HUMAN MARKER GENES AND AGENTS FOR DIAGNOSIS, TREATMENT AND PROPHYLAXIS OF CARDIOVASCULAR DISORDERS AND ARTHEROSCLEROSIS

Field of the Invention

The invention relates to novel targets for the screening of compounds useful in the treatment and prophylaxis or prevention of cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis. The invention also relates to novel compounds for use as a medicament for diseases or conditions involving Cardiovascular diseases, preferably disorders ^" of lipid metabolism and atherosclerosis. The invention furthermore relates to antagonists and expression-inhibitory compounds that target G-protein coupled receptors (GPCRs), kinases and proteases of the invention, 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 Cardiovascular diseases, preferably disorders of lipid metabolism and 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 present day treatment ofdyslipidemia, numerous well-controlled outcome studies of lipid-altering drug mono-therapy 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 include lipid-

lowering drugs: HMG-CoA reductase inhibitors (statins), PPAR-alpha 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 mono-therapy 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 mono-therapy, 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 needed (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.

Current approaches to 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 or Atherosclerosis.

The underlying technical problem of the present invention, therefore, can be seen as being the provision of novel screening methods, compounds, and molecular targets for the identification of compounds useful in the treatment and/or prophylaxis or prevention of Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis.

This problem is solved by the subject matter of the independent and dependent claims of the present patent application. .. ^'

Summary of the Invention

The invention relates to methods of screening compound libraries for compounds useful in the treatment and/or prophylaxis or prevention of Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis. The invention further relates to the molecular targets for use in said screening methods. Furthermore, the invention relates to kits and agents for use in screening methods of the invention, and to compounds found to bind to, or modulate, the molecular targets of the invention. In one aspect of the invention, it relates to methods of treatment of a subject in need, by administering agents that bind to, or modulate, targets of the invention. In another aspect 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 1 and 2, or of any one of the polypeptides encoded thereby, for the identification of compounds useful in the treatment and/or prophylaxis of Atherosclerosis. The invention furthermore 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 listed in table 1 and 2 in the manufacture of ,a medicament for the treatment and/or prophylaxis of Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis or a disease associated with Atherosclerosis. The invention furthermore relates to a method of reducing Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis in a subject, said

method comprising the step of administering to a subject in need a pharmaceutical composition according to the invention.

Brief Description of the Tables

Table 1-4:

The target list comprises screening data and gene specific information for 497 siRNAs targeting 467 different genes, selected as positives from the total number of screened genes (target genes). The selected genes were found positive by at least one of the three siRNAs tested per gene. As selection criteria, positive siRNAs showed an LDL-DiI uptake value of more than 2 standard deviations above the overall screen average value, ^■ corresponding to at least 314% of the unspecific control mean LDL-DiI uptake value measured in each screening plate.

The target list consists of 4 tables:

Table 1 contains numerical screening values for LDL-DiI uptake (column 3, "LDL-DiI mean %") and cell density (column 4, "proliferation mean %", values normalized to the unspecific control siRNA) as well as the gene symbol (column 6, "target symbol") and a functional classification (column 5, "Target Class(es)") of the target genes.

Table 2 contains complementary information on the target genes consisting of the gene symbol ("column3, "target symbol"), RefSeq number (column 4, "RefSeq accession"), Entrez Gene ID (column 5) and a functional description derived from NCBI (column 6, "Target description").

Table 3 indicates the nucleotide sequence of the sense strand of positive siRNAs (column 3, "siRNA sequence (21-mer)") and indicates the corresponding SEQ ID NO of each siRNA sequence.

Table 4 indicates the average expression, level of the target genes in 3 different cell types: HepG2 human hepatoma cell line (column 4), HuH human hepatoma cell line (column 6) and human primary hepatoma cells (column 8).

Each row indicates screening data and gene information specific to one single siRNA and its corresponding gene. Between the 4 tables, rows of the same number correspond to the same siRNAs, which are identified by a unique siRNA ID indicated in the second column ("siRNA ID") of all 4 tables. "

The first column of all 4 tables assigns serial numbers to all target genes. SiRNAs directed against the same gene have the same serial gene number. Genes with serial numbers 1 to 3 were independently identified by all 3 siRNAs tested against that gene. 24 genes with serial numbers 4 to 27 were identified by 2 of the 3 tested siRNAs. Genes with serial numbers above 27 were identified by a single positive siRNA.

Detailed Description of the Invention

A human druggable genome siRNA library was screened in a cellular assay using Huh7 hepatoma cells. Read-out was expression of LDL-R as measured by binding of LDL-DiI. Targets whose downregulation resulted in an upregulation of LDL-R expression were scored as hits (see examples).

A "functional variant" of a first polynucleotide or polypeptide, within _. the meaning of the invention, shall be understood as being a second polynucleotide or polypeptide of preferably high sequence identity to said first polynucleotide or polypeptide, but being different in length and sequence, due to the addition and/or deletion and/or substitution of nucleotides or amino acid residues from said first polynucleotide or polypeptide, said second polynucleotide or polypeptide still having essentially the same characteristic biological activity as has the first polynucleotide or polypeptide. Such characteristic biological activity can be catalytic activity, binding properties, or other biological activities of the original molecule.

"Reference level", within the meaning of the invention, shall be understood as being any reference level with which a measured level of, e.g., expression or activity can be compared to. Such reference levels can be obtained, e.g., from previous experiments or from literature.

"Wild-type level", with respect to an expression level of a gene, shall be understood as being an expression level typically observed in wild-type organisms, i.e. in not recombinantly modified organisms of the same species.

'.'Binding affinity" of a molecule A to a protein P, within, the meaning of the invention shall be understood as being the thermodynamic quantity that corresponds to the dissociation constant of the complex consisting of the molecule A and the protein P in a reaction A + P — > AP under standard conditions. In this case the binding affinity is [A] * [B] / [AB], wherein square brackets symbolize the concentration of the respective species.

A "reporter gene" for a target protein, within the meaning of the invention, shall be understood as being- a gene which is under control of a promotor which is influenced, directly or indirectly, by said target protein. Well known reporter genes are genes coding for fluorescent proteins under the control of a second messenger-dependent promotor.

"Nucleic acids", within the meaning of the invention, shall be understood as being all known nucleic acids such as DNA, RNA, peptide nucleic acids, morpholinos, and nucleic acids with backbone structures other than phosphodiesters, such as phosphothiates or phosphoramidates.

