirus (APC + MCMV), with AdFasL (FasL positive control) or with mouse cytomegalovirus and AdFasL (MCMV + AdFasL) were infected. The mice were treated with the APCs four times every three days and examined four weeks after the start of the APC therapy. Lungs, kidneys and liver were stained with hematoxilin and eosin and evaluated by three independent people. The bars in the illustration represent the mean ± standard deviation of the inflammatory response of the different organs for the differently treated mice. Lung-gld or lung-lpr: lung of B6-gld / gld or / pr // pr-mice; Leber-gld or Leber-lpr: liver of B6-gld / gld or pr // pr mice; Kidney-gld or kidney-lpr: kidney of B6-gld / gld or / pr // pr mice; APC-AdLacZ: antigen-presenting cells infected with an adenovirus that expresses LacZ; APC + MCMV: antigen presenting cells infected with mouse cytomegalovirus; APC-AdFasL: antigen-presenting cells infected with an adenovirus that expresses FasL; APC-AdFasL + MCMV: antigen-presenting cells infected with mouse cytomegalovirus and with an adenovirus that expresses FasL; * denotes mean values that differ significantly from the control group with regard to a confidence interval of 95% (P <0.05).

FIG. 5 graphically shows the result of an experiment to determine the amount of reactive T cells specific for mouse cytomegalovirus from cytomegalovirus (MCMV) -infected mice which, prior to infection with AdFasL-infected antigen presenting cells (APC) were treated. B6-pr // pr mice were infected with mouse cytomegalovirus and treated with different APCs as described in FIG. 4. Spleen cells were isolated from the MCMV-infected mice 4 weeks after APC therapy. The T cells were stimulated in vitro with MCMV-infected APCs and the IL-2-containing supernatant was isolated after 48 hours. APC-AdLacZ: antigen-presenting cells infected with an adenovirus that expresses LacZ; APC-AdFasL: antigen-presenting cells infected with an adenovirus that expresses FasL; APC-AdFasL + MCMV: antigen-presenting cells infected with mouse cytomegalovirus and with an adenovirus that expresses FasL; * denotes mean values that differ significantly from the control group with regard to a confidence interval of 95% (P <0.05).

FIG. 6 graphically shows the result of a reduced production of autoantibodies in cytomegalovirus (MCMV) -infected mice which were treated before the infection with AdFasL-infected antigen presenting cells (APC). B6-gld / gld mice were infected with mouse cytomegalovirus and treated with different APCs as described in FIG. 4. Sera were obtained from the MCMV-infected mice 4 weeks after APC therapy. RF IgGl: rheumatoid factor; ds-DNA IgGl: autoantibodies against double-stranded DNA; APC-AdLacZ: antigen-presenting cells infected with an adenovirus that expresses LacZ; APC-AdFasL: antigen-presenting cells infected with an adenovirus that expresses FasL; APC-AdFasL + MCMV: antigen-presenting cells infected with mouse cytomegalovirus and with an adenovirus that expresses FasL; * denotes mean values that differ significantly from the control group with regard to a confidence interval of 95% (P <0.05).

Figure 7 shows human macrophages infected with an adenovirus expressing LacZ. The virus-infected macrophages were identified with an X-Gal stain, which ß-galactosidase (LacZ) detects by means of their catalytic properties in the infected cells.

FIG. 8 graphically shows the results of an experiment to demonstrate the modulating influence of IL-10 and tumor necrosis factor (TNF) on the function of dentritic cells (DC) as antigen-presenting cells. Dentritic cells were generated from mononuclear cells of the peripheral blood by treatment with IL-4 and GM-CFS in vitro. The DCs were either treated with TNF or with IL-10 and then incubated with allogeneic T cells and the stimulation and proliferation of the T cells measured by the incorporation of ³ H-labeled thymidine. APC: antigen presenting cells; DC (TNF): Dentritic cells stimulated with tumor necrosis factor (TNF); DC (IL-10): Dentritic cells stimulated with interleukin-10 (IL-10); allo T cells: T cells of a donor with an allogeneic MHC pattern, ie MHC pattern deviating from the DCs. The X axis shows the amount of APCs that were used in the reaction. The Y axis shows the radioactive decays per minute (CPM) as a measure of the incorporation of the radioactively labeled nucleotide or as a measure of the stimulation of the T cells.

FIG. 9 graphically shows the results of an experiment for the detection of an allogene-specific suppression by tolerating antigen-presenting cells (APC). Dentritic cells were generated from mononuclear cells of the peripheral blood by treatment with IL-4 and GM-CFS in vitro. The DCs were treated with either TNF or IL-10 and then incubated with allogeneic T cells. Five days later, the T cells from this reaction were incubated with antigen-presenting cells from a third allogeneic donor and the stimulation and proliferation of the T cells were measured by the incorporation of ³ H-labeled thymidine. A, B and C denote donors with different (allogeneic) MHC patterns; MLR: Mixed Lymphocyte Reaction. The Y axis shows the radioactive decays per minute (CPM) as a measure of the incorporation of the radioactively labeled nucleotide or as a measure of the stimulation of the T cells. The composition of the first stimulation response (1st MLR) and the second response (2nd MLR) is shown under the individual bars.

FIG. 10 schematically shows the structure of the vectors (A) pcDNA3-TK-IRES-crmA and (B) pcDNA3-FasL-IRES-PLP, according to the invention. Coding reading frames such as the nucleic acid sequence for the Fas ligand (FasL), the proteolipid protein (PLP), the thymidine kinase (TK) and crmA, and for resistance proteins such as neomycin and ampicilin are shown with bright arrows. Regulatory active, eukaryotic promoter elements such as the CMV promoter and the SV40 promoter are represented by dark arrows, prokaryotic promoters such as the SP6 and the T7 promoter are represented by thin angled arrows. Interfaces for selected restriction endonucleases such as BamΑl, EcoRI, Xhόl and HincfiR are marked with the identification of the nuclease. Are marked by thin bars regulatory nucleic acid sequences such as the polyadenylation sequence of the SV40 virus (SV40polyA) and the LRES, the internal ribosome binding site.

The term "vector" or "gene transfer vector" used here denotes naturally occurring or artificially created organisms and constructs for the uptake, multiplication, expression or transfer of nucleic acids in cells. Vectors are, for example, viruses such as retroviruses, adenoviruses, adeno-associated viruses, smallpox viruses, alpha viruses or herpes viruses. Vectors are also, for example, bacteria, such as listeria, shigella or salmonella. Vectors are also, for example, liposomes or naked DNA, such as bacterial plasmids, virus-derived plasmids, phagemids, cosmids, bacteriophages or artificially produced nucleic acids such as artificial chromosomes.

The term "apoptosis receptor" as used herein denotes polypeptides which are located in the cytoplasmic membrane of cells and which, after interaction and activation by a specific ligand, induce apoptosis in the cell. An example of apoptosis receptors are polypeptides that belong to the subfamily of tumor necrosis factor receptors that are characterized by cytoplasmic death domains, e.g. CD95 / Fas / Apol, TRAIL-Rl, TRAIL-R2 or Apo3.

The term “ligand” or “apoptosis-triggering ligand” or “apoptosis ligand” used here denotes a membrane-bound polypeptide which can interact with apoptosis receptors. Binding of the ligands to the apoptosis receptors activates the receptors and induces apoptosis in the cells that carry the receptors. Apoptosis ligands are, for example, CD95L / FasL / ApolL, TRAIL or Apo3L.

The term "anti-apoptosis molecules" used here denotes polypeptides which inhibit apoptosis in the cell. These polypeptides can be of cellular or viral origin. Anti-apoptosis molecules also refer to nucleic acid molecules, comprising nucleic acids that are complementary to nucleic acids that code for polypeptides that trigger apoptosis.

The term "antigen" as used herein refers to polypeptides that comprise either a whole protein or parts of a protein, the single or multiple T cell epitopes include, and after proteolytic processing by the cell presented by MHC molecules and bound by T cell receptors.

The term "suicide enzyme" as used herein denotes polypeptides which convert low or non-toxic substrates into toxic substances or so that they can be used or converted by cell enzymes.

The term “IRES” used here denotes nucleic acid sequences of viruses which enable functionally active ribosomes to be bound independently of the cellular regulatory sequences such as, for example, the 5'-cap structure. IRES sequences are characterized by a strong secondary structure. IRES sequences have been described, for example, in picornaviruses.

An object of the present invention is to enable targeted, antigen-specific immunotherapy in cases of autoimmune diseases and diseases with immunopathogenesis. Individual T-cell clones with defined specificity for cellular or pathogen-specific proteins are to be eliminated and thus an immunological tolerance to an antigen is to be created or restored.

The invention relates to a gene transfer vector comprising at least one nucleic acid molecule, comprising a first nucleic acid sequence coding for one or more apoptosis-triggering ligands, a second nucleic acid sequence coding for one or more antigen (s) and optionally, a third nucleic acid sequence coding for a or more anti-apoptosis molecule (s), and optionally a fourth nucleic acid sequence, coding for one or more suicide enzyme (s). Preferred is a gene transfer vector comprising a nucleic acid molecule comprising the first three, or all four, or the first two and the fourth nucleic acid sequences. Also preferred is a gene transfer vector comprising two nucleic acid molecules, the first and second nucleic acid sequences occurring on a first nucleic acid molecule and the third and fourth nucleic acid sequences occurring on a second nucleic acid molecule. A gene transfer vector is particularly preferred, the first and second nucleic acid sequences being functionally linked to one another such that the expression of the second nucleic acid sequence is dependent on the expression of the first nucleic acid sequence, and / or the third and fourth nucleic acid sequences is functionally interconnected that the expression of the fourth nucleic acid sequence is dependent on the expression of the third nucleic acid sequence.

The gene transfer vectors according to the invention can be used for the therapy of autoimmune diseases and other diseases which are based on immunopathogenesis. Immunopathogenesis means damage to cells, tissues or organs by cellular or humoral immunomechanisms, i.e. H. by lymphocytes or antibodies or complement-mediated mechanisms. The vectors according to the invention can be used to modify cells of the body ex vivo by genetic engineering. These gene therapy vectors give the cells to be modified a number of new properties which make them suitable for treating autoimmune diseases and other diseases based on immunopathogenesis. For the purposes of this invention, suitable means that the modified cells are able to specifically attract the immune cells which are involved in the pathogenesis and to destroy them by inducing apoptosis.

The vectors according to the invention can be based on a large number of vector systems available today which are able to carry a number of different genes or functional areas and to expand the corresponding gene products in eukaryotic cells. In the case of vectors based on viral systems, nucleic acid sequences are packaged in these vectors with the aid of packaging cells or others in vztro systems. The vectors can then either actively enter the cells or be taken up by them. Non-viral vectors are introduced into the target cells using different methods of transfer based on physical and biological mechanisms. An important property of the vectors according to the invention is that no viral or other proteins related to the vector system are synthesized in the modified cells, which could interfere with the function of the cells modified by the vector systems. Expression of viral or other proteins would be harmful in the sense of the invention if the modified cells that produce these proteins are recognized and destroyed by immune cells. For the purposes of this invention, this detection would be harmful if it would impair the natural function of the immune system, viral or bacterial pathogens, degenerate cells, or other cells or pathogens normally recognized by the immune system. It is also harmful if this affects the efficiency of the cells that destroy immunopathogenic immune cells.

The cells modified by means of the vectors according to the invention, comprising the combination of nucleic acid sequences according to the invention, express antigens which are recognized by immunopathogenic immune cells. These immunopathogenic cells play a special role in the pathogenesis of a defined disease, since they specifically recognize (endogenous) antigens and coordinate an immune reaction against these antigens and the antigen-expressing cells. The antigens introduced into the cells with the vectors according to the invention can be specific for certain diseases or specific for the attacked organ, tissue or the attacked cell type. The vectors according to the invention additionally code for ligands which trigger apoptosis. These ligands that trigger apoptosis induce natural cell death in the immunopathogenic immune cells that recognize the antigens. The vectors according to the invention can code for one or more different ligands which trigger apoptosis. Of the modified cells, only those immune cells that recognize the artificially synthesized antigenic epitope and therefore physically come into contact with the modified cells are recognized and destroyed.

The nucleic acid sequences which are responsible for triggering apoptosis (ligands which trigger apoptosis) and those which code for the antigenic polypeptides (antigens) can be functionally coupled or linked at the transcriptional level in such a way that expression of the antigens is not without expression the ligands that trigger apoptosis is possible. The safety of the vectors according to the invention is greatly increased since this prevents the altered cells from being recognized by the diseased immune cells and thereby stimulated without the apoptosis being triggered in these cells at the same time.

The nucleic acid sequences of the vectors according to the invention can additionally carry genes or functional areas which prevent the cells modified by the vector according to the invention from initiating apoptosis and thus self-destructing themselves (anti-apoptosis molecules) via autocrine mechanisms. This greatly increases the efficiency of the vectors and the changed cells. The genes or functional areas either code for regulators of the activity of apoptosis-inducing factors or prevent their expression. The vectors according to the invention can optionally additionally comprise nucleic acid sequences which encode polypeptides which make it possible, if desired, to eliminate the genetically modified cells after re-infusion into the body (suicide genes). A functional coupling which is comparable to the functional coupling of the nucleic acid sequences which code for the antigen and the apoptosis-triggering ligands can be present for the nucleic acid sequences which code for the anti-apoptosis molecules and the suicide enzymes. This coupling ensures that cells that can no longer be eliminated in the body due to the anti-apoptosis molecules can be removed by the function of the suicide enzymes.

The nucleic acid sequences for the antiapoptosis molecules and the suicide enzymes can lie on the same nucleic acid molecule on which the nucleic acid sequences for the apoptosis-triggering ligands and the antigens lie, or they can lie on a different nucleic acid molecule.

