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Modulation of the expression of stat-1-dependent genes

Modulation of the expression of stat-1-dependent genes description/claims

The present invention relates to decoy-oligonucleotides and antisense-oligonucleotides having the nucleic acid sequence according to SEQ ID NO: 1 to 43 as well as the use thereof as medicaments.

It is a major aim of the decipherment of the human genome to identify morbid genes (due to the mode of action of their products) and morbid changes in the structure of these genes (polymorphisms) respectively and to assign them to a disease pattern. Therefore a causally determined therapy for most diseases has come into reach if it is accepted that these are caused by a defined number of gene products being expressed too strongly, too weakly or deficiently. In fact the usually singular genetic defect (monogenetic diseases) is already known for a set of hereditary diseases (e.g. cystic fibrosis) whereas the situation for other diseases (e.g. hypertension) turns out to be considerably more complex. The latter are obviously not the result of a single but multiple genetic defects (polygenetic disease) predetermining the affected persons to develop the disease in coincidence of certain environmental factors. Albeit this constraint the targeted intervention in the expression of one or multiple genes affords the opportunity of a cause- and not only a symptom-based therapy.

Transcription factors are DNA-binding proteins that attach to the promoter region of one or multiple genes inside the cell nucleus thereby regulating their expression, i.e. the regeneration of the proteins these genes are coding for. Besides the physiologically important role of controlling developmental and differentiation processes in the human body, transcription factors display a high potential for eliciting a disease particularly if they activate the gene expression at a wrong point of time. In addition (possibly the same) transcription factors can block genes with a protective function und act predisposing for the formation of a disease. Insofar the in the following described principle of an anti-transcription factor therapy aims at the inhibition of morbid genes and the activation of protective genes in contrast.

Inflammation is a defence reaction of the organism and its tissues against damaging stimuli aiming at the remediation of the damage or at least its local limitation and at abolishing the cause of damage (e.g. invaded bacteria or foreign substances). The elicitors of an inflammation can be micro-organisms (bacteria, viruses, fungi or parasites), foreign substances (pollen, crystals of asbestos or silicates), destruction of the tissue by mechanical impairment, chemical noxa and physical influences as well as elicitors from the body itself (collapsing tumour cells, extravasal blood, autoimmune reactions) or crystals of intra-bodily precipitated substances (uric acid, calcium oxalate and calcium phosphate, cholesterol).

The rapid activation of mastocytes (inside the tissue) or of basophile granulocytes in the blood is an example for the tripping of a very strong acute-inflammatory response and is discriminatory for immunological hypersensitivity reactions of the immediate type (humoral allergy type I). If the organism got into contact with an antigen (or an allergen, respectively, in the case of hypersensitivity) already beforehand B-lymphocytes had been sensitised as a reaction to this. The B-lymphocytes transform into plasmocytes in cooperation with previously sensitised CD4-positive type 2 T-helper cells (Th2 cells) and start producing antibodies of the IgE-type against the antigen. During this differentiation process the co-stimulation of the B-lymphocytes via the CD40-receptor by the Th2-cells expressing the respective ligand (CD154) is of crucial importance. When the antigen-loaded IgE-antibodies bind to the respective receptors (type Fcε) on the mastocytes these start to release different mediators of inflammation especially histamine, interleukin-8, leukotrienes and tumour necrosis factor-α (TNFα). Consequence of which is the attraction of professional inflammatory cells especially of eosinophile and neutrophile granulocytes and monocytes but also of T-lymphocytes on-the-spot (chemotaxis). At the same time a histamine dependent vasodilatation and increase of permeability of the endothelial cells coating the interior vascular wall takes place. Due to the vascular dilatation the flow velocity decreases facilitating the establishment of the physical contact between the attracted leukocytes and the endothelial cells. These endothelial cells being exposed to cytokines (e.g. TNFα) and thereby already activated display an intensified expression of selectins on their luminal surface (e.g. E-selectin) causing a rolling along the endothelial cells of the leukocytes and thereby the activation of further adhesion molecules (integrins; e.g. intercellular adhesion molecule-1 [ICAM-1] or vascular cell adhesion molecule-1 [VCAM-1]). The leukocytes can now adhere to the vascular wall (margination) and the histamine-related increase in permeability (loosening of the union of endothelial cells) favours their migration into the extravasal space (diapedese). At the same time augmented amounts of protein rich fluid (inflammatory exudate) attain the interstitial space forming an oedema. Circumjacent nerve endings are irritated by the increasing pressure in the tissue and by further mediators generated by the inflammatory cells and trigger pains making the damage of the tissue aware.

