EP1537209A2 - Correction fonctionnelle de la variante sp -786 /sp c/t du gene i enos /i humain - Google Patents

Correction fonctionnelle de la variante sp -786 /sp c/t du gene i enos /i humain

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Publication number
EP1537209A2
EP1537209A2 EP03750346A EP03750346A EP1537209A2 EP 1537209 A2 EP1537209 A2 EP 1537209A2 EP 03750346 A EP03750346 A EP 03750346A EP 03750346 A EP03750346 A EP 03750346A EP 1537209 A2 EP1537209 A2 EP 1537209A2
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Prior art keywords
dna
sequence
seq
oligonucleotides
enos
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EP03750346A
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German (de)
English (en)
Inventor
Marco Cattaruzza
Markus Hecker
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Avontec GmbH
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Avontec GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/13—Decoys
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to decoy oligonucleotides with the nucleic acid sequence according to SEQ FD NO: 1 to 34 and their use as medicaments.
  • the present invention further relates to a method for diagnosing the ⁇ 786 C / T variance in the eNOS gene.
  • a key goal of decoding the human genome is to identify disease-causing genes (due to the mode of action of their products) or disease-causing changes in the structure of these genes (polymorphisms) and to assign them to a clinical picture.
  • This means that causal therapy for a variety of diseases is within reach if one accepts that these are caused by a defined number of gene products that are expressed too strongly, too weakly or incorrectly.
  • the generally singular genetic defect (monogenetic disease) is already known for certain hereditary diseases (eg cystic fibrosis)
  • the situation for polygenetic diseases eg atherosclerosis
  • the coincidence of different genetic defects predestines the person concerned for the disease , but this only develops after exposure to certain environmental factors. Regardless of this, the targeted intervention in the expression of one or more genes offers the chance of cause-related and not merely symptom-related therapy, even in the case of polygenetic diseases.
  • Atherosclerosis with its primary complications of heart attack, cardiac and renal insufficiency and stroke, is responsible for more than 50% of all deaths in industrialized countries, with this trend continuing to increase. In 2020, atherosclerosis-related cardiovascular diseases will be the most common cause of death worldwide, not least because of increasing life expectancy. In addition to the suspected genetic predisposition, hypertension, hypercholesterolemia, smoking and diabetes (type I and II) are among the primary risk factors for atherosclerosis.
  • Atherosclerosis is a basic systemic disease that can manifest itself at any time in a previously unaffected vascular section.
  • Anti-inflammatory drugs such as glucocorticoids (e.g. prednisone), immune suppressants (e.g. cyclosporin A) and antimetabolites (e.g. azathioprine) sometimes have serious side effects.
  • glucocorticoids e.g. prednisone
  • immune suppressants e.g. cyclosporin A
  • antimetabolites e.g. azathioprine
  • myelotoxicity neurotoxicity
  • nephrotoxicity metabolic disorders up to the induction of diabetes mellitus, arterial hypertension, infections and malignancies.
  • chronic inflammatory or autoimmune diseases include chronic obstructive bronchitis and emphysema (summarized as chronic obstructive pulmonary cancer or COPD; prevalence in Germany 4-1%), psoriasis (2-3%>), rheumatoid arthritis (chronic polyarthritis; 0.8%) and insulin-dependent diabetes mellitus (type I; 0.5%).
  • COPD chronic obstructive bronchitis and emphysema
  • psoriasis (2-3%>
  • rheumatoid arthritis chronic polyarthritis
  • insulin-dependent diabetes mellitus type I; 0.5%).
  • the infiltration of these immune cells into the vascular wall also plays an important role in the pathogenesis of atherosclerosis.
  • Further examples of an overactivation of Thl cells are transplant rejection, contact dermatitis and persistent secondary diseases of bacterial or viral infections.
  • the object of the present invention is therefore to provide means for prevention, therapy or diagnosis of the diseases mentioned.
  • the object is achieved by the subject-matter defined in the patent claims.
  • FIG. 1 shows the sequence of the human nitrogen monoxide (NO) synthase (e ⁇ S) gene in the region of the T to C transition at position -786.
  • the underlined bases indicate the consensus binding sites for two known transcription factors.
  • WSS wall shear stress
  • FIG. 3 shows in a bar chart the restoration of the shear stress induction of the eNOS expression in cultured endothelial cells from donors with a ⁇ 786 C / C genotype, who for 4 hours with a C type (SEQ ID NO: 1) but not with a T- Type allele decoy oligonucleotide (SEQ ID NO: 3) had been pretreated (concentration in the medium of 10 ⁇ mol / 1).
  • WSS wall shear stress
  • FIG. 5 shows in bar graphs the effects of IL-10 (2 ng / ml) induced increased eNOS expression on CD154-stimulated (2 ⁇ 10 5 mouse myeloma cells ml) de novo synthesis of IL-12 p40 on (A) mRNA and ( B) Protein level over 6 hours in IL-10 receptor-expressing cultured endothelial cells from donors with ⁇ 786 ⁇ / T or " ⁇ 786 C / T genotype and their modulation by blocking the eNOS activity by nitroarginine.
  • FIG. 6 shows in a bar graph the lack of inhibition of CD154-stimulated IL-12 p40 expression in IL-10 receptor-expressing cultured endothelial cells from donors with a ⁇ 786 C / C genotype and their restoration by pretreating the cells (4 hours, 10 ⁇ mol / 1) with the C-type allele decoy oligonucleotide (SEQ ID NO: 5).
