EP1325122A2 - Modulation de la transcription de produits geniques pro-inflammatoires - Google Patents

Modulation de la transcription de produits geniques pro-inflammatoires

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Publication number
EP1325122A2
EP1325122A2 EP01986318A EP01986318A EP1325122A2 EP 1325122 A2 EP1325122 A2 EP 1325122A2 EP 01986318 A EP01986318 A EP 01986318A EP 01986318 A EP01986318 A EP 01986318A EP 1325122 A2 EP1325122 A2 EP 1325122A2
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Prior art keywords
irf
seq
inhibitor
cells
double
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English (en)
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Markus Hecker
Andreas H. Wagner
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Avontec GmbH
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Avontec GmbH
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===

Definitions

  • the present invention relates to inhibitors of the translation factor IRF-1, their use as a therapeutic agent and their use for the prevention or therapy of cardiovascular complications such as restenosis after percutaneous angioplasty or the stenosis of renal bypasses, chronic (transplant-arteriosclerosis or nasculopathy) or acute transplant rejection.
  • graft versus host disease GVHD
  • immunological hypersensitivity reactions especially bronchial asthma and atopic dermatitis
  • chronic recurrent inflammatory inflammation genes especially ulcerative colitis and Crohn's disease
  • psoriasis and sarcoidosis and autoimmune diseases especially diabetes mellitus, multiple sclerosis (multiple sclerosis) Lupus erythematosus), rheumatoid arthritis and nasculitis.
  • the vascular endothelium plays a key role in inflammatory diseases, as it is the primary site of interaction of circulating inflammatory cells with tissue.
  • inflammatory diseases e.g. rheumatoid arthritis
  • arteriosclerotic vascular wall lesions including transplant and renal bypass nasculopathy and restenosis after percutaneous angioplasty as well as in chronic inflammatory recurrent inflammation as well as in chronic inflammatory recurrent inflammation
  • Discussions e.g.
  • Lymphocytes and endothelial cells communicate via the CD40 / CD154 receptor / ligand system (also referred to as T ⁇ F receptor / ligand 5 system) with a consecutive increase in chemokine and adhesion molecule expression in the endothelium.
  • CD40 / CD154 receptor / ligand system also referred to as T ⁇ F receptor / ligand 5 system
  • the endothelial cells apparently release biologically active interleulcin-12 only after activation of the CD40 signaling pathway in an amount which is equal to the maximally stimulated monocyte (this are commonly considered the main source of interleukin-12).
  • erleukin-12 is the most important stimulus or differentiation factor for naive T-helper cells, which react with an increased formation of interferon- ⁇ or expression of CD 154 on their surface (these T-helper cells are then considered as THl cells).
  • Interferon- ⁇ in turn increases the expression of CD40 in the endothelial cells, so that a vicious cycle occurs in which endothelial cells, T helper cells and recruited monocytes mutually stimulate one another and keep the inflammatory reaction going.
  • CD40 / CD154 The inflammatory reaction-triggering stimulatory properties of CD40 / CD154 have been demonstrated in animal experiments, among others, in acute or chronic graft rejection (nasculopathy) and in Crohn's disease.
  • endothelial-leukocyte interaction via CD40 / CD154 not only the endothelial-leukocyte interaction via CD40 / CD154, but also, for example, the CD40 / CD154-mediated interaction of monocytes / macrophages or dendritic cells with THl cells or naive T helper cells play a role.
  • smooth vascular muscle cells can also express keratinocytes of the skin or synovial fibroblasts in joints CD40.
  • the activation of the CD40 signaling pathway in these cells is also important not only for the inflammatory reaction, but also leads to tissue restructuring processes such as the remodeling of the vascular wall in transplant nasculopathy, the skin changes in psoriasis or the erosions of the articular cartilage in the rheumatoid arthritis.
  • tissue restructuring processes such as the remodeling of the vascular wall in transplant nasculopathy, the skin changes in psoriasis or the erosions of the articular cartilage in the rheumatoid arthritis.
  • the co-stimulatory properties of CD40 / CD154 are important for the differentiation of B lymphocytes in antibody-producing plasma cells, which is triggered by contact with TH2 cells.
  • the B lymphocytes express CD40, the TH2 cells CD 154. Without this co-stimulation, the plasma cells primarily produce antibodies of the IgM type and hardly antibodies of the IgE or IgG type.
  • An excessive TH2 response i.e. the excessive production of IgE and IgG antibodies, plays an important role in primary allergic, chronic recurrent inflammatory disorders such as bronchial asthma, atopic denatitis and ulcerative colitis, but also in collagen diseases such as systemic lupus erythematosus ( SLE), whereby the formation of auto-reactive autoantibodies is in the Nordergrund in SLE and is therefore regarded as a generalized autoimmune effect.
  • SLE systemic lupus erythematosus
  • the distinction between autoimmune diseases and chronic recurrent inflammatory diseases are problematic, since a common predisposing factor is apparently the imbalance between a TH1 and a TH2-mediated cellular or humoral immune response.
  • results associated with the CD40 / CD154 signaling pathway represent inhibition of CD40 expression in the CD154 target cells.