The granulocytes which have migrated to the site of inflammation and the monocytes which have re-differentiated into macrophages attempt to eliminate the causers of the inflammation by phagocytosis and lysis respectively thereby triggering the release of inter alia proteolytic enzymes and oxygen radicals that may damage also the surrounding tissue. In particular the activation of the macrophages can account in many ways for the fact (e.g. by the release of further cytokines like interleukin-1 β or interleukin-6) that the entire organism is involved by the primarily local inflammatory response in terms of an acute phase response. Representative characteristics of an acute phase response are fatigue, lassitude and fever, an increased release of leukocytes from the bone marrow (leukocytosis), the detection of acute phase proteins in the blood (e.g. C-reactive protein), the stimulation of the immune system as well as weight loss due to a changed status of the metabolism.

If the cause of the inflammation can be eliminated the process of wound healing falls into line with the destroyed tissue being repaired. At best this amounts to an entire re-establishment (restitutio ad integrum), whereas bigger lesions or an excessive production of connective tissue (especially collagen) result in the formation of a scar which is possibly associated with considerable dysfunctions depending on the affected tissue. If the cause cannot be eliminated at once (foreign substances or wound infection) the wound healing is delayed at simultaneous increase of the immigration and activity of the phagocytes bringing about the doom of the tissue (necrosis) up to the formation of cavities (abscess). The result is almost always a scarred restructuring of the tissue with a respective loss of function. If the local limitation of the inflammation which is derived from the causative agent does not succeed, the inflammation spreads over the entire organism via the lymphatic system. The consequence is a sepsis with a possibly fatal upshot (septic shock).

Wound healing is also interfered with if the inflammatory and the healing process are in balance. The result is a chronic inflammation which may be fibrosing (excessive synthesis of collagen) or granulomatous (organisation of inflammatory cells into a granulation tissue) and usually brings about a continuous destruction and increasing constraint of functionality of the affected tissue respectively.

Besides the depicted common inflammatory response which may degenerate chronically there are inflammatory diseases that exhibit both common grounds and distinct differences with regard to the underlying pathogenesis. Two inflammatory diseases of such kind are for example complications after cardio-surgical interventions and the immunological incompatibility reactions which more space in this specification is dedicated to because of their enormous clinical relevance.

The balloon-tipped catheter based mechanical dilatation (percutaneous angioplasty) and the bypassing of arteriosclerotically stenosed arteries by means of venous bypasses respectively still constitute the therapies of choice in patients with coronary and peripheral circulatory disorders respectively in order to provide protection against an imminent infarction or organ failure. But the rate of re-occlusion (restenosis) of the arteries which were mechanically dilated and (in the majority of cases) treated with a metallic vascular support (stent) appears unacceptably high with 20-50% within 6 months. Also the rate of re-occlusion of aortocoronary and peripheral venous bypasses respectively with 50-70% after 5 years is more than dissatisfactory for the treated patients in particular against the background of the risk around the procedure and the postoperative risk respectively. Presumably because of the damage of the vascular wall (hereby both the endothelial and the smooth muscle cells being affected) the restenosis after angioplasty shows particularly in the early stage a pronounced inflammatory component being characterised inter alia by the infiltration of the vascular wall with professional inflammatory cells (above all monocytes and T-lymphocytes). The fibro-proliferating stenosis formation (intimal hyperplasia) in aortocoronary and peripheral venous bypasses respectively seems to be based also on a inflammatory reaction which in particular is caused by mechanical and physical noxa. It has been known for a long time also that the so called ischemia/refusion-related damage in the context of surgical interventions or organ transplantations is accompanied by an inflammatory-based tissue damage in which the interaction between endothelial cells and professional inflammatory cells (above all granulocytes but also monocytes and T-cells) as well as the release of tissue damaging substances (oxygen radicals, cytokines) play a quite crucial role.

In connection with the mentioned cardio-vascular complications it is important that there are protective mechanisms, above all in the endothelial and smooth muscle cells of the vascular wall, which help to limit the extent of the inflammatory response and the subsequent adaptive restructuring of the tissue. To this for example belongs the synthesis of nitric oxide (NO) by the NO-synthase in the endothelial cells. NO, probably featuring the endogenous antagonist of the oxygen radical superoxide, inhibits inter alia the expression of pro-inflammatory chemokines (e.g. monocyte chemoattractant protein-1, MCP-1) and of adhesion molecules (e.g. ICAM-1) in endothelial cells, the expression of receptors for growth factors in smooth muscle cells (e.g. endothelin B-receptor) as well as the release of growth factors from leukocytes. Insofar it is easy to comprehend that a mechanical damage just as a functional damage of the endothelium (e.g. by a cytokine-induced reduction of the expression of the NO-synthase in these cells) counteract the processes of inflammation and subsequent fibro-proliferating re-structuring of the vascular wall which form the basis for the mentioned cardio-vascular complications.