  • SEQ ID NO: 5 the C-type allele decoy oligonucleotide
  • FIG. 7 shows an example of the results of the real-time PCR / fluorescence resonance energy transfer (FRET) / DNA melting curve analysis developed for genotyping for the three variants of the ⁇ 786 C / T polymorphism of the human
  • FIG. 8 shows schematically two variants of the real-time PCR / FRET / DNA melting curve analysis for diagnosing the " C / T variance in the eNOS gene.
  • the circled numbers stand for the SEQ ID NO of the anchor or detection samples according to the invention (FL , Fluorescein; LC 640, LC-Red 640).
  • the term "decoy oligonucleotide” or “cis-element decoy” as used herein denotes a double-stranded DNA molecule which has a sequence which corresponds to or is similar to the natural nuclear binding sequence of a DNA-binding protein or protein complex in the genome and to which the protein or the protein complex binds in the cell.
  • the cis element decoy thus acts as a molecule for the competitive inhibition (better neutralization) of the protein or protein complex, which expression of the eNOS, a gene with a proven important protective function, if the C / C genotype is present blocked and thus predisposes to the formation of the diseases and complications defined in the patent claims.
  • C / T variance used here denotes the presence of a heterozygous or homozygous T to C transition at position -786 of the human eNOS gene. Accordingly, the term “C / C genotype” used here is the homozygous T to C transition and the C / T genotype is the heterozygous T to C transition.
  • NO nitrogen monoxide
  • the synthesis of nitrogen monoxide (NO) by the endothelial cells of the vascular wall plays an important role in the regulation of organ blood flow. and in maintaining vascular wall integrity.
  • the latter effect of NO is based in particular on the inhibition of the proliferation of smooth vascular muscle cells.
  • NO inhibits the expression of chemokines and cell adhesion molecules in the endothelium. This suppresses the recruitment, activation and transmigration of leukocytes circulating in the blood in inflammatory processes.
  • NO u. a. played an important role in the prevention of atherosclerosis, which, according to current understanding, is a chronic recurrent inflammatory disease of the vascular wall.
  • endothelial NO synthase eNOS
  • the physiologically most important stimulus for the activity of the enzyme is a change in the wall shear stress. This is the viscous tension exerted by the flowing blood on the endothelial cells that line the arteries and veins inside.
  • an increase in the wall shear stress triggered by a constriction of the blood vessel or an increase in the blood flow, leads to an increase in the NO synthase activity and consequently to an NO-mediated vasodilation, ie an increase in the Organ perfusion.
  • Interleukin-10 is an important anti-inflammatory cytokine for chronic recurrent inflammatory diseases such as Psoriasis, rheumatoid arthritis or Crohn's disease, which are caused by excessive activation of type 1 T helper cells (Moore et al. (2001) Annu. Rev. Immunol. 19, 683).
  • the infiltration and activation of Thl cells in the vascular wall also plays an important role in the pathogenesis of atherosclerosis (Daugherty and Rateri (2002) Circ. Res. 90, 1039).
  • IL-10 inhibits the formation of IL-12 in the antigen presenting cells communicating with the Thl cells.
  • endothelial cells are also able to express the MHC II molecules required for the antigen presentation on their surface and to release biologically active IL-12.
  • IL-12 itself is the most important factor for the differentiation of naive T helper cells to Thl cells and stimulates their proliferation (clonal expansion).
  • the synthesis of IL-12 in human endothelial cells can only be induced after costimulation via the CD40 receptor / CD40 ligand system (Lienenlüke et al. (2000) Eur. J. Immunol. 30, 2864).
  • the endothelial cells usually express the receptor (CD40) and activated Thl cells the CD40 ligand (also known as CD154).
  • the human eNOS gene has a number of polymorphisms in which a single base is exchanged (SNP for single nucleotide polymorphism), including a T to C transition in the promoter of the gene at position -786 (FIG. 1).
  • SNP single nucleotide polymorphism
  • FIG. 1 Linkage analyzes show that this base exchange does not occur in isolation, but is always associated with an A to G transition at position -922 and a T to A transition at position -1486.
  • T to C transition at position -786 which has now been elucidated by the inventors, the functional significance of these polymorphisms is not yet known (Wattanapitayakul et al. (2000) Trends Pharmacol. Sci. 22, 361).
  • coronary heart disease refers to the (manifest) atherosclerosis formed in the coronary arteries, which leads to a progressive narrowing of the affected vessels.
  • the consequences of this circulatory disorder in the coronary arteries are (in order of severity in ascending order) the stable angina pectoris (the formation of bypass arteries, so-called collaterals, at least partially compensates for the atherosclerosis-related circulatory disorder, the patients only become symptomatic, for example, when they are under physical stress Chest pain), the unstable angina pectoris (increasing blood vessel congestion caused by blood clots (thrombosis), the patients already have chest pain at rest, about 25% of them suffer a myocardial infarction within 4 weeks), the myocardial infarction itself (total occlusion of a coronary artery by a blood clot ) as well as sudden cardiac death (severe circulatory disorders due to acute closure of one or more coronary arteries with cardiac arrhythmia, which lead to cardiac arrest
  • the coronary heart disease and the coronary insufficiency triggered by it is primarily responsible for the manifestation of a heart muscle weakness (heart failure).
  • coronary artery disease continues to be the leading cause of death in western industrialized nations.
  • the discovery of genetic risk factors for the development of coronary artery disease is of great socio-economic importance.
  • the very rare spontaneous cramping of one or more coronary arteries differs from CAD in that spontaneous cramping is the extreme form of dynamic coronary stenosis (eccentric stenosis with constriction of the plaque-free patient wall) with unstable angina pectoris.
