[go: up one dir, main page]

WO2006092209A1 - Nouvelles cibles et composes utilises pour traiter un trouble cardio-vasculaire, une dyslipidemie et une atherosclerose et procedes pour identifier de tels composes - Google Patents

Nouvelles cibles et composes utilises pour traiter un trouble cardio-vasculaire, une dyslipidemie et une atherosclerose et procedes pour identifier de tels composes Download PDF

Info

Publication number
WO2006092209A1
WO2006092209A1 PCT/EP2006/001451 EP2006001451W WO2006092209A1 WO 2006092209 A1 WO2006092209 A1 WO 2006092209A1 EP 2006001451 W EP2006001451 W EP 2006001451W WO 2006092209 A1 WO2006092209 A1 WO 2006092209A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
polypeptide
activity
atherosclerosis
dyslipidemia
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2006/001451
Other languages
English (en)
Inventor
Ulrich Betz
Donatella D'urso
Petros Gatsios
Michael Seewald
Jochen Strayle
Helmuth Hendrikus Geradus Van Es
Marlijn Van Zutphen
Emir Mesic
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Galapagos NV
Original Assignee
Galapagos NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Galapagos NV filed Critical Galapagos NV
Publication of WO2006092209A1 publication Critical patent/WO2006092209A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/323—Arteriosclerosis, Stenosis

