WO2007000292A2

WO2007000292A2 - Novel targets and compounds useful in the treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia and atherosclerosis, and methods to identify such compounds

Info

Publication number: WO2007000292A2
Application number: PCT/EP2006/006117
Authority: WO
Inventors: Ulrich Betz; Donatella D'urso; Petros Gatsios; Michael Seewald; Jochen Strayle; Helmuth Hendrikus Gerardus Van Es; Marlijn Van Zutphen; Emir Mesic
Original assignee: Galapagos Nv
Priority date: 2005-06-29
Filing date: 2006-06-24
Publication date: 2007-01-04
Also published as: WO2007000292A3

Abstract

The present invention provides targets and methods for the screening for compounds useful in the prevention, amelioration or treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis. The invention also relates to the targets that were identified. Inhibiting or activating target genes of the invention, or their expression products, by using compounds identifiable by methods of the invention, is beneficial in the treatment and/or prophylaxis of diseases involving a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.

Description

Novel targets and compounds useful in the treatment and/or prophylaxis of A cardiovascular disorder, dyslipidemia and Atherosclerosis, and methods to identify such compounds

Field of the invention

The invention relates to novel targets in the screening for compounds useful in the treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis. The invent- tion 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 agonists that target G-protein coupled receptors (GPCRs), kinases, proteases, and other drugable classes and to methods for identifying such compounds. 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.

Background of the Invention

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 nations. 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.

With regard to treatment of dyslipidemia, numerous well-controlled outcome studies of lipid- altering drug monotherapy in >50000 subjects have consistently demonstrated a relative risk reduction (compared to placebo) of only 20-40% after 3-6 years of therapy. 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 hypercholesterolemic market. The majority of patients observed in monotherapy trials of lipid-altering drugs have not had their CHD prevented. This suggests that further absolute and relative CHD risk will only be achieved through extending the duration of lipid-altering therapy, achieving more aggressive lipid treatment goals or treating multiple lipid parameters. It may also be reasonable to conclude that the best way to further reduce

CHD risk is to aggressively correct the abnormality or abnormalities, which contribute most to the atherosclerotic process in the individual patient. This may occur through monotherapy, or through a multifactorial approach with the use of compounds addressing multiple risk factors. The US National Cholesterol Education Program (NCEP) has issued new guidelines that could significantly enhance the number of patients prescribed hypolipidemics in the US. The NCEP continues to identify LDL cholesterol as the primary target of therapy. Acceptable levels of LDL cholesterol as well as HDL cholesterol and triglycerides are more stringent than those in earlier guidelines. Therefore, additional lipid lowering therapies are necessary (e.g., currently, half of patients treated with statins do not reach the new target LDL level).

Taken together, the therapeutic strategies currently available for treating Atherosclerosis are not satisfactory. As a major drawback, their limited efficacy calls for additional strategies to identify new medicaments with improved efficacy against Atherosclerosis.

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. Molecular targets involved in high density lipoprotein (HDL) cholesterol metabolism include cholesteryl ester transfer protein (CETP) with effective inhibitors under development, ATP-binding cassette transporter (ABC) Al as well as scavenger receptor class B Type 1 (SRBl). Nuclear receptors as PPARs, LXR and FXR are also targets of investigational agents.

Because of the small number of available targets and because of the limited success in screening methods using available targets, a great need is felt in the art for promising targets and novel screening methods for compounds highly active in the treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.

Brief Description of the Drawings

Figure 1 shows a standard curve for secreted ApoBlOO [μg/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 for activators (average of all samples plus 2.3 times standard deviation).

Figure 5 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 for repressors (average of all samples minus 2.1 times standard deviation).

Figure 6 shows a standard curve for secreted ApoAl [μg/ml] over light absorbance at 450 nm.

Figure 7 shows an example of a plate used in the rescreen for ApoB 100 secretion activators. The dotted line represents the set cut-off value for activators (average of all samples plus 1.8 times standard deviation).

Figure 8 shows an example of a plate used in the rescreen for ApoB 100 secretion repressors. The dotted line represents the set cut-off value for repressors (average of all samples minus 2.1 times standard deviation).

Figure 9 shows the plate layout used in the 3 MOI test for dose response

Brief Description of the Tables

Table 1 describes diluting of ApoB 100 standard for the purposes of ApoB 100 standard curve.

Table 2 describes diluting of ApoAl standard for the purposes of ApoAl standard curve.

Table 3 shows for each of the 19 activator targets of the invention: SEQ ID NO; hit code; Hit type, describing designated activator or repressor activity; results of the primary screen (Example 2) biological duplicates); number of samples of the biological duplicate which scored over the cutoff value (average of all samples plus 2.3 times standard deviation) in the primary screen (Example 2); results of the secondary screen (re-screen Example 4, biological duplicates); number of samples of the biological duplicate which scored above the cutoff value (average of all samples plus 1.8 times standard deviation)in the re-screen; hit call (number of samples which came up as a hit in the primary- and the re-screen per target); drugable class; and target symbol.

Table 4 shows for each of the 19 activator targets of the invention: SEQ ID NO; hit code, target symbol; GenBank Accession number; and a brief description of the target.

Table 5 shows the results of ApoAl, and %viable cells measurements for biological duplicates for the 19 activators as determined in Example 4. Table 6 shows for each of the 19 activator targets of the invention the results of expression profiling experiments in HepG2, Huh cells, primary hepatocytes, and whole liver. 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 as determined in Example 5.

Table 7 shows for each of the 56 repressor targets of the invention: seqID; hit code; Hit type, describing designated activator or repressor activity; results of the primary screen (Example 2)biological duplicates); number of samples of the biological duplicate which scored under the cutoff value (average of all samples minus 2.1 times standard deviation) in the primary screen (Example 2); results of the secondary screen (re-screen Example 4, biological duplicates); number of samples of the biological duplicate which scored under the cutoff value (average of all samples minus 2.1 times standard deviation)in the re-screen; hit call (number of samples which came up as a hit in the primary- and the re-screen per target); drugable class; and target symbol.

Table 8 shows for each of the 56 repressor targets of the invention: seqID; hit code, target symbol; GenBank Accession number; and a brief description of the target.

Table 9 shows the results of, ApoA 1 , and %viable cells measurements for biological duplicates for the 56 repressors as determined in Example 4.

Table 10 shows for each of the 56 repressor targets of the invention the results of expression profiling experiments in HepG2, Huh cells, primary hepatocytes, and whole liver. 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 as determined in Example 5.

Table 1 1 shows for all 14 activator and 31 repressor hits the results of the 3 MOI test (dose response) for ApoBlOO secretion. It includes seq. ID, hit code, notation if a hit is a repressor or an activator, % of ApoBlOO secretion for biological duplicates of all 3 MOI's, hit calling for MOI effect (yes or no) and hit calling for ApoBlOO secretion (number of samples_Moii_δo which score below the cut-off (repressor hits) or above the cut-off (activator hits)).

Table 12 shows for all 14 activator and 31 repressor hits the results of the 3 MOI test (dose response) for ApoAl secretion and cell viability. It includes seq. ID, hit code, notation if a hit is a repressor or an activator, % of ApoAl secretion for biological duplicates of all 3 MOI's including the hit selection (number of samples_Monβo which score above the cut-off) and % of viable cells for biological duplicates of all 3 MOI's including the hit selection (number of samples_Moii₆o which score above the cut-off).