The term "to comprise", within the meaning of the invention, refers to nucleic acids in which the nucleic acids with the described sequences are functionally relevant, e.g. for diagnostic use or therapeutic use, such as vectors for therapeutic use or expression of corresponding proteins. Preferably, any additional nucleic acids upstream or downstream ^• of the sequence are not longer than 20 kb. The term "comprise" does not relate to large constructs accidentally including the sequence, such as genomic BAC or YAC clones.

"% identity" of a first sequence towards a second sequence, within the meaning of the invention, means the % identity which is calculated as follows: First the optimal global alignment between the two sequences is determined with the CLUSTALW algorithm [Thomson JD, Higgins DG, Gibson TJ. 1994. ClustalW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22: 4673-4680], Version 1.8, applying the following command line syntax: ,/clustalw -infile=./infile.txt -output= - outorder=aligned -pwmatrix=gonnet -pwdnamatrix=clustalw -pwgapopen=10.0

~pwgapext=0.1 -matrix=gonnet -gapopen=10.0 -gapext=0.05 -gapdist=8 -hgapresidues=GPSNDQERK -maxdiv=40. Implementations of the CLUSTAL W algorithm are readily available at numerous sites on the internet, including, e.g., http://www.ebi.ac.uk. Thereafter, the number of matches in the alignment is determined by counting the number of identical nucleotides (or amino acid residues) in aligned positions. Finally, the total number of matches is divided by the number of nucleotides (or amino acid residues) of the longer of the two sequences, and multiplied by 100 to yield the % identity of the first sequence towards the second sequence.

"Arteriosclerosis", within the meaning of the invention, is the thickening and hardening of the arteries due to the build-up of calcium deposits on the insides of the artery walls. Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis is a similar condition due to the build-up of fatty substances. Both conditions have similar effects on the circulation of the blood throughout the body. Heart disease, high blood pressure, stroke, and ischemia (starvation of the cells due to insufficient circulation) may be the result of arteriosclerosis and cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis. Within the context of this invention, "Atherosclerosis" shall be understood as encompassing both, Atherosclerosis and Arteriosclerosis as defined above.

The "nucleic acid expression vector" may be an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, particularly into a mammalian host . cell, is integrated into the host cell ■ genome and replicated together with the chromosome(s) into which it has been integrated. Preferably, the "nucleic acid expression vector" may be an expression vector which- is usually applied in gene therapeutic methods in humans, particularly a retroviral vector or an adenoviral vector.

The term "expression cassette" is defined herein to include all components which are necessary or advantageous for the expression of a specific target polypeptide. An "expression cassette" may include, but is not limited to, the nucleic acid sequence of - interest itself (e.g. encoding or corresponding to the siRNA or polypeptide of interest) and "control sequences". These "control sequences" may include, but are not limited to, a promoter that is operatively linked to the nucleic acid sequence of interest, a ribosome

binding site, translation initiation and termination signals and, optionally, a repressor gene or various activator genes. Control sequences are referred to as "homologous", if they are naturally linked to the nucleic acid sequence of interest and referred to as "heterologous" if this is not the case. The term "operably linked" indicates that the sequences are arranged so that they function in concert for their intended purpose, i.e. expression of the desired protein, or, in case of RNA, transcription of the desired RNA.

The term "antibody" as used herein includes both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F(ab) ₂ fragments that are capable of binding antigen or hapten. The present invention also contemplates "humanized" hybrid antibodies wherein amino acid sequences of a non-human donor- antibody exhibiting a desired antigen-specificity are combined with sequences of a human acceptor antibody. The donor sequences will usually include at least the antigen-binding amino acid residues of the donor but may comprise other structurally and/or functionally relevant amino acid residues of the donor antibody as well. Such hybrids can be prepared by several methods well known in the art.

The invention relates to

1. , Method for identifying a compound as being useful in the treatment 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 1, or a fragment, or a derivative thereof;

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

(c) determining a first level of an activity or property, said activity or property being affected by an activity or property of said target polypeptide; and

(d) selecting or discarding said candidate compound, based on a comparison of said first level of said activity or property with a reference level of said activity or property;

characterised in that

said disease is a disease selected from the group comprising cardiovascular diseases, disorders of lipid metabolism or atherosclerosis.

2. Use of a method of Count 1 for the screening for substances useful in the treatment or prophylaxis of A disease selected from the group comprising cardiovascular diseases, disorders of lipid metabolism 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 1, 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 or prophylaxis of A disease selected from the group comprising cardiovascular diseases, disorders of lipid metabolism 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 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 or prophylaxis of a disease, comprising the steps of

(a) contacting said compound with a target polypeptide selected from the group listed in Table 1, 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

said disease is A disease selected from the group comprising cardiovascular diseases, disorders of lipid metabolism or atherosclerosis.

22. . Use of a method of count 21 or 22 for screening for compounds, useful in the treatment or prophylaxis of A disease selected from the group comprising cardiovascular diseases, disorders of lipid metabolism or atherosclerosis.

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

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 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. Use of a compound that inhibits an activity and/or the expression of any of the polypeptides listed in Table 1 in the manufacture of a medicament for the treatment or prophylxis of A disease selected from the group comprising cardiovascular diseases, disorders of lipid metabolism or atherosclerosis.

29. Use of Count 28, wherein said compound is identified according to any one of the methods or uses of Counts 1 to 27.

30. Use of an agent inhibiting the expression of a polypeptide selected from the group listed in Table 1 for the preparation of a medicament for the treatment or prophylaxis of A disease selected from the group comprising cardiovascular diseases, disorders of lipid metabolism or atherosclerosis.

31. 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 small interfering RNA (siRNA) having the sequence of any of SEQ ID NO:1 to

497;

a microRNA (nu ^' RNA) suitable for inhibition of a polypeptide selected from the group listed in table 1 ; or

a short hairpin RNA (shRNA) suitable for silencing expression of a polypeptide selected from the group listed in table 1.

32. Use of Count 31, wherein the nucleotide sequence of said agent is present in a vector.

33. Use of Count 32, wherein the vector is an adenovirus, a retrovirus, an alphavirus, an adeno-associated virus (AAV), a lentivirus, a herpes simplex virus (HSV) or a sendai virus.

34. Use of any of Counts 31 to 33, wherein said agent is siRNA, and said siRNA comprises a sense strand of 17 to 31 nucleotides which is identical to a region of the coding sequence, or its complementary sequence, of any of the polypeptides of Table 1.

35. Use of Count 34, wherein the siRNA further comprises a cleavable loop region connecting the sense and the antisense strand.