Definitions

  • the invention relates to novel targets in the screening for compounds useful in the treatment of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • the invention relates to novel compounds for use as a medicament for diseases or conditions involving a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • the invention especially relates to antagonists and expression-inhibitory compounds that target G-protein coupled receptors (GPCRs), kinases and proteases, and to methods for identifying such compounds.
  • GPCRs G-protein coupled receptors
  • the invention further relates to methods for identifying these antagonists and expression-inhibitory compounds, and methods for diagnosing a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis or a susceptibility to such a condition.
  • Atherosclerosis is by far the single most important pathological process in the development of coronary heart disease (CHD), which is the single most common cause of morbidity and mortality in both men and women in developed countries.
  • CHD coronary heart disease
  • Atherosclerosis is a complex disease with multiple risk factors. It has been reported that 80-90% of patients who develop significant CHD and >95% of patients who experience fatal CHD have major atherosclerotic risk factors.
  • Hypercholesterolemia or raised blood cholesterol levels, is the most prevalent cardiovascular condition, with a total prevalent condition of 320 million patients in the 8 major pharmaceutical markets.
  • Standard therapy for atherosclerosis includes lipid-lowering drugs: HMG-CoA reductase inhibitors (statins), PPARalpha agonists (fibrates) and niacin.
  • Statins are the most recently launched class of anti- hypercholesterolemics and now dominate the hypercholesterolemia market.
  • NCEP National Cholesterol Education Program
  • the current approaches for the purpose of lowering low density lipoprotein (LDL) cholesterol and therefore preventing the progression of Atherosclerosis include Squalene Synthase Inhibitors, intestinal bile acid transport (IBAT) protein inhibitors and SREBP cleavage-activating protein (SCAP) activating ligands.
  • Other current approaches that affect lipid metabolism are microsomal triglyceride transfer protein (MTP) inhibitors, acylcoenzyme A : cholesterol acyltransferase (ACAT) inhibitors and nicotinic acid receptor (HM 74) agonists.
  • MTP microsomal triglyceride transfer protein
  • ACAT cholesterol acyltransferase
  • HM 74 nicotinic acid receptor
  • HDL high density lipoprotein
  • cholesteryl ester transfer protein CETP
  • ABS ATP-binding cassette transporter
  • SRBl scavenger receptor class B Type 1
  • Nuclear receptors as PPARs, LXR and FXR are also targets of investigational agents.
  • Figure 1 shows a standard curve for secreted ApoBlOO [ ⁇ g/ml] over light absorbance at 450 nm.
  • Figure 2 shows ApoBlOO secretion of HepG2 cells six days post infection with the respective adenovirus.
  • Figure 3 shows the position of positive and negative controls on the Z' infection control plates
  • Figure 4 shows an overview of all datapoints from the primary screen in which the standard deviations of the duplicate data points are indicated on the X- and Y-axis.
  • the dotted lines represent the threshold value (average of all samples minus 1.7 times standard deviation).
  • Figure 5 shows an overview of all datapoints from the secondary screen in which the standard deviations of the duplicate data points are indicated on the X- and Y-axis.
  • the dotted lines represent the threshold value (average of all samples minus 1.4 times standard deviation).
  • Figure 6 shows a standard curve for secreted ApoAl [ ⁇ g/ml] over light absorbance at 450 nm.
  • Figure 7 shows the plate layout used in the validation experiments of 250 hits for ApoBlOO and ApoAl secretion and cell-viability.
  • Figure 8 shows the plate layout used in the 3 MOI test.
  • Table 1 shows for each of the 250 targets of the invention: Target number; hit code; results of the primary screen (Example 2)(biological duplicates); number of samples of the biological duplicate which scored under the cutoff value of 1.7 in the primary screen; results of the secondary screen (re-screen Example 3, biological duplicates); number of samples of the biological duplicate which scored under the cutoff value of 1.4 in the re-screen; hit type (number of samples which came up as a hit in the primary- and the re-screen per target); drugable class; and target symbol.
  • Target number shows for each of the 250 targets of the invention: Target number; hit code, target symbol; GenBank Accession number; and a brief description of the target.
  • Table 3 shows for each of the 250 targets of the invention: SEQ ID NO under which the siRNA sequence is listed in the sequence listing; hit code; and the siRNA sequence.
  • Table 4 shows the results of ApoBlOO, ApoAl, and %viable cells measurements for biological duplicates as determined in Example 5.
  • Table 5 shows the results of expression profiling experiments in HepG2, Huh cells, primary hepatocytes, and whole liver as determined in example 6. Given are the probe IDs of the probes immobilized on the Affymetrix(R) Chip for the detection of the respective target expression, and average expression as determined in the respective cell line.
  • Table 6 describes diluting of ApoBlOO standard for the purposes of ApoBlOO standard curve.
  • Table 7 describes diluting of ApoAl standard for the purposes of ApoAl standard curve.
  • Table 8 shows an overview of the results of the ApoBlOO assay from the 3 MOI test.
  • Table 9 shows an overview of the results of the ApoAl assay from the 3 MOI test.
  • Table 10 shows an overview of the results of the MTS cell viability assay from the 3 MOI test.
  • Table 11 gives an overview of 18 adenoviral shRNA constructs taken to the on target analysis.
  • Table 12 shows an overview of the results from the on target analysis.
  • Table 13 gives an overview of the adenoviral shRNA constructs tested in the on target analysis.
  • Table 14 lists the targets selected for mRNA expression determination
  • Table 15 showes the primer sets used for mRNA expression determination
  • Table 16 shows an overview of the results of the mRNA expression determination for TMPRSS7
  • Table 17 shows an overview of the results of the mRNA expression determination for P2X4
  • Table 18 shows an overview of the results of the mRNA expression determination for LPLl
  • Table 19 shows an overview of the results of the mRNA expression determination for SFXN2
  • Table 20 shows an overview of the results of the mRNA expression determination for SLC22A3
  • the invention relates to compounds that are identified using the methods according to the invention.
  • the invention also relates to the use of any one of the target genes listed in Table 1, or of any one of the polypeptides encoded thereby, in the identification of a compound useful in the treatment of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis, as well as to the use of a known or novel compound that decreases the activity and/or the expression of a polypeptide encoded by any one of the target genes listed in Table 1 in the manufacture of a medicament for the treatment of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • the invention furthermore relates to a method of reducing a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis in a subject, said method comprising the step of administering a pharmaceutical composition according to the invention.
  • the invention also relates to methods for diagnosis of a pathological condition involving a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis in a subject, one of said methods comprising the steps of: (a) determining the nucleic acid sequence of at least any one of the target genes listed in Table 1 within the genomic DNA of said subject; (b) comparing the sequence from step (a) with the nucleic acid sequence obtained from a database and/or a healthy subject; (c) identifying any difference(s) related to the onset of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • the inventors of the present invention identified novel target genes that are involved in a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis by using the SilenceSelect collection (see WO03/020931) a so-called 'knock-down' library: A screen in which siRNA molecules were transduced into cells by recombinant adenoviruses. This screen was used in order to repress or inhibit the expression and activity of the corresponding gene and gene product in a cell. By identifying a siRNA that induce the desired phenotype in the screen for the repression of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis, a direct link is made to the corresponding target gene and target gene product. This target gene is subsequently used in methods for identifying compounds that can be used to prevent or treat a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • the invention thus relates to the novel identified link between certain polynucleotides or polypeptides present in a cell with a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis. It is disclosed for the first time that these polypeptides are involved in this process and that these polypeptides can be targeted by different kinds of compounds. These compounds are in turn applicable for therapeutics that are useful in the treatment of diseases such as a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • the invention furthermore provides methods and means for identifying even more and other novel compounds by applying the target polypeptides of the invention.
  • the invention relates to the fact that certain compounds (if identified by the methods provided, or already known to interact with the polypeptides) may now be applied for the treatment or prevention of occurrence of diseases involving a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis. For compounds that were already found in the past to bind to these polypeptides, this is a new application, which is also covered by the present invention.
  • the invention relates to:
  • Method for identifying a compound as having increased probability of being useful in the treatment of a disease comprising the steps of (a) providing a first cell expressing a target polypeptide selected from the group listed in Table 1, or a fragment, or a derivative thereof; (b) exposing said first cell to a candidate compound; (c) determining a first level of an activity or property, said activity or property being affected by an activity or property of said target polypeptide; and (d) selecting or discarding said candidate compound, based on a comparison of said first level of said activity or property with a reference level of said activity or property; characterised in that said disease is a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • Count 12 or 13 wherein said promoter is a cyclic AMP-responsive promoter, an NF-KB responsive promoter, a NF-AT responsive promoter, or a promoter responsive to transcription factors or to nuclear hormone receptors.
  • step (c) selecting said compound if binding is detected in step (b) or if a change in activity is detected in step (b);