The invention further relates to a gene transfer vector as a therapeutic agent. The invention further relates to the use of the gene transfer vectors for the production of a therapeutic agent for the prevention or therapy of autoimmune diseases, e.g. rheumatoid artritis, systemic lupus erythematosus, Sjogren's syndrome, polymyositis, dermatomyositis, polymyalgica rheumatica, temporal arteritis, spondyloarthropathies such as ankylosing spondylitis, Crohn's disease, ulcerative colitis, celiac disease, autoimmune nebiitis, autoimmune nebiitis, type I diabetes mellitus, autoimmune hepatitis i , Herpetiformis, pemphigus vulgaris, alopecia, multiple sclerosis, myastenia gravis, or for the prevention or therapy of chronic inflammatory processes that are based on immunopathogenesis, for example chronic inflammation after viral or bacterial infections such as chronic hepatitis in hepatitis B virus or hepatitis -C virus infections, or inflammation of the brain after infection with the measles virus, and for the prevention or therapy of graft rejection.

The invention further relates to the use of the gene transfer vectors for the ex vivo modification of eukaryotic cells, in particular animal or mammalian cells, in particular human cells. Retroviral vectors

The gene transfer vectors are preferably retroviral and in particular based on lentiviruses. These represent a suitable platform for the development of efficient vectors for the transfer of nucleic acids into cells. The insertion of a desired foreign gene into a suitable vector and the packaging into retroviral particles can be carried out using methods already described in detail and is state of the art. The recombinant viruses generated are then isolated and incubated in vivo or ex vivo with the desired target cells. A large number of different retroviral systems have been described so far and are suitable for transferring the combinations of nucleic acid sequences according to the invention. Retroviral and in particular lentiviral gene transfer vectors are therefore preferred for transferring the combinations of nucleic acid sequences according to the invention into eukaryotic cells.

Retroviral gene transfer vectors according to the invention can be based on different retroviruses such as, for example, the retroviruses of type B, C or D as well as spumaviruses and lentiviruses.

Representative representatives of suitable retrovirus families are, for example, those described in "RNA Tumor Viruses" on pages 2-7 and a large number of xenotrophic retroviruses, such as e.g. NZB-Xl, NZB-X2 and NZB9-1 and polytrophic retroviruses, e.g. MCF and MCF-MLV. These retroviruses can be found in inventory or collections such as the American Type Culture Collection ("ATCC"; Manassas, Va.), or can be isolated from biological material using common and published molecular biological techniques.

Retroviruses particularly suitable for the production of retroviral gene transfer vectors include representatives from the group of avian leukemia viruses, bovine leukemia viruses, mouse leukemia viruses, Mink-ce // ocw5-inducing viruses, mouse sarcoma viruses, Gibbon viruses. Leukemia viruses, feline leukemia viruses, reticuloendothelial viruses and Rous sarcoma viruses. Mouse leukemia viruses such as representatives 4070A and 1504A, Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC No. VR-590), Kirsten, are particularly suitable. Harvey's Sarcoma Virus and Rauscher (ATCC No. VR-998) and the Moloney Mouse Leukemia Virus (ATCC No. VR-190). The Rous sarcoma virus is also particularly suitable Bratislava, Bryan high titer (e.g., ATCC No. VR-334, VR-657, VR-726, VR-659, and VR-728), Bryan Standard, Carr-Zilber, Engelbreth-Holm, Harris, Prague (e.g., ATCC No. VR-772, and 45033) and Schmidt-Ruppin (e.g., ATCC No. VR-724, VR-725, VR-354).

In special applications described in the invention, constituents of the nucleic acid molecules of the retroviral gene transfer vectors can also originate from retroviruses other than those listed. For example, retroviral vector long terminal repeat (LTR) regions from the mouse sarcoma virus, the tRNA binding site from the Rous sarcoma virus, the packaging signal from the mouse leukemia virus, and the origin for the second strand DNA Synthesis derived from the avian leukemia virus.

Furthermore, nucleic acid molecules can be used for retroviral vectors which contain a 5 'LTR, a tRNA binding site, a packaging signal, one or more heterologous sequences, an origin of second-strand DNA synthesis and a 3' LTR, the nucleic acid molecule not being used for Gag / Pol or Env coding sequences. LTR contain three elements, the U5, R and U3 region. These elements contain a variety of signals which are important for the biological activity of retroviruses, for example promoter and Erc zαncer elements, which are located in the U3 region. LTRs can be clearly characterized within a provirus using the characteristic sequence duplications at the ends of the genome. The 5 'LTR preferably used in the present invention contain a 5' promoter element and a minimal LTR sequence which enables a reverse transcription and integration of the vector nucleic acid into the genome of the target cell. The 3 'LTR region contains a polyadenylation signal and LTR sequences which are required for the reverse transcription and integration of the vector nucleic acid into the genome of the target cell.

The tRNA binding site and the origin of the second strand DNA synthesis are required for biological activity and the identification of these components is state of the art. For example, retroviral tRNAs bind to a tRNA binding site using Watson-Crick base pairing and are packaged with the retroviral genome into the virus particles. The tRNA then serves the reverse transcriptase as a primer for the DNA synthesis. The tRNA binding sequence is located immediately downstream of the 5 'LTR and can be easily identified by its location. In the same way, the origin of the second strand DNA synthesis of great importance for the second strand DNA synthesis of retroviruses. This region, called the polyuridine tract, is located directly upstream of the 3 'LTR.

In addition to the 5 'and 3' LTR, the tRNA binding sequence and the origin of the second strand DNA synthesis, the nucleic acids can include retroviral gene transfer vectors

Contain packaging signal, and also one or more heterologous sequences, which are described in detail below.

For example, retroviral gene transfer vectors are used which have neither Gag / Pol nor Env-coding nucleic acid sequences. For example, retroviral

Gene transfer vectors without Gag / Pol or Env coding sequences are generated by the production of vector constructs that have an expanded packaging signal. The

The term "expanded packaging signal" defines a nucleotide sequence which exceeds the minimal sequence necessary for the specific packaging of nucleic acids. The use of the extended packaging sequence allows the production of higher titres

Virus stocks based on increased RNA packaging. For example, the minimum packaging signal of the Moloney Mouse Leukemia Virus (Mo-MLV) is one

Coded sequence that begins at the end of the 5 'LTR and contains the Pst I interface. The expanded packaging signal from Mo-MLV contains sequences beyond nucleotide 567, the start of the Gag / Pol gene (nucleotide 621) and ends beyond nucleotide 1560. Therefore, deletion of the sequence beyond nucleotide 567 allows a retroviral from the Mo-MLV

Gene transfer vector are produced, which has no extended packaging signal.

In addition, nucleic acid sequences can be used for retroviral gene transfer vectors in which the packaging signal has been partially or completely superimposed on the retroviral Gag / Pol sequence, but has been completely deleted or truncated upstream of the start codon of the Gag / Pol gene. Furthermore, nucleic acid sequences can be used for retroviral gene transfer vectors which contain a packaging signal which is expanded in the 5 'region before the start of the Gag / Pol gene. If these retroviral vectors are used, packaging cell lines should preferably be used for the production of the recombinant virus particles in which the 5 'terminal end of the Gag / Pol gene is modified in a Gag / Pol expression cassette in such a way that it Gene-modified codon usage that differs from the wild-type HΓV-1 gag sequence. In addition, nucleic acid sequences can be used for retroviral gene transfer vectors which have a 5 'LTR, a tRNA binding site, a packaging signal, an origin of the second strand DNA synthesis and a 3' LTR region, the nucleic acid sequence not having a retroviral nucleic acid sequence upstream of the 5 'LTR. These vectors do not have an encoding sequence upstream of the 5 'LTR. Furthermore, nucleic acid sequences can be used for retroviral gene transfer vectors which have a 5 'LTR, a tRNA binding sequence, a packaging signal, an origin of the second strand DNA synthesis and a 3' LTR, but which do not contain a retroviral packaging signal sequence downstream of the 3 'LTR. The term "packaging signal sequence" used here defines a sequence which is required for packaging an RNA genome.

Suitable packaging cell lines for establishing the above-mentioned retroviral gene transfer vectors are already available and have been widely used for the production of cell lines (also referred to as vector cell lines) for the production of recombinant vector particles.

Among the retroviral vectors, lentiviral vectors are particularly suitable for transferring the combinations of nucleic acid sequences according to the invention, since they are able to introduce and express nucleic acid sequences in a large number of resting and postmitotic cells, such as, for example, neuronal, liver, muscle and hematopoietic stem cells , The lentiviral vector particles can be generated by triple infection of mammalian cells with (i) a Gag / Pol expression vector, (ii) a transfer construct which contains the packaging signal, the foreign nucleic acid sequence (s) and the flanking LTRs, and (iii) an expression vector for a coat protein , Here, coat proteins of different amphotrophic or xenotrophic retroviruses and other viruses can be used, such as, for example, the coat proteins of the Moloney mouse leukemia virus (Mo-MLV) and the MLV isolate 4070A and the vesicular stomatitis virus (VSV) G glycoprotein or that Rabies G glycoprotein. Lentiviral vectors produced in this way are able to stably transfect a large number of different cells. Lentiviral vectors containing the central polyuridine tract and the terminator sequence of the HIV-Pol gene have an increased transduction efficiency based on an improved nuclear translocation of the vector. To improve the safety of lentiviral gene therapy vectors, different functional packaging constructs with deletions in the accessory genes Vif, Vpr, Vpu and Nef of HIV-1 and SIY-1 can be produced. Furthermore, functional lentiviral vectors can be developed that no longer need the viral transactivator protein Tat. In order to minimize the development of replication-competent recombinants, self-inactivating vector systems can also be developed in which, for example, a comprehensive area of the U3 region in the 5 'and / or 3' LTR including the TATA box and the binding sites for the transcription factors SP1 and NF-κB are deleted within the vector becomes. These modifications remove a large part of the viral transcription elements. The deletion of the U3 region also prevents possible interference between the promoter located in the LTR and internal promoters and drastically reduces the risk of activation of neighboring cellular genes at the integration site of the lentviral vector. In order to avoid the Rev dependence of the lentiviral Gag / Pol and Env genes, synthetically produced codon-adapted HIV or SIV Gag / Pol and Env genes are used, for example. Alternatively, constitutive transport elements (CTE) of other viruses, such as, for example, the CTE of the Mason-Pfitzer monkey virus or the monkey retrovirus type 1 (SRV-1) as well as the post-transcriptional regulatory element (PRE) of the hepatitis B virus and the post-transcriptional direct repeat (DR) element of the Rous sarcoma virus can be used to enable Rev-independent export of the HIV / SIV-Gag, Gag / Pol and Env gene transcripts. These methods allow the transactive Rev protein to be excluded from the lentiviral vectors, which contributes to increased safety of this vector type. A compilation of the current lentiviral vector systems is given, for example, in the review by Buchschacher and Wong Staal (Buchschacher et al. (2000) Blood 95: 2499-2504).

In addition to retroviral and lentiviral vectors, a large number of other viral and non-viral gene transfer vectors can be used, which can also be used to transfer the combinations of nucleic acid sequences according to the invention. Since these vector systems are to be used to generate therapeutically usable, genetically modified cell lines, the viral vector systems are modified for safety reasons in such a way that they are no longer able to replicate lytically. The gene transfer vectors are preferably vectors based on adenoviruses, adeno-associated viruses, poxviruses, alphaviruses or herpesviruses. Adenoviral vectors

The production of recombinant adenoviral vectors (Ad vectors), including the El / E3, El / E4, and "gutless" vectors is state of the art and can be carried out according to published protocols. For example, (1) the desired nucleotide sequence can be inserted into a pBHGl 1 plasmid in order to generate recombinant El / E3-deleted ad vectors after transfection of 293 cells and subsequent intracellular recombination; (2) the desired nucleotide sequence is first integrated into the El region of one of a large number of deleted Ad vectors, co-transfected with a Clael digested H5dl 1014 vector and the recombinant, El / E4 deleted Ad vectors after transfection of 293-E4 cells and subsequent intracellular homologous recombination are isolated and (3) the desired nucleotide sequence together with an appropriate amount of a "Stujfer" sequence, for example which, for example, originate from the DNA of the bacteriophage lambda, are first inserted into the ArAd plasmid in order subsequently to ensure efficient packaging of the recombinant "gutless" adenovirus vector genomes after transfection into 293 cells and infection with an H5.CBALP helper virus. The recombinant "gutless" adenovirus vector particles can be purified from contaminating helper viruses, for example, due to the lower density of the vector particles compared to the helper virus, by equilibrium sedimentation in a cesium chloride gradient.

Adeno-associated viruses

Furthermore, various developed adeno-associated virus (AAV) vector systems can be used for gene transfer. A detailed description of the construction of AAV vectors has been published and is state of the art.

In recombinant AAV vectors, all coding sequences are usually replaced by the desired heterologous nucleic acid sequences. The recombinant AAV are co-transfected by an AAV vector, which carries the desired gene, and a helper AAV plasmid, which has all the essential AAV genes, into adenovirus-infected cells, all of which are required for AAV replication and the production of vector particles Provide helper functions, manufactured. Disadvantages of this vector system for gene therapy use, however, are the low titer of recombinant vectors and possible contamination of the vectors with wild-type AAV and infectious helper viruses. Furthermore, the size of the in AAV Vectors to be integrated foreign sequences limited to 5 kb.

poxviruses

Alternative viral vector systems for the transfer of nucleic acids encoding a desired foreign gene are based on members of the poxvirus family, including the vaccinia virus and birdpox virus. Such vector systems are produced as described below by way of example for recombinant vaccinia viruses. First, the DNA encoding the desired gene is integrated into a suitable vector such that it is adjacent to a vaccinia promoter and a flanking vaccinia DNA sequence such as e.g. the coding sequence for the thymidine kinase (TK). This vector is then transfected into cells and these are simultaneously infected with vaccinia viruses. The insert is then recombined with the foreign gene into the viral genome by homologous recombination. The resulting TK-positive recombinants can be established by cultivating the viruses on cells in the presence of 5-bromodeoxyuridine and then isolating plaques.