  • CAD cardiovascular disease
  • vasospastic angina pectoris eccentric stenosis with constriction of the plaque-free patient wall
  • unstable angina pectoris eccentric stenosis with constriction of the plaque-free patient wall
  • .Patients with vasospastic angina pectoris are often smokers and usually have other vascular phenomena (migraines, Raynaud's disease).
  • the pathogenesis of vasospastic angina pectoris has not yet been clarified. Autonomic nervous system involvement is suspected.
  • the inventors were able to demonstrate that in cultured endothelial cells that had been isolated from the umbilical cord vein of donors with the C / C genotype, the typical increase in eNOS expression at mRNA as well as at protein level caused by an increase in wall shear stress does not occur ( Figure 2).
  • the transcription is controlled by proteins that bind to the starter region of a gene (promoter region).
  • the correct assembly of several of these transcription factors causes an activation of the RNA polymerase and initiates the transcription.
  • binding an incorrect transcription factor can prevent the formation of the transcriptosome and thus block the expression of the gene.
  • Decoy oligonucleotides mimic the sequence motif to which the target transcription factor binds in the starter region of its target gene (s) and neutralize it. As a result, the induction or repression of the transcription mediated by this transcription factor is prevented.
  • human endothelial cells express the receptor for IL-10 after exposure to various pro-inflammatory cytokines, ie under (simulated) inflammation conditions, as well as after treatment with vitamin D3.
  • the stimulation of the receptor ie incubation of the cells expressing IL-10 receptor - with IL-10, resulted in a significant increase in eNOS expression in the endothelial cells (FIG. 4); an effect that could be attributed to the activation of the transcription factor signal transducer and activator of transcription (STAT) -3 with the help of electrophoretic mobility shift analyzes and the use of appropriate decoy oligonucleotides.
  • IL-10 can develop its anti-inflammatory effect in Thl-weighted chronic inflammatory diseases, in particular the inhibition of IL-12 synthesis in the endothelial cells, by increasing the eNOS expression and the associated increased NO formation.
  • the STAT binding site in the promoter of the human eNOS gene, which is important for the IL-10 effect, is approximately 60 base pairs above the T to C transition at position -786.
  • ⁇ C / T polymorphism has an effect on the inducibility of eNOS expression by IL-10.
  • IL-10 exposure did not inhibit CD154-induced synthesis of IL-12 in these cells, but actually increased it (FIG. 6). Due to the spatial proximity, the formation of the transcriptosome in the region of the STAT binding site is inhibited by the inhibitory transcription factor binding at position -786.
  • the intra-articular application of a decoy oligonucleotide during the inflammatory flare can sustainably strengthen or enable the endogenous IL-10-mediated inhibition of the Thl cell response and thus weaken the inflammatory process and the associated cartilage destruction in the affected joint.
  • Thl-weighted chronic inflammatory diseases such as Crohn's disease
  • the tendency to show a significantly higher prevalence of the ⁇ 786 C / C genotype (Table 2).
  • the systemic application of a liposomally packaged decoy oligonucleotide is likely to provide the greatest therapeutic benefit.
  • the present invention therefore relates to the provision of a decoy oligonucleotide which is in is able to bind sequence-specifically to a protein or a protein complex (hereinafter referred to as "transcription factor” or “inhibitory transcription factor”).
  • the sequence of the decoy oligonucleotide used to prevent binding of the inhibitory transcription factor is the sequence to which the transcription factor binds in the promoter of the eNOS gene.
  • the cis-element decoy can also be larger than the 10-mer nucleus binding sequence and can be extended at the 5 'end and / or at the 3 J end.
  • Corresponding mutations in the area of the nucleus binding sequence eg 5'-CTAGCTGACT-3 ' lead to a complete loss of the binding of the transcription factor to the decoy oligonucleotide, visible from the lack of biological action (Table 3).
  • the present invention further relates to the provision of decoy oligonucleotides which are capable of binding to the transcription factor in a sequence-specific manner and which have one of the following sequences, only one strand of the decoy oligonucleotides being reproduced here and the complementary strand also being included :
  • the use of the decoy oligonucleotides or cis-element decoys according to the invention which contain a consensus core binding site for the inhibitory transcription factor represents the preferred method for specifically inhibiting the activity of this factor.
  • Squelching or also called neutralization
  • DNA fragments were used which contain specific binding sites for the transcription factor E2F (Mann et al. (1999) Lancet 354, 1493).
  • the present invention thus relates to a method for specifically inhibiting the activity of the inhibitory transcription factor which binds in the promoter region of the eNOS gene at position -786 ' , comprising the step of administering a decoy oligonucleotide according to the present invention.
  • the present invention further relates to the decoy oligonucleotides according to the invention as medicaments.
  • the present invention further relates to the use of the decoy oligonucleotides according to the invention for the manufacture of a medicament, in particular for the prevention or therapy of atherosclerosis and its secondary diseases (for example coronary heart disease with a heart attack and heart failure, cerebral circulatory disorders with a stroke or multi-infarct dementia, and peripheral arterial occlusive disease ), chronic inflammatory or autoimmune diseases such as rheumatoid arthritis (chronic polyarthritis), psoriasis including psoariasis arthritis, allergic contact eczema and atopic eczema (neurodermatitis), chronic inflammatory bowel diseases (especially Crohn's disease and ulcerative colitis), type I and II diabetes and the like Consequences (e.g.