Summary of the Invention

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 3 and

Table 7, or of any one of the polypeptides encoded thereby, in the identification of a compound useful in the treatment and/or prophylaxis 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 3 and Table 7 in the manufacture of a medicament for the treatment and/or prophylaxis 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 3 and Table 7 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.

Detailed Description of the Invention

The inventors of the present invention identified novel target genes that are involved in a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis by using the FlexSelect collection (see WO99/64582), a so-called arrayed adenoviral 'knock-in' library: A screen in which cDNA molecules were transduced into cells by recombinant adenoviruses. This screen was used in order to increase the expression and hence the activity of the corresponding gene and gene product in a cell. By identifying a cDNA 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. For detailed description, see examples. 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 and/or prophylaxis 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. Furthermore, 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 and/or modulate the activity of these polypeptides, this is a new application, which is also covered by the present invention.

The invention relates to:

1. Method for identifying a compound as being useful in the treatment and/or prophylaxis of a disease, comprising the steps of

(a) providing a first cell expressing a target polypeptide selected from the group listed in Table 3 or 7, 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.

2. Use of a method of Count 1 for the screening for substances useful in the treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.

3. Method of Count 1 or use of Count 2, wherein said host cell expresses said target polypeptide above wild-type level. 4. Method or use of any of Counts 1 to 3, wherein said target polypeptide expression is recombinant polypeptide expression.

5. Method or use of any of Counts 1 to 4, wherein said compound is selected if said first level of said activity or property is lower than said reference level of said activity or property.

6. Method or use of any of Counts 1 to 4, wherein said compound is selected if said first level of said activity or property is higher than said reference level of said activity or property.

7. Method or use of any of Counts 1 to 6, wherein said reference level is a level obtained from a second cell expressing the target polypeptide at a lower level as compared to said first cell.

8. Method or use of any of Counts 1 to 6, wherein said reference level is the level obtained with said first cell in the absence of the candidate compound.

9. Method or use of any of Counts 1 to 8, wherein said method further comprises contacting the host cell with a known agonist or antagonist of the target polypeptide before determining the first level.

10. Method or use of any of Counts 1 to 9, wherein said activity or property being affected by said activity or property of said target polypeptide is binding affinity of said compound to said target polypeptide.

11. Use of a method, said method comprising the steps of

(a) culturing a population of cells expressing a target polypeptide listed in Table 3 or 7, or a functional fragment or derivative thereof;

(b) determining a first level of expression and/or activity of said target protein in said population of cells;

(c) exposing said population of cells to a compound, or a mixture of compounds;

(d) determining a second level of expression and/or activity of said target polypeptide in said population of cells during or after said exposure of said population of cells to the compound, or the mixture of compounds; and

(e) comparing said first and said second level; for the screening for substances useful in the treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.

12. Method or use of any of Counts 1 to 11, wherein said first level of an activity or property is determined with a reporter, said reporter being controlled by a promoter responsive to at least one second messenger.

13. Method or use of Count 12, wherein said at least one second messenger is cyclic AMP, or Ca2+, or both.

14. Method or use of 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.

15. Method or use of any of any of Counts 12 to 14, wherein the reporter is luciferase or beta- galactosidase.

16. Method or use of any of Counts 1 to 15, wherein the compound is a low molecular weight compound.

17. Method or use of any of Counts 1 to 15, wherein the compound is a polypeptide.

18. Method or use of any of Counts 1 to 15, wherein the compound is a lipid.

19. Method or use of any of Counts 1 to 15, wherein the compound is a natural compound.

20. Method or use of any of Counts 1 to 15, wherein the compound is an antibody or a nanobody.

21. Method for identifying a compound as being useful in the treatment and/or prophylaxis of a disease, comprising the steps of

(a) contacting said compound with a target polypeptide selected from the group listed in Table 3 or 7, or a fragment, or a derivative thereof;

(b) detect binding of said compound to said target polypeptide or detect a change in activity of said target polypeptide;

(c) selecting said compound if binding is detected in step (b) or if a change in activity is detected in step (b); characterised in that

22. Use of a method of count 21 or 22 for screening for compounds, useful in the treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.

23. Method or use of any of counts 21 to 22, wherein binding is detected in vitro or in vivo.

24. Method or use of any of counts 21 to 23, wherein said target polypeptide is a recombinant polypeptide.

25. Method or use of any of counts 21 to 24, wherein said compound is selected if the numerical value of the binding affinity is equal to or lower than 10 micromolar.

26. Method or use of any of counts 21 to 25, wherein said compound is a low molecular weight compound.

27. Method or use of any of counts 21 to 25, wherein said compound is a polypeptide, or a lipid, or a natural compound, or an antibody or a nanobody.

28. Method or use according to counts 1 - 27, wherein for a target listed in table 3 an antagonist and for a target listed in table 7 an agonist is selected.

29. Use of a compound that inhibits an activity and/or the expression of any of the polypeptides listed in Table 3 in the manufacture of a medicament for the treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.

30. Use of a compound that increases an activity and/or the expression of any of the polypeptides listed in Table 7 in the manufacture of a medicament for the treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.

31. Use of Count 29 or 30, wherein said compound is identified according to any one of the methods or uses of Counts 1 to 28.

32. Use of an agent inhibiting the expression of a polypeptide selected from the group listed in Table 3 or of an agent increasing the expression of a polypeptide selected from the group listed in Table 7 for the preparation of a medicament for the treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.

33. Use of Count 30, wherein said agent is selected from the group consisting of: an antisense RNA encoding said polypeptide;

a ribozyme that cleaves the polyribonucleotide encoding said polypeptide;

an antisense oligodeoxynucleotide (ODN) enconding said polypeptide;

a small interfering RNA (siRNA) that is sufficiently homologous to a portion of the polyribonucleotide such that said siRNA is capable of inhibiting the polyribonucleotide that would otherwise cause the production of said polypeptide;

a micro RNA (miRNA) suitable for inhibition of a polypeptide listed in table 3; or

a short hairpin RNA (shRNA) suitable for silencing the expression of a polypeptide selected from table 3.

34. Use of Count 33, wherein the nucleotide sequence of said agent is present in a vector.

35. Use of Count 34, 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.

36. Use of any of Counts 33 to 35, 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 3 or 7.

37. Use of Count 36, wherein the siRNA further comprises a cleavable loop region connecting the sense and the antisense strand.

38. Method for diagnosing a pathological condition involving a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis, or a susceptibility to said condition in a subject, comprising

(a) obtaining a sample of the subject's mRNA corresponding to a polypeptide selected from the group listed in Table 3 or 7, or a sample of the subject's genomic DNA corresponding to a polypeptide of Table 3 or 7;

(b) determining the nucleic acid sequence of said mRNA or said genomic DNA;

(c) obtaining the nucleic acid sequence encoding said polypeptide of Table 3 or 7 from a public database; and (d) identifying any difference(s) between the nucleic acid sequences determined in step (b) and (c);

wherein 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).