Alternatively, other bird pox viruses, such as Fowlpox and Canarypox viruses can be used for gene transfer. Recombinant birdpox viruses that express immunogens from human pathogenic organisms can induce a protective immune response after administration in mammals. The use of birdpox viruses is particularly advantageous for use in humans and other mammals, since representatives of the birdpox genus replicate productively only in susceptible bird species, but not in mammalian cells. Methods for producing recombinant birdpox viruses are state of the art and are based on genetic recombination mechanisms, comparable to those that were previously described for the production of recombinant vaccinia viruses.

alphaviruses

Representatives of the genus of the alpha viruses, such as, for example, vectors derived from Sindbis and Semliki Forest viruses, can also be used for the transfer of nucleotide sequences of selected genes. The production and use of vectors based on the Sinbis virus is state of the art and has been published several times. bacteria

The gene transfer vectors are also preferably bacteria, in particular Listeria monocytogenes (Δmpl, ΔactA, ΔplcB), Shigella flexneri (ΔaroA, ΔvirG) and Salmonella thypimurium. A promising DNA delivery system for the recruitment and activation of antigen-specific cells uses bacterial suicide vectors based on attenuated Listeria monocytogenes (Δmpl, ΔactA, ΔplcB), Shigella flexneri (ΔaroA, ΔvirG) and Salmonella thypimurium isolate. The production and use of such bacterial gene transfer systems has been published in detail and is state of the art. For example, "suicide" strains of L. monocytogenes are able to specifically target professional antigen-presenting cells. After the bacteria have been absorbed into the cytoplasm of the target cell, they are destroyed, mediated by a Listeria-specific phage lysine, which leads to a release of the plasmid DNA transported by the bacteria, which subsequently arrives in the cell nucleus. The main advantages of this bacterial system are the oral application of the bacteria and the targeted introduction of plasmids into APCs, which play a central role in the induction of a cellular immune response. The suicide strains of the invasive bacteria Shigella flexneri and Salmonella thypimurium were attenuated by deleting genes that are essential for the production of metabolites of cell wall synthesis. These bacterial strains lyse after infection of mammalian cells due to the lack of these metabolites.

Plasmid DNA

The gene transfer vectors are preferably also plasmids. Naked plasmid DNA is suitable as a vector for the combinations of nucleic acid sequences according to the invention. These expression vectors contain, for example, the following essential elements: one or more strong constitutive and / or inducible promoters, a transcription terminator, such as that of bovine growth hormone, antibiotic resistance or another marker for selection of the transformed organism, the first, second, and / or third and / or fourth nucleic acid sequence according to the invention and an origin of replication which enables production in a suitable host organism, in particular a second generation of linear DNA plasmids, the so-called MLOGE transfection vectors, are suitable for the efficient transfer of the nucleic acid sequences according to the invention. MIDGE vectors consist of both strands of a DNA polymer that contain any number of the desired coding sequences and the promoter and necessary for expression of foreign genes Contains terminator sequences and are linked at both ends with loops of single-stranded deoxyribonucleotides in such a way that a covalently closed molecule is formed. In addition to the foreign genes desired for medical use with the regulatory units required for efficient expression, MLDGES do not contain any further coding sequences, such as are required for the amplification and selection of conventional DNA transfer vectors. For example, these vectors have no Ori sequences which contain potential integration sites and no coding sequences for antibiotics as selection markers which are under the control of promoters which can often not be switched off completely in mammalian cells.

The efficiency of this system for gene transfer and for improved foreign gene expression in mammals and human cells can be increased in particular by attaching a heterologous class of peptides which contain so-called nuclear localization sequences (NLS). For example, the core import of MIDGE-like constructs can be improved by the NLS of the SV-T antigen and the foreign gene expression can be increased. Furthermore, specific targeting can be achieved by coupling the MIDGES with tissue-specific ligands. Analogous to bare DNA, the MIDGES can also be coupled with a large number of non-viral gene transfer systems in order to ensure more efficient uptake into the target cell.

Furthermore, gene transfer vectors can be used which contain components of eukaryotic DNA transposons. Transposons are naturally occurring genetic elements that can move from one position to another within a chromosome. Representatives of the Tcl / marine family, e.g. the sleeping beauty transposon can be used to produce suitable vectors for use in mammalian cells. An advantage of these vectors is that multiple regulatory sequences can be integrated into the vectors. These vectors integrate into the host cell genome and allow long-lasting expression of the desired foreign sequences.

Systems for the transfer of nucleic acids into eukaryotic cells

Furthermore, a variety of methods for gene transfer in mammalian cells based on non-viral systems have been described. The non-viral vectors are often used in combination with particle-mediated gene transfer or with viral vector systems. In brief, the combinations of nucleic acid sequences according to the invention can be integrated into one or a combination of several conventional gene transfer vectors, which enable suitable control elements for an efficient expression of the desired foreign gene (s) with high yields. The vectors according to the invention can then be coupled to synthetic gene transfer molecules such as, for example, polymeric DNA-binding cations such as polylysine, protamine and albumin, or can be bound to ligands which convey specific targeting of cells, such as, for example, asialoorosomucoid, insulin, galactose, lactose or transferrin.

The efficiency of taking up naked DNA can also be increased by using biodegradable latex beads. Latex beads loaded with DNA are absorbed into the target cells with increased efficiency due to the endocytosis mediated by the latex beads. The efficiency of this method can be increased by increasing the hydrophobicity and the associated improved disaggregation of the beads in the endosome, which results in a more efficient release of the DNA into the cytoplasm.

In addition, various compositions of liposomes and immunostimulatory reconstituted influenza virosomes (ΓRTV, immunepotentiating reconstituted influenza virosomes) are also suitable as vehicles for the transfer of the vectors according to the invention into mammalian and human cells (US Pat. No. 5,879,685). In addition, foreign nucleic acid sequences can be integrated into a vector with suitable control sequences, bound to synthetic gene transfer molecules such as polymeric DNA-binding cations (e.g. polylysine, protamine and albumin) and coupled with cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose or transferrin. Another administration system is based on the packaging of sequences which contain the desired genes under the control of different tissue-specific or consumer promoters in liposomes. Furthermore, the transfer of the nucleic acids described above can be increased by association with a photopolymerized hydrogel material. Another common method for transferring nucleic acids is administration by means of a portable "particle gun", and the use of ionizing radiation to activate gene transfer. Other methods for optimizing the efficiency of viral vector transduction include variation of the "multiplicity of infection" (MOI), depletion of ions such as phosphations, addition of polycationic substances such as protamine sulfate, variation of the contact time, temperature, pH and common Centrifugation of cells and virus or vector stocks.

The gene transfer systems described can be used for the genetic manipulation of isolated human or mammalian cells, in particular antigen-presenting cells (monocytes, macrophages, dendritic cells, B cells). If retroviral gene transfer vectors are used, the cells can be transferred into the S phase by stimulation in order to enable the infection of these cells. Cells in the first quarter to half of the S phase have proven to be particularly sensitive to retroviral transfection.

Polypeptides with antigenic epitopes

The vectors according to the invention are characterized in that they comprise nucleic acid sequences which code for proteins or parts of proteins or polypeptides which are recognized by immune cells (antigens). Immune cells in the sense of the invention are lymphocytes with regulatory or cytolytic properties, such as CD8 ⁺ / CD4 ^" T cells or CD87DC4 ⁺ T cells or CD87CD47CD56" killer cells (NK cells). Proteins or polypeptides in the sense of this invention are proteins of human or animal cells. These proteins or polypeptides are either on the surface of the attacked cells, for example glycoproteins on cell membranes due to their function, or they are located inside the cells, for example regulatory proteins. These proteins or polypeptides are processed in the cell and presented to the body's immune cells in the context of class 1 or class 2 MHC molecules. The proteins or polypeptides in the sense of this invention are recognized by the own immune cells and lead to a stimulation of these cells, ie to an increase in the cells by the release of messenger substances (LFN-γ, IL2, TNF, etc.) or by the cytolytic Immune cell activity can be measured.

The vectors according to the invention are characterized in that they comprise nucleic acid sequences which code for the polypeptides which have one or more linear or structural epitopes. These epitopes can be presented by MHC molecules from immunopathogenic T cells are recognized. These peptide regions and epitopes can also be encoded and expressed in the context of other immunogenic or non-immunogenic polypeptides, for example as fusion proteins or in the form of exchangeable cassettes in proteins, the cassettes coding for regions or epitopes or combinations of epitopes of the proteins. For the purposes of this invention, epitopes of the proteins are those regions of the proteins or those amino acid sequences which are recognized by immune cells such as, for example, T cells or NK cells. These epitopes can be recognized by immune cells of healthy people as well as people with autoimmune diseases.

The vectors according to the invention can be used, for example, for the therapy of autoimmune diseases, of chronic inflammatory processes which are based on immunopathogenesis and of tissue and organ rejection reactions. For example, There are many autoimmune diseases known that involve auto-reactive T cells that recognize the body's own proteins and structures. The vectors according to the invention can comprise nucleic acid sequences which code and express for these endogenous proteins and structures. All diseases can be considered for therapy in which T cells are involved in the pathogenesis and the proteins and structures attacked by the T cells are identified.

A group of autoimmune diseases are rheumatological diseases. With rheumatoid artritis, for example, the joints are attacked; clinical complications include joint destruction, kidney damage and amyloidosis. Systemic lupus erythematosus affects and damages various organs and tissues such as the central nervous system and the kidney. Sjögren's syndrome affects exocrine glands such as salivary glands. Polymyositis and dermatomyositis are

Autoimmune diseases of the muscles and skin and lead to muscle weakness and paralysis. Polymyalgica rheumatica and arteritis temporaiis are inflammatory diseases of the vessels and cause muscle weakness and blindness. Spondyloarthropathies such as Bechterew's disease in turn affect the joints and lead to stiffening.

Another group of autoimmune diseases are a number of gastrointestinal diseases. Crohn's disease affects the entire intestinal tract and leads to bleeding, stenosis, fistulas, and not infrequently leads to the development of tumor diseases. colitis Ulcerative pain is an inflammatory disease of the colon and leads to perforation and bleeding. Celiac disease affects both the small and large intestines and results in weight loss. Autoimmune hepatitis is an inflammatory disease of the liver resulting in cirrhosis and liver transplantation.

Endocrinological diseases can also be due to autoimmune reactions. Type I diabetes mellitus is an inflammatory disease of the pancreas and leads to diabetes, vascular damage e.g. with impairment of the kidney, peripheral nervous system and eyes, and subsequently requires kidney and pancreas transplants. Adrenal insufficiency and thyroiditis are also autoimmune diseases with T cell pathogenesis. In addition, some skin diseases are assigned to the group of autoimmune diseases. Some examples are psoriasis, dermatitis herpetiformis, pemphigus vulgaris: infections can cause complications with these diseases. Alopecia leads to hair loss. Finally, there are also neurological diseases that have to be attributed to autoimmune reactions. Multiple sclerosis affects, for example, the central and peripheral nervous system, myastenia gravis the muscles. In both neurological autoimmune diseases, paralysis can occur as complications.

The vectors according to the invention can also be used to treat chronic inflammatory processes based on immunopathogenesis, for example chronic inflammation after viral or bacterial infections, such as chronic hepatitis in the case of hepatitis B virus or hepatitis C virus infections, or post-inflammation of the brain Measles virus infection.

The vectors according to the invention can also be used for the therapy of tissue and organ rejection reactions. The immune response against foreign structures on the surfaces of the cells of the organ to be transplanted, which are essentially formed by the MHC (major histocompatibility cow? / E) molecules which are present on all cells, generally include a transplantation of tissues and organs if the donor and recipient are incompatible or if the donor's immune response is not suppressed. Compatible in the sense of organ transplants means that donors and recipients are so extensive in the various alleles for class I and Class II MHC agree that there is no inflammatory reaction.

T cells normally recognize fragments of foreign proteins associated with the body's own MHC with their T cell receptors. T cells that recognize the body's own fragments of proteins with the body's own MHC are inactivated or are not activated in various ways. In the event of an immune response against an organ to be transplanted, the recipient's T cells recognize the combination of antigen and foreign MHC, even if the same antigen is not recognized in connection with its own MHC.

The vectors according to the invention, which are used in connection with a transplant for the purpose of inducing a tolerance to the organ to be transplanted or already transplanted, encode and express proteins and structures of the organ types to be transplanted or already transplanted. The vectors, which are used, for example, in connection with a pancreas transplant, code for characteristic proteins from the pancreas. Vectors that are used in kidney or liver transplantations code for characteristic proteins of liver or kidney cells. The encoded proteins also include those proteins that are not organ specific, such as endothelial cells, but are present in the transplanted organ and are a target of the inflammatory response in the event of organ rejection.

For three autoimmune diseases, multiple sclerosis, myastenia gravis, rheumatoid artritis, and diabetes mellitus type I, the selection of possible antigenic proteins which can be encoded and expressed by the vectors according to the invention is described in more detail by way of example.

multiple sclerosis

Multiple sclerosis (MS), a disease of the human central nervous system, is characterized by perivascular inflammation and demyelination. The accumulation of activated T cells in the early MS lesions as well as in the surrounding, still inconspicuous areas of the white marrow body underlines the role of cell-mediated immunity (T cells) in the development of multiple sclerosis. Studies on a generally accepted animal model, experimental autoimmune encephalomyelitis (EAE), by immunization with myelin components have shown that EAE can be transferred from one animal to the other by activated, myelin-specific T cells. As a rule, immune cells can be detected in a patient with multiple sclerosis against different components of the myelin, components of astrocytes, and proteins that do not come from cells of the central nervous system. In addition, different immune cells recognize different areas of a component (epitopes), with an increase in the number of recognized epitopes correlating with the worsening of the disease.

The myelin basic protein (MBP) is a separable and relatively easy to clean component of the myelin. About 30% of the myelin in the central nervous system (CNS) consists of MPB. MBP was the first protein component of myelin to have anti-inflammatory properties. By way of example, the vectors according to the invention encode and express the following epitopes, individually or in combination: AS 1 - 20, AS 7 - 26, AS 16 - 38, AS 38 - 55, AS 50 - 68, AS 61 - 82, AS 71 - 89, AS 83 - 102, AS 94 - 117, AS 108 - 131, AS 124 - 141, AS 131 - 145, AS 139 - 153, AS 148 - 162, AS 153 - 170, AS 80 - 102, AS 81 - 99, AS 82 - 100, AS 83 - 99, AS 85 - 99, AS 86 - 99, AS 159 - 169.