36. Vector comprising any of SEQ ID NO:1 to 497.

37. Use of a vector of Count 36 as a medicament.

38. Use of a vector of Count 37 for the manufacture of a medicament useful in the , treatment or prophylaxis of A disease selected from the group comprising cardiovascular diseases, disorders of lipid metabolism or atherosclerosis.

39. Use according to Count 37 or 38, wherein the vector is an adenoviral, retroviral, adeno-associated viral, lentiviral or a sendaiviral vector.

40. Method for diagnosing a pathological condition involving A disease selected from the group comprising cardiovascular diseases, disorders of lipid metabolism 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 1, or a sample of the subject's genomic DNA corresponding to a polypeptide of Table 1;

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

(c) obtaining the nucleic acid sequence encoding said polypeptide of Table 1 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 disease selected from the group comprising cardiovascular diseases, disorders of lipid metabolism or a disease selected from the group comprising cardiovascular diseases, disorders of lipid metabolism or atherosclerosis, or a susceptibility to such a condition in a subject is diagnosed, if such difference(s) are identified in step (d).

41. Method for diagnosing a pathological condition involving A disease selected from the group comprising cardiovascular diseases, disorders of lipid metabolism or atherosclerosis or a -susceptibility to such a condition in a subject, comprising

(a) determining the amount of, a polypeptide of Table 1 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.

One further embodiment of the invention is the use of the genes/proteins listed in table 1 as therapeutical targets in the field of cardiovascular diseases, preferably lipid metabolism disorders or atherosclerosis.

Furthermore, those targets listed in table 1 are preferred, which are highly expressed in HepG2 cells, Huh cells, primary hepatocytes, and whole liver cells. Those targets of Table 1, which show an average expression of above 1000 in HepG2 cells, Huh cells, primary hepatocytes, or whole liver cells, in Table 4, are preferred targets of the invention. Even more preferred are targets of Table 1, which. show an average expression of above 1000 in at least two, or three or (most preferred) four cell types, in a list of cell types consisting of HepG2 cells, Huh cells, primary hepatocytes, and whole liver cells, in Table 4.

According to a further preferred embodiment, the nucleic acid molecules may also have the antisense-sequence of any of the sequences of the invention. According to a further embodiment, fragments or functional variants of the nucleic acid molecules as described above may be used.

According to a further embodiment, the nucleic acid molecule comprises a nucleotide sequence which is capable of hybridizing with the nucleic acid sequences of the invention under conditions of medium/high stringency. In such hybrids, duplex formation and stability depend on substantial complementarity between the two strands of the hybrid and a certain degree of mismatch can be tolerated. Therefore, the nucleic acid molecules and probes of the present invention may include mutations (both single and multiple), deletions, insertions of the above identified sequences, and combinations thereof, as long as said sequence variants still have substantial sequence similarity to the original sequence which permits the formation of stable hybrids with the target nucleotide sequence of interest. Suitable experimental conditions for determining whether a given DNA or RNA sequence "hybridizes" to a specified polynucleotide or oligonucleotide probe involve pre- soaking of the filter containing the DNA or RNA to examine for hybridization in 5 x SSC (sodium chloride/sodium citrate) buffer for 10 minutes, and pre-hybridization of the filter in a solution of 5 x SSC, 5 x. Denhardf s solution, 0,5 % SDS and 100 mg/ml of denaturated sonicated salmon sperm DNA (Maniatis et al.,1989), followed by hybridization in the same solution containing a concentration of 10 ng/ml of a random ¹ primed (Feinberg, A.P. and Vogelstein, B. (1983), Anal. Biochem. 132:6-13), ³² P-dCTP- labeled (specific activity > 1 x 10 ⁹ cpm/μg) probe for 12 hours at approximately 45°C. The filter is then washed twice for 30 minutes in 2 x SSC, 0,5% SDS at at least 55 ⁰ C (low stringency), at least 60 ⁰ C (medium stringency), preferably at least 65°C (medium/high stringency), more preferably at least 70 ⁰ C (high stringency) or most preferably at least 75 ⁰ C (very high stringency). Molecules to which the probe hybridizes under the chosen conditions are detected using an x-ray film or a "phosphor imager". "Suitable conditions" for the production of the above double-stranded RNA-molecule are all in vivo or in vitro conditions that according to the state of art allow the expression of a first and a second RNA-strand with the above sequences and lengths that - when hybridized - form a double- stranded RNA-molecule. Particularly preferred ".suitable conditions" for the production of

the above double-stranded RNA-molecule are the "in vivo conditions" in a living human or animal cell or the "in vitro conditions" in cultured human or animal cells.

The isolated nucleic acid molecules of the invention, or their modulators/regulators may be used for treating or diagnosing Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis either in vitro or in vivo.

Treatment and/or prophylaxis of Artherosclerosis using said nucleic acid molecules can be achieved in different ways familiar to the person skilled in the art. For example, the isolated nucleic acid molecules may be inserted downstream of a strong promotor to overexpress the corresponding protein or polypeptide. Overexpression of the protein or polypeptide may lead to suppression of the endogenous protein's biological function. By introducing deletions or other mutations into the nucleic acids, or by using suitable fragments, it is possible to generate sequences encoding dominant-negative peptides or polypeptides. Such dominant-negative peptides or polypeptides can inhibit the function of the corresponding endogenous protein.

According to a further preferred embodiment, the invention relates to the use of the above identified nucleic acid molecules or functional variants thereof in form of RNA, particularly antisense RNA and double-stranded RNA, for the manufacture of a medicament for the treatment and/or prophylaxis of Artherosclerosis. Also ribozymes can be generated for the above identified sequences and used to degrade RNA transcribed from the corresponding endogenous genes.

Particularly preferred is the use of these RNA molecules in a therapeutic application of the RNAi technique, particularly in humans or in human cells. An RNAi technique particularly suited for mammalian cells makes use of double-stranded RNA oligonucleotides known as "small interfering RNA" (siRNA). Therefore, according to a further preferred embodiment, the invention relates to the use of nucleic molecules comprising small interfering RNA with a sequence corresponding to any of the sequences given in table 3.

These siRNA molecules can be used for the therapeutic silencing of the expression of the genes of the invention comprising nucleic acid sequences of the invention, in mammalian cells, particularly in human cells, particularly for the therapy of Artherosclerosis.

The inhibition of a specific target gene in mammals is achieved by the introduction of an siRNA-molecule having a sequence that is specific (see above) for the target gene into the mammalian cell. The siRNAs comprise a first and a second RNA strand, both hybridized to each other, wherein the sequence of the first RNA strand is a fragment of one of the sequences of the invention and wherein the sequence of the second RNA strand is the antisense-strand of the first RNA strand. The siRNA-molecules may possess a characteristic 2- or 3 -nucleotide 3 '-overhanging sequence. Each strand of the. siRNA molecule preferably has a length of 19 to 31 nucleotides.