39. 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) determining the amount of a polypeptide of Table 3 or 7 in a biological sample of said subject; and

(b) comparing the amount determined in (a) with a the amount of the polypeptide in a healthy subject;

wherein an increase or a decrease of the amount of said polypeptide compared to the amount present in a healthy subject is indicative of the presence of the pathological condition.

Whereas the method relates to uses, methods and substances relating to all targets listed in Table 3 and 7, some of the Targets 1 to 75 of Table 3 or 7 are more preferred within the context of the present invention. For example, those targets are preferred targets, which have exceeded the threshold values of 2.3 (targets of table 3) more than twice or lay more than twice times under - 2.1 (targets of table 7) in the screening runs reported in Table 3 or 7. Even more preferred targets are targets which have exceeded the threshold value more than three times in the screening experiments reported in Table 3 or 7 (i.e., targets with hit call "2-2" in Table 3 or 7).

Furthermore, those targets listed in Table 3 or 7 are preferred, which are highly expressed in HepG2 cells, Huh cells, primary hepatocytes, and whole liver cells. Those targets of Table 3 or 7, which show an average expression of above 200, more preferable 400 in HepG2 cells, Huh cells, primary hepatocytes, or whole liver cells, in Table 6 or 10, are preferred targets of the invention. Even more preferred are targets of Table 6 or 10, which show an average expression of above 200, more preferable 400 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 6 or 10. Furthermore, those targets listed in Table 3 or 7 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.

Furthermore, those targets listed in Table 3 or 7 are preferred targets of the invention, which are not suppressing ApoAl secretion from the HepG2 cells. Those targets of Table 3 or 7, which do not show suppression of ApoAl secretion of more than 10% (>90% remaining), in Table 5 or 9, are preferred targets of the invention.

Furthermore, those targets listed in Table 3 or 7 are preferred targets of the invention, which do not affect cell-viability. Those targets of Table 3 or 7, which do not show a lowering in cell viability of more than 10% (>90% remaining), in Table 5 or 9, are preferred targets of the invention.

Especially preferred targets are targets listed in table 3 or 7, which increase ApoAl secretion.

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 3 or increases the expression and/or activity of a polypeptide encoded by any one of the target genes of Table 7, 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 3 or 7, 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 for targets from table 3 or the second activity level is higher than the first activity level for targets from table 7.

It will be understood by a skilled person that a decrease in expression level will also result in a reduced 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 3 or 7 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 3 or 7, 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.

This means that compounds that were known to bind to the polypeptides of the invention may now be useful in the treatment and/or prophylaxis of diseases such as a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis, a utility which could not be envisioned before the present invention. Thus, 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 3 or 7, for the preparation of a medicament for the treatment and/or prophylaxis 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, Fluorometric Imaging Plate Reader (FLIPR®) 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. For high-throughput purposes, libraries of compounds can be used such as peptide libraries (e.g. LOPAP™, Sigma Aldrich, St. Louis), lipid libraries (BioMol, Hamburg), synthetic compound libraries (e.g. LOPAC™, Sigma Aldrich, St. Louis) or natural compound libraries (Specs, TimTec, Newark, DE).

In one embodiment of the methods of the present invention, 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. Preferably, 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. In another preferred embodiment, the reporter gene is selected from the group consisting of: alkaline phosphatase, GFP, eGFP, dGFP, luciferase and beta-galactosidase.

In another embodiment of the present invention, 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.

In yet another embodiment of the present invention, said polypeptide is a protease wherein the expression and/or activity

of said protease is measured by determining the level of cleavage of a substrate of said protease. Such protease assays are well known in the art.

In yet another embodiment of the present invention, 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. Such transporter assays are well known in the art.

In yet another embodiment of the present invention, 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. Such ion channel assays are well known in the art.

In yet another embodiment of the present invention, 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. Such PDE assays are well known in the art. In yet another embodiment of the present invention, 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 enzyme assays are well known in the art.

In yet another embodiment of the present invention, 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. Such 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.

For a proper identification it is preferred that 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 3 or increases the activity and/or the expression of a polypeptide encoded by any one of the target genes of Table 7 in the manufacture of a medicament for the treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis. Preferably, 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 drug candidates and there are multiple examples of commercially available peptides such as fertility hormones and platelet aggregation inhibitors.

In yet another embodiment, 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. Using 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 a polypeptide of table 3 or increases the activivty of a polypeptide of table 7. In another embodiment, 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).

In one specific embodiment of the present invention, 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 3, or a fragment, or a derivative thereof; and contacting the cell with a compound that inhibits the translation in the cell of the polynucleotide.

In another embodiment of the present invention, the compound is an expression activating agent that increases 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 7, or a fragment, or a derivative thereof; and contacting the cell with a compound that inhibits the translation in the cell of the polynucleotide.

Examples of 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. Preferably, said expression inhibitory agent comprises a nucleotide sequence of any one the genes listed in Table 3.

In one preferred embodiment, 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 3, and an antisense strand of 17-23 nucleotides complementary to the sense strand. It is well known in the art which domains are suitable for siRNA activity and how such siRNA molecules can be designed. Preferably, 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. Even more preferred is the nucleic acid sequence UUGCUAUA as described in WO 03/020931.

microRNAs (miRNAs) are evolutionarily conserved small non-protein-coding RNA gene products that regulate gene expression at the post-transcriptional level. In animals, mature miRNAs are ~22nucleotides long and are generated from a primary transcript through sequential processing by nucleases belonging to the RNAselϋ family.

An alternative to transfecting cells with chemically synthesized siRNAs are DNA-vector-mediated mechanisms to express substrates that can be converted into siRNA in vivo. In the first approach the sense and antisense strands of the siRNA are expressed from different, usually tandem promoters. Alternatively, short hairpin (sh)RNAs are expressed and processsed by Dicer into siRNAs. In general, chemically synthesized short interfering (si)RNA sequences that are effective at silencing gene expression are also effective when generated from short hairpin (sh)RNAs. However, the length of the stem and the size and composition of the loop are important for the efficiency of silencing.

In another preferred embodiment, if the compound comprises nucleic acids, 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. Such transfer may be achieved by a wide variety of means, for instance by direct transfection of naked isolated DNΛ, 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. Preferably, the nucleic acid compound is delivered by means of a (recombinant) vector such as a recombinant virus.

Preferably, 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. The generation and cloning procedures for such recombinant vectors are well known to the skilled person. Generally, recombinant vectors are being applied that are made replication-defective when introduced in host cells. These vectors are typically made on packaging cells, which cells provide the factors that are lacking from the recombinant vector to be replication-competent. 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.

Another aspect of the present invention relates to siRNA according to the invention for use as a medicament, preferably, wherein said use is in the treatment and/or prophylaxis 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. Preferably, 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 and/or prophylaxis 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 3 or 7. However, their possible role in the treatment and/or prophylaxis 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. Thus, 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 and/or prophylaxis of diseases in which a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis are involved. Thus, 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 role of the target genes that were identified in the course of the present invention in the pathway leading to a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis was unknown until the present invention. This finding now enables one to use this knowledge in methods for diagnosing a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis by checking the status of the gene, its expression and/or its activity. Therefore, 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 3 or 7 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. It also relates to methods for diagnosing a pathological condition involving a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis in a subject, said methods comprising the steps of: determining the expression and/or activity of a polypeptide encoded by any one of the target genes of Table 3 or 7 in a sample from said subject; and comparing the level determined in the first step with the expression or activity of said polypeptide in a sample from a healthy individual; wherein the increase of the level in the sample of said subject as compared to the healthy individual is indicative for the onset or presence of said pathological condition. Preferably, the pathological condition is a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.