  • One disadvantage of anti-CD 154 antibody treatment is the risk of hypersensitivity reactions (against the Antibodies), especially after repeated application and poor accessibility, at least for tissue-resistant epitopes (eg infiltrated T-lymphocytes), since the antibodies usually have to be applied via the bloodstream.
  • tissue-resistant epitopes eg infiltrated T-lymphocytes
  • there are no low molecular weight receptor antagonists for CD40 due to the trimerization of the receptor molecule after ligand binding, CD40 antibodies are more likely to activate the CD154 target cells.
  • Fig. 2 shows schematically the result of the time-dependent increase in the nuclear translocation of NFKB (p65 / p50 heterodimer), the p91 / p91 homodimer of Stat-1 and of IRF-1 in human endothelial cells, which was 0.5 hours (NFKB and Stat-1) or 3 hours (IRF-1) with TNF ⁇ (1000 U / ml), IFN ⁇ (1000 U / ml) and TNF ⁇ (100 U / ml) plus IFN ⁇ (1000 U / ml).
  • a pre-incubation (1 hour) with cycloheximide (Cx, 1 ⁇ M) shows that IRF-1 is expressed de ovo. Representative electrophoretic mobility shift analysis, comparable results were obtained in further experiments.
  • Fig. 4 shows schematically the effects of various cis-element decoys against Stat-1, NFKB and IRF-1 (10 ⁇ M, 4 h preincubation) on the CD40 protein content (a) determined with the aid of fluorescence activated cell sorting (FACS) in human Endothelial cells which were incubated for 24 hours with TNF ⁇ (100 U / ml) / IFN ⁇ (1000 U / ml) and the detection of the cell surface protein PECAM-1 (b), which is characteristic of endothelial cells.
  • FACS fluorescence activated cell sorting
  • FIG. 5 shows schematically the results of the effects of TNF ⁇ (2000 U / ml), IFN ⁇ (1000 U / ml) and TNF ⁇ (100 U / ml) plus IFN ⁇ (1000 U / ml) on the CD40 and IRF-1, respectively mRNA levels in human endothelial cells after 9 hours of incubation. Representative experiment, comparable results were obtained in further experiments.
  • FIG. 6 shows schematically the results for the time-dependent increase in CD40 or IRF-1 mRNA expression in human endothelial cells, which for 0, 0.5, 1.5, 3 and 9 hours with IFN ⁇ (1000 U / ml) were incubated. Representative experiment, comparable results were obtained in further experiments.
  • Fig. 7 shows schematically the specificity of the cis-element decoy effect on the CD40 mRNA expression in the human endothelial cells.
  • Preincubation (4 hours) with the cis element Decoy (IRF-ln cons, 10 ⁇ M) but not with the corresponding mutated control oligonucleotide (IRF-ln mut, 10 ⁇ M) inhibits CD40 mRNA expression in cells which are subsequently used for Incubated for 9 hours with TNF ⁇ (100 U / ml) and IFN ⁇ (1000 U / ml).
  • TNF ⁇ 100 U / ml
  • IFN ⁇ 1000 U / ml
  • FIG. 8 shows the inhibition of the cytoin-induced (100 U / ml) TNF ⁇ , 1000 U / ml IFN ⁇ ) expression of the IRF-1 protein (after 3 hours) and the CD40 mRNA (after 9 hours) in human endothelial cells which had previously been treated with an IRF-1 antisense oligonucleotide (AS; SEQ ID NO: 23) for 5 hours (concentration 0.2 ⁇ M).
  • AS IRF-1 antisense oligonucleotide
  • the left half of the picture shows the statistical summary of 3 experiments with different cell batches, the right half shows a representative Western blot or RT-PCR analysis, in (b) plus the densitometric evaluation ("intensity"), given in% the stimulated control and based on the internal standard ß-actin (* P ⁇ 0.05 compared to the stimulated control cells).
  • the corresponding missense (MS) and scrambled (SCR) control oligonucleotides did not influence the expression of IRF-1 or CD40.
  • FIG. 9 shows the electrophoretic mobility shift analysis of the uptake of various IRF-1 cis-element decoys (SEQ ID NO: 13, 17, 19 and 21) in cultured THP-1 cells and the subsequent neutralization of IRF-1.
  • the THP-1 cells were Element decoys preincubated for 1 hour and then stimulated for a further 3 hours with TNF ⁇ (100 U / ml) and IFN ⁇ (1000 U / ml).
  • TNF ⁇ 100 U / ml
  • IFN ⁇ 1000 U / ml
  • the result of the subsequent preparation and analysis of the samples is shown in the left half of the figure.
  • the right half of the picture shows the electrophoretic mobility shift analysis of a core extract of stimulated control cells obtained under identical experimental conditions, which was additionally treated with an anti-IRF-1 antibody as described in Krzesz et al. (1999) FEBS Lett. 453, 191 before the electrophoretic mobility shift analysis had been incubated (supershift analysis).
  • decoy-ODN or "cis-element decoy” or “double-stranded DNA oligonucleotide” used here denotes a double-stranded DNA molecule which has a sequence which corresponds to or is similar to the natural IRF-1 nuclear binding sequence in the genome and to which the transcription factor IRF-1 binds in the cell.
  • the cis element decoy thus acts as a molecule for the competitive inhibition of IRF-1.