  • said disease is a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • Binding is detected, if the binding constant [mol/liter] is determined to be equal or below 10 micromolar, or 1 micromolar, or 100 nanomolar, or, most preferred, 10 nanomolar.
  • a change in activity within the meaning of the invention, shall be understood as being, e.g., a difference in activity prior to and after addition of the test compound.
  • Count 28 Use of Count 28, wherein said compound is identified according to any one of the methods of Counts l to 27.
  • 30 Use of an agent inhibiting the expression of a polypeptide selected from the group listed in Table 1 for the preparation of a medicament for the treatment of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • ribozyme that cleaves the polyribonucleotide of said polypeptide
  • ODN antisense oligodeoxynucleotide
  • siRNA small interfering RNA
  • siRNA small interfering RNA having the sequence of any of SEQ ID NO: 1 to 250.
  • Count 32 Use of Count 32, wherein the vector is an adenovirus, a retrovirus, an alphavirus, an adeno-associated virus (AAV), a lentivirus, a herpes simplex virus (HSV) or a sendai virus.
  • AAV adeno-associated virus
  • HSV herpes simplex virus
  • Counts 31 to 33 wherein said agent is siRNA, and said siRNA comprises a sense strand of 17 to 23 nucleotides which is identical to a region of the coding sequence, or its complementary sequence, of any of the polypeptides of Table 1.
  • Count 34 Use of Count 34, wherein the siRNA further comprises a cleavable loop region connecting the sense and the antisense strand.
  • Count 38 or 39 wherein the vector is an adenoviral, retroviral, adeno- associated viral, lentiviral or a sendaiviral vector.
  • 41 Method for diagnosing a pathological condition involving a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis, or a susceptibility to said condition in a subject, comprising
  • step (d) identifying any difference(s) between the nucleic acid sequences determined in step (b) and (c);
  • a pathological condition involving a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis, or a susceptibility to such a condition in a subject is diagnosed, if such difference(s) are identified in step (d).
  • Method for diagnosing a pathological condition involving a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis or a susceptibility to such a condition in a subject comprising
  • a further embodiment of the invention are the screening methods described above, wherein the identified compounds are useful in the treatment of one or more diseases selected from the group consisting of cardiovascular disorders, dyslipidemia, and Atherosclerosis.
  • Targets 1 to 250 of Table 1 are more preferred within the context of the present invention.
  • those targets are preferred targets, which have exceeded the threshold values of -1.7 and -1.4 more than twice in the screening runs reported in Table 1 (i.e., Targets 1 to 194 of Table 1 are preferred).
  • Even more preferred targets are targets which have ⁇
  • those targets listed in Table 1 are preferred, which are highly expressed in HepG2 cells, Huh cells, primary hepatocytes, and whole liver cells.
  • those targets of Table 1 which show an average expression of above 1000 in HepG2 cells, Huh cells, primary hepatocytes, or whole liver cells, in Table 4, are preferred targets of the invention.
  • Even more preferred are targets of Table 1, which show an average expression of above 1000 in at least two, or three or (most preferred) four cell types, in a list of cell types consisting of HepG2 cells, Huh cells, primary hepatocytes, and whole liver cells, in Table 4.
  • those targets listed in Table 1 are preferred which are expressed in human liver or primary human hepatocytes according to expression analysis with Affymetrix technology using Mas5 algorithm generating a presence call.
  • those targets listed in Table 1 are preferred targets of the invention, which are not suppressing ApoAl secretion from the HepG2 cells.
  • those targets listed in Table 1 are preferred targets of the invention, which do not affect cell-viability.
  • targets of the invention are targets selected from the group consisting ofLPLl, P2X4, SFXN2, SCL22A3 and TMPRSS 7.
  • the invention further relates to methods for identifying a compound that decreases the expression and/or activity of a polypeptide encoded by any one of the target genes of Table 1, said method comprising the steps of providing a host cell expressing a polypeptide having an amino acid sequences selected from the group listed in Table 1, or a fragment, or a derivative thereof; determining a first activity level of the polypeptide; exposing the host cell to a compound; determining a second activity level of the polypeptide after exposing of the host cell to the compound; and identifying the compound, whereby the second activity level is lower than the first activity level.
  • the methods according to the invention may further comprise the step of exposing said cell to an agonist of the polypeptide to trigger the expression and/or activity of the polypeptide. Since expression or activity levels of the polypeptides as disclosed herein may be low in said population of cells, it is preferred that they exhibit levels high enough so that the effect of the identifiable compounds can be properly screened. To establish this, the polypeptides may be over-expressed in the population of cells. This can be achieved, for instance, through transfection of expression plasmids comprising the genes or functional parts or derivatives thereof that encode the target polypeptides of interest. Thus, the methods according to the invention may comprise a further step of over-expressing a polypeptide encoded by any one of the target genes of Table 1 in said population of cells.
  • the invention also relates to a method for identifying a compound that influences the expression or activity of a protein associated with a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis, said method comprising the steps of: contacting one or more compounds with a polypeptide comprising an amino acid sequence selected from the group listed in Table 1, or a derivative, or a fragment thereof; determining the binding affinity of the compound to the polypeptide, or its derivative or fragment; contacting a population of mammalian cells expressing the polypeptide with the compound that exhibits a numerical value of the binding affinity of preferably at most 10 micromolar (preferably in the nanomolar or picomolar range); and identifying the compound that influences the expression or activity of the protein.
  • the invention also relates to the use of compounds that bind with a numerical value for the binding affinity of preferably at most 10 micromolar to any one of the polypeptides listed in Table 1, for the preparation of a medicament for the treatment and/or prevention of diseases related to a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • the binding affinity of the compound with the polypeptide or polynucleotide can be measured by methods known in the art, such as using surface plasmon resonance biosensors (Biacore, Neuchatel), by saturation binding analysis with a labeled compound (using e.g. Scatchard Plots or Lindmo analysis), by differential UV spectrophotometer, fluorescence polarisation assay, Fluoro- metric Imaging Plate Reader (FOPR®) system, Fluorescence resonance energy transfer, and Bioluminescence resonance energy transfer.
  • the binding affinity of compounds can also be expressed in dissociation constant (Kd) or as IC50 or EC50.
  • the IC50 represents the concentration of a compound that is required for 50% inhibition of binding of another ligand to the polypeptide. ⁇
  • the EC50 represents the concentration required for obtaining 50% of the maximum effect in any assay that measures enzyme activity.
  • the dissociation constant, Kd is a measure of how well a compound binds to the polypeptide, it is equivalent to the compound concentration required to saturate exactly half of the binding-sites on the polypeptide.
  • Compounds with a high binding affinity have low Kd, IC50 and EC50 values, i.e. in the range of 100 nM to 1 pM; a moderate to low affinity binding relates to a high Kd, IC50 and EC50 values, i.e. in the micromolar range. Binding affinities may be determined in in vivo settings as well as in in vitro settings.
  • libraries of compounds can be used such as peptide libraries (e.g. LOP APTM, Sigma Aldrich, St. Louis), lipid libraries (BioMol, Hamburg), synthetic compound libraries (e.g. LOP ACTM, Sigma Aldrich, St. Louis) or natural compound libraries (Specs, TimTec, Newark, DE).
  • peptide libraries e.g. LOP APTM, Sigma Aldrich, St. Louis
  • lipid libraries BioMol, Hamburg
  • synthetic compound libraries e.g. LOP ACTM, Sigma Aldrich, St. Louis
  • natural compound libraries Specs, TimTec, Newark, DE.
  • said polypeptide is a GPCR, wherein the expression and/or activity of said GPCR is measured by determining the level of any one of the second messengers cyclic AMP, Ca2+ or both.
  • the level of the second messenger is determined with a reporter gene under the control of a promoter that is responsive to the second messenger. More preferably, the promoter is a cyclic AMP-responsive promoter, " an NF-KB responsive promoter, or a NF-AT responsive promoter.
  • the reporter gene is selected from the group consisting of: alkaline phosphatase, GFP, eGFP, dGFP, luciferase and beta-galactosidase.
  • said polypeptide is a kinase or a phosphatase wherein the expression and/or activity of said kinase or phosphatase is measured by determining the level of phosphorylation of a substrate of said kinase or phosphatase.
  • said polypeptide is a protease wherein the expression and/or activity
  • protease assays are well known in the art.
  • said polypeptide is a transporter, wherein the expression and/or activity of said transporter is measured by measuring the transfer rate of a substrate of the said transporter either from extracellular to intracellular or vice versa.
  • transporter assays are well known in the art.
  • said polypeptide is an ion channel, wherein the expression and/or activity of said ion channel is measured by determining a change in the electrical membrane potential of test cells after activation of said channel.
  • ion channel assays are well known in the art.
  • said polypeptide is a phosphodiesterase (PDE), wherein the expression and/or activity of said PDE is measured by determining the cleavage rate of cAMP or cGMP.
  • PDE phosphodiesterase
  • said polypeptide is an enzyme other than GPCR, protease or PDE or kinase or phosphatase, wherein the expression and/or activity of said enzyme is measured by determining its ability to catalyze its specific biochemical reaction. Multiple en ⁇ yme assays are well known in the art.
  • said polypeptide is a Nuclear Hormone Receptor (NHR), wherein the expression and/or activity of said NHR after addition of its ligand is measured by determining the expression of a reporter gene present in a test cell expressed under the control of a promotor responsive to said NHR.
  • NHR assays are well known in the art.
  • the present invention relates to methods to identify compounds, wherein it is preferred that the compound is selected from the group consisting of: a small molecule compound (such as a low- molecular weight compound), an antisense RNA, an antisense oligodeoxynucleotide (ODN), a siRNA, a ribozyme, a shRNA, an antibody, a nanobody, a peptide, a polypeptide, a nucleic acid, a lipid, and a natural compound.