Diagnostic methods of the invention are preferably performed in a sample, ex vivo.

The expression or activity of 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. It can however not be excluded that 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 expression or activity of an Atherosclerosis-associated protein is affected, as used herein, if the expression or activity of the protein is increased upon incubation with the compound. Although a rise of those levels may differ and may be multifold, it is held here that an increase 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 increase of said expression and/or activity that is comparable to an increase of 30% (or more) in vivo. It can however not be excluded that levels found in vitro do not perfectly correlate with levels found in vivo, such that a slightly increased level in vitro may still result in a higher increase in vivo when the compound is applied in a therapeutic setting. It is therefore preferred to have increased in vitro levels of at least 10%, more preferably more than 30%, even more preferably more than 50% and most preferably an increase between 50% and 100%.

Reduction as used herein may be achieved in different ways. The compounds may target the polypeptides directly and inhibit their activity. The compounds may also target the transcript- tion/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.

It is to be understood that the term "Atherosclerosis-associated protein" refers to a protein that is involved in the onset, treatment and/or prophylaxis or amelioration of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis in a patient. Preferred Atherosclerosis-associated proteins are the proteins listed in Table 3 or 7.

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.

The activity of an Atherosclerosis-associated protein can be determined by using fluorogenic small peptide substrates. The specificity of these substrates, however, 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 buildup 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.

Within the context of this invention, "atherosclerosis" shall be understood as encompassing both, atherosclerosis and arteriosclerosis as defined above.

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.

Myocardial infarction (MI) is generally caused by an abrupt decrease in coronary blood flow that follows a thrombotic occlusion of a coronary artery previously narrowed by arteriosclerosis. MI prophylaxis (primary and secondary prevention) is included as well as the acute treatment of MI and the prevention of complications.

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.

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 and/or prophylaxis 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. 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. Although the formation of these lesions is a chronic process, occurring over decades of an adult human life, the majority of the morbidity associated with atherosclerosis occurs when a lesion ruptures, releasing thrombogenic debris that rapidly occludes the artery. When such an acute event occurs in the coronary artery, myocardial infarction can ensue, and in the worst case, can result in death.

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. 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.

Thus, a need exists for therapeutic methods and agents to treat cardiovascular pathologies, such as atherosclerosis and other conditions related to coronary artery disease.

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.

Too high or too low levels of fats in the bloodstream, especially cholesterol, herein referred to as "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. Examples of disorders of lipid metabolism are 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 FV 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.

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.

The term "expression" relates to both endogenous expression and over-expression by for instance transfection or stable transduction.

The term "agonist" refers to a ligand that stimulates the receptor the ligand binds to in the broadest sense.

The term "polypeptide" relates to proteins, proteinaceous molecules, fractions of proteins, peptides, oligopeptides, enzymes (such as kinases, proteases, GPCRs etc.).

The term "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 substiruents 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.

The term "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. Preferably, fragments of a polypeptide are 3, 5, 8, 12, 20, 50, 100 amino acids long.

The term "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. The term "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'-0-(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.

The term "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. Preferably, fragments of a polynucleotide are 10, 20, 50, 100, 300 nucleotides long.

A "reference level" for a protein activity or expression level, within the meaning of the invention, 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.

EXAMPLES

Example 1. Development of a high-throughput screening method for the detection of suppression or induction of ApoBlOO secretion from HepG2 cells

Principle of the assay

ApoBlOO, synthesized in the liver, is an essential structural component of very low density lipoproteins (VLDL's) and its metabolic products, intermediate density lipoproteins (IDL's) and low density lipoproteins (LDL's). 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 major risk factor for atherosclerosis.

An assay was developed using HepG2 cells (human hepatocytic cell-line) as a model system to screen for suppression or induction of secretion of ApoBl OO-associated lipoproteins from these cells. HepG2 cells were seeded in 96 well plates and 1 day after plating infected with cDNA expressing adenoviruses (Ad-cDNA) from the FLeXSelect collection (arrayed adenoviral cDNA library, a collection of adenoviruses mediating the expression of various human cDNAs, in which every well (384-well plates) contains a single virus type. Further details about the concept of arrayed adenoviral libraries can be found in WO99/64582 (Arrayed adenoviral libraries for performing functional Genomics)). Ad-cDNA's from the FLeXSelect collection can be divided into 2 groups depending on type of fiber they contain: Ad5C01- or Ad5C20-cDNA's as described in WO02/24933. In the case of Ad5C01-cDNA's individual Ad-cDNA infections were performed.

Library Ad5C20-cDNA's however were co-infected with Ad5C01Att01/A011200-MCP_vl virus, which increases the transduction efficiency of viruses with Ad5C20- fiber type in HepG2 cells. Medium refreshments were performed 2 and 4 days post infection. ApoBlOO levels were measured in the supernatants of HepG2 cells 6 days after the start of the infection using ELISA.

siRNA adenoviruses (Ad-siRNA) from the SilenceSelect™ collection (see WO03/020931) were also used for the development of this assay.

Control- and standard-viruses

Ad5C01Att01/A150100-ApoB100_v6; Ad-siRNA, target sequence: 5'-

GAGGCAGCTTCTGGCTTGC. Cloned using Sapl -sites into vector and virus generated as described in WO03/020931. Ad5C01Att01/A150100-ApoAl_v2; Ad-siRNA, target sequence: 5'-

GGACCTGGCCACTGTGTAC. Cloned using Sap 1 -sites into vector and virus generated as described in WO03/020931.

Ad5C01AttOl/A15O10O-eGFP_v6; Ad-siRNA, target sequence: 5'- GAACGGCATCAAGGTGAAC. Cloned using Sapl-sites into vector and virus generated as described in WO03/020931.

Ad5C01Att01/A150100-empty; Ad-siRNA empty virus (generated from A150100, as described in WO03/020931), Ad5C01 -fiber as described in WO02/24933.

Ad5C20Att01/A150100-empty; Ad-siRNA empty virus (generated from A150100, as described in WO03/020931 ), Ad5C20-fiber as described in WO02/24933.

Ad5C01 Att01/A010800-eGFP_vl ; Ad-cDNA referred to as pIPspAdAptό-eGFP in WO02/070744.

Ad5C20Att01/A010800-eGFP_vl ; Ad-cDNA referred to as pIPspAdAptό-eGFP in WO02/070744.