  • the inventors were able to elucidate the transcription factors involved in the inflammation-related, cytoldn-mediated increase in CD40 receptor expression in human endothelial cells. Surprisingly, it has been found that the transcription factors are nuclear
  • NF -KB Factor KB
  • Stat-1 Signal Transducer and Activator of Transcription-1
  • TNF ⁇ Tumor necrosis factor- ⁇
  • IFN ⁇ interferon- ⁇
  • Transcription factors are not latently present in the cell, but first have to be synthesized de novo, usually after exposure to interferon- ⁇ and activation of the transcription factor Stat-1.
  • oligonucleotides are used in human cells in cell culture, the cytokine-induced CD40 expression (both with monostimulation with IFN ⁇ and with a combination of IFN ⁇ and TNF ⁇ ) is inhibited.
  • the induction of IRF-1 precedes the induction of CD40, so that an antisense oligonucleotide blockade of IRF-1 expression inhibits cytokine-induced CD40 expression to the same extent as the decoy oligonucleotides. Switching off the IRF-1 activity in cells results in a highly significant and selective inhibition of CD40 expression in these cells.
  • the endothelium-leukocyte interaction in particular the interaction of TH1 and endothelial cells, is weakened and represents the basis for the success of the therapy.
  • This also applies analogously to the weakening of the CD40 / CD154-mediated Interaction of naive T helper cells with antigen-presenting cells (e.g. monocytes, dendritic cells), of TH2 cells with B lymphocytes, and of other CD40-expressing cells (e.g. smooth muscle cells, keratinocytes, fibroblasts) with CD154-expressing cells ( THl cells, activated platelets).
  • antigen-presenting cells e.g. monocytes, dendritic cells
  • TH2 cells e.g. smooth muscle cells, keratinocytes, fibroblasts
  • CD154-expressing cells e.g. smooth muscle cells, keratinocytes, fibroblasts
  • One aspect of the present invention is therefore to provide an inhibitor of the activity of the transcription factor IRF-1 as a therapeutic agent.
  • Proteins, including IRF-1 can be inhibited in their activity in various ways.
  • Anti-IRF-1 antibodies, natural or synthetic substances that cause IRF-1 interaction with the DNA, i.e. reducing transactivation activity can be used.
  • the de novo synthesis of IRF-1 could also be inhibited by blocking Stat-1 or the signaling pathways leading to Stat-1 activation (Janus kinases).
  • a preferred method for the specific inhibition of IRF-1 activity is the use of double-stranded DNA oligonucleotides, also called cis-element decoy or decoy-ODN, which contain a binding site for IRF-1.
  • cis-element decoy or decoy-ODN double-stranded DNA oligonucleotides
  • decoy-ODN double-stranded DNA oligonucleotides
  • the sequence of a nucleic acid used to prevent binding of the transcription factor IRF-1 is the sequence to which IRF-1 naturally binds in the cell.
  • the cis-element decoy can also be larger than the 13-mer binding sequence and can be extended at the 5 'end and / or at the 3' end. Corresponding mutations in the region of the binding sequence lead to the loss of the binding of STAT-1 to the decoy oligonucleotide.
  • the DNA oligonucleotide according to the invention comprises not only the sense or forward sequence but also the complementary antisense or reverse sequence.
  • Preferred DNA oligonucleotides according to the invention have the following 13-mer binding sequences for IRF-1:
  • the cis element decoy can also have a sequence different from the above sequence and can be longer than a 13-mer.
  • the affinity for binding a nucleic acid sequence to IRF-1 can be determined using the Electrophoretic Mobility Shift Assay (EMSA) (Sambrook et al. (1989) Molecular Cloning. Cold Spring Harbor Laboratory Press; Krzesz et al. (1999) FEBS Lett. 453 , 191) can be determined.
  • ESA Electrophoretic Mobility Shift Assay
  • This test system is suitable for the quality control of nucleic acids intended for use in the method of the present invention or the determination of the optimal length of a binding site. It is also suitable for the identification of other sequences which are bound by IRF-1.
  • EMSA For an EMSA, intended for the isolation of new binding sites, the best suited are purified or recombinantly expressed versions of IRF-1, which are used in several alternating rounds of PCR amplification and selection by EMSA (Thiesen and Bach (1990) Nucleic Acids Res 18, 3203).
  • Genes which are known to contain IRF-1 binding sites in their promoter or enhancer regions and which are therefore putative targets for specific squelching by the method of the present invention include the CD40 gene and other pro- inflammatory genes e.g. cyclooxygenase-2, subunits of NADPH oxidase ( ⁇ 67phox and gp91phox), the inducible isoform of nitrogen monoxide (NO) synthase, interleukins 6, 8 and 12 as well as the adhesion molecules RANTES (soluble in T-lymphocytes, regulated upon activation, normal T-cell expressed, presumed secreted) and VCAM-1 (vascular cell adhesion olecule-1, also called CD 106).
  • pro- inflammatory genes e.g. cyclooxygenase-2, subunits of NADPH oxidase ( ⁇ 67phox and gp91phox), the inducible isoform of nitrogen monoxide (NO) synthase, interleukins 6,
  • the method of the present invention modulates the transcription of a gene or genes in such a way that the gene or genes, e.g. CD40, not or reduced expression.