With more than 50%, the myelin proteolipid protein (PLP) is the largest component of myelin in the central nervous system. PLP is a membrane protein with highly hydrophobic properties. Proliferation experiments predominantly identified CD4 ⁺ PLP-specific T cells in the blood of people with multiple sclerosis. By way of example, the vectors according to the invention encode and express the following epitopes, individually or in combination: AS 40-60, AS 89-106, AS 103-120, AS 125-143, AS 139-154, AS 1-275, AS 95-117, AS 139 - 151 and AS 185 - 206.

The myelin oligodendrocyte protein (MOG) is a smaller component (0.01 - 0.05%) of the myelin. MOG has a molecular weight of 26 - 28 kDa and is made up of an immunoglobulin-like variable domain and two hydrophobic (potential) transmembrane domains. Studies in animal models where MOG induces both a T cell-mediated inflammatory response and demyelinating antibodies, and studies in multiple sclerosis identified glycoprotein as an important antigen in autoimmune demyelinating diseases of the central nervous system. The vectors according to the invention encode and express the following epitopes of the MOG, individually or in combination: AS 1- 22, AS 35 - 55, AS 36 - 45, AS 34 - 56, AS 43 - 57, AS 64 - 96, AS 92 - 106, AS 134 - 148, AS 1 - 26, AS 14 - 39, AS 27 - 50, AS 38 - 60, AS 50 - 74, AS 63 - 87, AS 76 - 100, AS 89 - 113, AS 101 - 125, AS 162 - 178, AS 168 - 182, AS 14-36.

The myelin-associated oligodendrocytic basic protein (MOBP) is one of the main components of myelin. Patients with relapsing multiple sclerosis show a cellular immune response to MOBP. By way of example, the vectors according to the invention encode and express the following epitopes, individually or in combination: AS 1-19, AS 11-29, AS 21-39, AS 31-49, AS 37-60, AS 41-59, AS 51-69, AS 83-99, AS 1-60, AS 27-50.

The oligodendrocyte-specific protein (OSP) represents about 7% of the total myelin. OSP is a transmembrane protein with a length of 207 amino acids. A comparison of the tertiary structure of the OSP showed homology to peripheral myelin protein 22 in the CNS. Antibodies against OSP could be detected in the spinal fluid of people suffering from multiple sclerosis with a relapsing course. T cells with a specificity for OSP could be shown in humans. By way of example, the vectors according to the invention encode and express the following epitopes, individually or in combination: AS 52-71, AS 72-91, AS 82-101, AS 102-121, AS 132-151, AS 142-161, AS 182-201, AS 192 - 207.

The myelin-associated glycoprotein (MAG) is a component of myelin in the central and peripheral nervous system. MAG is a membrane protein with a molecular weight of 100 kDa, composed of five extracellular immunoglobulin-like domains, a single transmembrane domain and a cytoplasmic domain. Two isoforms (L and S-form) formed by alternative splicing are described, which are detected at different times of myelin formation. MAG is located in the periaxonal membranes of the myelin-forming Schwann cells and oligodendrocytes and is associated with glia-axon interactions. By way of example, the vectors according to the invention encode and express the following epitopes, individually or in combination: AS 20-34, AS 124-137, AS 354-377, AS 570-582. The proteins described below are further targets for pathogenic immune cells. The glycoprotein PO as a component of the peripheral nervous system and Schwann cells. PO has a molecular weight of 30 kDa and represents over 50% of the mass of the compact myelin in the peripheral nervous system. The peripheral myelin protein 22 (PMP-22 / PAS-II) has a molecular weight of 22 kDa. PMP-22 is not specific for Schwann cells, but is also expressed in other tissues such as the lungs, stomach and heart. pl70k / SAG (Schwann Cell Membrane Glycoprotein) is a glycoprotein with a molecular weight of 170 kDa. SAG is produced by myelinating and non-myelinating Schwann cells. Oligodendrocyte myelin glycoprotein (OMgp) is a glycoprotein with homology to MBP that is only expressed in the central nervous system. Schwann Cell Myelin Protein (SMP) is another glycoprotein that is produced by Schwann cells and was discovered in chickens. The glycoprotein has 44% homology to MAG. Other polypeptides that have been identified as targets for autoimmune reactive cells are the Transaldolase, SIOSS, Alpha B Crystallin, 2 ', 3' Cyclic Nucleotide 3 'Phosphodiesterase (CNP) and GFAP. The vectors according to the invention also code, for example, for these proteins or fragments thereof, in particular for regions or combinations of regions which carry T cell epitopes.

Myastenia gravis Myastenia gravis, an autoimmune disease that causes progressive muscle weakness, is caused by autoantibodies to the acetylcholine receptor on muscle cells. The production of autoantibodies depends on specific T helper cells. 10% of patients with myastenia gravis suffer from thymus epithelial tumors. The tumor cells synthesize mRNAs that code for the - and ε subunits of the acetylcholine receptor, and may thus incorrectly sensitize T cells in the thymus or circulating T cells to epitopes of the acetylcholine receptor. However, the mechanism of tolerance induction in the maturing thymocytes may also be disturbed. Areas in the subunits of the acetylcholine receptor which are epitopes for B and T cells have been described several times. By way of example, the vectors according to the invention encode and express subunits of the acetylcholine receptor or fragments of these subunits, in particular the following areas, individually or in combination: AS cd - 437, AS α3 - 181, AS α37 - 114, AS 37 - 181, AS α62 - 90, AS 73 - 90, AS α75 - 90, AS α75 - 115, AS αl30 - 178, AS αl38 - 167, AS αl44 - 156, AS l49 - 158, AS αl49 - 163, AS αl46 - 160, AS ε201 - 219.

Rheumatoid arthritis

Rheumatoid artritis was one of the first systemic diseases that was attributed to autoimmune mechanisms. Essentially two aspects of rheumatoid artritis suggest a fundamental immune system disorder as the cause of the disease. First, the often very massive infiltration of lymphocytes, including CD4 ⁺ T cells, in the inflamed hypertrophic synovial tissue, and second, the production of large amounts of rheumatoid factor from B cells and plasma cells in the synovium. Rheumatoid factors are antibodies that are directed against structuring regions of the IgG heavy chain. The specific tissue damage in the joints and in "extra-articular structures" is attributed to the inflammatory "pannus" and accumulations of granular cells, which are referred to as rheumatoid nodes. The most important argument in favor of the involvement of T cells in the development of rheumatoid artritis is the strong association of the disease with certain MHC class II haplotypes and the observation that the disease in the mouse model can be adaptively transmitted by isolated T cells , A wide variety of antigens have been described as possible targets for autoreactive immune cells, including structures of connective tissue such as collagen, proteoglycans and antigens on chondrocytes, heat shock proteins and exogenous viral or bacterial antigens. The vectors according to the invention code by way of example for these proteins or fragments thereof, in particular for regions or combinations of regions which carry T cell epitopes, for example type II collagen (CII).

Diabetes mellitus type 1 Diabetes mellitus type 1 is an autoimmune disease with multifactorial causes, including genetic predisposition. The destruction of the insulin-producing ß cells is caused by T lymphocytes. Both CD4 ⁺ and CD8 ⁺ T cells are involved in the pathogenesis and both subtypes are equally necessary for the development of the inflammatory response. Experiments with NOD mice, which are an animal model for diabetes, identified CD8 ⁺ T cells as the functional effector cells. The disease could be transmitted by transferring CD8 ⁺ T cells from prediabetic NOD mice to SCID-NOD mice, which, although they have the genetic predisposition but no immune cells. A number of possible autoantigens in diabetes have been identified. Antibodies against various autoantigens have been detected in prediabetic patients and those with recently diagnosed diabetes. In addition, CD4 ⁺ and CD8 ⁺ T cells with different specificity were detected. The gene therapy vectors within the scope of the invention described here carry functional areas or genes which code and express for antigenic proteins, protein fragments or epitopes and combinations of epitopes which represent targets for CD4 ⁺ and / or CD8 ⁺ T cells. Antigenic proteins in the sense of this invention are those proteins which are recognized by T cells of persons with diabetes in connection with MHC class I or MHC class II on syngeneic cells.

One target of the autoimmune mechanisms in type I diabetes is tyrosine phosphatase IA-2. Autoantibodies to the protein, which are almost always directed against the protein's intracellular domain, are detectable in patients with diabetes. In people with a genetic predisposition to diabetes, autoantibodies against IA-2 are a clear marker of a rapid worsening of the disease. As an antigenic target for T cells in diabetes, the vectors can encode and express the entire IA-2 protein or protein fragments. By way of example, the vectors according to the invention encode and express the following regions, individually or in combination, which have been identified as epitope-bearing regions in connection with MHC-DR4: AS 654-674, AS 709-732, AS 753-771, AS 797-817, AS 854 - 872, AS 955 - 975.

Among the possible autoantigens in type I diabetes, insulin and its precursor, proinsulin, are the only proteins that are only synthesized in the beta cells. Autoantibodies to proinsulin can be detected in prediabetic and diabetic patients. Diabetes can be generated in NOD mice by transfer of proinsulin-specific T cells, which shows the importance of proinsulin as a target of the cellular immune response. As an antigenic target for T cells in diabetes, the vectors can encode and express the entire alpha chain, the entire beta chain, the connecting peptide, the entire proinsulin protein or protein fragments, individually or in combination. By way of example, the vectors according to the invention encode and express the following regions, which have been identified as epitope-bearing regions: AS 1-15 (pl-15) and AS 35-50 (p35-50), from the region of the connecting peptide between the alpha and beta chains ; AS 6 - 80 (a6-80), from the area of the alpha chain; AS 9 - 23 and AS 10 - 25 (b 10-25), from which Area of the beta chain.

Glutamic acid decarboxylase 65 (GAD65) is one of the target structures for autoimmune reactions in patients with type I diabetes. Mononuclear cells in the blood of patients react to the protein with cell division and over 70% of the patients also have antibodies against GAD65. A combination of antibodies against GAD65 and IA-2 in people who are genetically predisposed to diabetes due to their MHC type is a strong indicator of developing type I diabetes. The vectors can be used as an antigenic target for T cells in diabetes encode and express the entire GAD65 protein or protein fragments. By way of example, the vectors according to the invention encode and express the following regions, individually or in combination, which have been identified as epitope-bearing regions in connection with MHC-DQ8: AS 1-60, AS 51-120, AS 101-115, AS 111-180, AS 171 - 240, AS 206 - 220, AS 231 - 300, AS 291 - 360, AS 351 - 420, AS 411 - 480, AS 431 - 445, AS 461 - 475, AS 471 - 530, AS 521 - 585, AS 536 - 550, AS 121-140, AS 201 - 220, AS 231 - 250, AS 471 - 490.GAD65 is also a target of CD8 ⁺ T cells. Vectors can therefore also encode and express the region AS 15-23. Other possible areas are AS 505 - 519 and AS 521 - 535.

The heat shock protein Hsp60 represents another target for activated immune cells in type I diabetes. The vectors encode and express, for example, the area of AS 437-460

(Peptide 277), which was identified as a T cell epitope in NOD mice. In people with diabetes and in healthy controls, a number of epitopes or areas in the HspόO-

Protein containing T cell epitopes identified. The vectors according to the invention code, by way of example, for the entire Hsp60 protein or fragments, in particular for regions described below or combinations of regions which carry T cell epitopes: AS 1 -

20, AS 16 - 35, AS 31 - 50, AS 46 - 65, AS 61 - 80, AS 106 - 125, AS 121 - 140, AS 136 -

155, AS 151 - 170, AS 166 - 185, AS 195 - 214, AS 240 - 259, AS 255 - 275, AS 271 - 290,

AS 286 - 305, AS 301 - 320, AS 346 - 365, AS 421 - 440, AS 436 - 455, AS 466 - 485, AS

511 - 530.

Other targets of the autoimmune reaction in type I diabetes are proteins in the cell membrane of beta cells that have not yet been characterized. The island cell protein ICA69 and a protein with a molecular weight of 38 kDa in the secretory granules were used as a further antigen (Roep et al (1991) Lancet 337: 1439-1441). The vectors according to the invention code by way of example for these proteins or fragments thereof, in particular for regions or combinations of regions which carry T cell epitopes.

Expression of apoptosis-inducing ligands

The vectors according to the invention are characterized in that they comprise nucleic acid sequences which code for one or more ligands which trigger apoptosis. Apoptosis is a process controlled at the level of the genes, which is involved in the regulation of homeostasis, the development of tissues and organs and the removal of cells of the immune system that are no longer necessary. Apoptosis is also involved in the elimination of cells that have undergone changes due to damage to their chromosomes or due to infection with viral pathogens. Normal cells are protected from apoptosis by so-called "survival signals". However, proapoptotic signals, which are triggered by damage or infection, initiate a sequence of processes that end with the death of the cells. Cell death by apoptosis is characterized by a compression of the chromatin , a fragmentation of the chromosomal DNA, a kind of blistering of the membrane, a shrinkage of the cell, and finally a decay of the dead cell in the membrane-enclosed vesicle (apoptotic body).

There are essentially two signaling pathways in the cell that trigger apoptosis. Both ways react to different triggers. The path relevant for the present invention leads to a stimulation of apoptosis receptors on the surface of the cells, such as CD95 / Fas / Apol, TRAIL or Apo3, and mediation by adapter molecules, such as FADD and TRADD, for the activation of the regulatory active caspase 8 ( FLICE, initiator caspase) and subsequent caspases such as Caspase 3 (effector caspase), which trigger apoptosis. On the other hand, other signals such as DNA damage, lack of growth factors, lack of cell attachment or activated oncogenes lead to the induction of a signal cascade in which factors from mitochondria such as cytochrome C, APAFl and factors of the Bcl family are involved. This cascade leads to the activation of caspase 9 (initiator caspase) and subsequently to activation of caspase 3.