The siRNAs can be introduced into the mammalian cell by any suitable known method of cell transfection, particularly lipofection, electroporation or microinjection. The RNA oligonucleotides can be generated and hybridized to each other in vitro or in vivo according to any of the known RNA synthesis methods.

In another embodiment, the invention relates to the use of a nucleic acid molecule as defined above, wherein the nucleic acid molecule is contained in at least one nucleic acid expression vector which is capable of producing a double-stranded RNA-molecule comprising a sense-RNA-stand and an antisense-RNA-strand under suitable 'conditions, wherein each RNA-strand, independently from the other, has a length of 19 to 31 nucleotides.

In this alternative method (also described in Tuschl, Nature Biotechnology, Vol. 20, pp. 446-448), vector systems capable of producing siRNAs instead of the siRNAs themselves are introduced into the mammalian cell for down-regulating gene expression. The preferred lengths of the RNA-strands produced by such vectors correspond to those preferred for siRNAs in general (see below).

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 RNAseIII 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.

The coding sequence of interest may, if necessary, be operably linked to a suitable terminator or to a poly-adenylation sequence. In the case of RNA, particularly siRNA, "coding sequence" refers to the sequence encoding or corresponding to the relevant RNA strand or RNA strands.

Further, the vector may comprise a DNA sequence enabling the vector to replicate in the ^■ mammalian host cell. Examples of such a sequence — particularly when the host cell is a mammalian cell - is the SV40 origin of replication.

A number of vectors suitable for expression, in mammalian cells are known in the art and several of them are commercially available. Some commercially available mammalian expression vectors which may be suitable include, but are not limited to, pMClneo

(Stratagene), pXTl (Stratagene), pSG5 (Stratagene), pcDNAI (Invitrogen), EBO-pSV2- neo (ATCC 37593), pBPV-l(8-2) (ATCC 37110), pSV2-dhfr (ATCC 37146). Preferred are all suitable gene therapeutic vectors known in the art.

In a particularly preferred embodiment of the invention the vector is a retroviral vector. Retroviruses are RNA- viruses possessing a genome that after the infection of a cell, such as a human cell, is reversely transcribed in DNA and subsequently is integrated into the genome of the host cell. Retroviruses enter their host cell by receptor-mediated endόcytosis. After the endocytosis into the cell the expression of the retroviral vector may be silenced to ensure that only a single cell is infected. The integration of the viral DNA into the genome is mediated by a virus-encoded protein called integrase, wherein the integration locus is not defined. Retroviral vectors are particularly appropriate for their use in gene therapeutic methods, since their transfer by receptor-mediated endocytosis into the host cell, also known to those skilled in the art as "retroviral transduction" is particularly

efficient. A person skilled in the art also knows how to introduce such retroviral vectors into the host cell using so called "packaging cells".

In another particularly preferred embodiment of the invention, the vector is an adenoviral vector or a derivative thereof. Adenoviral vectors comprise both replication-capable and replication-deficient vectors. The latter include vectors deficient in the El gene.

The recombinant vector is preferably introduced into the mammalian host cells by a suitable pharmaceutical carrier that allows transformation or transfection of the mammalian, in particular human cells. Preferred transforrήation/transfection techniques include, but are not limited to liposome-mediated transfection, virus-mediated transfection and calcium phosphate transfection.

In a preferred embodiment, the invention relates to the use of a vector system capable of producing siRNAs as defined above, wherein the nucleic acid corresponding to the siKNA is contained in at least one nucleic acid expression vector comprising a first expression cassette containing the nucleic acid corresponding to the sense-RNA-strand under the control of a first promoter and a second expression cassette containing the nucleic acid corresponding to the antisense-RNA-strand under the control of a second promoter.

In the above mentioned vector system, the vector comprises two individual promoters, wherein the first promoter controls the transcription of the sense-strand and the second promoter controls the transcription of the antisense strand (also described in Tuschl, Nature Biotechnology, Vol. 20, pp. 446-448). Finally the siRNA duplex is constituted by the hybridisation of the first and the second RNA-strand.

The promoter used in the aforementioned "expression cassettes" may be any DNA sequence which shows transcriptional activity in a host cell of choice, preferably in a mammalian host cell, particularly in a human host cell. The promoter may be derived from genes encoding proteins either homologous or heterologous to the host cell. ^■

As a promoter in general every promoter known in the prior art can be used that allows the expression of the gene of interest under appropriate conditions in a mammalian host cell, in particular in a human host cell. Particularly promoters derived from RNA polymerase III transcription units, which normally encode the small nuclear RNAs (snRNAs) U6 or the

human RNAse P RNA Hl, can be used as promoters to express the therapeutic siRNAs. These particularly preferred promoters U6 and Hl RNA which are members of the type III class of Polymerase III promoters are - with the exception of the first transcribed nucleotide (+1 position) - only located upstream of the transcribed region.

In a preferred, embodiment, the invention relates to the use of a vector system capable of

, producing siRNAs for the above identified nucleic acid sequences, wherein the sequence is contained in at least one nucleic acid expression vector comprising an expression cassette containing the sequence of the sense-RNA-strand and of the antisense-RNA-strand under the control of a promoter leading to a single-stranded RNA-molecule and wherein the single-stranded RNA-molecule is capable of fomiing a back-folded stem-loop-structure.

, In this vector system (also described in Tuschl, Nature Biotechnology, Vol. 20, pp. 446- 448), only a single RNA-strand is produced under the control of a single promoter, wherein the RNA strand comprises both the sense- and of the antisense-strand of the final double- stranded siRNA molecule. This structure leads to a back-folding of the RNA-strand by hybridisation of the complementary sense- and antisense-sequences under stem- loop formation. Finally the intracellular processing of this fold-back stem-loop-structure gives rise to siRNA.

In another preferred embodiment according to the present invention, the "nucleic acid expression vector" comprises an expression cassette containing the sequence of the sense- RNA-strand and of the antisense-RNA-strand both under the control of a single promoter leading to a single-stranded RNA-molecule. This single-stranded RNA-molecule is hereby capable to form a back-folded stem-loop-structure. These expressed "hairpin RNA- molecules" subsequently give rise to siRNAs after intracellular processing.