  • a small molecule compound such as a low- molecular weight compound
  • an antisense RNA such as a low- molecular weight compound
  • ODN antisense oligodeoxynucleotide
  • siRNA siRNA
  • a ribozyme a shRNA
  • the compound has a numerical value for the binding affinity to the polypeptide of at most 10 micromolar, but preferably less than 1 micromolar, more preferably less than 100 nanomolar, even more preferably less than 10 nanomolar, and most preferably less than 1 nanomolar.
  • the invention relates to the use of a compound that decreases the activity and/or the expression of a polypeptide encoded by any one of the target genes of Table 1 in the manufacture of a medicament for the treatment of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • said compound is identifiable according to any one of the methods of the present invention.
  • the compound that is identified according to the present invention can be a low molecular weight compound.
  • Low molecular weight compounds i.e. compounds with a molecular weight of 500 Dalton or less, are likely to have good absorption and permeation in biological systems and are consequently likely to be successful drug candidates (Lipinski, et al. 2001, Advanced Drug Delivery Reviews. 46(1-3): 3-26).
  • the compound may also be a peptide. Peptides can be excellent drag candidates and there are multiple examples of commercially available peptides such as fertility hormones and platelet aggregation inhibitors.
  • the compound is a natural compound.
  • Natural compounds are generally seen as compounds that have been extracted from e.g. plants or that are synthesized on the basis of a natural occurring molecule. Using natural compounds in screens has the advantage that one is able to screen more diverse molecules. Natural compounds have an enormous variety of different molecules. Synthetic compounds do not exhibit such variety of different molecules.
  • the compound may also be a lipid.
  • lipids as candidate compounds can increase the chance of finding a specific antagonist for the polypeptides of the present invention.
  • the compound may also be a polyclonal or monoclonal antibody that interacts with a polypeptide involved in the cascade leading to diseases such as a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis, wherein it is preferred that the antibody is reactive with a polypeptide of the invention, wherein said antibody inhibits the activity of the polypeptides.
  • the compound may be a nanobody, the smallest functional fragment of naturally occurring single- domain antibodies (Cortez-Retamozo V, et al., 2004, Cancer Res. 64(8): 2853-7).
  • the compound is an expression inhibitory agent that inhibits the expression and/or the translation of the nucleic acid encoding the polypeptide, comprising providing a host cell expressing a polynucleotide selected from the group listed in Table 1, or a fragment, or a derivative thereof; and contacting the cell with a compound that inhibits the translation in the cell of the polynucleotide.
  • expression inhibitory agents are an antisense oligonucleotide, a ribozyme, an antisense oligodeoxynucleotide and a siRNA.
  • the expression inhibitory agent must be sufficiently homologous to a portion of the polyribonucleotide such that the expression inhibitory agent is capable of inhibiting the polyribonucleotide that would otherwise cause the production of the polypeptide.
  • said expression inhibitory agent comprises a nucleotide sequence of any one the genes listed in Table 1. Suitable nucleotide sequences applicable in methods of the invention are, for example, those listed in Table 3.
  • said compound is an siRNA comprising a sense strand of 17-23 nucleotides homologous to a nucleotide sequence of any of the target genes of Table 1, and an antisense strand of 17-23 nucleotides complementary to the sense strand.
  • the siRNA further comprises a loop region connecting the sense and the antisense strand, wherein it is preferred that the loop region comprises a cleavable nucleic acid sequence.
  • the nucleic acid sequence UUGCUAUA as described in WO 03/020931.
  • Most preferred are the siRNA sequences listed in Table 3.
  • the compound can be modified to confirm resistance to nucleolytic degradation or to enhance the activity, cellular distribution, or cellular uptake, wherein it is further preferred that the modification comprises a modified internucleoside linkage, a modified nucleic acid base, a modified sugar, and/or a chemical linkage of the oligonucleotide to one or more moieties or conjugates.
  • the population of cells may be exposed to the compound or the mixture of compounds through different means, for instance by direct incubation in the medium, or by nucleic acid transfer into the cells.
  • transfer may be achieved by a wide variety of means, for instance by direct transfection of naked isolated DNA, or RNA, or by means of delivery systems, such as recombinant vectors.
  • Other delivery means such as liposomes, or other lipid-based vectors may also be used.
  • the nucleic acid compound is delivered by means of a (recombinant) vector such as a recombinant virus.
  • the vector is a recombinant vector selected from the group consisting of: an adenovirus, a retrovirus, an alphavirus, an adeno-associated virus (AAV), a lentivirus, a herpes simplex virus (HSV) or a sendai virus.
  • AAV adeno-associated virus
  • HSV herpes simplex virus
  • sendai virus a recombinant vector selected from the group consisting of: an adenovirus, a retrovirus, an alphavirus, an adeno-associated virus (AAV), a lentivirus, a herpes simplex virus (HSV) or a sendai virus.
  • AAV adeno-associated virus
  • HSV herpes simplex virus
  • sendai virus ai virus
  • An example is the deletion of the functional part of the El -region from recombinant adenoviruses.
  • This part of the adenoviral genome is typically used to introduce the gene of interest, or in the case of the present invention, to introduce the nucleic acid compound such as a nucleic acid comprising the sequence of for instance a siRNA molecule.
  • the packaging cell provides for the functional factors of the El -region such that the recombinant vector can be produced in the packaging cells but will be replication-defective in cells that do not harbour the functional El -region, such as host cells that are targeted with the recombinant vector. Such cells may be the cells in the population of cells as used herein.
  • siRNA according to the invention for use as a medicament, preferably, wherein said use is in the treatment of a pathological condition involving a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • the invention further relates to vectors comprising a siRNA according to the invention.
  • said vector is selected from the group consisting of: an adenovirus, a retrovirus, an alphavirus, a lentivirus, an adeno-associated virus, a herpes simplex virus or a sendai virus.
  • the compounds of the present invention may be used in the treatment of diseases in which a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis play an important role. Some compounds may be known to interact with or to act upon the target genes or their products as listed in Table 1. However, their possible role in the treatment of diseases such as a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis was unknown until the present invention.
  • the invention relates also to a pharmaceutical composition comprising a compound according to the present invention, or to a vector according to the invention, and a pharmaceutically acceptable solvent, diluent, excipient and/or carrier.
  • the invention also relates to methods for treatment, prevention and/or amelioration of a pathological condition involving a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis, said methods comprising the step of administering a pharmaceutical composition according the invention.
  • the compounds may be used directly for the treatment of diseases in which a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis are involved.
  • the invention also relates to methods for treating a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis in a subject, said method comprising the step of administering a pharmaceutical composition according to the invention.
  • the oligonucleotides listed in Table 3 were identified in this invention as compounds that were able to reduce an activity of the target protein, said activity being known by the person skilled in the art to be a suitable disease model of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis. More specifically, the oligonucleotides were shown to inhibit the expression and/or activity of a protein involved in a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis. Therefore, polynucleotide sequences comprising any of the oligonucleotide sequences listed in Table 3 (SEQ ID NO:1 - SEQ ID NO:250)can be used as a medicament.
  • polynucleotide sequences comprising any of the oligonucleotide sequences listed in Table 3, can be used for the manufacture of a medicament for the treatment of a disease, preferably a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • the oligonucleotide sequences listed in Table 3 are used as expression inhibitory compounds.
  • the expression inhibitory compounds comprising any of the oligonucleotide sequences listed in Table 3 can be used to prevent or ameliorate a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis in a patient. Therefore, they can be used as a medicament. Moreover, they can be used for the manufacture of a medicament for the treatment of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • the oligonucleotide sequences listed in Table 3 can be used as siRNAs to inhibit the expression and/or activity of a protein involved in a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • siRNAs comprising any of the oligonucleotide sequences listed in Table 3 can thus be used as a medicament.
  • siRNAs comprising any of the oligonucleotide sequences listed in Table 3 can be used for the manufacture of a medicament for the treatment of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • the compounds containing any of the oligonucleotides listed in Table 3, can be modified to confer resistance to nucleolytic degradation or to enhance the activity, cellular distribution, or cellular uptake of said oligonucleotides.
  • the population of cells may be exposed to the compound or the mixture of compounds through different means.
  • the compound is delivered by means of a (recombinant) vector such as, for example, a recombinant adenovirus.
  • a vector comprising any of the oligonucleotides listed in Table 3, can therefore also be used as a medicament. Furthermore, a vector comprising any of the oligonucleotides listed in Table 3, can be used for the manufacture of a medicament for the treatment of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • the invention also relates to methods for the diagnosis of a pathological condition involving a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis in a subject, said methods comprising the steps of: determining the nucleic acid sequence of at least any one of the target genes of Table 1 within the genomic DNA of said subject; comparing the sequence from the first step with the nucleic acid sequence obtained from a database and/or a healthy subject; and identifying any difference(s) in sequence related to the onset of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • the pathological condition is a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.