The apoptosis receptors belong to a subfamily of so-called "death receptors", which form part of the tumor necrosis factor (TNF) receptor superfamily. The members of this Family are characterized by two to four copies of cysteine-rich extracellular domains. The apoptosis receptors, in turn, have intracellular "death domains" (DD), which are essential for the transmission of the apoptosis signal. The apoptosis signal is transmitted to the caspases by adapter proteins, which finally initiate apoptosis over several stages.

The apoptosis-triggering ligands are membrane-bound or soluble proteins, for example CD95L / FasL / ApolL, Apo2L / TRAIL or Apo3L, which interact with specific receptor molecules, such as CD95 / Fas / Apol, TRAIL or Apo3, on another or the same cell, and thereby trigger apoptosis in the receptor-carrying cells. The ligands that trigger apoptosis belong, for example, to the superfamily of tumor necrosis factors (TNF). Members of the TNF family are characterized by structural and biochemical properties. They are type II transmembrane proteins, with the exception of lymphotoxin (LT) α and β, which can also be converted into soluble ligands by proteolytic cleavage. The ligands appear as trimers with three identical subunits, which, by interacting with their specific apoptosis receptors, cause these receptors to trimerize.

Caspases are a group of cysteine proteases. So far, 14 caspases have been identified in mammals (including humans). Caspases recognize motifs consisting of 4 amino acids, which they cut on the carboxyl side of the amino acid aspartate. Caspases are synthesized as zymogens, i.e. they are precursor proteins with a very low activity, which are activated by proteolytic cleavage. The activated enzymes are heterotetramers, each consisting of two identical cleaved subunits. Some caspases are activated by autoproteolytic cleavage, others by additional caspases. The so-called initiator caspases (caspase 8 and caspase 9) start the avalanche of self-reinforcing caspase activity by activating so-called effector caspases (caspase 3) through proteolytic cleavage.

Adapter proteins represent a connection between the effectors (caspases) and the regulators (receptors Bcl-2 family) of apoptosis. The adapters interact physically via homotypic interactions with the three groups of factors via so-called death domains (DD), death ejfector domains ( DED) and caspase recruitment domains (CARD). The vectors according to the invention comprise nucleic acid sequences which code for ligands which trigger apoptosis. The vectors code, for example, for the ligand CD95L, which binds to the receptor molecule CD95 / Fas / Apo-1. CD95 is a glycosylated membrane protein (type I) with a molecular weight of approximately 45-52 kDa (335 amino acids). Differential splicing of the messenger RNA also creates several soluble forms of the protein. The expression of the CD95 molecule is stimulated by interferon-γ (IFN-γ), by TNF and by the activation of T cells. Under natural conditions, the CD95-mediated apoptosis is triggered by the binding with the natural ligand CD95L (FasL, Apo-IL). CD95L is a membrane protein related to TNF (type II), which can also occur as a soluble form due to proteolytic cleavage and can bind to CD95. The interaction of CD95L with CD95 leads to a Ca ^{2 ~} -independent (ie different from the triggering by Perforin / Granzyme) apoptosis of the CD95-carrying cells.

Furthermore, the vectors according to the invention can comprise nucleic acid sequences which code for the protein TRAIL (APO-2L) which binds to the specific receptor molecules TRAIL-Rl (DR4), TRAIL-R2 (KILLER, DR5, TRICK2), TRAIL-R3 (LIT, DcRl ) and TRAIL-R4 (TRUNDD, DcR2) binds. TRAIL has been detected naturally on a number of cells, such as type II interferon-stimulated monocytes, cytomegalovirus-infected fibroblasts, type I interferon and antigen-stimulated T cells or NK cells. TRAIL induces apoptosis in a number of transformed tumor cell lines and activated T cells. The mRNA encoding the TRAIL-R2 receptor could be detected in a wide variety of tissues, such as spleen, thymus and lymphocytes in peripheral blood. Cells and tissues expressing mRNA for TRAIL-R2 are sensitive to TRAIL-mediated apoptosis.

Furthermore, the vectors according to the invention can comprise nucleic acid sequences which code for the APO-3 ligand (Apo3L / TWEAK). Apo3L is a type II transmembrane protein with a length of 149 amino acids. The extracellular region of ApoL3 shows high homology to TNF. Apo3L mRNA was detected in a wide variety of lymphoid and non-lymphoid tissues. ApoL3 binds to the receptor molecule Apo3 (DR3, WSL-1, TRAMP, LARD) and induces apoptosis in the receptor-bearing cells. The receptor for Apo3L is primarily expressed on the cell surface of lymphocytes, for example on unstimulated resting lymphocytes in peripheral blood (PBL), phytohaemagglutinin (PHA) -treated PBL, CD4 ⁺ T cells, CD8 ⁺ T cells and B cells. The induction of apoptosis via Apo3L is mediated by FADD / MORT1. ApoL3-mediated apoptosis is blocked by the viral caspase inhibitors CrmA of the cowpox virus and by the p35 protein of baculoviruses.

Inhibition of apoptosis in the genetically modified cells

The vectors according to the invention are characterized in that they optionally comprise nucleic acid sequences which code for one or more anti-apoptosis molecules. An interaction of CD95, TRAIL, TRAMP, TNF or lymphotoxin with their specific receptors can not only take place between different cells, but can also lead to apocytosis on one cell and thus autocrine. An example of this is the apoptosis of activated immune cells, the so-called activation-induced cell death, which leads to the removal of immune cells that are no longer required. T cells that have been stimulated to multiply via their T cell receptor express CD95 on their cell surface, which interacts with CD95L on the membrane.

In order to prevent such autocrine induction or a paracrine induction by other cells, for example in cell culture during the modification of the starting cells, in the cells which have been modified with the vectors according to the invention, the vectors can comprise nucleic acid sequences which code for intracellular inhibitors of apoptosis ( anti apoptosis molecules). These inhibitors either interact with the different receptor molecules in the cell membrane, with the adapter molecules that transmit the death signal between the receptors and the caspases, or with the caspases themselves. These inhibitors either come from the cells themselves and are involved in the regulation of apoptosis. Or they are viral proteins that prevent apoptosis in virus-infected cells until sufficient virus progeny are produced and released.

The induction of apoptosis can be inhibited at the various stages of the cascade of receptors, adapters and caspases. Viruses in particular have invented numerous strategies to defend themselves against apoptosis as an immune mechanism. The present invention uses these viral and cellular mechanisms to protect the cells altered by the vectors from autocrine induction of apoptosis. The vectors according to the invention can comprise, for example, nucleic acid sequences which code for adenoviral proteins from the E3 region of the virus. These proteins inhibit membrane-bound biochemical processes that are induced when TNFR is activated. The protein E3-14.7K inhibits the release of arachidonic acid by phospholipase A2 (PLA) as a result of stimulation via the TNF receptor. The adenoviral proteins E3-14.5K and E3-10.4K form the RTD complex, which prevents translocation of PLA ₂ from the cytoplasm to the cell membrane as a result of stimulation of the TNFR. RTD also causes rapid internalization and lysosomal degradation of membrane-bound CD95.

Furthermore, the vectors according to the invention can comprise nucleic acid sequences which code for proteins which have high homology to the DED domains. These proteins are called FLEPs or the viral proteins are called vFLIPs. Through a homotypic interaction of the FLIPs / vFLIPs with the adapter protein FADD via the DED domains, the signal cascade between receptors and FADD on the one hand and Caspase 8 (FLICE) on the other hand is blocked and apoptosis is thereby prevented.

Furthermore, the vectors according to the invention can comprise nucleic acid sequences which code for the proteins described below. The proteins MC 159 (Bertin et al. (1997) Proc Natl Acad Sei USA 94: 1172-1176) and MC160 of the Molluscum Contagiosum Virus bind to FADD and thus prevent recruitment of Caspase 8 and FADD. The proteins BORFE2 (E 1.1) of herpesvirus BHV-4 and E8 of the horse herpesvirus EHV-2 (Bertin et al. (1997) Proc Natl Acad Sei USA 94: 1172-1176) bind the inactive procaspase 8 and prevent an interaction with FADD and thus an activation. The proteins K13 from HHV-8 and ORF71 from herpes virus Saimiri also have DED domains and work in the same way. Furthermore, viral proteins can be encoded which bind signal factors (FADD, TRADD, TRAF) of the apoptosis cascade and thereby inactivate them. The adenovirus protein E1B-19K, which is homologous to the cellular protein Bcl-2, interacts with FADD and thereby blocks the signal transmission of TNFR and CD95. In addition, E1B-19K is also able to functionally replace Bcl-2 and to prevent activation via the mitochondria (via Caspase 9). The LMP-1 protein from Epstein-Barr virus interacts with various TRAF molecules (TNFR- associated factors) and thereby blocks the signal transmission from the TNFR. LMP-1 also binds TRADD. Probably due to a modified binding site for TRADD, however, the apoptosis signal cascade is not triggered. LMP-1 additionally induces the expression of the antiapoptotic proteins A20, Bcl-2 and Mcl-1. The vectors according to the invention also express, for example, the LT protein of SV40, which mediates resistance to Fas-induced apoptosis via a protein kinase C-mediated route. The vectors according to the invention code, for example, for the polyoma proteins ST and MT, both of which mediate resistance to CD95- and TNFR-mediated apoptosis. The ST protein achieves this by binding and inhibiting the PP2A protein. The MT protein directly activates signaling pathways that promote cell survival. This includes the PI3 kinase, which subsequently phosphorylates the proapopic protein Bad and thus inactivates it.

Inhibitors of caspases can also be encoded on the vectors according to the invention in order to prevent autocrine induction of apoptosis in the changed cells. The p35 protein of the Baculoviruses Autographica californica nuclear polyhedrosis Virus (AcNPV) and Bombyx mori NHV (BmNPV) is synthesized in the early phase of virus multiplication and prevents apoptosis by a number of different stimuli. p35 blocks the induction of apoptosis by TNF and CD95 ligands. p35 is cleaved by a series of caspases (caspase 1, 3, 6, 7, 8 and 10). The cleavage products are not released, but remain bound and form a stable inhibitory complex with the caspases. p35 can, for example, be encoded by the vectors according to the invention. The vectors according to the invention can also code for viral proteins which have homology to cellular serpins. Serpins are chymotrypsin-like serine proteases and inhibit a number of different caspases. Cowpox virus CrmA inhibits CD95L and TNFR-induced apoptosis by blocking caspases 1 and 8. The SPI-1 and SPI-2 proteins of the rabbit pox virus and the protein B13R of the vaccinia virus show high homology to CrmA and also inhibit apoptosis by CD95L and TNF. The proteins Serp-1 and Serp-2 of the myxomavirus and SPI-4 of the rabbit pox virus have corresponding antiapoptotic properties. In addition, homologous proteins can be encoded into cellular cLAPs, such as, for example, vIAPs, or cellular proteins, such as, for example, FLAME-1 or I-FLICE.

Cydia pompnella granulosis virus (CpGP), Orgyiapse audotsugata polyherdosis virus (OpMNPV) and AcNPV code for viral IAPs (vIAPs) that are in the signaling cascade above of p35 act. vIAPs bind and inhibit inactive procaspases and caspase 8, but cannot inhibit caspases that have already been activated like p35. cIAPs interact with TRAF molecules, but can also prevent apoptosis through non-TNFR-associated processes. A conserved RTNG finger motif and at least one so-called BIR motif (baculovirus IAP repeat) are necessary for the antiapoptotic activity. FLAME-1 is a cellular protein that inhibits apoptosis by the CD95 / TNF receptor (Srinivasula et al. (1997) J Biol Chem 272: 18542-18545). FLAME-1 has high homology to Caspase 10 and Caspase 8 (FLICE). Two neighboring regions are located in the amino terminal area and show homology to the DED domains of FADD, which enable homotypic interactions with other DED proteins. A third neighboring region shows homology to the functional caspase domain of caspases 8 and 10. FLAME-1 interacts directly with FADD, caspase 8 and caspase 9, but has no caspase activity. FLAME-1 therefore acts as a dominant negative repressor of apoptosis by CD95 / TNF receptor. The inhibitory effect results from the blocking of the receptor complex from CD95 / TNR receptor / FADD by the functionally inactive FLAME-1 protein. I-FLICE is another cellular inhibitor (Hu et al. (1997) J Biol Chem 272: 17255-17257) which can be encoded and expressed by the vectors according to the invention in order to prevent apoptosis in the gene therapy-modified cells. I-FLICE shows structural homologies to FLICE / Caspase 8 and Caspase 10. In the amino terminal area there are two neighboring domains with homology to FADD's DED domains. In the carboxy-terminal area there is a domain with homology to the caspase domain. However, I-FLICE shows no caspase activity and acts as a dominant negative inhibitor of apoptosis by CD95 / TNF receptor. In contrast to FLAME-1, I-FLICE only binds to FLICE / Caspase 8 and Caspase 10, but not to FADD. I-FLAME is therefore not recruited by binding CD95L / TNF to the CD95 / TNR receptor / FADD receptor complex. The inhibitory effect is caused by complexing and thereby inactivating caspases 8 and 10.

Inhibition of apoptosis in the cells modified by the vectors according to the invention can also be produced by inhibiting the expression of the proteins involved in the induction (apoptosis receptors, adapters and caspases), for example using an antisense approach. The antisense RNAs encoded and synthesized by the vectors according to the invention are either directed exclusively against a protein in the apoptosis signal chain and inhibit apoptosis by a certain route, for example by membrane-associated receptors or by a mitochondrial-mediated route. Alternatively, the antisense KNA includes different areas that are specific for different targets in the signal cascade to trigger apoptosis. These different areas in the antisense RNA are specific for receptor proteins, adapter proteins, and / or caspases. The vectors of the invention either synthesize a single antisense RNA or a combination of different ones

which are specifically directed, for example, against individual apoptosis receptors, caspases or adapter molecules. Alternatively, the vectors express a single antisense RNA, which contains several regions, each of which is specific for individual apoptosis receptors, caspases or adapter molecules, and in combination prevents the expression of several proteins involved in apoptosis.

In connection with this invention, the vectors carry, for example, functional regions which synthesize antisense RNA to apoptosis receptors in eukaryotic cells. By blocking the expression of receptor molecules that start proapoptotically effective signal cascades, autocrine stimulation of apoptosis in the cell modified with the gene therapy vector is blocked at the earliest stage.