In a preferred embodiment of the invention the nucleic acid expression vector that gives rise to the expression of siRNAs according to the present invention is first introduced into therapeutic, non-toxic virus particles or virus-derived particles that are suitable for gerie therapeutic applications and that can infect mammalian, in particular human target cells, such as packaging cells etc.

In a preferred embodiment, the first and the second RNA strand of the siRNA may have, independently from the other, a length of 19 to 25 nucleotides, more preferred of 20 to 25 - nucleotides, and most preferred of 20 to 22 nucleotides.

In another preferred embodiment, the first and the second RNA strand of the siRNA may have, independently from the other, a length of 26 to 30 nucleotides, more preferred of 26 to 28 nucleotides, and most preferred of 27 nucleotides.

In another aspect, the invention relates to the use of isolated proteins or polypeptides comprising a sequence selected from the group consisting of

(a) a sequence as disclosed by the corresponding accession number in table 2;

(b) a sequence that exhibits a sequence identity with any of the sequences according to (a) of at least 90 % over at least 100 residues,

(c) or functional variants of the sequences defined in (a) or (b),

for the manufacture of a medicament for the treatment and/or prophylaxis of Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis. Proteins, polypeptides and peptides can be introduced into the cells by various methods known in the art. For example, amphiphilic molecules may be membrane permeable and can enter cells directly. Membrane-bound proteins or polypeptides (usually lipophilic molecules or containing transmembrane domains) may insert directly into cell membranes and can thus exert their biological function. Other ways of introduction or intracellular uptake include microinjection, lipofection, receptor-mediated endocytosis, or the use of suitable carrier-molecules, particularly carrier-peptides. Suitable carrier-peptides include or can be derived from HIV-tat, antennapedia _r related peptides (penetratins), galparan _. (transportan), polyarginine-containing peptides or polypeptides, Pep-1, herpes simplex virus VP-22 protein. Another possible introduction method is to introduce nucleic acid vectors capable of expressing such proteins, polypeptides or peptides

Suitable methods to produce isolated polypeptides are known in the art. For example, such a method may comprise transferring the expression vector with an operably linked nucleic acid molecule encoding the polypeptide into a suitable host cell, cultivating said host cells

under conditions which will permit the expression of said polypeptide or fragment thereof and, optionally, secretion of the expressed polypeptide into the culture medium. Depending on the cell-type different desired modifications, e.g. glycosylation, can be achieved.

The proteins, polypeptides and peptides may also be produced synthetically, e.g. by solid phase synthesis (Merrifield synthesis).

The polypeptides used in the invention may also include fusion polypeptides. In such fusion polypeptides another polypeptide may be fused at the N-terminus or the C-terminus of the polypeptide of interest or fragment thereof. A fusion polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding another polypeptide to a nucleic acid sequence (or a portion thereof) of the present invention, Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences so that they are in frame and the expression of the fusion polypeptide is under control of the same promotor(s) and terminator.

Expression of the polypeptides of interest may also be performed using in vitro produced synthetic mRNA. Synthetic mRNA can be efficiently translated in various cell- free systems, including but not limited to, wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems including, but not limited to, microinjection into frog oocytes, preferably Xenopus laevis oocytes.

Treatment and/or prophylaxis of Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis, using said isolated proteins or polypeptides, can be achieved by different ways familiar to the person skilled in the art: Overexpressiόn of the protein or polypeptide may lead to suppression of the endogenous protein's biological function. By introducing deletions or other mutations, or by using suitable fragments, it is possible to generate sequences encoding dominant-negative peptides or polypeptides. Such dominant-negative peptides or polypeptides can inhibit the function of the corresponding endogenous protein. For example, functional variants or mutants can be generated which consist only of binding domains but are enzymatically inactive (i.e. partially lacking their biological function). Such dominant-negative molecules may interfere with the biological function of the endogenous proteins or polypeptides by binding to intracellular binding partners and thus blocking activation of the endogenous molecule.

In another aspect, the invention relates to the use of an antibody which is directed against at least one polypeptide comprising a sequence as defined above for the manufacture of a medicament for the treatment and/or prophylaxis of Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis.

The term "antibody" as used herein includes both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F(ab) ₂ fragments that are capable of binding antigen or hapten. The present invention also contemplates "humanized" hybrid antibodies wherein amino acid sequences of a non-human donor antibody exhibiting a desired antigen-specificity are combined with sequences of a human acceptor antibody. The donor sequences will usually include at least the antigen-binding amino acid residues of the donor but may comprise other structurally and/or functionally relevant amino acid residues of the donor antibody as well. Such hybrids can be prepared by several methods well known in the art.

Antibodies specifically binding to proteins of the invention, or suitable fragments thereof, particularly in humanized form, may be used as therapeutic agents in a method for treating Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis. The use of said antibodies may also include the therapeutical inhibition of the above identified nucleic acid molecules or their corresponding polypeptides. In particular, this use may be directed to Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis. The antibodies or fragments may be introduced into the body by any method known in the art. Delivery of antibodies, particularly of fragments, into live cells may be performed as described for peptides, polypeptides and proteins. If the antigen is extracellular or an extracellular domain, the antibody may exert its function by binding to this domain, without need for intracellular delivery.

Antibodies can be coupled covalently to a detectable label, such as a radiolabel, enzyme label, luminescent label, fluorescent label or the like, using linker technology established for this purpose. Labeling is particularly useful for diagnostic purposes (see below) or for monitoring the distribution of the antibody within the body or a neoplastic tumor, e.g. by computed tomography, PET (positron emission tomography), or SPECT (single photon, emission computed tomography).

Ea another aspect, the invention relates to the use of an isolated nucleic acid molecule comprising a nucleic acid with a sequence selected from the group of sequences consisting of:

a) the nucleic acid sequences presented by the corresponding accession number in table 2;

b) nucleic acid sequences encoding polypeptides that exhibit a sequence identity with the protein encoded by a nucleic acid according to a) of at least 90 % over at least 100 residues and/or which are detectable in a computer aided search using the BLAST sequence analysis programs with an e-value of at most 10 ^"5 ,

c) sequences of nucleic acid molecules which are capable of hybridizing with the nucleic acid molecules with sequences corresponding to (a) or (b) under conditions of medium or high stringency,

d) the antisense-sequence of any of the sequences as defined in (a), (b) or (c),

e) functional variants of (a), (b), (c) or (d),

f) RNA sequences corresponding to any of the sequences as defined in (a), (b),

(c), (d), or (e),

for the manufacture of a medicament for the activation of Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis.