  • Diagnostic methods of the invention are preferably performed in a sample, ex vivo.
  • an Atherosclerosis-associated protein is affected, as used herein, if the expression or activity of the protein is reduced upon incubation with the compound. Although a reduction of those levels may differ and may be multifold, it is held here that a reduction of 30% (or more) in a patient (in vivo) is a preferred level. Thus the influence on the expression or activity of the Atherosclerosis-associated protein as used herein refers to a preferred reduction of said expression and/or activity that is comparable to a reduction of 30% (or more) in vivo.
  • levels found in vitro do not perfectly correlate with levels found in vivo, such that a slightly reduced level in vitro may still result in a higher reduction in vivo when the compound is applied in a therapeutic setting. It is therefore preferred to have reduced in vitro levels of at least 10%, more preferably more than 30%, even more preferably more than 50% and most preferably a reduction between 50% and 100%, which would mean an almost complete disappearance of the expression or activity of the Atherosclerosis-associated protein.
  • the compounds may target the polypeptides directly and inhibit their activity.
  • the compounds may also target the transcription/translation machinery involved in the transcription and/or translation of the polypeptide from its encoding nucleic acid.
  • the compounds may furthermore target their respective DNA 's and mRNA's thereby preventing the occurrence of the polypeptide and thereby diminishing their activity. It is thus to be understood that the compounds that are identified by using the methods of the present invention may target the expression, the activity, etc. of the polypeptides at different levels, finally resulting in an altered expression or activity of an Atherosclerosis-associated protein.
  • Atherosclerosis-associated protein refers to a protein that is involved in the onset, treatment or amelioration of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis in a patient.
  • Preferred Atherosclerosis-associated proteins are the proteins listed in Table 1.
  • the activity of the Atherosclerosis-associated protein is believed to be causative and/or to correlate with the progression of various diseases associated with Atherosclerosis. These diseases include, but are not limited to, coronary heart disease, stroke, myocardial infarction and circulatory disturbances, high blood pressure and angina. Patients suffering from such diseases may benefit from treatment with compounds of the present invention.
  • the expression of an Atherosclerosis-associated protein can be determined by methods known in the art such as Western blotting using specific antibodies, or ELISAs using antibodies specifically recognizing a particular Atherosclerosis-associated enzyme.
  • an Atherosclerosis-associated protein can be determined by using fluorogenic small peptide substrates.
  • the specificity of these substrates is often limited. In general, the use of these substrates is limited to the testing of purified proteases in biochemical assays, to avoid interference of other proteases.
  • Arteriosclerosis is the thickening and hardening of the arteries due to the build-up of calcium deposits on the insides of the artery walls. Atherosclerosis is a similar condition due to the build- up of fatty substances. Both conditions have similar effects on the circulation of the blood throughout the body. Heart disease, high blood pressure, stroke, and ischemia (starvation of the cells due to insufficient circulation) may be the result of arteriosclerosis and atherosclerosis.
  • arteriosclerosis shall be understood as encompassing both, atherosclerosis and arteriosclerosis as defined above.
  • cardiovascular disorder The following conditions are understood to be a cardiovascular disorder:
  • Heart failure is defined as a pathophysiological state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failures such as high-output and low- output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause.
  • MI Myocardial infarction
  • Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen.
  • This group of diseases includes stable angina, unstable angina and asymptomatic ischemia.
  • Arrhythmias include all forms of atrial and ventricular tachyarrhythmias, atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexitation syndrome, ventricular tachycardia, ventricular flutter, ventricular fibrillation, as well as bradycardic forms of arrhythmias. ⁇ JL X. ⁇
  • Hypertensive vascular diseases include primary as well as all kinds of secondary arterial hypertension, renal, endocrine, neurogenic, others.
  • the genes may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications arising from cardiovascular diseases.
  • Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders.
  • PAOD peripheral arterial occlusive disease
  • acute arterial thrombosis and embolism inflammatory vascular disorders
  • Raynaud's phenomenon Raynaud's phenomenon
  • Atherosclerosis is a cardiovascular disease in which the vessel wall is remodeled, compromising the lumen of the vessel.
  • the atherosclerotic remodeling process involves accumulation of cells, both smooth muscle cells and monocyte/macrophage inflammatory cells, in the intima of the vessel wall. These cells take up lipid, likely from the circulation, to form a mature atherosclerotic lesion.
  • the formation of the atherosclerotic lesion can be considered to occur in five overlapping stages such as migration, lipid accumulation, recruitment of inflammatory cells, proliferation of vascular smooth muscle cells, and extracellular matrix deposition.
  • stages such as migration, lipid accumulation, recruitment of inflammatory cells, proliferation of vascular smooth muscle cells, and extracellular matrix deposition.
  • Each of these processes can be shown to occur in man and in animal models of atherosclerosis, but the relative contribution of each to the pathology and clinical significance of the lesion is unclear.
  • Cardiovascular diseases include but are not limited to disorders of the heart and the vascular system like congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, peripheral vascular diseases, and atherosclerosis.
  • dyslipidemia can cause long-term problems.
  • the risk to develop atherosclerosis and coronary artery or carotid artery disease (and thus the risk of having a heart attack or stroke) increases with the total cholesterol level increasing. Nevertheless, extremely low cholesterol levels may not be healthy.
  • hyperlipidemia abnormally high levels of fats (cholesterol, triglycerides, or both) in the blood, may be caused by family history of hyperlipidemia), obesity, a high-fat diet, lack of exercise, moderate to high alcohol consumption, cigarette smoking, poorly controlled diabetes, and an underactive thyroid gland), hereditary hyperlipidemias (type I hyperlipoproteinemia (familial hyperchylomicronemia), type II hyperlipoproteinemia (familial hypercholesterolemia), type III hyperlipoproteinemia, type IV hyperlipoproteinemia, or type V hyperlipoproteinemia), hypolipoproteinemia, lipidoses (caused by abnormalities in the enzymes that metabolize fats), Gaucher's disease, Niemann-Pick disease, Fabry's disease, Wolman's disease, cerebrotendinous xanthomatosis, sitosterolemia, Refsum's disease, or Tay-Sachs disease.
  • hyperlipidemia abnormally high levels of fats (cholesterol, triglycer
  • Kidney disorders may lead to hypertension or hypotension. Examples for kidney problems possibly leading to hypertension are renal artery stenosis, pyelonephritis, glomerulonephritis, kidney tumors, polycistic kidney disease, injury to the kidney, or radiation therapy affecting the kidney. Excessive urination may lead to hypotension.
  • expression relates to both endogenous expression and over-expression by for instance transfection or stable transduction.
  • agonist refers to a ligand that stimulates the receptor the ligand binds to in the broadest sense.
  • polypeptide relates to proteins, proteinaceous molecules, fractions of proteins, peptides, oligopeptides, enzymes (such as kinases, proteases, GPCRs etc.).
  • derivatives of a polypeptide relates to those peptides, oligopeptides, polypeptides, proteins and enzymes that comprise a stretch of contiguous amino acid residues of the polypeptide and that retain the biological activity of the protein, e.g. polypeptides that have amino acid mutations compared to the amino acid sequence of a naturally-occurring form of the polypeptide.
  • a derivative may further comprise additional naturally occurring, altered, glycosylated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally occurring form of the polypeptide.
  • It may also contain one or more non-amino acid substituents compared to the amino acid sequence of a naturally occurring form of the polypeptide, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence.
  • fragment of a polypeptide relates to peptides, oligopeptides, polypeptides, proteins and enzymes that comprise a stretch of contiguous amino acid residues, and exhibit substantially a similar, but not necessarily identical, functional activity as the complete sequence.
  • fragments of a polypeptide are 3, 5, 8, 12, 20, 50, 100 amino acids long.
  • polynucleotide refers to nucleic acids, such as double stranded, or single stranded DNA and (messenger) RNA, and all types of oligonucleotides. It also includes nucleic acids with modified backbones such as peptide nucleic acid (PNA), polysiloxane, and 2' ⁇ O-(2- methoxy)ethylphosphorothioate.
  • PNA peptide nucleic acid
  • polysiloxane polysiloxane
  • 2' ⁇ O-(2- methoxy)ethylphosphorothioate 2' ⁇ O-(2- methoxy)ethylphosphorothioate
  • derivatives of a polynucleotide relates to DNA-molecules, RNA- molecules, and oligonucleotides that comprise a stretch or nucleic acid residues of the polynucleotide, e.g. polynucleotides that may have nucleic acid mutations as compared to the nucleic acid sequence of a naturally occurring form of the polynucleotide.
  • a derivative may further comprise nucleic acids with modified backbones such as PNA, polysiloxane, and 2'-O-(2-methoxy)ethyl-phosphoro- thioate, non-naturally occurring nucleic acid residues, or one or more nuclei acid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl-, chloro-, and methanocarbanucleo- sides, or a reporter molecule to facilitate its detection.
  • nucleic acids with modified backbones such as PNA, polysiloxane, and 2'-O-(2-methoxy)ethyl-phosphoro- thioate, non-naturally occurring nucleic acid residues, or one or more nuclei acid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl-, chloro-, and me
  • fragment of a polynucleotide relates to oligonucleotides that comprise a stretch of contiguous nucleic acid residues that exhibit substantially a similar, but not necessarily identical, activity as the complete sequence.