  • Decreased or suppressed expression in the context of the present invention means that the transcription rate is reduced in comparison to cells which are not treated with a double-stranded DNA oligonucleotide according to the invention.
  • Such a change can be determined, for example, by Northern blot (Sambrook et al., 1989) or RT-PCR analysis (Sambrook et al., 1989).
  • Such a reduction is typically at least a 2-fold, in particular at least a 5-fold, in particular at least a 10-fold reduction.
  • the loss of activation can be calibrated, for example, if IRF-1 acts as a transcription activator on a specific gene and therefore squelching the activator leads to loss of expression of the target gene.
  • the method of the present invention enables the inhibition of the expression of a gene, provided that this is blocked by a constitutively active or (after corresponding stimulation of the cell) an activated transcription factor.
  • a constitutively active or (after corresponding stimulation of the cell) an activated transcription factor is an example of this.
  • an activated transcription factor is the inhibition of the expression of the prepro-endothelin-1 gene in native endothelial cells of the jugular vein of the rabbit by a cis-element decoy against the transcription factor CCAAT / enhancer binding protein (Lauth et al., J. Mol. Med ., (2000), 78, 441).
  • the expression of genes can be inhibited, the products of which have a protective effect, e.g. against inflammatory diseases.
  • the cis element decoy which is used in the present invention contains one or more, preferably 1, 2, 3, 4 or 5, particularly preferably 1 or 2, binding sites to which IRF-1 specifically binds.
  • the nucleic acids can be produced synthetically, by enzymatic methods or in cells. The individual processes are state of the art and known to the person skilled in the art.
  • the length of the double-stranded DNA oligonucleotide is at least as long as a sequence used that specifically binds IRF-1.
  • the double-stranded DNA oligonucleotide used is usually between about 13-65 bp, preferably between about 13-65 bp 26 bp and particularly preferably between 18-23 bp.
  • oligonucleotides are rapidly degraded by endo- and exonucleases, in particular DNases and RNases in the cell. Therefore, the DNA oligonucleotides can be modified to stabilize them against degradation, so that a high concentration of the oligonucleotides in the cell is maintained over a longer period of time. Typically, such stabilization can be obtained by introducing one or more modified intemucleotide bonds.
  • a successfully stabilized DNA oligonucleotide does not necessarily contain a modification to every hite ⁇ iucleotide bond.
  • the intemucleotide bonds at the respective ends of both oligonucleotides of the cis-element decoy are preferably modified.
  • the last six, five, four, three, two or the last or one or more emucleotide bonds within the last six intemucleotide bonds can be modified.
  • various modifications of the intemucleotide bonds can be introduced into the nucleic acid and the resulting double-stranded DNA oligonucleotides can be tested for sequence-specific binding to IRF-1 using the routine EMSA test system.
  • Modified cis-element decoys which still show sufficient binding can be selected, with sufficient binding meaning at least about 50% or at least about 75%, and particularly preferably about 100%, of the binding of the unmodified nucleic acid.
  • Cis-element decoys with modified intemucleotide binding which still show sufficient binding, can be checked whether they are more stable in the cell than the unmodified cis-element decoys.
  • the cells transfected with the cis-element decoys according to the invention are examined at various times for the amount of the cis-element decoys then still present.
  • a cis-element decoy marked with a fluorescent dye (eg Texas red) or a radioactively marked (eg 32 P) cis-element decoy is preferably used, followed by digital fluorescence microscopy or autoradiography or scintigraphy.
  • a successfully modified cis-element decoy has a half-life in the cell that is higher than that of an unmodified one Cis-element decoys, preferably at least about 48 hours, more preferably at least about 4 days, most preferably at least about 7 days.
  • Modified intemuldeotide phosphate residues and / or non-phosphorus bridges in a nucleic acid that can be used in a method of the present invention include, for example, methylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, phosphate ester, while non-phosphorus ether nucleotide analogs, for example Contain siloxane bridges, carbonate bridges, carboxymethyl ester bridges, acetamidate bridges and / or thioether bridges.
  • a further embodiment of the invention is the stabilization of nucleic acids by introducing structural features into the nucleic acid which increase the half-life of the nucleic acid.
  • Such structures containing hairpin and bell DNA are disclosed in US 5,683,985.
  • modified hitemuldeotide-phosphate residues and / or non-phosphorus bridges can be introduced together with the structures mentioned.
  • the resulting nucleic acids can be tested for binding and stability in the test system described above.
  • the binding sequence can be present not only in a cis-element decoy, but also in a vector.
  • the vector is a plasmid vector and in particular a plasmid vector that is able to replicate autosomally, thereby ensuring the stability of the introduced double-stranded nucleic acid increased.
  • Another aspect of the present invention is a double-stranded DNA oligonucleotide which is capable of binding to the transcription factor IRF-1 in a sequence-specific manner and preferably has one of the following sequences, only one strand of the double-stranded DNA oligonucleotide being shown here and the complementary strand is also included:
  • Double-stranded DNA oligonucleotides of the present invention have a length, modifications, and possibly a repetition of the specific binding site, as described in detail above.