In another aspect, the invention relates to the use of a an isolated peptide or polypeptide comprising a peptide or polypeptide with a sequence selected from the group consisting of:

(a) a sequence as disclosed by the corresponding accession number in table 2;

(b) a sequence that exhibits a sequence identity with any of the sequences according to (a) of at least 90 % over 100 residues.

(c) functional variants of the sequences defined in (a) or (b),

for the manufacture of a medicament for ,_ the treatment and/or prophylaxis of Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis.

In another aspect, the invention relates to the use of an antibody which is directed against at least one peptide or polypeptide with a sequence as defined above for the manufacture of a medicament for the treatment and/or prevention of cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis.

Expression of RNA or polypeptides may be achieved by introduction of genomic DNA or cDNA containing suitable promoters, preferably constitutive or homologous promoters. Alternatively, any suitable nucleic acid expression vector can be used. The encoded protein or polypeptide may be full-length or a fragment or peptide with a similar biological function.

The proteins, polypeptides or peptides may also be generated by any known in vivo or in vitro method and introduced directly into the cells.

It is known that suitable antibodies can be used to activate the biological function of target proteins they bind to. Activation may occur by inducing conformational changes upon binding to the target protein. Another possibility is that the antibody binds two or more target proteins and brings them into sufficiently close physical proximity to induce interaction of the target proteins. The latter mode of activation is particularly known for membrane-bound dimeric receptors.

With respect to the specific embodiments relating to the used nucleic acids, peptides, polypeptides, proteins, and antibodies the same applies as defined above for the other uses of the invention.

In another embodiment, the invention relates to a medicament containing an isolated nucleic acid molecule, peptide, polypeptide, or antibody selected from the group consisting of ^■

. a) nucleic acid molecules or nucleic acid expression vectors as defined above,

b) a peptide or polypeptide comprising a sequence as defined above,

c) an antibody directed against at least one peptide or polypeptide according to (b).

Preferably this isolated nucleic acid molecule is an RNA molecule and preferably is double-stranded. Particularly the isolated nucleic acid molecule is an siRNA molecule according to the present invention.

The following considerations for medicaments and their administration apply also to the medicaments of the invention as to the above disclosed uses.

The medicament preferably comprises additionally a suitable pharmaceutically acceptable carrier, preferably virus-particles or virus-derived particles that may harbour the viral vectors, transfection solutions comprising liposomes, particularly cationic liposomes, calcium phosphate etc. Preferably a carrier is used, which is capable of increasing the efficacy of the expression vector or virus particles containing the expression vector to enter the mammalian target cells. The medicament may additionally comprise other carrier substances, preferably starch, lactose, fats, stearin acid, alcohol, physiological NaCl- solutions or further additives, in particular stabilizers, preservatives, dyes and flavourings.

The medicaments may also comprise other suitable substances. For example, RNA or siRNA containing medicaments may contain substances which stabilize double-stranded RNA molecule and/or which enable the double-ístranded RNA molecule or DNA expression vector to be transfected or to be injected into the human or animal cell.

Administration can be carried out by known methods, wherein a nucleic acid is introduced into a desired cell in vitro or in vivo. For therapeutic applications, the medicament may be in form of a solution, in particular an injectable solution, a cream, ointment, tablet, suspension, granulate or the like. The medicament may be administered in any suitable way, in particular by injection, by oral, nasal, rectal application. The medicament may particularly be administered parenteral, that means without entering the digestion apparatus, for example by subcutaneous injection. The medicament may also be injected intravenously in the form of solutions for infusions or injections. Other suitable administration forms may be direct administrations on the skin in the form of creams, ointments, sprays and other transdermal therapeutic substances or in the form of inhalative substances, such as nose sprays, aerosoles or in the form of microcapsules or implantates.

The optimal administration form and/or administration dosis for a medicament either comprising double-stranded RNA molecules with the above sequences or comprising

nucleic acid vectors capable to express such double-stranded RNA molecules depend on the type and the progression of the disease to be treated.

In another embodiment of the invention, an activator or an inhibitor of a protein of the invention can be administered to a patient in need.

Preferably, the activator or inhibitor is administered in pharmaceutically effective amount. As used herein, a "pharmaceutically effective amount" of an activator or inhibitor is an amount effective to achieve the desired physiological result, either in cells treated in vitro or in a subject treated in vivo. Specifically, a pharmaceutically effective amount is an amount sufficient to positively influence, for some period of time, one or more clinically defined pathological effects associated with Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis. The pharmaceutically effective amount may vary depending on the specific activator or inhibitor selected, and is also dependent on a variety of factors and conditions related to the subject to be treated and the severity of the disease. For example, if the activator or inhibitor is to be administered in vivo, factors such as age, weight, sex, and general health of the patient as well as dose response curves and toxicity data obtained in .pre-clinical animal tests would be among the factors to be considered. If the activator or inhibitor is to be contacted with cells in vitro, one would also design a variety of pre-clinical in vitro studies to asses parameters like uptake, half-life, dose, toxicity etc. The determination of a pharmaceutically effective amount for a given agent (activator or inhibitor) is well within the ability of those skilled in the art. Preferably, the activator or inhibitor is present in a concentration of 0,1 to 50% per weight of the pharmaceutical composition, more preferably 10 to 30%.

An inhibitor, activator, or drug according to the present invention may also be a "small molecule". Small molecules are molecules which are not proteins, peptides antibodies or nucleic acids, and which exhibit a molecular weight of less than 5000 Da, preferably less than 2000 Da, more preferably less than 2000 Da, most preferably less than 500 Da. Such small molecules may be identified in high throughput procedures/screening assays starting from libraries. Such methods are known in the art. Suitable small molecules can also be designed or further modified by methods known as combinatorial chemistry.

In another aspect, the present invention relates to the use of an isolated nucleic acid molecule comprising a sequence as defined above or the use of a ligand binding specifically at least one polypeptide comprising a sequence as defined above for the in vitro diagnosis of Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis.

The diagnostic use of the above identified nucleic acid molecules and probes may include, but is not limited to the quantitative detection of expression of said target genes in biological probes (preferably, but not limited to tissue samples, cell extracts, body fluids, etc.), particularly by quantitative hybridization to the endogenous nucleic acid molecules comprising the above-characterized nucleic acid sequences (particularly cDNA, RNA)

The invention further relates to methods for diagnosis a pathological condition involving Atherosclerosis in a subject, said methods comprising the steps of: (a) determining the nucleic acid sequence of one of the target genes listed in table 1 and table 2 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; and identifying any difference(s) related to the onset of Atherosclerosis.