The vectors according to the invention can code, for example, for CD95-specific antisense RNAs and block the expression of CD95 / Fas. Blocking CD95 prevents induction of apoptosis via CD95L / FasL. The gene therapy vectors encode and express, for example, TNFR-specific antisense RNAs which block expression of TNFR and protect the cell from TNF-mediated apoptosis. Alternatively, the vectors encode and express, for example, TRAIL-R1 or TRALL-R2-specific antisense RNAs which prevent expression of the receptors TRAIL-R1 or TRAIL-R2. This makes the cells resistant to TRAIL-mediated apoptosis. Alternatively, the gene therapy vectors encode and express TRAMP receptor-specific antisense RNAs which prevent expression of the TRAMP receptor in the altered cells. This makes the cells resistant to TRAMP-mediated apoptosis. The vectors according to the invention can further comprise nucleic acid sequences which encode, for example, antisense RNA to adapter proteins in eukaryotic cells. Since these adapter molecules function one level below the apoptosis receptors and one adapter molecule is used by several apoptosis receptors, blocking the expression of a single adapter protein can block more than just one apoptosis signaling pathway. By blocking a single adapter molecule, resistance to different ligands that trigger apoptosis can be achieved. The vectors according to the invention either synthesize a single antisense RNA or a combination of different antisense RNAs which are specifically directed against individual adapter proteins. Alternatively, the vectors express an antisense RNA which contains individual regions which are in each case specific for an adapter protein and, in combination, prevents the expression of several adapter proteins.

In the context of this invention, the gene therapy vectors can encode, for example, FADD-specific antisense RNAs which prevent expression of FADD. By inhibiting FADD expression, signal transmission by the apoptosis receptors CD95, TNFR, TRAIL-R1, TRAIL-R2 and TRAMP is blocked. This makes the cells resistant to induction of apoptosis via CD95L, TNF, TRAIL and CD3. The vectors can also code, for example, for TRADD-specific antisense RNAs which prevent expression of TRADD. TRADD is specifically involved in triggering apoptosis by TNF / TNFR. Inhibition of the expression of TRADD specifically blocks the triggering of apoptosis by TNF / TNFR. The vectors can also encode APAF1 -specific antisense RNAs. APAFl is an adapter protein that plays a central role in triggering apoptosis via the mitochondrial-associated pathway. APAFl is released together with cytochrome C from the mitochondria and associates with dATP in the cytoplasm of the cell to form a trimeric complex that activates caspase 9. Inhibition of the synthesis of APAFl leads to a blockade of apoptosis via the mitochondrial-associated pathway.

In connection with this invention, the gene therapy vectors can also encode antisense RNA against caspases, for example caspase 1, 3, 8 or 9, in eukaryotic cells. The vectors according to the invention either synthesize a single antisense RNA or a combination of different antisense RNAs that are specific against individual caspases are directed. Alternatively, the vectors express an antisense RNA which contains individual regions which are each specific for one caspase and, in combination, prevents the expression of several caspases.

suicide enzymes

The vectors according to the invention are characterized in that they optionally comprise nucleic acid sequences which code for suicide enzymes by means of which genetically modified cells can be used at any time, e.g. in vivo, can be eliminated. The suicide genes encode and express, for example, enzymes which convert biological substrates (prodrugs) which are supplied to the body from the outside into toxic substances or modify the substances in such a way that the enzymes of the cell use them as substrates. Alternatively, suicide enzymes code for substances that are toxic per se, but the expression of these genes in the genetically modified cell is strictly controlled. In connection with this invention, the genes for substances which are toxic per se are strongly repressed in the cell and are not synthesized. By adding biological or chemical substances, the genes that code for the toxic proteins are activated and the cells die. The vectors described in connection with this invention preferably code for suicide genes which convert non-toxic or little toxic prodrugs into toxic substances.

The prodrugs used must have significantly lower toxicity than the activated substances and must be good substrates for the activating enzymes. In addition, these substances must be sufficiently chemically stable under physiological conditions and have good pharmacological and pharmacokinetic properties. Depending on the type, some prodrugs are absorbed into the cells and converted intracellularly into the toxic substance. Other prodrugs are activated extracellularly. Accordingly, the prodrugs or the activated toxic substances must be easily absorbed by the cells.

The vectors according to the invention encode and express, for example, the thymidine kinase (TK) of the herpes simplex virus (HSV). The TK of HSV phosphorylates acyclovir to acyclovir diphosphate, which is further phosphorylated by cellular kinases. The thymidine kinase of the eukaryotic cell cannot phosphorylate due to its substrate specificity, which is why non-infected cells or HSV-TK-negative cells are resistant to guanosine analogues. By systemic administration of acyclovir or gancyclovir selectively the HSV-infected or tymidine kinase-positive cells, which have been modified, for example, with the therapy vectors of this invention, are eliminated.

The vectors according to the invention can also code for the thymidine kinase of varicella zoster virus (VZV). The TK of VZV is comparable in effect to the TK of HSV, with the difference that the TK of VZV uses 6-methoxipurin arabinonucleoside.

The vectors mentioned in this invention can also encode enzymes which activate the following prodrug / enzyme systems: carboxylesterase (CA) activates irinotecan; Cytosine deaminase (CD) activates 5-fluorocytosine (5-FC); Carboxypeptidase G2 (CPG2) activates 2-chloroethyl-2-mesyloxyethyl-aminobenzoyl-L-glutamic acid (CMDA) and CJS278 ^H as well as the self-activating products doxorubicin and daunorubicin; Cytochrome P450 (Cyt 450) activates cyclophosphamide (CP), ifosfamide (IF), ipomeanol and 2-aminoanthracene (2-AA); Deoxycytidine kinase (dCK) activates cytosine arabinose (ara-C); Nitroreductase (NR) activates CB1954 (5-aziridinyl-2,4-dinitrobenzamide); Purine nucleoside phosphorylase (PNP) activates 6-methylpurin-2'-deoxyribonucleoside (6-MePdR); Thymidine phosphorylase (TP) activates 5'-deoxy-5-fluorouridine (5'-DFUR); Xanthine guanine phosphoribosyl transferase (XGPRT) activates 6-thioxanthine (6-TX) and 6-thioguanine (6-TG). Furthermore, the vectors according to the invention can code for the bacterial uracil phosphoribosyl transferase that activates 5-fluorouracil, or for a fusion protein made of cytosine deaminase (FCY1) and uracil phosphoribosyl transferase (FUR1) from Saccharomyces cerevisiae, which activates fluorocytosine (5) ,

Vectors for gene therapy according to the invention

The invention relates to a gene transfer vector comprising at least one nucleic acid molecule, comprising a first nucleic acid sequence coding for one or more apoptosis-triggering ligands, a second nucleic acid sequence coding for one or more antigen (s) and optionally, a third nucleic acid sequence coding for a or more anti-apoptosis molecule (s), and optionally a fourth nucleic acid sequence, coding for one or more suicide enzyme (s). A gene transfer vector is particularly preferred, the first and second nucleic acid sequences being functionally linked to one another in such a way that the expression of the second nucleic acid sequence is dependent on the expression of the first nucleic acid sequence, ie the expression of the antigens is physically coupled to and from the expression of the ligands which trigger apoptosis dependent. Furthermore, a gene transfer vector is particularly preferred, the third and fourth nucleic acid sequences being so functionally linked to one another that the expression of the third nucleic acid sequence is dependent on the expression of the fourth nucleic acid sequence, ie the expression of the anti-apoptosis molecules is always linked to and from the expression of the suicide enzymes dependent.

Antigen-presenting cells (APCs) can be treated with one of the vectors according to the invention for the treatment of diseases such as, for example, autoimmune diseases or diseases which are based on immunopathogenesis or diseases which are based on rejection of transplanted tissues or organs. APCs can be treated, for example, with a gene transfer vector comprising nucleic acid sequences coding for the antigens, ligands which trigger apoptosis, antiapoptosis molecules and for suicide enzymes. Furthermore, the APCs can be treated with any type of combination of vectors, for example with several of the vectors according to the invention, which code for different antigens. The APCs can also be treated, for example, with a combination of vectors according to the invention which code for antigens and vectors according to the invention which code for anti-apoptosis molecules. Combinations of vectors are useful if, for example, the individual nucleic acid regions of the vectors are so large that they negatively influence, for example, the production or use of the vector, or if APCs are to be treated with vectors which encode different antigens, apoptosis ligands, anti-apoptosis molecules or suicide enzymes ,

Control elements for the expression of gene information

The gene transfer vectors according to the invention comprise nucleic acids which, in addition to the first to fourth nucleic acid sequences described above, can contain further sequences and functional regions, for example for controlling and controlling the expression of genes, for example in mammalian cells. These sequences and functional regions can be promoters and / or promoter elements, preferably viral promoter sequences for the expression of gene sequences in mammalian cells. Some examples of viral promoter sequences are the SV40 early promoter, the mouse mammary tumor virus (MMTV) LTR promoter, the human immunodeficiency virus type 1 (HIV-1) LTR promoter, the adenovirus major / αte promoter (Ad MLP) and herpes - Simplex virus (HSV) promoter. In addition, promoters of non-viral genes, such as promoters of murine 3-phosphoglycerate kinase, human ubiquitin C and murine metallotheionein, are also suitable for the efficient expression of gene sequences in mammals. The expression can be carried out by a consumable or regulatable (inducible) promoter. For example, a glucocorticoid-inducible promoter can be used in certain cell types, such as hormone-stimulable cells.

The expression rates can usually be increased by combining the above-mentioned promoter elements with so-called E / z / zα «cer elements. Here, viral EMÄαHcer elements often have a particular efficiency, since they usually have a broader host spectrum than enhancers from mammalian cells. Very efficient representatives of viral enhancers include the SV40 early gene enhancer and the promoter / enhancer combinations from the LTR of Rous sarcoma virus and the human cytomegalovirus. In addition, adjustable enhancer elements can be used, e.g. are only active in the presence of inductors, such as hormones or metal ions.

These sequences and functional regions can also be leader sequences and / or processing sequences, e.g. a protease cleavage site, preferably the adenoviral three-part leader sequence, and a multiplicity of zeαer sequences of mammalian proteins, such as the leader of the erytropoietin gene and the iPA leader, in order to mediate an efficient secretion of foreign proteins from mammalian cells.

These sequences and functional regions can also be transcription termination and polyadenylation sequences. Some very efficient poly-A signals for use in mammalian expression vectors are derived, for example, from the bovine growth hormone, the mouse β-globin, the SV40 early transcription unit and the herpes simplex thymidine kinase gene. Prokaryotic transcription terminators are described in detail and their incorporation shows diverse positive effects on gene expression. In eukaryotes identified a consensus sequence with the nucleotide sequence ATC AAA (A7T) TAG GAA GA in the termination region of 9 genes.

These sequences and functional regions can also be translation control elements. An optimal Kozak sequence (CC (A / G) CcaugG) promotes the translation initiation of eukaryotic mRNAs. The purines A or G in position -3 and the G directly above the start codon are of particular importance for optimal translation initiation.

Furthermore, the expression efficiency of cDNA gene sequences which do not contain any introns can be increased significantly by 10-20 times in some cases by fusion of an intron in the 5 'region of the ORF. A particular effectiveness in different cells has, for example for a large number of different introns, a synthetic intron SIS, which was generated by the fusion of an adenovirus splice donor with an immunoglobulin gene splice acceptor, or a SV40 19S late mRNA intron ,

Furthermore, the translation initiation at the correct start codon can be severely impaired by the presence of additional AUG codons in the 5 ^' untranslated region. Such an inhibition can be minimized by the presence of a translation termination codon in-frame with the AUG located above. In addition, the translation is often affected by the tendency of defined sequences in the 5 ^Λ untranslated regions (UTR) to form secondary structures. Furthermore, destabilizing motifs within foreign gene sequences can have a negative effect on the expression rates. Representatives of such sequence sequences are examples of AU-rich sequences in the 3 "UTR area of many unstable mammalian mRNAs. Very efficient destabilizing sequence motifs are UUAUUUAUU or UUAUUUA (U / A) (U (JA). To increase the expression of the desired foreign genes, these sequence motifs should be removed or be deactivated.

Furthermore, the expression efficiency of the desired nucleic acid sequence can be increased by selecting and using suitable, host-specific codons (codon usage).

Typically, an expression vector includes a combination of a promoter, a polyadenylation signal, and a transcription termination sequence. Enhancers, introns with functional splice donors and acceptor sites as well as leader sequences can also, if required to be installed modularly in the constructs. Expression constructs are often contained in a replicon, such as in extrachromosomal elements (eg plasmids), which are able to stably survive in a host, such as a mammalian cell. Mammal replication systems include those that are derived from animal viruses and require transactive factors for replication. For example, plasmids which contain the replication systems of papovaviruses, such as SV40 or polyomaviruses, replicate in the presence of the associated viral T antigen in an externally high copy number. Other examples of mammalian replicons include those derived from the bovine papilloma virus and the Epstein-Barr virus. Furthermore, a replicon can contain two replication systems which ensure survival in the host, for example a replication system for gene expression in mammals and a system for the amplification of the vector in bacteria. The plasmid pMT2 is an example of such a mammalian / bacterial shuttle vector.

Regulation of the Expression of the Apoptosis-Triggering Ligands The nucleic acid sequences which code for apoptosis-triggering ligands can be regulated in such a way that the expression of the ligands can be switched on and off. The expression of the ligands which trigger apoptosis can be switched off completely in transduced cells. This is of particular importance for the production of stable vector-producing cell lines or for the production of the bait cells. Since most cells have constitutive apoptosis receptors on the surface, including the packaging cell lines that can be used to produce viral gene transfer vectors and the cells to be transduced, antigen-presenting cells, transduction of these cells with the gene for an apoptosis-triggering ligand would lead to paracrine and autocrine induction of apoptosis. Switching off the expression of the ligand in the packaging lines or in the transduced cells in culture prevents an unspecific interaction between these cells and enables cultivation.