  • fragments of a polynucleotide are 10, 20, 50, 100, 300 nucleotides long.
  • a “reference level” for a protein activity or expression level shall be understood as being any level of a protein activity or an expression level, with which another level of protein activity or expression level can be compared.
  • Reference levels can be determined in separate experiments (e.g., by measurements in healthy or diseased individuals) or can be taken from literature.
  • a reference level e.g., can also be calculated from a series of measurements in a current experiment, such as the mean of multiple measurements in a series of measurements.
  • a "wild type" level of expression is the level of expression of a gene in an organism not genetically modified by recombinant DNA technology, or by purposeful manipulation of the expression pattern by conventional means, such as, e.g., multiple rounds of mutation and selection.
  • Example 1 Development of a high-throughput screening method for the detection of suppression ofApoBlOO secretion from HepG2 cells
  • ApoBlOO synthesized in the liver, is an essential structural component of very low density lipoproteins (VLDLs) and its metabolic products, intermediate density lipoproteins (IDLs) and low density lipoproteins (LDLs).
  • VLDLs very low density lipoproteins
  • IDLs intermediate density lipoproteins
  • LDLs low density lipoproteins
  • ApoBlOO is required for the intracellular assembly and secretion of these lipoproteins, and serves as a ligand for LDL receptor-mediated clearance of these lipoproteins from the plasma.
  • Hepatic overproduction of ApoBlOO-containing lipoproteins is a maj or risk factor for atherosclerosis .
  • HepG2 cells human hepatocytic cell-line
  • Ad-siRNA siRNA adenoviruses
  • Target sequence 5'-GAGGCAGCTTCTGGCTTGC. Cloned using Sapl- sites into vector and virus generated as described in WO03/020931.
  • Target sequence 5'- GGACCTGGCCACTGTGTAC. Cloned using Sapl- sites into vector and virus generated as described in WO03/020931.
  • A150100-empty; empty virus (generated from A150100, as described in WO03/020931).
  • AO 10800-eGFP_vl AO 10800-eGFP_vl ; referred to as pIPspAdApt6-eGFP in WO 02/070744
  • HepG2 cells were obtained from ATCC (HB-8065). The cells were cultured in Tl 75 flasks (Nunc; Cat. No. 159910) at 37°C in a humidified incubator at 10% CO 2 .
  • the medium that is used for culruring the cells is RPMI 1640 + L-Glutamine (Gibco; Cat. No. 21875-034) + 10% Fetal Bovine Serum heat inactivated (ICN; Cat. No. 29-167- 54 100% NHT) + 10 mM HEPES buffer (Gibco; Cat. No. 15630-056). Cells were passed 1:2 twice a week after reaching ⁇ 70% confluency, using the following protocol:
  • HepG2 cells After trypsinization of HepG2 cells (see above) the upper part of cell suspension from a 15 ml tube was used for the seeding. HepG2 cells were seeded on day 0 at 40000 cells/well in a 96-well PLL- coated plate (Becton Dickinson; Cat. No. AA356516) in 130 ⁇ l HepG2 medium. They were infected the next day (day 1) with control viruses, diluted with HepG2 medium in order to achieve an MOI of 160 virus particles/cell (VP/cell, determined as described by Ma et al. (2001, J Virol Methods, 93:181-8).
  • VP/cell 160 virus particles/cell
  • the volume of each virus dilution to be added per well was 20 ⁇ l to get a total volume of 150 ⁇ l (virus containing) medium in each well.
  • the (virus containing) medium was taken off the cells and 120 ⁇ l of fresh, pre-warmed HepG2 medium (without virus) was added to each well. This medium refreshing step was repeated on day 5.
  • supernatants were harvested (110 ⁇ l from each well) and stored at - 80 0 C.
  • Total Human Apolipoprotein B (ApoB) ELISA Assay obtained from ALerCHEK, Inc. (Cat. No. A70102) was used.
  • ApoB standard provided in the kit (2.640 ⁇ g/ml) and HepG2 medium, dilutions were made for the standard curve (see Table 6). 100 ⁇ l of 7x in HepG2 medium diluted supernatant or standard curve - Zo - dilution (in duplicate) was transferred to the ELISA plate.
  • the primary screen of the SilenceSelect library was performed in 3 infection batches. Besides the first infection batch (Pilot screen) for which only one ELISA batch was performed, the other two infection batches were split in 2 ApoBlOO ELISA batches performed on two different days.
  • Example 1 For screening of the SilenceSelect library, the optimized protocol described in Example 1 was used. However, there were a few adjustments made to this protocol.
  • day 1 12.38 ⁇ l Ad-siRNA virus from each well of the SilenceSelectTM collection (WO 03/020931), stored in 384 well plates (estimated titer of 2 x 10 9 viral particles per ml) was transferred with the aid of a 96 channel dispenser (Tecan Freedom 200 equipped with TeMO96, TeMO384 and RoMa, Tecan AG, Switzerland, further referred to as Tecan) to individual wells of a 384-well plate containing 65 ⁇ l fresh HepG2 medium, thus diluting the viruses 6.25 times.
  • a 96 channel dispenser Tecan Freedom 200 equipped with TeMO96, TeMO384 and RoMa, Tecan AG, Switzerland, further referred to as Tecan
  • Ad- siRNA viruses were nominated as hits if at least one of the two data points (duplicates on independent assay plates) scored under the threshold. Threshold was set at average minus 1.7 times standard deviation of all data points per plate.
  • Z'-factor analysis was performed for all ELISA batches. This means that for each ELISA batch to be performed, 5 Z' infection plates were taken along during the infection process. Each Z' infection plate contained 15 wells with A150100-ApoB100_v6 as a positive control and 45 wells with A150100-eGFP_v6 as a negative control (see Figure 3).
  • the dilutions of the viruses for the Z' plate were made manually using the known virus titers in order to achieve a MOI of 160 VP/cell and were aliquoted in a 96 well v-bottom plate (Greiner; Cat. No. 651180) according to the layout from Figure 3 at 160 ⁇ l per well.
  • the Tecan was used to infect the cells with these diluted viruses the same way as described for the library plates. This was followed by the medium refreshments on day 3 and 5 and harvesting of supernatants on day 7 (performed as described earlier).
  • ApoBlOO ELISA's of the Z' plate samples were performed as described for the library plates. However, in these cases standard curve dilutions were added manually in rows A and H of the ELISA plates (100 ⁇ l per well). Concentrations of ApoBlOO were calculated as described in Example 1. These values were used to calculate the Z'-factor for each of the 5 ELISA batches according to the following formula: 1 - ((3 * STDEV (neg. controls) + 3 * STDEV(pos. controls)) / ABS(AVERAGE(neg. controls) - AVERAGE(pos. controls))) as described by Zhang et al. (1999, J. Biomol. Screening 4, 67-73).
  • This Z'-factor had to be equal to or higher than 0.15 for an ELISA batch to be considered as valid. All ELISA batches with a Z'-factor lower than 0.15 were repeated and, if necessary, the complete infection batch was repeated.
  • a total of 443 hits were isolated which scored under the threshold. Of these 443 hits, 170 hits scored positive in duplicate and 273 hits scored in single.
  • An overview of all data points from the primary screen is provided in Figure 4, in which the "standard deviation" of the duplicate data points are indicated on the X-axis and Y-axis. The threshold (average of all samples minus 1.7 times standard deviation) is indicated by dotted lines.
  • the ApoBlOO-screen of the 443 hits was repeated in a re-screen. Therefore, the original virus material of these hits (in matrix tubes) together with 480 viruses that scored negative in the primary screen were rearranged in 10 matrix tube boxes with hits in rows A, C, E, and G and neutral viruses in rows B, D, F, and H. To propagate these viruses, 4 x 10 4 PerC6.E2A cells were seeded in 200 ⁇ l of DMEM (Gibco Cat. No. 41966-029) containing 10% non-heat inactivated FBS (ICN; Cat. No.
  • Re-screening of 443 hits and 480 neutral viruses from the primary screen was performed in the same way as the primary screen using propagated viruses from 96 well plates instead of the original batch of viruses from 384 well plates.
  • the threshold was set at average minus 1.4 times standard deviation of data points from rows B, D, F, and H (neutral controls) per plate.
  • the next step in the validation of the 250 hits from the rescreen was retesting of these hits for ApoBlOO secretion including an ApoAl secretion assay and a cell-viability (MTS) assay.
  • ApoBlOO secretion including an ApoAl secretion assay and a cell-viability (MTS) assay.
  • MTS cell-viability
  • CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (further referred to as MTS assay) (Promega; Cat. G5421) was used.
  • MTS assay CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay
  • enzymatic activity of dehydrogenase in metabolically active cells is measured. It is performed according to the protocol provided in the kit.
  • remainders of supernatant after harvesting (day 7), were removed using multichannels and fresh, pre-warmed HepG2 medium was added to the cells (100 ⁇ l per well).
  • One conditioned medium plate (uninfected samples) was freeze/thawed 3 times to be used as a cell lysate (dead cells) control.
  • 10 ⁇ l of this solution was added to each well and plates were incubated for 1 hour at 37°C 10% CO2 in the dark.
  • the conversion of MTS into aqueous, soluble formazan in metabolically active cells is measured by the amount of 490 nm absorbance (using FLUOstar Galaxy, BMG), which is directly proportional to the number of living cells in measured samples.
  • % viable HepG2 cells (A490sample / A490uninfected HepG2 cells) * 100.
  • Total Human Apolipoprotein Al (ApoAl) ELISA Assay obtained from ALerCHEK, Inc. (Cat. A70101) was used.
  • ApoAl standard provided in the kit (0.335 ⁇ g/ml) and HepG2 medium, dilutions were made for the standard curve (see Table 7). 100 ⁇ l of supernatant diluted 3x (in HepG2 medium) or standard curve dilution (in duplicate) was transferred to the ELISA plate.
  • the propagated virus material in matrix tubes (see Example 3) together with two control viruses A150100-ApoB100_v6 and A150100-ApoAl_v2 (diluted in HepG2 medium to achieve a titer of 2 x 109 viral particles per ml and aliquoted in matrix tubes) were rearranged in 4 matrix tube boxes (for layout see Figure 7).
  • the screening of these viruses together with 5 z' plates was performed according to the protocol described in Example 1, 2 and 4 with the following adjustments. After performing MTS assay, measured absorbance values were divided by the average signal of the complete plate and multiplied by 100 to get percentages of viable cells.
  • ApoAl ELISA was performed using a 96 channel dispenser (Tecan), Plate Washer (Tecan) and Multidrops (Labsystems), like being used in the ApoBlOO ELISA described in Example 2. In both ApoBlOO and ApoAl ELISA, each plate contained a standard curve (row H, Figure 7). Concentrations of ApoBlOO and ApoAl were calculated for all samples as described in Example 1 and 4.
  • the threshold was set at 40%. All hits with ApoBlOO secretion (at least one of the two data points, duplicates on independent assay plates) equal to or below this threshold were considered as confirmed ApoBlOO hits. A total number of 244 confirmed ApoBlOO hits was isolated in this experiment (see Table 4).