  • diabetic nephropathy, retinopathy and vasculopathy multiple sclerosis, sarcoidosis, collagenosis and vasculitis (including glomerular epithitis), acute and chronic rejection transplanted organs, graft versus host disease (GVHD) and the ischemia / reperfusion damage of organs after a surgical intervention, the vasculopathy of venous bypasses, (pre-) eclampsia or pregnancy-induced hypertension, arterial hypertension and its secondary diseases (e.g.
  • left heart hypertrophy aneurysm formation with the risk of bulging hemorrhages and arteries) and arterioles
  • pulmonary hypertension chronic renal failure
  • chronic obstructive pulmonary diseases COPD
  • bacterial infections and their complications e.g. Helicobacter pylori gastritis, tuberculous pericarditis, Lyme disease with subsequent Borrelia arthritis or neuroborreliosis complications
  • complications infections with cytomegalovirus, hepatitis B and C herpes and HI (human immunodeficiency) yives such as portal hypertension and fibrosis or opportunistic infections, in particular Pneumocystis carinii pneumonia.
  • decoy oligonucleotides according to the invention effectively neutralize the inhibitory transcription factor in vitro, it is crucial for the therapeutic effectiveness that the DNA molecule is quickly and sufficiently absorbed into the target cell.
  • the cis element decoy according to the invention should not exceed a certain length, since this is limiting for the transport into the target cell.
  • Any decoy oligonucleotide with a length of at least 10 bp is suitable up to a length of approximately 30 bp, preferably up to a length of approximately 27 base pairs, particularly preferably up to a length of approximately 23, particularly preferably with a length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, - 20, 21, 22 or 23 base pairs.
  • the decoy oligonucleotide according to the invention not only comprises the sense or forward sequence but also the complementary antisense or reverse sequence.
  • Preferred decoy oligonucleotides according to the invention have a 10-mer core binding sequence for the inhibitory transcription factor, as is contained in SEQ ID NO: 33.
  • the cis element decoy can also have a sequence that deviates from the above sequence and can be longer than a 10-mer.
  • the sequences as contained in SEQ JD NO: 1 to SEQ JD NO: 34 are particularly preferred. This list of the preferred sequences is not exhaustive. It will be apparent to those skilled in the art that a variety of sequences can be used as a transcription factor inhibitor as long as they meet the above conditions listed for the 10-mer consensus core binding sequence and an affinity for the transcription factor.
  • the affinity for the binding of a nucleic acid sequence to this transcription factor can be determined by 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 the transcription factor.
  • 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. eNOS, are increasingly expressed.
  • Increased expression in the context of the present invention means that the transcription rate is increased in comparison to cells which are not treated with a decoy oligonucleotide according to the invention.
  • Such an increase can be determined, for example, by Northern blot (Sambrook et al., 1989) or RT-PCR (Sambrook et al., 1989).
  • Such an increase is typically at least a 2-fold, in particular at least a 5-fold, in particular at least a 10-fold inhibition of gene expression.
  • oligonucleotides are rapidly broken down in the cell by endo- and exonucleases, in particular DNases and RNases.
  • the nucleic acids can therefore be modified in order to stabilize them against degradation, so that a high concentration of the oligonucleotides is maintained in the cell over a longer period of time.
  • stabilization can be obtained by introducing one or more modified internucleotide linkages.
  • a successfully stabilized DNA oligonucleotide or decoy oligonucleotide does not necessarily contain a modification to every intemucleotide linkage.
  • the internucleotide 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 internucleotide linkages can be within the last six internucleotide linkages be modified.
  • various modifications of the internucleotide linkages can be introduced into the nucleic acid and the resulting decoy oligonucleotides can be tested for sequence-specific binding to the inhibitory transcription factor 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 internucleotide binding which still show sufficient binding, can be checked whether they are more stable in the cell than 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 35 S) 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 cis-element decoy, preferably at least about 48 hours, more preferably at least about 4 days, most preferably at least about 7 days.
  • Modified internucleotide-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 internucleotide analogs , for example 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 intemucleotide 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.
  • a cis-element decoy of the present invention is rapidly taken up into the cell. Adequate uptake is characterized by modulating the expression of one or more genes that are controlled by the target transcription factor (e.g. eNOS).
  • 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 diagnostic method according to the invention which preferably comprises a real-time PCR and subsequent melting curve analysis or a restriction fragment length polymorphism analysis (RFLP analysis).
  • the diagnostic methods according to the invention in patients who have to undergo a surgical intervention in which, for example, the blood supply to one or more organs is briefly interrupted or one or more autologous vascular grafts are transferred (e.g. aortocoronary venous bypass surgery) during and during Following the surgical intervention, a treatment with the decoy oligonucleotides according to the invention and / or with drugs which increase the NO synthesis capacity of the vascular endothelium or increase the bioavailability of nitrogen monoxide in the organism, such as angiotensin conversion enzyme (ACE) inhibitors, HMG- CoA reductase inhibitors, antioxidants or Nitric oxide releasing vasodilators.
  • ACE angiotensin conversion enzyme
  • genotyping with the diagnostic methods according to the invention should also be carried out with donors of organs in order to transfer the graft between explantation and implantation (ie ex vivo) and / or the organ recipient for a period of at least 3 days, more preferably at least about 2 weeks most preferably to be treated for at least about 6 months with the decoy oligonucleotides according to the invention and / or the medicaments mentioned above.
  • the diagnostic methods according to the invention can be used to estimate the individual risk of a person to develop one of the diseases associated with the _786 C / T variance of the eNOS gene and the carriers of the ⁇ 786 C / C genotype in the sense of a prevention strategy , especially with a view to avoiding further risk factors.