All previous attempts to check the restenosis after angioplasty medicamentously have not achieved the desired effect in the majority of patients. At present two local principles of therapy are favoured: the already approved vascular brachytherapy, a method for checking the cell growth by short-time radioactive irradiation of the dilated vascular section and the drug-eluting stents which are still in the clinical trial. This method comprises polymer coated stents which are “impregnated” by growth inhibiting medicaments (cytostatic and immunosuppressive agents) and release them slowly during a period of several weeks. Most recent clinical studies prove that both therapeutic approaches are not exempt from to some extent serious problems (e.g. in-stent-thrombosis running the danger of an infarction) despite of encouraging results at the beginning.

Besides the already delineated immunological type I-incompatibility reaction there are in principle four other forms of allergy and dysfunctions in the immune regulation respectively. The type I-reaction itself can in principal be sorted into two phases after allergisation was accomplished: the rapid release and regeneration of vascularly active inflammatory mediators from IgE-spiked mastocytes and the late reaction which is mediated by the attracted eosinophile and neutrophile granulocytes. The complete type I-reaction can take place either locally or systemically in dependence on the exposure to the allergen. Allergens in the respiratory air elicit reactions in the respiratory tract, typically accompanied by mucosal oedemas and hypersecretion (allergic rhinopathy, hay fever) as well as bronchospasm (asthma) whereas allergens in the nourishment elicit gastrointestinal symptoms like nausea, vomitus and diarrhoea. The skin reacts on allergens with itching and urticaria as well as atopic dermatitis (neurodermatitis) But if the allergen gains direct access to the bloodstream (e.g. infusion of blood products, medicaments) or if the exposure to the allergen is especially strong, a systemic immediate reaction results possibly entailing a life-threatening decrease of the blood pressure (anaphylactic shock).

In the case of the type II-reaction antigenically active cells (e.g. extraneous blood cells) or extracellular proteins (e.g. medicament-induced changes at the surface of a cell naturally produced in the body) take centre stage. After allergisation the second contact leads to the production of allergen-specific antibodies of the IgG- and IgM-type which bind to the allergenic cell in great quantities (opsonisation). Hereby the complement system (formation of a membrane attacking complex) and a special subpopulation of lymphocytes, the natural killer cells (NK-cells), are activated. The result is a destruction of the allergenic cell by cytolysis. A similar reaction is elicited when auto-antibodies attach to structures that are naturally produced in the body such as the basal membrane of the glomerular capillaries and thereby eliciting a rapidly progressive glomerulonephritis with imminent renal insufficiency. Besides the type 1 T-helper cells (Th1-cells, see below) the activated NK-cells are the main producers of interferon-γ, a cytokine that massively intensifies the inflammatory response in particular by the activation of macrophages.

The type III-reaction is characterised by the formation and deposition of immune-complexes (antigen-antibody-complexes) with subsequent activation of the complement system and phagocytes (granulocytes, macrophages). They circulate in the blood and successively deposit mainly in the capillaries of the renal glomeruli but also in the joints or in the skin. The hereby elicited inflammatory response may bring about a (immune-complex-) glomerulonephritis, pains in the joints as well as urticaria. Infections can also elicit a systemic type III-reaction if the immune system fails to eliminate the causative agent (e.g. streptococci). Representative local type III-reactions are the so called Arthus-reaction in the skin after an immunisation or the exogenous allergic alveolitis in the case of the deposition of antigen-antibody-complexes in the lung (e.g. bird-breeder's lung). The systemic lupus erythematodes is a type III-reaction as well but in terms of an autoimmune disease due to the formation of auto-antibodies.