Ad5C01Att01/A011200-MCP_vl ; Ad-cDNA; cDNA was PCR amplified from a mixed human fetal liver/placenta cDNA library using primers CD46-FOR: aaggcgcgccATGGAGCCTCCCGGCCGCCGCGAGT and CD46-REV2: atgcggccgcCTATTCAGCCTCTCTGCTCTGCT. The resulting 1.2 kb fragment was digested with the restriction enzymes Ascl and CciNI (Notl isoschizomer). AOl 1200 (pIPsp Adapt 10/Zeo(as)- 3kb) vector was digested with the same enzymes, gel-purified and used to ligate to the digested MCP fragment. The resulting clone is identical to ntl 57-131 1 of NM_153826 (Homo sapiens membrane cofactor protein CD46,trophoblast-lymphocyte cross-reactive antigen MCP, transcript variant d). Virus was generated as described in WO99/64582.

Development of the assay

HepG2 cells were obtained from ATCC (HB-8065). The cells were cultured in Tl 75 flasks (Nunc; Cat. 159910) at 37°C in a humidified incubator at 10% CO₂. The medium that is used for culturing the cells (further referred to as the HepG2 medium) is RPMI 1640 + L-Glutamine (Gibco; Cat.

21875-034) + 10% Fetal Bovine Serum heat inactivated (ICN; Cat. 29-167-54 100% NHI) + 10 mM HEPES buffer (Gibco; Cat. 15630-056). Cells were passed 1 :2 twice a week after reaching ± 70% confluency, using the following protocol: After washing the cells twice with 10 ml PBS (Gibco; Cat. 10010-015) 2 ml Trypsin-EDTA (Gibco; Cat. 25300-054) was added per T175 flask and cells were incubated for 5 minutes. After this incubation 8 ml of HepG2 medium was added to the detached cells to inactivate trypsin-EDTA. Cells were resuspended by pipetting up and down 5-6 times using a 10 ml pipette and transferred to a 15 ml Falcon tube (Greiner; Cat. 188261). After 2-3 minutes (to allow cell-clumps to settle) the upper portion was used for passing: 5 ml cell suspension + 20 ml fresh, pre-warmed HepG2 medium in a new Tl 75 flask.

In a series of experiments, carried out in 96-well plates, several parameters were optimized for the assay to detect suppression or induction of ApoBlOO secretion: cell seeding density, plate type for optimal adherence of the cells, multiplicities of infection (MOI) of control viruses, infection efficiency of Ad5C01-cDNA's (using Ad5C01Att01/A010800-eGFP_vl) and Ad5C20-cDNA's

(using Ad5C20Att01/A010800-eGFP_vl with or without Ad5C01Att01/A011200-MCP_vl), duration of infection, toxicity, refreshing of medium between the day of infection and the day of readout, ApoBlOO- and ApoAl -detection method and cell viability (MTS) assay.

Using control viruses described above, the following infection protocols resulted in high transduction efficiency without loss of cell viability:

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. AA356516) in 100 μl HepG2 medium. They were infected the next day (day 1) with Ad5C01- or Ad5C20- control viruses. Ad5C01 -viruses were diluted with HepG2 medium in order to achieve an MOI of 160 virus particles/cell (VP/cell, determined as described by Ma et al., J Virol Methods 2001; 93:181-8). The volume of Ad5C01-virus dilutions to be added per well was 50 μl to get a total volume of 150 μl (virus containing) medium in each well. In the case of Ad5C20-viruses, a co-infection with Ad5C01Att01/A011200-MCP_vl was performed. Therefor Ad5C20-viruses were diluted with HepG2 medium in order to achieve an MOI of 320 VP/cell after adding 25 μl of virus dilution per well. Ad5C01 AttOl/AOl 1200-MCP_vl virus was diluted with HepG2 medium in order to achieve an MOI of 40 VP/cell after adding 25 μl of virus dilution per well. Ad5C20-virus infections were performed by adding 25 μl Ad5C20-virus dilution plus 25 μl Ad5C01Att01/A01 1200-MCP_vl virus dilution to the same well (total volume of (virus containing) medium in each well is 150 μl).

After 2 days (day 3), the (virus containing) medium from all wells was taken off the cells and 120 μl of fresh, pre-warmed HepG2 medium (without virus) was added to each well. This medium refreshing step was repeated on day 5. On day 7 (6 days after infection) supernatants were harvested (110 μl from each well) and stored at -8O⁰C.

Using Ad5C01Att01/A150100-ApoB100_v6 as a positive control for suppression of ApoBlOO secretion, the following protocol resulted in the highest dynamic range for the measuring of ApoBlOO concentrations: For measuring of ApoBlOO concentrations in harvested HepG2 supernatants "Total Human Apolipoprotein B (ApoB) ELISA Assay" obtained from ALerCHEK, Inc. (Cat. A70102) was used. Using ApoB standard provided in the kit (2.640 μg/ml) and HepG2 medium, dilutions were made for the standard curve (see Table 1). 100 μl of 7x in HepG2 medium diluted supernatant or standard curve dilution (in duplicate) was transferred to the ELISA plate. After 45 minutes incubation at room temperature all samples were decanted from the ELISA plate and the wells were washed 5 times with 200 μl per well of 1 :15, in distilled water (Gibco; Cat. 15230-089) diluted, wash buffer (provided in the kit). After washing the plate, 100 μl of HRP conjugated goat anti-ApoB (from the kit) was added to each well. This was incubated for 45 minutes at room temperature followed by washing the plate the same way as described above. After washing, 100 μl of TMB/peroxide substrate (provided in the kit) was added per well. After incubation (15 minutes, room temperature in the dark), the reaction was terminated by adding 100 μl of 0.5 N sulfuric acid (provided in the kit) per well. The absorbance was read at 450 nm using FLUOstar (Galaxy, BMG). The signal measured in a blanc sample (HepG2 medium) was subtracted from all other values and these corrected values were used in the calculations. The unknown concentrations were interpolated from the standard curve (polynomial curve, see Figures 1 and 2).

Example 2. Screening of 4735 adenoviral cDNA expressing vectors (FLeXSelect collection) in the ApoBlOO assay

The primary screen of the FLeXSelect library was performed in one infection batch which was split in two ApoBlOO ELISA batches performed on two different days.

For screening of the FLeXSelect library, the optimized protocol described in Example 1 was used. However, there were a few adjustments made to this protocol.

Ad5C01-cDNA infections:

On the day of infection (day 1), 12.38 μl Ad5C01-cDNA from the FLeXSelect collection, stored in 384 well plates (estimated titer of 2 x 10⁹ 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 μl fresh HepG2 medium, thus diluting the viruses 6.25 times. After pipetting it up and down 3 times by the Tecan, 21 μl of each diluted virus was transferred to individual wells of the 96 well plates containing HepG2 cells seeded (using Multidrop, Labsystems) on day 0. All Ad5C01- cDNA's were screened in duplicate on independent assay plates. Ad5C20-cDNA infections:

On the day of infection (day 1), Ad5C01 AttOl/AOl 1200-MCP_vl virus was manually diluted with HepG2 medium in order to achieve an MOI of 40 VP/cell after adding 5 μl of virus dilution per well.

23.53 μl Ad5C20-cDNA from each well of the FLeXSelect collection, stored in 384 well plates, was transferred with the aid of Tecan to individual wells of a 384-well plate containing 50 μl fresh HepG2 medium, thus diluting the viruses 3.125 times. After pipetting it up and down 3 times by the Tecan, 21 μl of each diluted virus was transferred to individual wells of the 96 well plates containing HepG2 cells (seeded on day 0) in duplicate on independent assay plates. In addition, 5 μl of diluted Ad5C01 AttOl/AOl 1200-MCP_vl virus (see above) was added by Tecan to each well containing Ad5C20-cDNA viruses co-infecting the cells in order to get higher transduction efficiency.