  • the diagnostic methods according to the invention can be used to deliver carriers of the C / C genotype - as an alternative or in addition to the treatment with the decoy oligonucleotides according to the invention - to therapy with drugs which increase the NO synthesis capacity of the endothelial vessel or the bioavailability of Increase nitric oxide in the organism, such as Angiotensin conversion enzyme (ACE) inhibitors, HMG-CoA reductase inhibitors, antioxidants or nitric oxide releasing agents
  • ACE Angiotensin conversion enzyme
  • the present invention thus relates to methods for diagnosing a "786 C / T variance in the human eNOS gene, comprising the steps: adding DNA oligonucleotides to a patient's DNA sample, wherein a DNA oligonucleotide has a sequence that is upstream is the -786 position of the eNOS gene and corresponds to the sense strand, and another DNA oligonucleotide has a sequence that is downstream of the -786 position of the eNOS gene and corresponds to the antisense strand, performing a polymerase chain reaction (PCR), performing a D ⁇ A cleavage with a restriction enzyme which has a recognition sequence which is at least 4 nucleotides long and which contains the sequence 5'-CCGG-3 'but not the sequence 5'-CTGG-3', and detection of the DNA fragments obtained by DNA cleavage
  • the DNA oligonucleotides serve as primers for the PCR.
  • the eNOS sequence means the sequence listed under the GenBank Accession Number L10693 (gi: 348219), which is also referred to here as the sense strand and corresponds to the strand which contains the RNA sequence of the eiVOS gene product.
  • the start is labeled +1, so position -786 is upstream of the start point.
  • the antisense strand is the strand complementary to the sense strand, which is also referred to as the matrix.
  • the DNA oligonucleotides can have any length that is required for a PCR.
  • the DNA oligonucleotides can furthermore have such sequences from the eNOS gene or the 5 'region of the eNOS gene which allow a PCR to be carried out.
  • the primers must be spaced far enough apart so that the amplificates and the cleaved amplificates can be detected, but not to the extent that no more amplificates are generated.
  • the primers should be selected in such a way that DNA fragments with such a different size result from DNA cleavage and are easy to detect.
  • the primers preferably have the sequences according to SEQ JD NO: 35 and 36.
  • the restriction enzyme Hpall and the detection method are preferably agarose gel electrophoresis or capillary electrophoresis.
  • the present invention further relates to a kit for carrying out the method according to the invention, comprising the above DNA oligonucleotides (primers), reagents for carrying out a PCR, preferably including the Taq polymerase, a restriction enzyme and reagents for carrying out a DNA cleavage.
  • the present invention further relates to a method for the rapid determination of the " C / T variance of the human eNOS gene, comprising the following steps: addition of DNA oligonucleotides to a patient's DNA sample, wherein a DNA oligonucleotide has a sequence which is located upstream of the -786 position of the e ⁇ S gene and corresponds to the sense strand (primer 1), another fluorescent dye-modified DNA oligonucleotide has a sequence which comprises the -786 position of the eNOS gene and the sense or corresponds to the antisense strand and is complementary to the "786 C variant of the e / VCW gene promoter (detection sample), a further fluorescent dye-modified DNA oligonucleotide has a sequence which corresponds to the sense or antisense strand (anchor sample) and where the 3 'end of the anchor sample is 1-5 nucleotides upstream of the 5' end of the detection sample, if the anchor and
  • Primers 1 and 2 can be of any length required for PCR.
  • the primers can also have such sequences from the eNOS gene or the 5 'region of the eNOS gene which allow PCR to be carried out.
  • the primers must be spaced far enough apart so that it is possible to detect the amplified products and, if appropriate, the cleaved amplified products, but not to the extent that no more amplified products are produced.
  • Primer 1 preferably has the sequence 5'-CTGGGAACTGTAGTTTCCCTAG-3 'according to SEQ JD NO: 56 and primer 2 has the sequence 5'-ACCCTGTCATTCAGTGACGCAC-3' according to SEQ JD NO: 57.
  • Both the anchor sample and the detection sample used in the method are preferably complementary to the sense strand and have the sequence according to SEQ JD NO: 37 (5'- GGGTCAGCCGGCCAGGGAA-3 ') for the detection sample and the sequence according to SEQ JD NO: 38 ( 5'-AGCTTGATGCCCTGGTGGGAG-3 ') for the anchor sample.
  • the detection sample is covalently coupled to a fluorescent dye at the 3 'end (eg fluorescein, excitation wavelength 494 nm).
  • the anchor sample is covalently coupled at the 5 'end to a fluorescent dye that differs from that of the detection sample (eg LC Red-640, emission wavelength 640 nm).
  • the anchor sample is phosphorylated at the 3 'position of the last deoxyribose, preferably with PO 4 3 ⁇ .
  • the anchor sample has a melting temperature of 5-10 ° C above the melting point of the detection sample.
  • the dye combination is selected so that the excised dye of the detection sample is suitable for efficiently exciting the dye of the anchor sample using FRET so that it emits light of a defined wavelength as long as both samples are in close proximity to one another. Both the detection sample and the anchor sample are not suitable for PCR, since both are not enzymatic due to their chemical modifications in the 3 'or 5' position Allow chain extensions.
  • both the anchor sample and the detection sample used in the method are complementary to the antisense strand and have the sequence according to SEQ JD NO: 59 (5'-TTCCCTGGCCGGCTGA-3 ') for the detection sample and the sequence according to SEQ JD NO: 58 (5'-GCTCCCACCAGGGCATCAAGCT-3 ') for the anchor sample.