  • the optimal length of the cis-element decoy is selected in order to optimize the binding to IRF-1 and the uptake into the cell.
  • a double-stranded DNA oligonucleotide that is shorter than 12 bp binds only weakly to its target protein, while a double-stranded DNA oligonucleotide that is longer than 22 bp, although it binds strongly, is absorbed into the cell with low efficiency .
  • the binding strength can be determined by EMSA, while the uptake of the double-stranded nucleic acid can be analyzed with the aid of a fluorescent dye (e.g. Texas red) or radioactively labeled (e.g. 32 P) cis-element decoy and subsequent digital fluorescence microscopy or autoradiography or scintigraphy.
  • a cis-element decoy of the present invention can be stabilized as described above.
  • a preferred embodiment of the present invention are cis-element decoys which contain a palm-dromic binding site and therefore comprise at least two transcription factor binding sites in a short double-stranded nucleic acid.
  • the palindromic sequence does not necessarily result in a higher binding of IRF-1, but is absorbed faster (more efficiently) by the target cells.
  • the shorter cis-element decoys according to the invention are palindromic only at the ends because of the long (centrally arranged) binding sequence and the repetitive G / CAAA motifs.
  • the binding sequence may also be preferred rather be arranged at the edges, as is the case with some of the preferred cis-element decoy sequences.
  • a cis-element decoy of the present invention is rapidly taken up into the cell. Adequate uptake is characterized by the modulation of one or more genes that can be modulated by IRF-1.
  • the cis-element decoy of the present invention preferably modulates the transcription of a gene or genes after about 4 hours of contact with the cell, more preferably after about 2 hours, after about 1 hour, after about 30 minutes and most preferably after about 10 minutes.
  • a typical mixture used in such an experiment contains 10 ⁇ mol / 1 cis-element decoy.
  • the present invention further relates to a method for modulating the transcription of at least one gene in CD40-expressing cells, in particular in endothelial cells, monocytes, denditric cells, B-lymphocytes, smooth muscle cells, keratinocytes or fibroblasts, the method comprising the step of contacting the aforementioned Cells with a mixture containing one or more double-stranded nucleic acid (s), which are able to bind sequence-specifically to the transcription factor IRF-1, comprises.
  • a preferred method is use in endothelial cells that are part of a graft. Typically, the method is applied to a graft in vivo or ex vivo prior to implantation.
  • the grafts can be treated prior to implantation by ex vivo application of the method of the present invention or after implantation by in vivo application of the method.
  • the treated graft is (thin) intestine, heart, liver, lung, kidney and pancreas or a combination of several organs.
  • the treatment of the organs, more precisely the pearl incubation of their blood vessels with the cis-element decoys according to the invention can be carried out ex vivo by rinsing out the solution immediately before the implantation.
  • the organ can be stored in a corresponding preservation solution (cooled) at the same time (e.g. University of Wisconsin Solution, Brettschneider HTK solution).
  • the mixture containing the cis-element decoys according to the invention is mixed with the target cells (eg endothelial cells, monocytes, denditric cells, B-lymphocytes, smooth muscle cells, Keratinocytes or fibroblasts).
  • the target cells eg endothelial cells, monocytes, denditric cells, B-lymphocytes, smooth muscle cells, Keratinocytes or fibroblasts.
  • the goal of this h-touching is to transfer the cis-element decoys that bind IRF-1 into the target cell (ie, the CD40-expressing cell). Therefore, nucleic acid modification and / or additives or adjuvants which are known to increase membrane penetration can be used in the present invention (Uhlmann and Peyman (1990) Chem. Rev. 90, 544).
  • a mixture according to the invention contains only nucleic acid and buffer.
  • a suitable concentration of the cis-element decoys is in the range of at least 0.1 to 100 ⁇ mol / L, preferably 10 ⁇ mol / L, one or more suitable buffers being added.
  • Such a buffer is Tyrode solution containing 144.3 nmol / 1 Na + , 4.0 mmol / 1 K + , 138.6 mmol / 1 Cl " , 1.7 mmol / 1 Ca 2+ , 1.0 mmol 1 Mg 2+ , 0.4 mmol / 1 HPO 4 2 ' , 19.9 mmol / 1 HCO 3 " , 10.0 mmol / 1 D-glucose.
  • the mixture additionally contains at least one additive and / or auxiliary.
  • Additives and / or adjuvants such as lipid, cationic lipids, polymers, liposomes, nanoparticles, nucleic acid aptamers, peptides and proteins that are bound to DNA, or synthetic peptide-DNA molecules are intended, for example, to introduce nucleic acids into the cell to direct the mixture to only one subgroup of cells to prevent degradation of the nucleic acid in the cell to facilitate storage of the nucleic acid mixture prior to use.
  • Examples of peptides and proteins or synthetic peptide-DNA molecules are e.g. Antibodies, antibody fragments, ligands, adhesion molecules, all of which can be modified or unmodified.
  • Additives that stabilize the cis-element decoys in the cell are, for example, nucleic acid-condensing substances such as cationic polymers, poly-L-lysine or polyethyleneimine.
  • the mixture used in the process of the present invention is preferably applied topically by injection, catheter, suppository ("suppository"), aerosols (nasal or oral spray, inhalation) trocars, projectiles, pluronic gels, polymers that are persistent Release medication, or any other device that provides local access allows. Ex vivo use of the mixture used in the method of the present invention also allows local access.