Medium refreshment and harvesting of supernatants:

Two and four days post infection (day 3 and 5) the medium was manually replaced by fresh pre- warmed HepG2 medium as described in Example 1. Six days post infection (day 7), supernatants from all plates were harvested using the Tecan. For this, 75 μl supernatant from each 96 well plate was transferred to individual wells of a 384 well plates (supernatants of four 96 well plates were transferred to one 384 well plate). These harvest plates were stored at -80⁰C.

ADOBI OO ELISA'S:

Supernatants harvested on day 7 and stored in 384 well plates were transferred with the aid of the

Tecan, 20 μl per well, to individual wells of a 96 well v-bottom plate (Greiner; Cat. 651180) containing 120 μl fresh HepG2 medium, diluting them 7 times. After pipetting up and down 3 times, 100 μl of each diluted supernatant was transferred to individual wells of the 96 well ApoBlOO ELISA plate. All other steps were performed as described in Example 1. For all wash steps a Plate Washer (Tecan) was used. Adding of HRP conjugated goat anti-ApoB,

TMB/peroxide substrate and 0.5 N sulfuric acid was done using Multidrops (Labsystems). Readout was performed on a FLUOstar (Galaxy, BMG).

Analysis of the results was performed using A₄₅₀ values (absorbance measured at 450 nm). Ad- cDNA viruses were nominated as activator hits if at least one of the two data points (duplicates on independent assay plates) scored above the threshold which was set at average plus 2.3 times standard deviation of all data points per plate. Ad-cDNA viruses were nominated as repressor hits if at least one of the two data points (duplicates on independent assay plates) scored below the threshold which in this case was set at average minus 2.1 times standard deviation of all data points per plate.

To assure the quality of all infection and ELISA batches, Z'-factor analysis was performed for all ELISA batches. This means that for both ELISA batches that were performed, 5 Z' infection plates were taken along during the infection process. Each Z' infection plate contained 15 wells with Ad5C01Att01/A150100-ApoB100_v6 as a positive control for suppression of ApoBlOO secretion and 45 wells with Ad5C01Att01/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. 651180) according to the layout from Figure 3, 160 μl per well. The Tecan was used to infect the cells with these diluted viruses the same way as described for the Ad5C01-cDNA infections. 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 μl per well). Concentrations of ApoBlOO were calculated as described in Example 1. These values were used to calculate the Z'-factor for all 5 ELISA batches according to the following formula: l-((3*STDEV(neg.controls)+ 3*STDEV(pos.controls))/ABS(AVERAGE(neg.controls)- AVERAGE(pos.controls))). This Z'- factor had to be equal to or higher than 0.15 for an ELISA batch to be considered as valid.

In the primary screen a total of 136 activator hits were isolated which scored above the threshold (average plus 2.3 times standard deviation of all data points per plate). Of these hits, 29 scored positive in duplicate and 107 hits scored in single. There were also 144 repressor hits which scored under the threshold (average minus 2.1 times standard deviation of all data points per plate) of which 44 in duplicate and 100 in single. An overview of all datapoints from the primary screen is provided in Figure 4 and 5, in which the "standard deviation" of the duplicate data points are indicated on the X-axis and Y-axis. The threshold is indicated by dotted lines (for activators: average of all samples plus 2.3 times standard deviation (Figure 4) and for repressors: average of all samples minus 2.1 times standard deviation (Figure 5)). Example 3. Optimization of ApoAl detection method and MTS cell viability assay

The next step in the validation of the 136 activator- and 144 repressor hits from the primary screen, was rescreening of these hits for ApoBlOO secretion including an ApoAl secretion assay and a cell-viability (MTS) assay.

Measuring of HepG2 cell viability and detection of ApoAl levels in HepG2 supernatants were optimized in several experiments. Ad5C01Att01/A150100-ApoAl_v2 was used as a positive control for suppression of ApoAl secretion. These optimization experiments resulted in the following protocols.

For the measuring of cell viability on day 7 (6 days post infection) "CellTiter 96^® AQueous Non- Radioactive Cell Proliferation Assay" (further referred to as MTS assay) (Promega; Cat. G5421) was used. In this assay enzymatic activity of dehydrogenase in metabolically active cells is measured. It is performed according to the protocol provided in the kit. For the purpose of this assay, remainders of supernatant after harvesting (day 7), were removed using multichannels and fresh, pre- warmed HepG2 medium was added to the cells (100 μl per well). One conditioned medium plate (uninfected samples) was freeze/thawed 3 times to be used as a cell lysate (dead cells) control. After preparing the MTS/PMS solution (50 μl PMS solution + 1 ml MTS solution for each plate), 10 μl of this solution was added to each well and plates were incubated for 1 hour at 37⁰C 10% CO2 in 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. These absorption values were used to calculate percentages of viable cells in all samples with uninfected HepG2 cells as a reference (= 100% viable cells) according to the following equation: % viable HepG2 cells = (A490_sampι_e / A490_{uninfected H}e_Po2 _ceils) * 100.

For the measuring of ApoAl concentrations in HepG2 supernatants "Total Human Apolipoprotein Al (ApoAl) ELISA Assay" obtained from ALerCHEK, Inc. (Cat. A70101) was used. Using

ApoAl standard provided in the kit (0.335 μg/ml) and HepG2 medium, dilutions were made for the standard curve (see Table 2). 100 μl of supernatant diluted 3x (in HepG2 medium) or standard curve dilution (in duplicate) was transferred to the ELISA plate. After 120 minutes incubation at room temperature all samples were decanted from the ELISA plate and the wells were washed 5 times with 200 μl per well of 1 :15, in distilled water (Gibco; Cat. 15230-089) diluted, wash buffer

(provided in the kit). After washing the plate, 100 μl of HRP conjugated goat anti- ApoAl (from the kit) was added to each well. This was incubated for 120 minutes at room temperature followed by washing the plate the same way as described above. After washing, 100 μl of TMB/peroxide substrate (provided in the kit) was added per well. After incubation (15 minutes, room temperature in dark), the reaction was terminated by adding 100 μl of 0.5 N sulfuric acid (provided in the kit) per well. The absorbance was read at 450 nm using FLUOstar (Galaxy, BMG). The signal measured in blanc sample (HepG2 medium) was subtracted from all other measured values and these corrected values were used in the calculations. The unknown concentrations were interpolated from the standard curve (polynomial curve, see Figure 6).

Example 4. Propagation and the rescreen of the 136 activator- and 144 repressor hits from the ApoBlOO primary screen including ApoAl- and cell-viability assay

To further validate the results obtained in the primary screen, the ApoBlOO-screen of the 136 activator- and 144 repressor hits was repeated in a rescreen. This rescreen also included ApoAl secretion assay and a cell-viability (MTS) assay.