  • ApoBlOO hits were classified as prioritized hits if at least one of the two data points (duplicates on independent assay plates) scored positive for ApoAl secretion (ApoAl secretion > 65%) and cell viability (%viable cells > 70%).
  • a total of 138 confirmed ApoBlOO hits were classified as prioritized hits (see Table 4) and will be subjected to further validation steps.
  • Example 6 Determination of the expression level in HepG2, Huh, primary hepatocytes, and whole liver cells.
  • RNA 6000 Nano Chips from Agilent Technologies.
  • Sample preparation for hybridization was performed using "Once-Cycle cDNA Synthesis Kit” (Affymetrix) followed by "Gene Chip Expression 3 '-Amplification for IVT Labeling Kit” (Affymetrix).
  • Gene Chip Scanner 3000 + equipment Affymetrix
  • human Gene Chips HG- Ul 33 Plus 2
  • Affymetrix were used for signal detection. Signals were analyzed primarily using GCOS software (Affymetrix) and subsequently with GeneData software.
  • EXPCATl target detected in human liver
  • EXPCAT2 no probe on Affymetrix Chipset HG-Ul 33 A/B or not detected in any sample
  • EXPCAT3 detected in HepG2 cells but not in human liver
  • EXPCAT4 not detected in human liver and HepG2 cells but in another sample
  • targets from category EXPCATl and EXPCAT2 were prioritized.
  • EXPCAT2 genes were included in cases where there was no probe for the gene on the Affymetrix chip or if the gene's expression was negative in all samples (indicating that the probe did not work correctly). Further prioritization (manual selection) of targets from category EXPCATl and EXPCAT2 was based on their novelty, relevancy, drugability etc. 99 hits prioritized hits were selected to be taken to the next validation, the 3 MOI test (see Table 5).
  • Example 7 Screening for compounds useful in the treatment of Atherosclerosis using a cell based assay.
  • the recombinant CHO-Kl(ATCC No.: CCL-61) screening cell line expresses constitutively the calcium sensitive photoprotein Aequorin. After reconstitution with its cofactor Coelenterazin and increasing intracellular calcium concentration Aequorin is able to emit light (Rizzuto R, Simpson AW, Brini M, Pozzan T.; Nature 358 (1992) 325-327). Additionally, after transfection with a recombinant expression plasmid containing the full length cDNA for human CysLTR2, the screening cell line is stably expressing the CysLTR2 protein (Heise et.al., JBC 215 (2000) 30531- 30536).
  • the CysLTR2 screening cell line is able to react on stimulation with known CysLTR2 agonists (i.e. Leukotriene D4 and Leukotriene C4) with an intracellular Ca +4" release and resulting luminescence can be measured with appropriate luminometer (Milligan G, Marshall F, Rees S, Trends in Pharmacological Sciences 17 (1996) 235-237). Preincubation with CysLTR2 antagonists diminish the Leukotriene D4 or Leukotriene C4 induced Ca 1+ release and consequently the resulting luminescence.
  • Cells are seeded into 384 well cell culture plates and preincubated for 48 hours in culture medium (DMEM/F12 with Glutamax, Gibco Cat.# 61965-026; 10% Fetal Calf Serum, Gibco Cat.# 10270- 106; 1,4 mM Natriumpyruvat, Gibco Cat.# 11360-039; 1,8 mM Natriumbicarbonate, Gibco Cat.# 25080-060; 10 mM HEPES, Gibco Cat.# 15290-026) under standard cell culture conditions (96% humidity, 5% v/v CO 2 , 37 0 C).
  • culture medium DMEM/F12 with Glutamax, Gibco Cat.# 61965-026; 10% Fetal Calf Serum, Gibco Cat.# 10270- 106; 1,4 mM Natriumpyruvat, Gibco Cat.# 11360-039; 1,8 mM Natriumbicarbonate, Gibco Cat.# 25080-060; 10 mM HEPES, Gibco Cat.# 15290-0
  • Culture medium is replaced by Tyrode buffer (containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 20 mM Glucose, 20 mM HEPES) plus Coelenterazin (50 ⁇ M) and incubation is continued for additional 3-4 hours.
  • Reference agonists Leukotriene D4, Leukotriene C4 or putative agonists are added to the cells and luminescence is measured subsequently.
  • 15 min preincubation with putative antagonists is allowed before Leukotriene D4 ( 3 x 10 "8 M) stimulus.
  • Example 8 Screening for compounds useful in the treatment of Atherosclerosis usins a cell-free
  • the screening method for the identification of inhibitors of the human Phosphodiesterase 4B (PDE4B; NM_002600) using a cell-free biochemical assay will be taken as an example.
  • PDE4B (GenBank/EMBL Accession Number: NM_002600, Obernolte et al. Gene. 1993 129, 239- 247) is expressed in Sf9 insect cells using the Bac-to-BacTM baculovirus expression system. Cells are harvested 48 h after infection and suspended in lysis buffer (20 ml/11 culture, 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM MgC12, 1.5 mM EDTA, 10% Glycerin, 20 ⁇ L protease inhibitor cocktail set in [CalBiochem, La Jolla, CA USA]).
  • the cells are disrupted by sonication at 4°C and cell debris is removed by centrifugation at 15,000 x g at 4°C for 30 minutes.
  • the supernatant is designated PDE4B cell extract and is stored at -8O 0 C.
  • test substances are dissolved in DMSO and serial dilutions in DMSO are performed. 2 ⁇ l of the diluted test compounds are placed in wells of microtiter plates (Isoplate; Wallac Inc., Atlanta, GA). 50 ⁇ l of a dilution of the PDE4B cell extract (see above) is added.
  • the dilution of the PDE4B cell extract will be chosen that during the incubation with substrate the reaction kinetics is linear and less than 70% of the substrate is consumed (typical dilution 1: 150 000; dilution buffer: 50 mM Tris/HCl pH 7.5, 8.3 mM MgC12, 1.7 mM EDTA, 0.2% BSA).
  • the substrate, [5',8-3H] adenosine 3', 5'-cyclic phosphate (1 ⁇ Ci/ ⁇ l; Amersham Pharmacia Biotech., Piscataway, NJ) is diluted 1:2000 in assay buffer (50 mM Tris/HCl pH 7.5, 8.3 mM MgC12, 1.7 mM EDTA).
  • the reaction starts by addition of 50 ⁇ l (0.025 ⁇ Ci) of the diluted substrate and incubates at room temperature for 60 min.
  • the reaction is stopped by addition of 25 ⁇ l of a suspension containing 18 mg/ml yttrium scintillation proximity beads in water (Amersham Pharmacia Biotech., Piscataway, NJ.).
  • the microtiter plates are sealed, left at room temperature for 60 min, and are subsequently measured in a Microbeta scintillation counter (Wallac Inc., Atlanta, GA).
  • IC50 values will be determined by plotting the substrate concentration against the percentage PDE4B inhibition.
  • a hit is classified as a prioritized hit if:
  • a total of 48 confirmed ApoBlOO hits were classified as prioritized hits (see Table 8, 9 and 10).
  • 16 target genes were chosen to be taken to the next validation step. This target selection was based on different criteria (i.e. their novelty, relevancy, data obtained in previous screens/tests, potential to make small compounds against it etc.)
  • the functional ApoBlOO knock-down experiment was followed by the isolation of KNA from the selected samples, selected based on their functional data (ApoBlOO, ApoAl and cell viability, see Table 12 and 13). RNA isolations were performed on the remaining cells from the same samples of which the supernatant had been used for the functional ApoBlOO knock-down experiment. The samples taken along were all biological duplicates of all 3 MOI's of samples listed in Table 13. Biological duplicates of A150100-ApoB100_v6 and A150100-ApoAl_v2 controls (all 3 MOI's) were taken along in 5-fold. Isolations were performed using the SV96 Total RNA Isolation System (Promega cat.# Z3505) according to the manufacturers protocol.
  • RNA concentration was determined using RiboGreen RNA quantitation assay (Molecular Probes cat.# R-11491) according to the manufacturers protocol. The average RNA concentration was ⁇ 55 ng/ ⁇ l.
  • RNA quality control was performed by ethidium bromide agarose-gel analysis. From each construct the MOI 160 RNA sample was loaded on 1% agarose gel (3 gels in total). After electrophoresis, gels were stained with ethidium bromide. All RNA samples showed the same expected pattern after this analysis.
  • RNA quality control was followed by the DNase digest- and reverse transcriptase step in which RNA was converted to cDNA. This was done using Deoxyribonuclease I Amplification Grade kit (Invitrogen, cat.# 18068-015) and ImProm-IITM Reverse Trascription System kit (Promega, cat.# A3800) according to the following protocol.
  • 8 ⁇ l RNA ( ⁇ 440 ng) was incubated with 1 ⁇ l 1Ox DNase I Reaction Buffer and 1 ⁇ l DNase I Amp Grade (1 U/ ⁇ l) for 15 minutes at room temperature.
  • 1 ⁇ l 25 mM EDTA was added to each reaction mix and this was incubated for 15 minutres at 65°C and 1 minute on ice.
  • This DNase digesting step was followed by the reverse transcriptase step initiated by adding of 2.2 ⁇ l Oligo(dT) i5 primer (0.5 ⁇ g/ ⁇ l) to each reaction mix and two incubation steps (5 minutes at 70 0 C and 5 minutes on ice). After this, 39.6 ⁇ l of reverse transcription master mix was added to each reaction mix.
  • Reverse transcription mastermix contained per sample: 14.5 ⁇ l nuclease-free water, 10.6 ⁇ l ImProm-IITM reaction buffer (5x), 8 ⁇ l MgCl 2 (25 mM), 2.6 ⁇ l dNTP mixture (10 mM), 1.3 ⁇ l recombinant RNasin ® ribonuclease inhibitor and 2.6 ⁇ l ImProm-IITM reverse transcriptase. Adding of reaction mix was followed by two incubation steps (5 minutes at 25°C and 60 minutes at 42°C). Reverse transcription step was completed by addition of 35 ⁇ l nuclease free water to each reaction mix. This cDNA was used in the TaqMan RealTime-PCR RNA measurements.
  • the primer labelled with FAM (carboxyfluorescein succinimidyl ester) as the reporter dye and TAMRA (carboxytetramethylrhodamine) as the quencher is used as a probe.
  • the following reagents were prepared in a total of 25 ⁇ l : Ix TaqMan buffer A, 5.5 mM MgCl 2 , 200 nM of dATP, dCTP, dGTP, and dUTP, 0.025 U/ ⁇ l AmpliTaq GoldTM, 0.01 U/ ⁇ l AmpErase and GOI upper and lower primers each at 200 nM, 200 nM GOI FAM/TAMRA-labelled probe, and 5 ⁇ l of template cDNA.
  • Thermal cycling parameters were 2 min at 5O 0 C, followed by 10 min at 95°C, followed by 40 cycles of melting at 95 0 C for 15 sec and annealing/extending at 6O 0 C for 1 min.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Urology & Nephrology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Obesity (AREA)
  • Diabetes (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Cardiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des cibles et des procédés pour cribler des composés utilisés pour prévenir, améliorer ou traiter un trouble cardio-vasculaire, une dyslipidémie et/ou une athérosclérose. Cette invention concerne également les cibles qui ont été identifiées. L'utilisation de composés identifiables par les procédés selon cette invention pour inhiber des gènes cibles de l'invention ou leurs produits d'expression est bénéfique dans le cadre d'un traitement de maladies impliquant un trouble cardio-vasculaire, une dyslipidémie et/ou une athérosclérose.
PCT/EP2006/001451 2005-03-02 2006-02-17 Nouvelles cibles et composes utilises pour traiter un trouble cardio-vasculaire, une dyslipidemie et une atherosclerose et procedes pour identifier de tels composes Ceased WO2006092209A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US65831505P 2005-03-02 2005-03-02
US60/658,315 2005-03-02