The expression of the ligands is preferably switched off using the methods described below. 1. Using the RevTet system from ClonTech, USA. 2. Another possibility, which is based on the principle of double infection with an expression vector and a regulation vector, is based on the use of bacterial regulation systems in eukaryotes. The gene for the apoptosis-inducing ligand is under the control of the strong prokaryotic T7 promoter. Since neither in the packaging line If T7 polymerase is still present in the cells to be transduced later, the ligand is not expressed. The co-transduction of the gene for the T7 polymerase by means of a second regulatory vector leads to the expression of the ligand in the double-transduced cells. Another suitable system the Cre-loxP system of the bacteriophage Pl

Co-expression of antigens and apoptosis-inducing ligands or anti-apoptosis molecules and suicide enzymes

The vectors according to the invention preferably expand the antigens or the components of antigens which are recognized by immune cells only in connection with the apoptotically active ligands. This co-expression is achieved by coupling the nucleic acid sequences on a common transcript. This prevents the transduced cells from being transfected by the Presentation of components of the antigen stimulate specific T cells in vivo without destroying them

An internal πbosomal entry point (IRES) can be used to express two or more genes under the transcriptional control of a consumable or regulable promoter. For example, the IRES of picornaviruses and the hepatitis C virus, the BiP (immunoglobulin heavy chain binding protein) are used. , or retroviral 7i? ES sequences are used

A further possibility for the expression of two or more genes under the transptional control of a strong promoter is the use of sequences which cause a shift in the πbosomal reading frame during the translation, whereby a stop codon is ignored. Usually, these frame shift-SignÜQ on the RNA level require that the ribosomes are placed at a specific position in the -1 reading frame (m 5 ^" orientation) and in the new reading frame perform the translation. Such -1 frameshift signals were used in a large number of different virus families , such as, for example, retroviruses, corona viruses, astroviruses, totiviruses, podoviruses, siphoviruses, luteoviruses and dianthoviruses. The frame shift sequence of the rous sarcoma virus (RSV) consists of two essential components of a homopolymeric shpperv sequence, consisting of the sequence sequence (AAAUUUA) a RNA secondary structure located a few nucleotides downstream. By comparing the s / z / τpery sequences of different viruses, the following s ppery consensus sequence was created a 7-nucleotide sequence that contains 2 homopolymeric triplets (X-XXY-YYZ) (Brierley (1995) J Gen Virol 76 (Pt 8): 1885-1892).

In addition to vector-specific nucleic acid sequences which, for example, enable the vectors to multiply in bacteria or eukaryotic cells, or vectors comprise regulatory nucleic acid sequences which enable expression of the coding regions, or nucleic acid sequences which enable the vector to be packaged or the nucleic acid to be packaged in a vector , for example also nucleic acids which code for one or more suicide enzymes and for one or more anti-apoptosis molecules. The expression of the antiapoptosis molecules is always linked to the expression of the suicide enzymes and is dependent on them.

Target cells for treatment with the therapeutically active nucleic acids Antigen-presenting cells (APC) For the therapy of autoimmune diseases or of diseases with immunopathogenesis, syngeneic antigen-presenting cells are used by the persons who are to be treated to produce the bait cells. The MHC pattern of the anti-gene-presenting and the reactive cells is thus identical and only those T cells which react autoagressively or which recognize foreign antigens in connection with their own MHC are attracted and eliminated. In the case of treatment or prevention of graft rejection, antigen-presenting cells of the organ donor are cleaned. These cells are allogeneic (different MHC pattern) with respect to the recipient. Here the T cells must be recognized and eliminated, which recognize cellular antigens with foreign MHC from the donor.

Lymphocytes, accessory cells and effector cells are the most prominent representatives of the acquired immune system. Lymphocytes are able to specifically recognize foreign antigens and to stimulate a specific humoral and cell-mediated immune response. Different subpopulations of lymphocytes are known which differ in the type of antigen recognition and the specific effector functions. B lymphocytes are the producers of antibodies. They recognize extracellular and antigens presented on the surface of cells and differentiate into antibody-secreting plasma cells after contact with an antigen. T lymphocytes, the mediators of the cell-mediated immune response, can be divided into several subtypes, of which the CD4 ⁺ T helper cells and the CD8 ^{^} cytotoxic T cells are most significant. Helpers and cytotoxic T cells have a restricted specificity for antigens. You only recognize peptide antigens that are presented together with MHC class II or MHC class I on the surface of a body's own cell. After a specific recognition of a specific MHC class II / peptide complex, T helper cells secrete cytokines which stimulate T cells and other immune cells, such as B cells and macrophages, to proliferate and differentiate. Cytotoxic T cells (CTL) lyse cells that show peptides of foreign proteins together with MHC class I proteins on their surface. In contrast, the so-called suppressor T cells are a subtype of T helper cells that produce cytokines that suppress certain immune functions. A third class of lymphocytes, the natural killer (NK) cells, are part of the innate immune response to fight viruses and intracellular pathogens.

A very important importance for the control of the immune system is possessed by antigen presenting cells (APCs). These cells take up foreign antigens, process them into small peptides and present them on their surface together with MHC proteins.

There are two classes of APCs. Professional APCs present the generated ones

Peptides on MHC class I and II proteins and also express costimulatory proteins such as B7.1 and B7.2. The most important representatives of these APCs are dendritic cells and macrophages, but also B cells. These cells stimulate T helpers and cytotoxic T cells that recognize peptides complexed with their MHC class II or MHC class I using their T cell receptor. On the other hand, non-professional APCs that present MHC / peptide complexes but no costimulatory proteins are only recognized by already activated T cells. The professional APC are the main target cells into which the vectors according to the invention are to be introduced preferentially ex vivo.

The purification of different populations of blood lymphocytes has been described in a large number of publications and is state of the art. Mononuclear cells can be purified from peripheral blood, for example, using Ficoll-Hypaque density gradient centrifugation. Furthermore, other methods based on the antibody-mediated recognition of immune cells can be used for the positive and negative selection of cell populations. Examples of such methods are immunomagnetic selection, "panning" for immobilized monoclonal antibodies, antibody / complement-mediated cell lysis and Cell sorting of fluorescence-labeled cells. A suitable surface marker for the selection of T cells is CD3. Specific T helper cells are selected using the CD4 and cytotoxic T cells using the CD8 marker. Additional T cell subpopulations can be selected using the markers CD30, CD45RA (naive T cells) and CD45RO (activated T cells and memory T cells). Activated T cells can be separated using the marker protein CD69. Monocytes can be purified using specific antibodies (AK) against the surface markers CD 14 and CD 11b, natural killer cells using anti-CD 16 AK and B cells using the markers CD 19 and CD22. APCs can be purified using specific antibodies against HLA-DR.

Suitable methods for separating T cells from mononuclear cell populations are based e.g. on the rosetting of T cells with red blood cells from sheep (SRBC). This method also allows the extraction of non-rosetting cell populations (B-lymphocytes, monocytes, macrophages). SRBCs treated with neuraminidase or 2-aminoethylisothiouronium bromide (AET) are preferred because they have an increased binding of T cells. The different T cell populations can be positively or separated using the previously described methods based on the recognition of specific surface markers by antibodies.

As an example, B cells can be purified very efficiently using CD19-specific antibodies using the methods already described. The methods of cell sorting using FACS and the use of immunomagnetic beads are particularly suitable. Monocytes can be exemplified by adherence to e.g. L-leucine methyl ester matrices, gradient sedimentation by colloidal silica particles and flow cytometry. However, in the former methods the monocytes are activated. Another very suitable method for cleaning large amounts of inactivated monocytes is countercurrent centrifugation using an elutriator (counterflow centrifugal elutriation, CCE).

Natural killer cells can be obtained by way of example from Ficoll-Hypaque gradient-purified lymphocyte populations by negative selection using anti-CD3, anti-CD5, anti-CD19, anti-CD 14 and anti-erythrocyte antibodies. Immune cells can also be generated from other cell populations by cytokine stimulation. For example, white blood cells as well as differentiated progenitor cells and stem cells can be stimulated by a variety of growth factors. In particular, the cytokines IL-3, IL-4, IL-5, IL-6, IL-9, GM-CSF, M-CSF, and G-CSF produced by activated T helper cells and activated macrophages stimulate myeloid stem cells, which then lead to Differentiate pluripotent stem cells, granulocyte-monocyte progenitor cells, eosinophil progenitor cells, basophil progenitor cells, megakaryocytes and rythroid progenitor cells. The differentiation is modulated by growth factors such as GM-CSF, H-3, IL-6, IL-1 1, and erythropoietin (EPO). Pluripotent stem cells then differentiate into lymphoid stem cells, bone marrow stromal cells, precursor T cells, precursor B cells, thymocytes, T helper cells, cytotoxic T cells and B cells. This differentiation is modulated by growth factors such as IL-3, IL-4, IL-6, IL-7, GM-CSF, M-CSF, G-CSF, IL-2, and IL-5. Granulocyte-monocyte precursors differentiate into monocytes, macrophages, and neutrophils. This differentiation is modulated by the growth factors GM-CSF, M-CSF, and IL-8. Eosinophilic precursors differentiate into eosinophils. This process is modulated by GM-CSF and IL-5. The differentiation of basophilic precursors in mast cells and basophils is stimulated by GM-CSF, IL-4, and IL-9. Megakaryocytes produce blood platelets after stimulation with GM-CSF, EPO and IL-6. Differentiate erythroid progenitor cells, stimulated by EPO, into red blood cells. The purity of the cell populations obtained is checked by FACS analysis using specific antibodies.

The proportion of dendritic cells in the blood is less than 0.2%. Therefore, a direct purification of this cell population is not necessary. However, larger amounts of dendritic cells can be generated with the help of cytokines from CD14-positive blood monocytes. In order to differentiate dendritic cells from monocytes, the cytokines TNF-α, GM-CSF and IL4 are used in suitable concentrations. This method has been published several times and is state of the art.

It is possible to record all of the mononuclear cells in cell culture medium and to stimulate them non-specifically. The B cells are stimulated for proliferation, for example, by means of cross-linking antibodies against the B cell receptor or by means of lectins (Pokeweed Mitogen) or IL-4. For example, T lymphocytes can be preferentially incubated can be stimulated with a CD3-binding agent, such as the monoclonal antibody OKT-3. A CD3 binding agent is a ligand that binds CD3 molecules to the surface of cells. The ligand can be an antibody, such as OKT-3, which is able to cross-link two or more CD3 molecules. This cross-linking induces the proliferation and activation of CD3 + cells, such as T-lymphocytes. The activation of T lymphocytes by CD3-binding agents can also be increased by varying certain parameters, such as the concentration of the binding agent, the incubation time, the number of cells, the incubation temperature, and the binding affinity of the agent, the affinity, and the efficiency of the cell activation become. T cells in peripheral blood monocytes can also be stimulated by phytohemaglutinin (PHA). Other lymphocyte stimulators include Tetanus Toxoid, Concanavalin A (Con A), Ionomycin and PMA.

Furthermore, nucleic acids which contain unmethylated CpG motif sequences with the sequence motif Pur-Pur-CG-Pyr-Pyr are particularly suitable for activating mammalian B cells, monocytes, macrophages and dendritic cells. CpG-containing bacterial or insect DNA, but also CpG-containing synthetic oligodeoxynucleotides (ODNs) with a minimum length of 8 nucleotides can be used to stimulate the above-mentioned cell populations. An increased effect can be achieved by using chemically modified and thereby stabilized oligonucleotides, such as phosphorothioate-linked ODNs. Mammalian immune cells, such as B cells, monocytes, macrophages and dendritic cells, are activated immediately after contact with CpG-containing nucleic acids, which is reflected in the increased surface expression of MHC class II molecules and costimulatory molecules as well as the increased transcription of defined cytokine mRNAs and the secretion of pro-inflammatory cytokines such as TNF-α, IFN-γ, IL12, and IL6. In particular, the modulation of the immunological properties of CpG-containing ODNs can be achieved by targeted selection or modification of the sequences flanking the CpG motifs.

The proliferation of the cells to be transduced later is necessary, on the one hand, to enable, for example, integration of the retroviral vectors into the cellular genome. On the other hand, proliferation of cells after transduction with a retroviral vector increases the likelihood that these cells will be recognized by matching T cells. For the therapy of autoimmune diseases or diseases with immunopathogenesis, syngeneic antigen-presenting cells are used by the persons who are to be treated for the production of the bait cells. The MHC pattern of the antigen-presenting and the reactive cells is thus identical and only those T cells which react autoagressively or which recognize foreign antigens in connection with their own MHC are attracted and eliminated. In the case of treatment or prevention of transplant rejection, antigen-presenting cells are purified from the peripheral blood of the organ donor. These cells are allogeneic (different MHC pattern) with respect to the recipient. Here the T cells must be recognized and eliminated, which recognize cellular antigens with foreign MHC from the donor.

Cultivation of APCs

The mammalian or human cells can be cultivated in a suitable nutrient medium to which at least one defined growth factor has been added. A variety of growth factors have been described that promote the growth of different cell types. Typical representatives of such growth factors are cytokine mitogens such as IL-2, IL-10, IL-12, and IL-15, which e.g. promote the growth and activation of lymphocytes. Other cell types are particularly affected by a different class of growth factors, such as hormones, including the human pregnancy hormone chorionic gonadotropin (hCG) and human growth hormone. The selection of suitable growth factors for defined cell populations is described in detail and is state of the art.

The following exemplary embodiments serve to explain the invention and are not to be interpreted as restrictive.