  • the detection sample is covalently coupled to a fluorescent dye (eg LC Red-640) at the 5 'end.
  • the anchor sample (SEQ JD NO: 58) is covalently coupled at the 3 'end to a fluorescent dye that differs from that of the detection sample (eg fluorescein).
  • the detection sample is also phosphorylated at the 3 'position of the last deoxyribose, preferably with PO 4 .
  • the anchor sample has a melting temperature of 5-10 ° C above the melting point of the detection sample.
  • the combination of dyes is selected so that the excised dye of the anchor sample is suitable to efficiently excite the dye of the detection sample by FRET in such a way that it emits light of a defined wavelength as long as both samples are in close proximity to one another.
  • the anchor sample preferably has a sequence according to SEQ LD NO: 38 or 58 and the detection sample has a sequence according to SEQ JD NO: 37 or 59.
  • DNA oligonucleotides for example, with other sequences are used as detection or anchor samples can be listed as specifically here.
  • the detection and anchor samples used in the method according to the invention do not necessarily have to bind the same strand, but for example the detection sample can bind the sense strand and the anchor sample the antisense strand or vice versa. In such a case, it is not necessary to select the sequences of the samples so that the samples are positioned in the immediate vicinity.
  • the above-described primers 1 and 2 and the detection After the PCR amplification, mediated by primers 1 and 2, of a fragment of the eiVOS 'prornoter which contains position -786 of the gene, the temperature of the mixture is raised so that all DNA double strands denature the temperature is lowered again so that detection and anchor samples can be scanned with the sense or Antisense strands of the amplified fragment hybridize, depending on whether the sample combination is complementary to the sense or antisense strand (see FIG. 8).
  • the melting curve is obtained by exciting the dye of the detection sample and continuously measuring the fluorescence of the anchor sample while increasing the temperature (for example about 0.1 ° C per second).
  • This takes advantage of the fact that the transfer of the excitation energy of the dye of the detection sample to that of the anchor sample only works as long as both samples have bound to the sense strand.
  • the T and the C variant by the temperature at which the detection sample melts off the sense strand, that is to say the FRET-induced fluorescence decreases significantly. This is the case for fragments of the ⁇ 786 T ⁇ variant due to the GT mismatch occurring about 12 ° C earlier than for sense strands of the
  • the melting curve is obtained by excitation of the dye of the anchor sample and continuous measurement of the fluorescence of the detection sample with a simultaneous increase in the temperature (for example about 0.1 ° C per second).
  • a simultaneous increase in the temperature for example about 0.1 ° C per second.
  • a fluorescence-based real-time PCR device (for example the LightCycler from Röche) is preferably used for the detection.
  • the person skilled in the art can see that not only the determination of a genotype in a PCR, but also the determination of more than one genotype in a PCR, in a so-called multiplex PCR, can be carried out simultaneously.
  • the present invention further relates to DNA oligonucleotides with the sequence according to SEQ JD NO: 35 to 38 and 56 to 59.
  • the present invention furthermore relates to a kit for carrying out the method according to the invention, comprising the above primers 1 and 2, two further DNA oligonucleotides (detection sample and anchor sample), reagents for carrying out a PCR, preferably including Taq polymerase, and reagents for carrying out a melting curve analysis ,
  • the present invention further relates to a method for modulating the transcription of at least one gene in cells, in particular in eNOS-expressing cells such as endothelial cells, epithelial cells of the lungs, kidneys and the female genitourinary tract, heart muscle cells, thrombocytes and neuronal cells (hippocampus), the method includes the step of contacting said cells with a mixture comprising one or more decoy oligonucleotides according to the invention which are capable of binding to the inhibitory transcription factor in a sequence-specific manner.
  • a preferred method is e.g. intra-articular injection of the nucleic acid-containing mixture into one or more joints in patients with rheumatoid arthritis.
  • the mixture containing the cis-element decoys according to the invention is brought into contact with the target cells (e.g. endothelial cells).
  • the goal of this contacting is to transfer the cis-element decoys that bind the inhibitory transcription factor to the target cell (e.g., the eNOS-expressing endothelial cell). Therefore, nucleic acid modification and / or additives or adjuvants 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 brought into contact with the target cells essentially contains only nucleic acid and buffer.
  • concentration range suitable for decoy oligonucleotides is 0.1 to 100 ⁇ mol / 1, preferably 0.5 to 25 ⁇ mol / 1 and particularly preferably 10 ⁇ mol / 1.
  • One or more suitable buffers can be added.
  • Such a buffer is a modified Ringer's solution containing 145 mmol / 1 Na + , 5 mmol 1 K + , 11 mmol / 1 Cl " , 2 mmol / 1 Ca 2+ , 1 mmol / 1 Mg 2+ , 10 mmol / 1 Hepes, 145 mmol / 1 isethionate, 10 mmol / 1 D-glucose, pH 6.5.
  • the mixture additionally contains at least one additive and / or auxiliary.
  • Additives and / or adjuvants such as lipids, 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 a subset of cells, to prevent degradation of the nucleic acid in the cell, and to facilitate storage of the nucleic acid mixture before 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 method of the present invention is preferably applied topically by injection, infusion, catheter, pluronic gels, sustained-release polymers, or any other device that allows local access.
  • the ex vivo use of the mixture (infusion or incubation) used in the method of the present invention also allows local access.
  • genomic DNA was isolated from the blood of patients (approx. 2 ml) and this by means of PCR amplification and subsequent RFLP analysis (see below) for variance.
  • CHD coronary artery disease
  • RFLP analysis RFLP analysis
  • two existing collectives could be used.