Publications (1)

Publication Number Publication Date
WO2006092209A1 true WO2006092209A1 (fr) 2006-09-08

Family

ID=36251854

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2006/001451 Ceased WO2006092209A1 (fr) 2005-03-02 2006-02-17 Nouvelles cibles et composes utilises pour traiter un trouble cardio-vasculaire, une dyslipidemie et une atherosclerose et procedes pour identifier de tels composes

Country Status (1)

Country Link
WO (1) WO2006092209A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014140376A1 (fr) * 2013-03-15 2014-09-18 Vib Vzw Domaines variables uniques anti-récepteur du mannose des macrophages pour leur utilisation dans des maladies cardiovasculaires

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002018424A2 (fr) * 2000-09-01 2002-03-07 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
WO2002046458A2 (fr) * 2000-12-07 2002-06-13 Aventis Pharma S.A. Acides nucleiques des genes humains abca5, abca6, abca9 et abca10, vecteurs contenant de tels acides nucleiques et leurs utilisations
WO2005013947A2 (fr) * 2003-07-29 2005-02-17 Universitätsklinikum Münster Moyens et procedes de traitement d'une maladie liee a un exces de transport de l'hyaluronane a travers la bicouche lipidique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002018424A2 (fr) * 2000-09-01 2002-03-07 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
WO2002046458A2 (fr) * 2000-12-07 2002-06-13 Aventis Pharma S.A. Acides nucleiques des genes humains abca5, abca6, abca9 et abca10, vecteurs contenant de tels acides nucleiques et leurs utilisations
WO2005013947A2 (fr) * 2003-07-29 2005-02-17 Universitätsklinikum Münster Moyens et procedes de traitement d'une maladie liee a un exces de transport de l'hyaluronane a travers la bicouche lipidique

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014140376A1 (fr) * 2013-03-15 2014-09-18 Vib Vzw Domaines variables uniques anti-récepteur du mannose des macrophages pour leur utilisation dans des maladies cardiovasculaires
JP2016512220A (ja) * 2013-03-15 2016-04-25 ブイアイビー ブイゼットダブリュＶｉｂ Ｖｚｗ 心血管疾患において使用するための抗マクロファージマンノース受容体単一可変ドメイン
US9617339B2 (en) 2013-03-15 2017-04-11 Vib Vzw Method of imaging a cardiovascular disease with an anti-macrophage mannose receptor immunoglobulin single variable domain

Similar Documents

Publication Publication Date Title
Song et al. Long noncoding RNA CASC11 promotes hepatocarcinogenesis and HCC progression through EIF4A3‐mediated E2F1 activation
Luo et al. MicroRNA-26 governs profibrillatory inward-rectifier potassium current changes in atrial fibrillation
Gou et al. miR-210 has an antiapoptotic effect in pulmonary artery smooth muscle cells during hypoxia
US20160313349A1 (en) Method of diagnosis and treatment
EP2972322B1 (fr) Cibles et composés moléculaires, et méthodes utilisées pour les identifier, dans le cadre du traitement de la fibrose
Wang et al. LncRNA SNHG7 promotes cardiac remodeling by upregulating ROCK1 via sponging miR-34-5p
EP1877798A2 (fr) Gènes marqueurs humains et agents de diagnostic, de traitement et de prophylaxie des troubles cardio-vasculaires et de l'athérosclérose
US8299044B2 (en) Apoptosis inducer for cancer cell
CN111630166A (zh) 条件性-siRNA及其在治疗心肌肥大中的用途
Rydén et al. Insulin action is severely impaired in adipocytes of apparently healthy overweight and obese subjects
Yang et al. Circ_0001052 promotes cardiac hypertrophy via elevating Hipk3
Qi et al. miR-129-5p restores cardiac function in rats with chronic heart failure by targeting the E3 ubiquitin ligase Smurf1 and promoting PTEN expression
EP2229946A2 (fr) Utilisation de la protéine stimulatrice de croissance KIAA1524
Zhang et al. Medium‐dose formoterol attenuated abdominal aortic aneurysm induced by EPO via β2AR/cAMP/SIRT1 pathway
WO2016152352A1 (fr) Biomarqueur spécifique du mélanome et son utilisation
JP5686730B2 (ja) 心臓肥大を抑制、遅延、および／または予防するための方法、および医薬組成物
US20210147842A1 (en) Methods and compositions for inhibiting necroptosis in neurovascular and/or neurodegenerative diseases or disorders
Yi et al. [Retracted] MiR‐579 Inhibits Lung Adenocarcinoma Cell Proliferation and Metastasis via Binding to CRABP2
WO2006092209A1 (fr) Nouvelles cibles et composes utilises pour traiter un trouble cardio-vasculaire, une dyslipidemie et une atherosclerose et procedes pour identifier de tels composes
WO2007000292A2 (fr) Nouvelles cibles et composes utiles pour le traitement et/ou la prophylaxie de troubles cardio-vasculaires de la dislipidemie, et de l'atherosclerose, et procedes d'identification de tels composes
US20140243387A1 (en) Methods for improving cardiac contractility
Chen et al. Linc-OIP5 working as a ceRNA of miR-616 promotes PON1 expression in HUEVC cells
EP2646572B1 (fr) Diagnostic et traitement d'une insuffisance cardiaque chronique
US20160077079A1 (en) The children's hospital of philadelphia
US7846910B2 (en) Pharmaceutical composition, composition for screening therapeutics preventing and treating beta amyloid accumulation in brain comprising GCP II (glutamate carboxypeptidase II) as an active ingredient and method for screening using the same

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

WWW Wipo information: withdrawn in national office

Country of ref document: RU

122 Ep: pct application non-entry in european phase

Ref document number: 06707043

Country of ref document: EP

Kind code of ref document: A1

WWW Wipo information: withdrawn in national office

Ref document number: 6707043

Country of ref document: EP