In contrast to the hypersensitivity reactions mentioned before the type IV-reaction is not humoral but cell constrained and reaches its maximum usually not until after several days (delayed type of reaction or delayed type hypersensitivity). Elicitors are mainly proteins, invaded foreign organisms (bacteria, viruses, fungi and parasites), other foreign proteins (e.g. wheat-derived gliadin in the case of celiac disease) as well as haptens (medicaments, metals [e.g. nickel in the case of contact dermatitis], cosmetics and plant components). The primary rejection of transplanted organs is also a type IV-reaction. The antigen is phygocytised by (tissue) macrophages, processed and presented to naive T-helper cells (CD4-positive); the allergisation of the T-helper cells takes several days. At the second contact the in such a way sensitised T-helper cells alter in Th1-cells; thereby the CD154-mediated co-stimulation of the antigen-presenting cell (this one expresses the CD40-receptor) plays an important role because this signalling pathway triggers the release of interleukin-12 from the macrophages. Interleukin-12 initiates the differentiation and proliferation of the T-helper cells. The Th1-cells on their part excite the formation of monocytes in the bone marrow by certain growth factors (e.g. GM-CSF), recruit these by means of certain chemokines (e.g. MIF) and activate them by the release of IFNγ. The hence resulting very strong inflammatory response may destroy tissue normally produced in the body (e.g. tuberculosis) or transplanted tissue in a large scale. Moreover CD8-positive cytotoxic T-cells are involved in the transplant rejection (cytolysis) with the CD8-positive cytotoxic T-cells being able to recognise their target (the foreign cell surface) and to “arm” themselves accordingly only by a preceding antigen-presentation like the CD4-positive Th1-cells.

A dysfunction of the immune regulation similar to a type IV-reaction forms the basis for e.g. the rheumatoid arthritis or the multiple sclerosis (auto-reactive Th1-cells) as well as for diabetes mellitus (auto-reactive cytotoxic T-cells). T-cells being directed against certain antigens of the causative agent (e.g. streptococci) which cross-react with auto-antigens (produced in the body; molecular mimicry) might potentially play a role at these autoimmune diseases besides bacterial super-antigens (e.g. the causative agent of TBC) and the according genetic predisposition (MHC-proteins, Th1/Th2-imbalance). In contrast, type V-reactions may be evoked inter alia by activating or blocking auto-antibodies of hormone- (e.g. thyrotropin in the case of Basedow's disease) or neurotransmitter-receptors (e.g. acetylcholine in the case of myasthenia gravis).

Comparable with the transplant rejection—yet in the reverse sense—is the graft versus host disease (GVHD) which appears in the course of allogenic bone marrow transplantations (between genetically non identical individuals) in about 40% of the recipients. During the acute-phase lasting up to three months the T-cells of the donor which have been transfused with the stem cells attack the host organism. The resulting possibly severe inflammation response becomes manifest preferably in the skin, the gastrointestinal tract and in the liver.

For the treatment of acute inflammatory diseases in dependence on to the assumed cause usually non-steroidal antiphlogistics (NSAIDs, inter alia inhibition of the synthesis of prostaglandins) and/or anti-infectious agents (devitalisation of bacteria, fungi or parasites) and antiviral chemotherapeutics respectively, contingently also glucocorticoids (general inhibitors of gene expression) in a local application, are utilised. In the case of severe or chronically recurring inflammatory diseases glucocorticoids or immunosuppressive agents (inhibition of the T-cell-activation) or cytostatics such as methotrexate are systemically administered. This also applies to the transplantation of organs and bone marrow respectively. Despite of their undisputable therapeutic effect a systemic administration of the mentioned pharmaceuticals can evoke severe side effects especially when permanently used. So for example up to 25% of the patients who take methotrexate for 2 or more years develop a severe cirrhosis of the liver. More recent active agents that are used in particular with chronically recurring inflammatory diseases block the pro-inflammatory effect of TNFα: antibodies directed against the cytokine itself and its receptor respectively, low-molecular antagonists of the receptor as well as a recombinantly produced, soluble receptor protein that traps the cytokine. But there is a growing number of indications for an increased incidence of infectious diseases during the therapy with the receptor protein (inter alia tuberculosis), and about 40% of the patients do not seem to respond to the therapy at all (non-responder). Also for the approved humanised TNFα-antibody there are according warning notices concerning the incidence of infections ranging up to sepsis 2-4 years after the start of the therapy. Moreover both active agents are contraindicated during an acute incident. In addition low-molecular antagonists of the receptor are approved for leukotrienes which are mainly used in the therapy of asthma as well as inhibitors of the cyclooxigenase-2, a new group of non-steroidal antiphlogistics (NSAIDs) with considerably reduced gastrointestinal side effects in comparison to the classical NSAIDs. Moreover there is a series of further—usually humanised—antibodies or antisense-oligonucleotide based approaches against adhesion molecules of leukocytes and endothelial cells respectively, cytokine receptors of T-helper cells or IgE-antibodies which are residing in different phases of the clinical trial. To refrain from the glucocorticoids and the anti-infectious agents as a group, the mentioned pharmaceuticals have in common to be directed specifically against a target molecule which is of relevance for the therapy.

The present invention is therefore based on the problem to provide substances for the prevention or therapy of excessive inflammatory responses and for the herewith associated implications for morbidity and mortality of the affected patients.

The problem is solved by the subject-matter defined by the patent claims.

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