Animal data

Transfection of murine antigen-presenting cells (APC) with the Fas ligand gene by means of adenoviral gene transfer. Peritoneal lavage was carried out in Fas (CD95) -deficient C57BL (B6) mice and the peritoneal macrophages were obtained. 5xl0 ⁶ macrophages were cultivated per well of a 6-well plate in complete DMEM medium for 24 hours and then with an adenoviral vector which led to the expression of the Fas ligand (FasL, CD95L) gene (AdFasL), or a control vector (AdLacZ, expression of the β-galactosidase) transfected After 48 hours, the transfected macrophages were tested for the expression and functionality of FasL. The Facs analysis showed that approximately 90% of the macropages transfected with AdFasL (AdFasL-APC) expressed the FasL gene on the cell surface, while no relevant FasL expression on the macrophages that had been transfected with AdLacZ (AdLacZ -APC), was ascertainable (FIG. 1A) In order to investigate whether the FasL expression is also functional, AdFasL-transfected macrophages were used in a cytotoxicity test with Fas-expanding A20 target cells. This showed a concentration-dependent cytolysis of the target cells in the cocultures with AdFaL-APC, while none or only a narrow spontaneous lysis was detectable in the cocultures of AdLacZ-APC and A20 target cells (FIG. 1B)

Inhibition of allogeneic proliferation of T cells by APC expressing Fas ligand

To determine whether the FasL expression on the AdFasL-APC modulates the interaction with T cells and whether an allogene-specific T cell response can be suppressed, AdFasL-APC and AdLacZ-APC from B6-lpr / lpr mice (H- 2D ^b) genenert and in a mixed lymphocyte reaction (MLR) with T cells from Fas-deficient MRL-lpr / lpr mice (H-2D ^k) or Fas-expnmierenden MRL - cocultured + / + control mice (H-2D ^k) The Allogeneic T-cell proliferation was determined by the inco-formation of [ ³ H] -thymιdιn. It was found that the allogeneic proliferation of the H-2D ^k T-cells in the co-cultures with H-2D ^b APC-FasL was significantly reduced compared to that Co-cultures of H-2D ^k T cells with H-2D ^b APC-LacZ (Fig. 2A). By using the Fas-deficient T cells of MRL-lpr / lpr mice, it was also possible to demonstrate that this suppression effect was based on the interaction of Fas with Fas ligand, that is, it required the functional expression of the two molecules (FIG. 2B)

Therapeutic application Fas ligand-expressing antigen-presenting cells in a mouse model with virus-induced autoimmune disease

Infection of Fas ligand deficient C57 / BL6 (B6) -gld / gld mice with the munit cytomegahevims (MCMV) induces a chronic autoimmune inflammatory response. In order to check whether antigen-specific suppression of the T cells by AdFasL-APC can also be achieved in vivo, AdFasL-APC and AdLacZ-APC were tested therapeutically in a mouse model with chronic autoimmune disease. The mouse model used was tested intraperitoneal infection of Fas-deficient B6-lpr / lpr mice or FasL-deficient Bβ-gld / gld mice with lxl 0 ⁶ PFU of the mouse cytomegalovirus (MCMV) induces a chronic inflammatory reaction in various organs, while B6- + / + Mice do not show any relevant organ changes after the elimination of virus-infected cells Hessen. The cause of the chronic inflammation in B6-lpr / lpr and B6-gld / gld mice was not a delayed elimination of virus-infected cells, but a persistent activation of T cells, which were mainly responsible for the inflammatory infiltrates in the organs, which is why this mouse model was outstandingly suitable for checking the effectiveness of the new therapy concept. In addition, the chronic inflammation showed a clear autoimmune component, since more autoantibodies could be detected. 3 shows the histological severity of the inflammatory reaction in the lungs. Kidney and liver over time in MCMV-infected B6 - + / + and FasL-deficient B6-gld / gld mice.

Significant improvement in MCMV-induced chronic inflammatory response by treatment with Fas ligand-expressing APC

28 days after MCMV infection, B6-gld / gld and B6-lpr / lpr mice were treated with AdLacZ-APC or AdFasL-APC (1 x 10 ⁶ APC ip, every 3rd day for 12 days). In addition, part of this APC was pulsed with MCMV in vitro prior to administration. The organs were removed 4 weeks after the start of treatment and the severity of the inflammatory reaction was determined (FIG. 4). There was a significant improvement in inflammation in the AdFasL-APC treated Bβ-gld / gld mice, which was exacerbated by prior pulsing of the APC with MCMV. It could also be demonstrated that the observed therapeutic effect was Fas-mediated, since no improvement was detectable in Fas-deficient B6-lpr / lpr mice.

Significant reduction of MCMV-specific T cells in the spleen after treatment with APC expressing Fas ligand

At the same time, the spleens were also removed from the B6-gld / gld mice and the spleen cells were cocultivated with MCMV-pulsed APC from B6 mice for 48 hours in a mixed lymphocyte reaction (MLR). There was a significant reduction in IL-2 secretion in the spleen cells of the animals which had been treated with APC-AdFasL (FIG. 5). Thus an antigen-specific suppression of the T cells by AdFasL-APC in vivo be demonstrated.

Reduced production of autoantibodies in MCMV-infected B6-gld / gld mice by treatment with Fas ligand-expressing APC Since MCMV-infected B6-gld / gld mice develop high concentrations of autoantibodies, it was investigated whether autoantibody production was achieved by the administration of AdFasL-APC can be influenced. For this purpose, the levels of rheumatoid factor IgGl and anti-double-stranded DNA IgGl autoantibodies were determined in the serum of mice that had been treated with either AdLacZ-APC, AdFasL-APC or MCMV-pulsed AdFasL-APC. A significant reduction in autoantibody production was found in the animals treated with AdFasL-APC, the suppression being increased by pulsing the AdFasL-APC with MCMV (FIG. 6).

Example with primary human cells Effective transfection of primary human macrophages with an adenoviral vector

Primary monocytes obtained by leukapheresis were differentiated into macrophages in vitro over 7 days, or by adding IL-4 and GM-CSF to the culture medium into dendritic cells (DC). The macrophages and DC were then transfected with AdLacZ in different concentrations. It was found that both macrophages and DC, in contrast to other cells (e.g. fibroblasts or murine macrophages), are significantly more difficult to transfect. Using a high MOI (500), however, successful transfection was detectable in approximately 30% of the macrophages after 72 hours by detection of the β-galactosidase by means of an x-Gal staining (FIG. 7). The transfection rate was significantly increased by using different cytokines and lipofectamine.

Inhibition of Allogeneic T Cell Proliferation by "Tolerogenic" Dendritic Cells Independent of adenoviral transfection, a 'tolerogenic' DC phenotype was also investigated. Other research groups had shown that by adding IL-10 to DC culture on day 5 a suppressive DC phenotype is generated, while the addition of TNF leads to a strongly activating DC phenotype at the same time, which is attributed among other things to the different expression of costimulatory molecules. It was therefore investigated whether these two DC populations were located in the Differentiate ability, an allogeneic To induce T cell stimulation in the MLR (Fig. 8). IL-10 matured DCs were shown to induce less proliferation of allogeneic T cells than DCs treated with TNF. This effect could be increased by adding an anti-Fas antibody (clone CH-11).

Evidence of an allogene-specific suppression by tolerating APC

In order to investigate whether the observed suppression of T cell proliferation is allogene-specific, the T cells were obtained 5 days after the start of the MLR and used in a second MLR against APC from a third donor ("third party") (FIG. 9) Allogeneic-specific suppression of the T cells could also be demonstrated for the IL-10 matured DC, since the response of the T cells, which had initially been co-cultivated with IL-10 matured DC, to the APC of the third donor was unaffected.

Construction of vectors according to the invention

The vectors pcDNA3-TK-IRES-crmA (FIG. 10A) and pcDNA3-FasL-IRES-PLP (FIG. 10B) are examples of vectors according to the invention.

pcDNA3-FasL-IRES-PLP (FIG. 10A) comprises nucleic acid sequences which code, for example, for the apoptosis-triggering ligand FasL and, for example, for the antigen proteolipid protein (PLP). Both areas are linked by an LRES sequence in such a way that the antigen is translated by the same mRNA as the ligand that triggers apoptosis. The vector is based on the cloning vector pcDNA3. The nucleic acid regions coding for FasL and for PLP were transcribed from RNA into cDNA by polymerase chain reaction (PCR). Methods for isolating the RNA from cells and the use of PCR to rewrite specific RNA molecules in cDNA are state of the art. The oligonucleotides which were used as primers for the PCR comprise, at their 5 'ends, interfaces for endonucleases which were subsequently used for the cloning of the cDNAs into the vector pcDNA3. The area coding for FasL was first cloned into the vector pcDNA3 via the interfaces for Hindϊll and BamHI. Subsequently, the fragment coding for PLP was used via them interfaces of BamHI and EcσRI. Finally, the nucleic acid fragment comprising the IRΕS was cloned between FasL and PLP via the recognition sequence for BamHI. The techniques for isolating nucleic acids, the cleavage of nucleic acids and the purification of nucleic acid cleavage products, as well as the ligation of individual nucleic acid fragments and the multiplication of the artificially generated ones Nucleic acids in bacteria are state of the art.

pcDNA3-TK-IRES-crmA (Fig. 10B) comprises nucleic acid sequences encoding a suicide enzyme such as thymidine kinase and an anti-apoptosis molecule such as crmA. The expression of crmA is coupled to the expression of TK by an ERES sequence in such a way that crmA can only be expressed together with the TK. The vector is based on the cloning vector pcDNA3 and the cloning strategy and the production of the vector according to the invention is comparable to that of pcDNA3-FasL-ERES-PLP. The region coding for the thymidine kinase was first cloned into the vector pcDNA3 via the interfaces for Hz «dIII and BamKl. Then the fragment coding for crmA was used via them interfaces of BamΑl and Xhol. Finally, the nucleic acid fragment comprising the ERES was cloned between FasL and PLP via the recognition sequence for BamΑl. The nucleic acid sequences of the two vectors pcDNA3-FasL-ERES-crmA and pcDNA3-TK-ERES-PLP are listed as SEQ ED NO: 1 and SEQ ID NO: 2, respectively.

Claims

claims

1. A gene transfer vector comprising at least one nucleic acid molecule comprising a) a first nucleic acid sequence coding for one or more apoptosis-triggering ligands, b) a second nucleic acid sequence coding for one or more antigen (s) and optionally, c) a third Nucleic acid sequence, coding for one or more anti-apoptosis molecule (s), and optionally d) a fourth nucleic acid sequence, coding for one or more suicide enzyme (s).

2. Gene transfer vector according to spoke 1, wherein the first and second nucleic acid sequence on one nucleic acid molecule and the third and fourth nucleic acid sequence on a further nucleic acid molecule, or the first three nucleic acid sequences, or all four nucleic acid sequences, or the first two and the fourth nucleic acid sequences occur on a nucleic acid molecule ,

3. gene transfer vector according to one of claims 1 or 2, wherein the first and second nucleic acid sequence and / or the third and fourth nucleic acid sequence are so functionally linked to one another that the expression of the second nucleic acid sequence of the

Expression of the first nucleic acid sequence is dependent, and / or that the expression of the third nucleic acid sequence is dependent on the expression of the fourth nucleic acid sequence.

4. Gene transfer vector according to one of claims 1 to 3, wherein the vectors viruses, in particular retroviruses, adenoviruses, adeno-associated viruses, poxviruses, alphaviruses or Heφesviruses, or bacteria, in particular listeria, shigella or salmonella, or liposomes, plasmids, phagemids, cosmids , Bacteriophages or artificial chromosomes.

Gene transfer vector according to one of claims 1 to 4, wherein the first nucleic acid sequence encodes CD95L / FasL / ApolL, TRAIL or Apo3L.

Gene transfer vector according to one of claims 1 to 5, wherein the second nucleic acid sequence for one or more epitopes of the myelin basic protein, myelin proteolipid protein, myelin proteolipid protein, myelin-associated basic protein on oligodendrocytes, oligodendrocyte-specific protein, myelin-associated glycoproteins, gly PO, peripheral myelin protein 22, pl70k / SAG, Schwann Cell Myelin Protein, Transaldolase, SlOOß, Alpha B Crystallin, 2 ', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP), GFAP, the α or ε subunits of the acetylcholine receptor , Type II collagen encoding tyrosine phosphatase IA-2, proinsulin, GAD65, Hsp60 or ICA69.

7. Gene transfer vector according to one of claims 1 to 6, wherein the third nucleic acid sequence for E3-14.7K, E3-14.5K, E3-10.4K, FLEP, vFLIP, MC159, MC160, BORFE2, E8 of the equine herpes virus EHV-2, Cl 3 encoded by HHV-8, ORF71 by Heφesvirus Saimiri, E1B-19K, LMP-1, LT protein by SV40, polyoma proteins ST and MT, inhibitors of caspases or antisense RNAs.

8. A gene transfer vector according to any one of claims 1 to 7, wherein the fourth nucleic acid sequence for thymidine kinase, carboxylesterase, cytosine deaminase, carboxypeptidase G2, cytochrome P450, deoxycytidine kinase, nitroreductase, purine nucleoside phosphorylase, thymidine phosphinolanase, phosphoribosyl

Transferase, bacterial uracil phosphoribosyl transferase, fusion protein from the cytosine deaminase and uracil phosphoribosyl transferase from Saccharomyces cerevisiae are encoded.

9. Nucleic acid sequence according to SEQ LD: 1 or SEQ ID NO: 2.

10. Gene transfer vector according to one of claims 1 to 9 as a therapeutic agent.

11. Use of the gene transfer vector according to one of claims 1 to 9 for the production of a therapeutic agent for the prevention or therapy of autoimmune diseases, in particular rheumatoid artritis, systemic lupus erythematosus, Sjogren's syndrome,

Polymyositis, dermatomyositis, polymyalgica rheumatica, arteritis temporaiis, spondylarthropathies, ankylosing spondylitis, Crohn's disease, ulcerative colitis, celiac disease, autoimmune hepatitis, type I diabetes, adrenal insufficiency, thyroiditis, Psoriasis, dermatitis, Heφetiformis, Pemphigus Vulgaris, Alopecia, Multiple Sclerosis, Myastenia Gravis, or for the prevention or therapy of chronic inflammatory processes that are based on immunopathogenesis, especially chronic inflammation after viral or bacterial infections, especially chronic hepatitis in hepatitis B Vims or hepatitis C virus infections, or inflammation of the brain after

Infection with the Masem-Vims, or for the prevention or therapy of graft rejection.

12. Use of the gene transfer vectors according to one of claims 1 to 10 for the ex vivo modification of animal or mammalian cells, in particular human cells.