Expression of the endogenous genes or their corresponding proteins can be analyzed in vitro in tissue samples, body fluids, and tissue and cell extracts. Expression analyis can be performed by any method known in the art, such as RNA in situ hybridization, PCR (including quantitative RT-PCR), and various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay techniques.

The diagnostic use may also include the detection of mutations in endogenous genes corresponding to the above identified nucleic acid sequences.

Suitable nucleic acid probes may be synthesized by use of DNA synthesizers according to standard procedures or, preferably for long sequences, by use of PCR technology with a selected template sequence and selected primers. The probes may be labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels. Typical radioactive labels include ³² P, ^l2 \, ³⁵ S, or the like. A probe labeled with a radioactive isotope can be constructed from a DNA template by a conventional nick

translation reaction using a DNase and DNA polymerase. Non-radioactive labels include, for example, ligands such as biotin or thyroxin, or various luminescent or fluorescent compounds. The probe may also be labeled at both ends with different types of labels, for example with an isotopic label at one end and a biotin label at the other end. The labeled probe and sample can then be combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs. Such nucleic acid probes may also be used for other than diagnostic purposes, e.g. for the identification of further homologs or orthologs.

"Ligands" binding specifically to said polypeptides are known in the art. Such ligands include proteins or polypeptides, for example intracellular binding partners, antibodies, molecular affinity ^" Bodies, and small molecules. Specifically binding ^' ligarids can be identified by standard screening assays known in the art (see also below), for example by yeast two-hybrid screens and affinity chromatography. A specifically binding ligand does not need to exert another function such as inhibiting or activating the molecule with which it interacts.

In a preferred embodiment, the ligand is an antibody binding specifically at least one polypeptide comprising a sequence as defined above.

"Specific binding" according to the present invention means that the polypeptide to be identified (the target polypeptide) is bound with higher affinity than any other polypeptides present in the sample. Preferred is at least 3 times higher affinity, more preferred at least 10 times higher affinity, and most preferred at least 50 times higher affinity. Non-specific binding ("cross-reactivity") may be tolerable if the target polypeptide can be identified unequivocally, e.g. by its size on a Western blot.

Preferably the specifically binding ligands can be labeled, e.g. with fluorescent labels, enzymes, molecular tags (e.g. GST, myc-tag or the like), radioactive isotopes, or with labeled substances, e.g. labeled secondary antibodies. For MRI (magnetic resonance imaging), the ligands may be chelated with gadolinium, superparamagnetic iron oxide or lanthanides. For PET (positron emission tomography) or SPECT (single photon emission computed tomography) commonly used isotopes include ¹¹ C, ¹⁸ F, ¹⁵ O, ¹³ N, ⁸⁶ Y, ⁹⁰ Y, and ¹⁶ Co.

Diagnostic kits may comprise suitable isolated nucleic acid or amino acid sequences of the above identified genes or gene products, labelled or unlabelled, and/or specifically binding ligands (e.g. antibodies) thereto and auxiliary reagents as appropriate and known in the art. The assays may be liquid phase assays as well as solid phase assays (i.e. with one or more reagents immobilized on a support). The diagnostic kits may also include ligands directed towards other molecules indicative of the disease to be diagnosed.

In another aspect, the invention relates to the use of an isolated nucleic acid molecule or a nucleic acid expression vectors as defined above or of an antibody which is directed against at least one polypeptide comprising a sequence as defined above, in a screening assay for the identification and characterization of drugs that are useful in the treatment and/or prophylaxis ^" of Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis.

"Screening assay" according to the present invention relates to assays which allow to identify substances, particularly potential drugs, useful in the treatment and/or prophylaxis of Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis, by screening libraries of substances. "Screening assay" according to the present invention also relates to assays to screen libraries for substances capable of binding to the nucleic acids, polypeptides, peptides or antibodies defined above. Suitable libraries may, for example, include small molecules, peptides, polypeptides or antibodies.

Suitable drugs include "interacting drugs", i.e. drugs that bind to the polypeptides or nucleic acids identified above. Such interacting drugs may either inhibit or activate the molecule they are bound to. Examples for interacting substances are peptide nucleic acids comprising sequences identified above, antisense RNAs, siRNAs, ribozymes, aptamers, antibodies and molecular affinity bodies (CatchMabs, Netherlands). Such drugs may be used according to any aspect of the present invention, including use for the manufacture of medicaments and methods of treatment and/or prophylaxis of Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis. It is known that such interacting drugs can also be labeled and used as ligands for diagnosis of a disease associated Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis.

The term "expression vector" as used herein does not only relate to RNA or siRNA expressing vectors, but also to vectors expressing peptides, polypeptides or proteins. The transfer of the expression vector into the host cell or host organism hereby may be performed by all known transformation or transfection techniques, including, but not limited to calcium phosphate transformation, lipofection, microinjection. The expression vector may be any known vector that is suitable to allow the expression of the nucleic acid sequence as defined above. Preferred expression vectors possess expression cassettes comprising a promoter that allows an overexpression of the RNA, peptide or polypeptide as defined above. After the transfer of the expression vector into the host cell/host organism one part of the host cells or host organisms are cultured in the presence of at least one candidate of an Jiώibitor- or activator-molecule and under culture conditions that allow the expression, preferably the overexpression of the RNA, peptide or polypeptide as defined above. The other part of the transfected host cells are cultured under the same culture conditions, but in the absence of the candidate of an inhibitor- or activator- molecule.

In another preferred embodiment, the screening method for the identification and characterization of an interacting molecule useful in the treatment and/or prophylaxis of Cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis from a library of test substances comprises the following steps:

a) recombinantly expressing a polypeptide encoded by a nucleic acid molecule sequence as defined above in a host cell,

b) isolating and optionally purifying the recombinantly expressed polypeptide of step (a),

c) . optionally labelling . of the test substances and/or labelling of the recombinantly expressed polypeptide,

d) immobilizing the recombinantly expressed polypeptide to a solid phase,

e) contacting of at least one test substance with the immobilized polypeptide,

f) optionally one or more washing step_s, and

g) detecting the binding of the at least one test substance to the immobilized polypeptid at the solid phase.

h) performing a functional assay.

Step a) includes the recombinant expression of the above identified polypeptide or of its derivative from a suitable expression system, in particular from cell-free translation, bacterial expression, or baculuvirus-based expression in insect cells.

Step b) comprises the isolation and optionally the subsequent purification of said recombinantly expressed polypeptides with appropriate biochemical techniques that are familiar to a person skilled in the art.