The original virus material of hits from primary screen (in matrix tubes) together with 488 viruses that scored negative in the primary screen were rearranged in 8 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 10⁴ PerC6.E2A cells were seeded in 200 μl of DMEM (Gibco Cat. 41966-029) containing 10% non- heat inactivated FBS (ICN; Cat. 29-167-54) and 1 ml MgC12 4.9 mol/1 (preparation: 996.17 g MgCL₂.6H₂O (Sigma; Cat. M2393) in 1000 ml milliQ H₂O, autoclaved) into each well of a 96 well plate and incubated overnight at 39⁰C in a humidified incubator at 10% CO2. Subsequently, 2 μl of crude lysate from the Ad-cDNA stocks in matrix tubes was added and incubation was proceeded at 34⁰C in a humidified incubator at 10% CO₂ for 7 to 10 days. All hits were propagated in duplicate using the Tecan. After CPE (cytopathogenic effect) evaluation, the plates were frozen at -80⁰C. After thawing the two lysates were pooled and were frozen at -8O⁰C for storage.

Rescreening of 136 activator- and 144 repressor hits together with 488 neutral viruses from the primary screen for ApoBlOO secretion was performed in the same way as the primary screen, only this time using propagated viruses from 96 well plates instead of the original batch of viruses from

384 well plates. The second difference was that in the rescreen 90 μl of supernatant was harvested from each well instead of 75 μl. To assure the quality of all infection and ELISA batches, Z'-factor analysis was performed as described in Example 2.

For the ApoBlOO rescreen the threshold for activator hits was set at average plus 1.8 times standard deviation of data points from rows B, D, F, and H (neutral controls) per plate. For the repressor hits, threshold was set at average minus 2.1 times standard deviation of data points from rows B, D, F, and H per plate. A total of 19 activator hits were isolated which scored above the threshold. 11 of these hits scored positive in duplicate and 8 hits scored in single. There were also 56 repressor hits which scored under the threshold of which 43 in duplicate and 13 in single. An example of ApoBlOO rescreen results for a plate is provided in Figure 7 and 8, in which the "standard deviation" of 96 single data points are indicated on the Y-axis. The threshold is indicated by dotted lines (for activators: average of all samples plus 1.8 times standard deviation, Figure 7 and for repressors: average of all samples minus 2.1 times standard deviation, Figure 8). ApoAl secretion assay and cell-viability (MTS) assay were performed as described in Example 3 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. After performing ApoAl assay, measured absorbance values were divided by the average signal of the complete plate and multiplied by 100 to get percentages of secreted ApoAl.

Ad-cDNA (ApoBlOO confirmed) rescreen hits were classified as prioritized hits if average of the two data points (duplicates on independent assay plates) scored positive for ApoAl secretion (ApoAl secretion > 70%) and cell viability (%viable cells > 70).

A total of 19 activator and 52 repressor hits were classified as prioritized hits (see table 3 - 10) and will be subjected to further validation steps.

Example 5. Determination of the expression level in HepG2, Huh, primary hepatocytes, and whole liver cells.

The expression levels of targets of the invention were determined using standard methods known to the person skilled in the art. Whereas it is not necessary to perform additional expression profiling experiments in order to practise the invention, some experimental details relating to the expression profiling experiments are provided for information purposes:

Preparation of total RNA was carried out using Trizole (Invitrogen) according to the manufacturer's instruction. The RNA quality was checked by gel-run and the integrity of ribosomal RNA bands using "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) and human Gene Chips "HG- U 133 Plus 2" (Affymetrix) were used for signal detection. Signals were analyzed primarily using GCOS software (Affymetrix) and subsequently with GeneData software. Based on the results of the expression analysis, targets of 19 activator and 56 repressor hits tested in this expressional analysis were classified into 4 different categories: EXPCATl (target detected in human liver), EXPCAT2 (no probe on Affymetrix Chipset HG-U133 A/B or not detected in any sample), EXPCAT3 (detected in HepG2 cells but not in human liver) and EXPCAT4 (not detected in human liver and HepG2 cells but in another sample).

Based on these expression profiles one part of 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. Eventually 14 activator and 31 repressor hits were selected to be taken to the following validation steps (see Figure 6 and 10).

Example 6. Validation of 14 activator and 31 repressor hits for dose response (3 MOI test).

An additional validation step for 14 activator and 31 repressor hits was testing of the adenoviral constructs for dose dependency. For this purpose, virus material used in the rescreen was repropagated as described in Example 4. 14 activator and 31 repressor (prioritized) hits were rearranged in new matrix tube boxes and together with the positive- and negative controls used for the infections according to the plate layout shown in Figure 9.

Infections were performed manually as described in Example 1, using exact titers in order to get required MOI's (see Figure 9). Due to the presence of positive and negative cotrols in each plate,

Z'-factor plates were not included in the 3 MOI test. All other steps from the 3 MOI test were performed as described in Example 2 and 3. In both ApoBlOO and ApoAl ELISA, each plate contained a standard curve (row H, Figure 9). Concentrations of ApoBlOO and ApoAl were calculated for all samples as described in Example 1 and 3. Using these concentrations, percentages of ApoBlOO and ApoAl secretion were calculated per plate according to the following equations: %ApoB100 secretion =

/ [ApoBl

* 100 and %ApoAl secretion = ([ApoAl ]_s-m_Pie(μg/mi) / [ApoAl]Ai5oioo-A_PoBioo_v6<μg/ιn])) * 100. For the calculations of percentages viable cells A490 absorption values were used with average A490 value of the plate as a reference (= 100% viable cells) according to the following equation: % viable HepG2 cells = (A490_samp,_e / A490_{average p}i_ate) * 100.

The prioritization of the hits in the 3 MOI test was performed according to the following criteria: Ia) for the KI repressors: [ApoB100]_Moi 320 ≤ [ApoB100]_Moi i«o and [ApoB100]_Moι 160 ≤ [ApoB100]_Moi so (dose response, based on average values of biological duplicates)

Ib) for the KI activators: [ApoB100]_MOI 320 ≥ [ApoB100]_Moi ieo and [ApoB100]_Moi ieo ≥ [ApoB100]_Moi so (dose response, based on average values of biological duplicates)

2a) for the KI repressors: % ApoBl 00 secretion (MOI 160) < 50%

2b) for the KI activators: % ApoBlOO secretion (MOI 160) > 200%

3) % ApoAl secretion (MOI 160) > 70%

4) % cell viability (MOI 160) ≥ 70%

5) Positive reading as defined by criterion 1 (based on averages of biological duplicates) and criteria 2, 3 and 4 (for at least one of the two independent measurement points in the assay).

A total of 1 activator and 5 repressor FLeXSelect hits were prioritized in the 3 MOI test. Two of the prioritized repressor hits are independent constructs overexpressing the same target cDNA (see Table 1 1 and 12).

Example 7. Screening for compounds useful in the treatment and/or prophylaxis of Atherosclerosis using a cell based assay.

The screening method for the identification of agonists or antagonists of the human cysteinyl leukotriene receptor 2 (CysLTR2; NM 020377) using a cell based assay will be taken as an example.

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 etal., JBC 275 (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⁺* release and consequently the resulting luminescence.