  • Another aspect of the present invention is to provide an inhibitor of IRF-1 expression as a therapeutic agent.
  • This inhibitor is preferably a single-stranded nucleic acid molecule, a so-called antisense oligonucleotide.
  • Antisense oligonucleotides can inhibit the synthesis of a target gene on three different levels, in the transcription (prevention of hnRNA synthesis), the processing (splicing) of the hnRNA to the mRNA and the translation of the mRNA into protein on the ribosomes.
  • the ner driving to inhibit the expression of genes by means of antisense oligonucleotides is well known in the art and to those skilled in the art.
  • the antisense oligonucleotide against IRF-1 used in the method according to the invention preferably has the sequence 5'-CGAGTGATGGGCATGTTGGC-3 '(SEQ ID ⁇ O: 23) and bridges the start codon.
  • Further preferred sequences for antisense oligonucleotides are 5'-GATTCGGCTGGTCGC-3 '(SEQ ID NO: 24), 5'-TAATCCAGATGAGCCC-3' (SEQ ID NO: 25) and 5'-GGAGCGATTCGGCTGGT-3 '(SEQ ID NO: 26).
  • the antisense oligonucleotide can be a single-stranded DNA molecule, RNA molecule or a DNA-RNA hybrid molecule.
  • the antisense oligonucleotide may also have one or more modified intemucleotide linkages, e.g. those described above for the Cis Element Decoy.
  • Another aspect of the present invention is an antisense oligonucleotide that specifically inhibits IRF-1 expression and preferably has one of the following sequences:
  • Another aspect of the present invention is furthermore the use of the antisense oligonucleotides and / or double-stranded DNA molecules according to the invention for the production of a medicament for the prevention and / or therapy of cardiovascular complications such as resteosis after percutaneous angioplasty or the stenosis of vein bypasses, the chronic (transplant - arteriosclerosis or vasculopathy) or acute graft rejection, graft versus host disease (GVHD), immunological hypersensitivity reactions (allergies) especially bronchial asthma and atopic dermatitis, chronic recurrent inflammatory diseases especially ulcerative colitis and Crohn's disease, in particular psoriasis, and autoimmune genes, and autoimmune diseases and sarcoma, and autoimmune genes, as well as anemia and autoimmune diseases Diabetes mellitus, multiple sclerosis, collagenosis (eg systemic lupus erythematosus), rheumatoid arthritis and vasculitis ..
  • a particular advantage of this therapeutic approach is the simultaneous weakening of the THl and TH2 cell response, in which the CD40 / CD154 signaling pathway has a co-stimulatory effect.
  • the TH1 cell reaction e.g. psoriasis
  • the TH2 cell reaction is dampened (e.g. atopic dermatitis) or vice versa.
  • Human endothelial cells were isolated from umbilical cord veins by treatment with 1.6 U / ml dispase in Hepes-modified Tyrode solution for 30 min at 37 ° C. and on gelatin-coated 6-hole tissue culture dishes (2 mg / ml gelatin in 0.1 M HC1 for 30 min. At ambient temperature) in 1.5 ml M199 medium containing 20% fetal calf serum, 50 U / ml penicillin, 50 ⁇ g / ml streptomycin, 10 U / ml nystatin, 5 mM HEPES and 5 mM TES, 1 ⁇ g / ml Heparin and 40 ⁇ g / ml endothelial growth factor, cultured.
  • the total endothelial RNA was isolated using the Qiagen RNeasy Kit (Qiagen, Hilden, Germany), followed by cDNA synthesis with a maximum of 3 ⁇ g RNA and 200 U Superscript TM II reverse transcriptase (Gibco Life Technologies, Düsseldorf, Germany) in a total volume of 20 ⁇ l according to the manufacturer's instructions.
  • cDNA loading 5 ⁇ l (approximately 75 ng cDNA) of the resulting cDNA solution and the primer pair (Gibco) for elongation factor 1 (EF-1) PCR with 1 U Taq DNA polymerase (Gibco) in a total volume of 50 ⁇ l used.
  • EF-1 served as the internal standard for PCR.
  • PCR products were separated on 1.5% agarose gels containing 0.1% ethidium bromide and the intensity of the bands was determined densitometrically using a CCD camera system and the One-Dscan gel analysis software from Scanalytics (Billerica, MA, USA) determined to adjust the volume of the cDNA in subsequent PCR analyzes.
  • Electrophoretic Mobility Shift Analysis The nuclear extracts and [ 32 P] -labeled double-stranded consensus oligonucleotides (Santa Cruz Biotechnologie, Heidelberg, Germany), non-denaturing polyacrylamide gel electrophoresis, autoradiography and supershift analysis were carried out as in Krzesz et al. (1999) FEBS Lett. 453, 191.
  • Oligonucleotides with the following single-stranded sequences were used (binding sequences are underlined): NFKB, 5'-AGTTGAGGGGACTTTCCCAGGC-3 '(SEQ ID NO: 35); STAT-1, 5'-CATGTTATGCATATTCCTGTAAGT G-3 '(SEQ ID NO: 36); IRF-1,5'-GGAAGCGAAAATGAAATTGACT-3 '(SEQ ID NO: 19).