  • anonymized (numerical code) DNA samples from patients in the rheumatism clinic of the University of Göttingen and the rheumatology department of the Freiburg University Hospital were tested for the variance.
  • Both groups offer a typical distribution of patients with rheumatoid arthritis, ie the affected people are approximately 80% female.
  • the age profile ranged from 20 years to 80 years with peaks in the number of patients at 35-40 years (early onset) and 60-70 years (late onset).
  • DNA from the umbilical artery of newborns from Göttingen and the surrounding area was used as a comparison collective for the distribution of genotypes. These samples were also anonymized and the children's parents had previously given a written declaration of consent for the corresponding use of the umbilical cord. Anonymous genomic DNA samples were available for the genotyping of patients with multiple sclerosis or Crohn's disease (Charite Berlin University Hospital, Institute of Neuro-Immunology and the University of Göttingen, Immunology Department).
  • the C variant of the human e / V ⁇ S gene promoter has an additional restriction interface for the restriction enzyme Hpa II and is therefore accessible to a classic restriction fragment length polymorphism analysis (RFLP analysis).
  • RFLP analysis restriction fragment length polymorphism analysis
  • the above-mentioned variance is also available in real-time PCR followed by FRET analysis.
  • genomic DNA obtained from blood with the aid of the QIAamp DNA Mini-Kit from Qiagen, Hilden was used with the aid of OS promoter-specific primers (forward primer 5'-GAGTCTGGCCAACACAAATCC-3 '(SEQ JD NO : 35); reverse primer 5'-GACCTCTAGGGTCATGCAGGT-3 ') (SEQ JD NO: 36) by polymerase chain reaction (PCR) a DNA fragment (657 base pairs (bp); from position -1135 to -456 of the human eNOS gene) and then subjected to a specific hydrolysis by the restriction endonuclease Hpa JJ.
  • OS promoter-specific primers forward primer 5'-GAGTCTGGCCAACACAAATCC-3 '(SEQ JD NO : 35); reverse primer 5'-GACCTCTAGGGTCATGCAGGT-3 ') (SEQ JD NO: 36) by polymerase chain reaction (PCR) a DNA fragment (657 base pairs (bp); from position
  • the resulting DNA fragments were analyzed by agarose gel electrophoresis. Depending on the genotype, 2 or 3 smaller DNA fragments result from the 657 bp fragment. While the T variant of the promoter has only one Hpa II interface in the section under consideration and thus breaks down into two fragments (284 bp and 373 bp) after hydrolysis by Hpa JJ, the additional interface of the C variant causes 3 fragments arise (46 bp - not detectable in the electrophoresis system used - 284 bp and 327 bp).
  • the three possible genotypes ( ⁇ C / C, T / T and C / T) were clearly identified by electrophoresis using the following fragment patterns: ⁇ 786 T / T: 284 and 373 bp; ⁇ 786 C / C: 284 and 327 bp; and _786 C / T: 284, 327 and 373 bp.
  • genomic DNA obtained from blood with the aid of the QIAamp DNA Mini-Kit from Qiagen, Hilden
  • eNO £ promoter-specific primers forward primer 5'-CTGGGAACTGTAGTTTCCCTAG-3 '(SEQ ID NO: 56 ); reverse primer 5'-ACCCTGTCATTCAGTGACGCAC-3 '(SEQ JD NO: 57) by PCR in a real-time PCR device (LightCycler from Röche) a DNA fragment (137 bp); from position - 848 to -711 des human eNOS gene, GenBank Accession ⁇ o. L10693) amplified.
  • Human endothelial cells were isolated from umbilical cord veins from previously genotyped donors 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 minutes at ambient temperature) in 1.5 ml M199 medium (Gibco Life Technologies, Düsseldorf, Germany), containing 20%.
  • fetal calf semen 50 U / ml penicillin, 50 ⁇ g / ml streptomycin, 10 U / ml nystatin, 5 mmol / 1 HEPES and 5 mmol / 1 TES, 1 ⁇ g / ml heparin and 40 ⁇ g / ml endothelial growth factor.
  • vWF von Willebrandt factor
  • FACS fluorometric detection
  • the mouse myeloma cell line P3xTBA7 (stably transfected with human CD40 ligand) and P3x63Ag8.653 (untransfected control cells) were in RPMI 1640 medium (Life Technologies) containing 10% fetal calf serum, 50 U / ml penicillin, 50 ⁇ g / ml streptomycin and 10 U / ml of nystatin.
  • the application of wall shear stress (generally 30 dyn / cm 2 for 24 to 36 hours) to the cultured endothelial cells was carried out using a cone-plate viscometer that can be placed in the incubator, as described in Schubert et al. (2000) Circ. Res. 87, 1188 described, with the exception of the dimension of the culture dishes (3.5 cm in contrast to 10.0 cm diameter).
  • cDNA amounts in the different samples 5 ⁇ l of the 1:10 diluted cDNA solution (corresponding to an RNA equivalent of 25-125 ng) of a PCR reaction (1 U Taq DNA polymerase ([Life Technologies] ) with specific primers for the constitutively expressed gene products glyceraldehyde phosphate dehydrogenase (GAPDH) and ribosomal protein L32 (rpl32), using GAPDH and rpl32 as internal calibration standards.
  • GAPDH glyceraldehyde phosphate dehydrogenase
  • rpl32 ribosomal protein L32
  • PCR products were contained on 1.5% agarose gels containing 0 , 1% ethidium bromide was separated 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) in order to adjust the volume of the cDNA in subsequent PCR analyzes.