Cells were seeded into 384 well cell culture plates and preincubated for 48 hours in culture medium (DMEM/F12 with Glutamax, Gibco CatJ 61965-026; 10% Fetal Calf Serum, Gibco CatJ 10270-106; 1 ,4 mM Natriumpyruvat, Gibco CatJ 11360-039; 1,8 mM Natriumbicarbonate, Gibco

CatJ 25080-060; 10 mM HEPES, Gibco CatJ 15290-026) under standard cell culture conditions (96% humidity, 5% v/v CO₂, 37⁰C). Culture medium is replaced by Tyrode buffer (containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 20 mM Glucose, 20 mM HEPES) plus Coelenterazin (50 μM) 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. For antagonist screening, 15 min preincubation with putative antagonists is allowed before Leukotriene D4 ( 3 x 10 ^s M) stimulus.

Example 8. Screening for compounds useful in the treatment and/or prophylaxis of Atherosclerosis using a cell-free assay.

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) was expressed in Sf9 insect cells using the Bac-to-BacTM baculovirus expression system. Cells were harvested 48 h after infection and suspended in lysis buffer (20 ml/11 culture, 50 mM Tris-HCl, pH 7.4, 50 mM NaCI, 1 mM MgC12, 1.5 mM EDTA, 10% Glycerin, 20 μL protease inhibitor cocktail set III [CalBiochem, La JoUa, CA USA]). The cells were disrupted by sonication at 4⁰C and cell debris were removed by centrifugation at 15,000 x g at 4⁰C for 30 minutes. The supernatant is designated PDE4B cell extract and is stored at -8O⁰C.

For determination of the in vitro effect of test substances on the PDE4B reaction, test substances are dissolved in DMSO and serial dilutions in DMSO are performed. 2 μl of the diluted test compounds are placed in wells of microtiter plates (Isoplate; Wallac Inc., Atlanta, GA). 50 μl 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 μCi/μl; 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 μl (0.025 μCi) of the diluted substrate and incubates at room temperature for 60 min. The reaction is stopped by addition of 25 μl 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.

Table 1:

Table 2:

A1* dilution is not used for making a standard curve and further calculations

Table 3:

TABLE 4:

TABLE 5:

TABLE 6:

TABLE 7:

TABLE 8:

TABLE 9:

TABLE 10:

Table 11:

Ul

4-

TABLE 12:

Ul

Claims

Claims:

(b) exposing said first cell to a candidate compound;

characterised in that

2. Use of a method of Claim 1 for the screening for substances useful in the treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.

3. Method of Claim 1 or use of Claim 2, wherein said host cell expresses said target polypeptide above wild-type level.

4. Method or use of any of Claims 1 to 3, wherein said target polypeptide expression is recombinant polypeptide expression.

5. Method or use of any of Claims 1 to 4, wherein said compound is selected if said first level of said activity or property is lower than said reference level of said activity or property.

6. Method or use of any of Claims 1 to 4, wherein said compound is selected if said first level of said activity or property is higher than said reference level of said activity or property.

7. Method or use of any of Claims 1 to 6, wherein said reference level is a level obtained from a second cell expressing the target polypeptide at a lower level as compared to said first cell.

8. Method or use of any of Claims 1 to 6, wherein said reference level is the level obtained with said first cell in the absence of the candidate compound.

9. Method or use of any of Claims 1 to 8, wherein said method further comprises contacting the host cell with a known agonist or antagonist of the target polypeptide before

^' 5 determining the first level.

10. Method or use of any of Claims 1 to 9, wherein said activity or property being affected by said activity or property of said target polypeptide is binding affinity of said compound to said target polypeptide.

11. Use of a method, said method comprising the steps of

10 (a) culturing a population of cells expressing a target polypeptide listed in Table 3 or

7, or a functional fragment or derivative thereof;

(c) exposing said population of cells to a compound, or a mixture of compounds;

15 (d) determining a second level of expression and/or activity of said target polypeptide in said population of cells during or after said exposure of said population of cells to the compound, or the mixture of compounds; and

(e) comparing said first and said second level;

for the screening for substances useful in the treatment and/or prophylaxis of a 20 cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.

12. Method or use of any of Claims 1 to 11, wherein said first level of an activity or property is determined with a reporter, said reporter being controlled by a promoter responsive to at least one second messenger.

13. Method or use of Claim 12, wherein said at least one second messenger is cyclic AMP, or 25 Ca2+, or both.

14. Method or use of Claim 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.

15. Method or use of any of any of Claims 12 to 14, wherein the reporter is luciferase or beta- galactosidase.

16. Method or use of any of Claims 1 to 15, wherein the compound is a low molecular weight compound.

17. Method or use of any of Claims 1 to 15, wherein the compound is a polypeptide.

18. Method or use of any of Claims 1 to 15, wherein the compound is a lipid.

19. Method or use of any of Claims 1 to 15, wherein the compound is a natural compound.

20. Method or use of any of Claims 1 to 15, wherein the compound is an antibody or a nanobody.

(c) selecting said compound if binding is detected in step (b) or if a change in activity is detected in step (b);

characterised in that

22. Use of a method of claim 21 or 22 for screening for compounds, useful in the treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.

23. Method or use of any of claims 21 to 22, wherein binding is detected in vitro or in vivo.

24. Method or use of any of claims 21 to 23, wherein said target polypeptide is a recombinant polypeptide.

25. Method or use of any of claims 21 to 24, wherein said compound is selected if the numerical value of the binding affinity is equal to or lower than 10 micromolar.

26. Method or use of any of claims 21 to 25, wherein said compound is a low molecular weight compound.

27. Method or use of any of claims 21 to 25, wherein said compound is a polypeptide, or a lipid, or a natural compound, or an antibody or a nanobody.

28. Method or use according to claims 1 - 27, wherein for a target listed in table 3 an antagonist and for a target listed in table 7 an agonist is selected.

31. Use of Claim 29 or 30, wherein said compound is identified according to any one of the methods or uses of Claims 1 to 28.

32. Use of an agent inhibiting the expression of a polypeptide selected from the group listed in

Table 3 or of an agent increasing the expression of a polypeptide selected from the group listed in Table 7 for the preparation of a medicament for the treatment and/or prophylaxis of a cardiovascular disorder, dyslipidemia, and/or Atherosclerosis.

33. Use of Claim 30, wherein said agent is selected from the group consisting of:

an antisense RNA encoding said polypeptide;

a ribozyme that cleaves the polyribonucleotide encoding said polypeptide;

an antisense oligodeoxynucleotide (ODN) enconding said polypeptide;

34. Use of Claim 33, wherein the nucleotide sequence of said agent is present in a vector.

35. Use of Claim 34, 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.

36. Use of any of Claims 33 to 35, wherein said agent is siRNA, and said siKNA 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 3 or 7.

37. Use of Claim 36, wherein the siRNA further comprises a cleavable loop region connecting the sense and the antisense strand.

(b) determining the nucleic acid sequence of said mRNA or said genomic DNA;

(c) obtaining the nucleic acid sequence encoding said polypeptide of Table 3 or 7 from a public database; and

(d) identifying any difference(s) between the nucleic acid sequences determined in step (b) and (c);

(a) determining the amount of a polypeptide of Table 3 or 7 in a biological sample of said subject; and (b) comparing the amount determined in (a) with a the amount of the polypeptide in a healthy subject;