  • Double-stranded dODN were from the complementary single-stranded phosphorothioate-linked oligonucleotides (Eurogentec, Cologne, Germany) as in Krzesz et al. (1999) FEBS Lett. 453, 191.
  • the cultured human endothelial cells were preincubated for 4 hours at a concentration of 10 ⁇ M of the respective dODN. These were the conditions that had already been optimized based on EMSA and RT-PCR analysis. Thereafter, the medium containing dODN was generally replaced by fresh medium.
  • the single-stranded sequences of the dODN were as follows (underlined letters denote phosphorothioate-linked bases, all in 5 '- 3' direction): NF-KB, AGTTGAGGGGACTTTCCCAGGC (SEQ ID NO: 35);
  • STAT-1 CATGTTATGCATATTCCTGTAAGTG (SEQ ID NO: 36); IRF-1, GGAAGCGAAAATGAAATTGACT (SEQ ID NO: 19;
  • the sequence of the IRF-1 antisense oligonucleotide was 5'-CGAGTGATGGGC-ATGTTGGC-3 '(SEQ ID NO: 23).
  • a missense oligonucleotide (IRF-1 MS, 5'-CGAGTGGTAGACGTATTGGC-3 '(SEQ ID NO: 38)) and a scrambled oligonucleotide (IRF-1 SCR, 5'-GAGCTGCTGAGGTCGTTGAG-3 c (SEQ ID NO : 39)) used.
  • the endothelial cells to be analyzed were first washed three times with 1 ml of FACS buffer (PBS, 2% fetal calf serum, sterile filtered) and then taken up in 2 ml of FACS buffer. After centrifugation (300xg, 5 min., + 4 ° C) and determination of the total cell number (Neubauer cell chamber), the fluorescence-labeled antibody (Pharmingen, San Diego, USA) was added according to the manufacturer (20 ⁇ l / 10 6 cells) and incubated for 30 min at + 4 ° C in the dark. The mixture was then washed with 2 ml of FACS buffer and centrifuged for 10 min at 300xg and + 4 ° C.
  • FACS buffer PBS, 2% fetal calf serum, sterile filtered
  • the supernatant was decanted, the cell pellet resuspended in 1 ml Cell-Fix (PBS, 1%> formaldehyde) and stored in the dark at + 4 ° C until measurement (EPICS®XL-MCL, Coulter, Krefeld, Germany).
  • the following antibodies were used: CD40, R-phycoerythrin (R-PE) - and fluorescein isothiocyanate (F ⁇ TC) conjugated; PECAM-1 (CD31), fluorescein isothiocyanate (F ⁇ TC) conjugated.
  • the corresponding R-PE and FITC-conjugated isotype controls were used to determine non-specific cell-antibody bindings.
  • Protein were denatured using a 10% polyacrylamide gel electrophoresis
  • BioTrace TM polyvinylidene fluoride transfer membrane (Pall Corporation, Rossdorf, Germany) transferred.
  • a polyclonal primary antibody directed against the C-terminus was used to detect CD40 protein.
  • the protein bands were determined after the addition of a peroxidase-coupled anti-rabbit IgG
  • the stem combination Brown Norway donor on Lewis recipient was used for allogeneic transplantation.
  • the transplant was rejected after 7 days without nmunsuppression.
  • the Lewis to Lewis transplant served as syngeneic controls.
  • the aorta was clamped proximal and distal to the outlet of the mesenteric artery, the portal vein was cut at the level of the liver hilum and the vascular bed of the small intestine was rinsed with cold University of Wisconsin (UW) solution until there were no macroscopic blood residues in the vascular bed, in the last step the intestinal lumen was also rinsed with cold UW solution and the intestine was removed with an aortic segment and kept in cold UW solution until implantation (duration up to 120 minutes).
  • UW University of Wisconsin
  • the abdomen was opened in the midline under ether inhalation anesthesia.
  • the aorta and vena cava were shown and simultaneously clamped off.
  • the vessel was connected end-to-side in continuous suturing using an 8-0 nylon thread.
  • the aortic segment bearing the mesenteric artery on the infrarenal aorta and the portal vein on the infrarenal vena cava were anastomosed.
  • the terminal ileum of the donor intestine was also connected end-to-side to the terminal ileum of the recipient intestine using a 6-0 nylon thread.
  • the oral end of the donor intestine was closed by ligature xmd the abdomen was closed in two layers continuously. Postoperatively, the animals received Temgesic in the drinking water for analgesia.
  • the intravital microscopy was carried out with an Axiotech Vario 100 microscope from Zeiss (Göttingen), equipped with an HBO 100 mercury lamp for epifluorescence measurements. With the use of 10x, 20x and 40x (water immersion) lenses, resolutions of 243x, 476x and 933x were calibrated. The microscopic images were recorded with a CCD video camera (CF 8/1, Kappa) and recorded on a video tape for later evaluation.
  • CF 8/1, Kappa CCD video camera
  • the rats Seven days after the transplant, the rats (6 animals per group) became deeper Diethyl ether anesthesia x examined intravital microscopically.
  • the trachea was cannulated to make breathing easier.