  • IL-12p40 -281 bp 30 cycles, 62 ° C, 5'-GTACTCCACATTCCTACTTCT-3 '(SEQ JD NO: 41), 5'-TTTGGGTCTATTCCGTTGTGTC-3' (SEQ JD NO: 42) IL-10 receptor - 565 bp , 32 cycles, 59 ° C, 5'-GGACACCCATCCCAAATCAGTC-3 '(SEQ FD NO: 43), 5'-CACGGTGAAATACTGCCTGGTG-3' (SEQ ID NO: 44)
  • GAPDH - 571 bp 19 cycles, 58 ° C, 5'-TCACCATCTTCCAGGAGCG-3 '(SEQ ID NO: 45), 5'- CTGCTTCACCACCTTCTTGA-3' (SEQ JD NO: 46) rpl32 - 368 bp, 20 cycles, 60 ° C, S ⁇ -GTTCATCCGGCACCAGTCAG-S SEQ JD NO: 47), 5'- ACGTGCACATGAGCTGCCTAC-3 '(SEQ ID NO: 48)
  • NM 000603, 129 bp, position 1151 to 1279 were generated using the forward primer 5 '-GGATGTGGCTGTCTGCATGGAC-3' (SEQ JD NO: 60) and the reverse primer 5 ' -TGGTCCACGATGGTGACTTTG 3 '(SEQ JD NO: 61) amplified.
  • the cDNA for GAPDH GenBank Accession No. BT006893, 138 bp. Position 525 to 662
  • the program to amplify both gene products consisted of a 15 minute incubation at 95 ° C followed by 40 cycles of 5 s at 95 ° C, 25 s at 58 ° C and 10 s at 72 ° C.
  • the melting curve was programmed with a slope of 0.2 ° C / s from 65 to 95 ° C and the fluorescence intensity was measured after each cycle.
  • a standard curve for both gene products was used for the quantitative analysis serial dilutions of the corresponding PCR fragments (10 2 to 5xl0 8 copies / reaction) also measured.
  • the gene products were cloned into the plasmid pCR-TOPO (Invitrogen, Düsseldorf, Germany).
  • Double-stranded decoy oligonucleotides were derived from the complementary single-stranded phosphorothioate-linked oligonucleotides (Eurogentec, Cologne, Germany) as described in Krzesz et al. (1999) FEBS Lett. 453, 191.
  • the cultured human endothelial cells were pretreated for 4 hours with the respective decoy oligonucleotide in a concentration of 10 ⁇ mol / 1 and added to the incubation medium in the same concentration after changing the medium.
  • the single-stranded sequences of the oligonucleotides were as follows (underlined letters denote phosphorothioate-linked bases):
  • Decoy-Oligoukleotide shown in Table 3 to neutralize the transcription factor binding to position -786 of the eNOS-G ns.
  • the sequence of the STAT-3 decoy oligonucleotide corresponds to position -858 to -838 in the human eNOS gene.
  • Both STAT-1 and STAT-3 bind to the oligonucleotide SIE.
  • additional supershift analyzes were carried out with a monoclonal mouse anti-human supershift antibody (Santa Cmz biotechnology) (addition in a concentration of 10 ⁇ g / ml to the core extract for 1 hour at room temperature).
  • the human umbilical cord vein endothelial cells were subsequent freezing in liquid nitrogen and thawed at 37 ° C. Protein extracts were, as in Hecker et al. (1994) Biochem J. 299, 247. 20-30 ⁇ g protein were separated using a 10% polyacrylamide gel electrophoresis under denaturing conditions in the presence of SDS according to the standard protocol and transferred to a BioTrace TM polyvinylidene fluoride transfer membrane (Pall Corporation, Roßdorf, Germany). Monoclonal mouse anti-human antibodies (BD Pharmingen, Heidelberg, Germany; 1: 5000 and 1: 3000 dilution) were used for the immunological detection of the eNOS and the IL-10 receptor.
  • the protein bands were added after adding a peroxidase-coupled anti-mouse IgG (1: 3000, Sigma, Deisenhofen, Germany) using the chemiluminescence method (SuperSignal Chemiluminescent Substrate; Pierce Chemical, Rockford, IL, USA) and subsequent autoradiography ( Hyperfilm TM MP, Amersham Pharmacia Biotech, Buckinghamshire, England).
  • IL-12 p40 ELISA The enzyme-linked antibody binding assay (ELISA) is a standard method for the quantitative analysis of proteins. As in Lienenlüke et al. (2000) Eur. J. Immunol. 30, 2864 described for the detection of the IL-12 p40 subunit in the supernatant of the incubated human endothelial cells.

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Abstract

La présente invention concerne des oligonucléotides leurres ayant la séquence d'acide nucléique définie par SEQ ID NO:1 à 34, et leur utilisation en tant que produit pharmaceutique. L'invention a également pour objet un procédé pour diagnostiquer la variante -786C/T du gène eNOS.
EP03750346A 2002-09-12 2003-09-12 Correction fonctionnelle de la variante sp -786 /sp c/t du gene i enos /i humain Withdrawn EP1537209A2 (fr)

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DE10242319A DE10242319A1 (de) 2002-09-12 2002-09-12 Funkionelle Korrektur der-786C/T-Varianz des humanen eNOS-Gens
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PCT/DE2003/003028 WO2004027062A2 (fr) 2002-09-12 2003-09-12 Correction fonctionnelle de la variante -786c/t du gene enos humain

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US8466187B2 (en) 2007-09-18 2013-06-18 Thermolife International, Llc Amino acid compositions
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