  • a polyurethane catheter was placed in the carotid artery to permanently check blood pressure and simplify the application of dyes.
  • the body temperature of the animals was kept constant by a heatable plate.
  • the animals were opened by means of a ventral-median incision, the descending colon was outsourced, a small incision was made antimesenterically and the intestine was fastened in a special holder to facilitate microscopy. In order to prevent the tissue from drying out, the intestine was permanently wetted with Ringer's solution.
  • the intestinal microcirl circulation was visualized by the injection of 0.8 ml 0.5%> FITC- (fluorescein isothiocyanate) coupled dextran.
  • FITC- fluorescein isothiocyanate
  • the various parameters were quantified as follows: The perfusion index resulted from the perfused Mulcosa areas (in%>) + 0.5x of all irregularly perfused Mulcosa areas (in% > ).
  • the functional capillary density was determined by a computer-assisted image analysis (CAP-IMAGE software, Zeintl, Heidelberg).
  • the leukocytes were marked by the injection of 0.2 ml of 0.1% Rhodamine-6 G (Sigma, Heidelberg) and microscopic postcapillary venules in the submu osa.
  • Adherent leukocytes (“stickers”) were defined as leukocytes that adhered to the endothelium in a vessel segment of 100 ⁇ m in length for at least 20 seconds. The number of stickers / mm 2 endothelial surface was calculated. The endothelial surface was obtained from the surface calculation for a cylinder.
  • the mulcosal functional capillary density was reduced in the control group as well as in the group treated with mutated control oligonucleotide to 10% of the values of syngene transplanted small intestines without rejection.
  • the functional capillary density was increased by a factor of 4 in small intestines treated with the Stat-1 cis element Decoy.
  • the blood flow (flow rate of the erythrocytes) in these animals was 10-fold and the perfusion index 3-fold.
  • the stasis index was reduced by 60% and the number of leukocytes adhering to the endothelium by 25%. Only the latter parameter was not statistically significantly changed. Overall, the rejection-related reduction in intestinal perfusion and thus the degeneration of the graft was significantly reduced in the group treated with the cis element Decoy.

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Abstract

L'invention concerne des inhibiteurs du facteur de transcription IRF-1, leur utilisation comme agent thérapeutique ainsi que pour la prévention ou la thérapie de complications cardiovasculaires telles que la resténose après une angioplastie percutanée ou la sténose de pontages veineux, le rejet chronique d'implants (artériosclérose ou vasculopathie liées à une transplantation) ou le rejet aigu d'implants, la réaction du greffon contre l'hôte, les hyperréactivités immunologiques (allergies), notamment l'asthme bronchique et la dermatite atopique, les maladies inflammatoires récurrentes chroniques, notamment la colite ulcéreuse et la maladie de Crohn, le psoriasis et la sarcoïdose, ainsi que les maladies auto-immunes, notamment le diabète sucré, la sclérose en plaques, les collagénoses (par exemple le lupus érythémateux systémique), la polyarthrite rhumatoïde et les vasculatarités.
EP01986318A 2000-10-06 2001-10-04 Modulation de la transcription de produits geniques pro-inflammatoires Withdrawn EP1325122A2 (fr)

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CA2471967A1 (fr) * 2002-01-03 2003-07-31 Board Of Regents, The University Of Texas System Oligonucleotides antisens wt1 inhibant le cancer du sein
DE10240417A1 (de) * 2002-09-02 2004-03-11 Avontec Gmbh Decoy-Oligonukleotid-Hemmung der CD40-Expression
JP2007512847A (ja) * 2003-12-03 2007-05-24 アネシヴァ， インコーポレイテッド Ｈｉｆオリコヌクレオチドデコイ分子
PL2158316T3 (pl) 2007-05-11 2015-10-30 Adynxx Inc Ekspresja genowa oraz ból
CN102803284B (zh) * 2009-06-08 2015-11-25 米拉根医疗公司 用于miRNA抑制剂和模拟物的化学修饰基序
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WO2015095636A2 (fr) * 2013-12-19 2015-06-25 The Children's Hospital Of Philadelphia Leurres du facteur de régulation de l'interféron 1 (irf1) et procédés d'utilisation de ceux-ci
CN103751804B (zh) * 2014-01-23 2015-09-30 武汉大学 干扰素调节因子4(irf4)基因在冠状动脉粥样硬化性心脏病中的应用
JP6705807B2 (ja) 2014-08-15 2020-06-03 エーダイニクス インコーポレイテッド 疼痛を治療するためのオリゴヌクレオチドデコイ
CN104258419A (zh) * 2014-09-29 2015-01-07 武汉大学 干扰素调节因子1基因在治疗动脉粥样硬化中的应用
WO2016113646A1 (fr) * 2015-01-15 2016-07-21 Koninklijke Philips N.V. Tomodensitométrie ifr
CN106620719A (zh) * 2016-10-20 2017-05-10 武汉大学 干扰素调节因子1及其抑制剂在治疗脂肪肝和ⅱ型糖尿病中的功能和应用
WO2020101880A1 (fr) * 2018-11-14 2020-05-22 Oklahoma Medical Research Foundation Compositions et procédés pour le traitement du lupus érythémateux dissémine
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