Alternatively, these screening assays may also include the expression of derivatives of the above identified polypeptides which comprises the expression of said polypeptides as a fusion protein or as a modified protein, in particular as a protein bearing a "tag"-sequence. These "tag" -sequences consist of short nucleotide sequences that are ligated 'in frame' either to the N- or to the C-terminal end of the coding region of said target gene. Commonly used. -tags to label recombinantly expressed genes are the poly-Histidine-tag which encodes a homopolypeptide consisting merely of histidines, particularly six or more histidines, GST (glutathion S-transferase), c-myc, FLAG ^® , MBP (maltose binding protein), and GFP. In this context the term "polypeptide" does not merely comprise polypeptides with the nucleic acid sequences of SEQ ID No. 1 to 31, their naturally occuring homologs, preferably orthologs, more preferably human orthologs, but also derivatives of these polypeptides, in particular fusion proteins or polypeptides comprising a tag-sequence.

These polypeptides, particularly those labelled by an appropriate tag-sequence (for instance a His-tag or GST-tag), may be purified by standard affinity chromatography protocols, in particular by using chromatography resins linked to anti-His-tag-antibodies or to anti-GST-antibodies which are both commercially available. Alternatively, His-tagged molecules may be purified by metal chelate affinity chromatography using Ni-ions. Alternatively to the use of 'label-specific' antibodies the purification may also involve the use of antibodies against said polypeptides. Screening assays that involve a purification step of the recombinantly expressed target genes as described above (step 2) are preferred embodiments of this aspect of the invention.

In an - optional - step c) the compounds tested for interaction may be labelled by incorporation of radioactive isotopes or by reaction with luminescent or fluorescent compounds. Alternatively or additionally also the recombinantly expressed polypeptide may be labelled.

In step d) the recombinantly expressed polypeptide is immobilized to a solid phase, particularly (but not limited) to a chromatography resin. The coupling to the solid phase is thereby preferably established by the generation of covalent bonds.

In step e) a candidate chemical compound that might be a potential interaction partner of the said recombinant polypeptide or a complex variety thereof (particularly a drug library) is brought into contact with the immobilized polypeptide.

In an - optional - step f) one or several washing steps may be performed. As a result just compounds that strongly interact with the immobilized polypeptide remain bound to the solid (immobilized) phase.

In step g) the interaction between the polypeptide and the specific compound is detected, in particular by monitoring the amount of label remaining associated with the solid phase over background levels.

Such interacting molecules may be used without functional characterization for diagnostic purposes as described above. .

In another aspect, the invention relates to a method for the preparation of a pharmaceutical composition wherein an inhibitor or activator of cell cycle progression is identified according to any of the screening methods described above, synthesized in adequate amounts and formulated into a pharmaceutical composition.

Suitable methods to synthesize the inhibitor or activator molecules are known in the art. For example, peptides or polypeptides can be synthesized by recombinant expression (see also above), antibodies can be obtained from hybridoma cell lines or immunized animals. Small molecules can be synthesized according to any known organic synthesis methods.

Similarly, said inhibitor or activator may be provided by any of the screening methods described above and formulated into a pharmaceutical composition.

Another embodiment of the invention is the use of the screening methods of the invention in the field of cardiovascular diseases, preferably disorders of lipid metabolism and atherosclerosis.

The following examples illustrate the present invention without, however, limiting the same thereto.

Unless otherwise specified, the manipulations of nucleic acids and polyp eptides/-proteins can be performed using standard methods of molecular biology and immunology (see, e.g. Maneates et al. (1989), Molecular cloning: A laboratory manual, Cold Spring Harbour Lab., Cold Spring Harbour, NY; Amusable, F.M. et al. (eds.) "Current protocols in Molecular Biology'^ John Wiley and Sons, 1995; Tijssen, P., Practice and Theory of Enzyme Immunoassays, Elsevier Press, Amsterdam, Oxford, New York, 1985).

Examples

EXAMPLE 1: Generation ofdsRNA molecules for RNAi experiments

siRNA of a given siRNA sequence were synthesized by Ambion, Inc. (Austin, Texas, USA), using standard methods known to the person skilled in the art of siRNA synthesis.

EXAMPLE 2: Cell seeding and transection of cells

Huh human hepatoma cells, cultivated in RPMI (Gibco/Invitrogen) medium containing 10% FBS, 1% non-essential amino acid solution (Gibco/Invitrogen), 1% Penicillin/Streptomycin solution (Gibco/Invitrogen), 1% Glutamine (Gibco/Invitrogen) and 1% Hepes pH 8 (Gibco/Invitrogen), were treated with siRNAs at a final concentration of 10OnM using a lipofection based transfection protocol.

24 h before transfection, Huh cells were disattached from the flask by incubation with 3ml Trypsine solution (Gibco/Invitrogen) for 5min at 37'C. Cells were harvested by adding 10ml of RPMI medium to the flask. 4000 cells/well were seeded in black, optical 96well plates (Costar/Cprning) in a volume of lOOul/well. To allow homogenous settling of the cells, important for an even intra well distribution of the cells, the plates were left for 30min at RT before they were transferred to an incubator with 37'C and 5% CO ₂ .

On the day of transfection, for each siRNA the transfection mix was prepared as follows: 4 μl of a 10 μM stock of siRNA was diluted with 64 μl of Opti-MEM (Invitrogen Inc.), and 1.6 μl Oligofectamine transfection reagent (Invitrogen) were diluted with 9.6 μl of Opti- MEM. For complex formation, both solutions were gently mixed and incubated for 20 min at RT. Culture medium was removed from the cells and 80 μl of fresh medium (DMEM ₅ Invitrogen) were added, followed by addition of 20 μl of transfection mix to each of replicate 3wells per siRNA. Cells were incubated at 37 ⁰ C for 4 hours and 50 μl of fresh medium, supplemented with 30 % fetal calf serum were added. 24h after addition of the transfection mix the complete medium described above, was replaced by RPMI medium, containing 2% Lipoprotein deficient serum (LPDS) instead of the 10% FBS.

As an internal control and for intra plate normalization each 96well screening plate, transfected with 88 different sample siRNAs contained the following 8 control wells: 2

wells with siRNAs directed against HMGCR, 2 wells against SQLE, 3 wells with unspecific control siRNAs sharing no complete sequence homology with any coding sequence in the human transcriptome and 1 well without any siRNA.

The 3 replicate wells, assayed per siRNA were situated on 3 different screening plates (inter plate triplicates).

EXAMPLE 3: Primary Screen

Cell staining and fluorescence microscopy based screening readout

Cell staining:

For a primary readout, the expression level of the LDL receptor (LDLR) was measured by an indirect assay, quantifying the amount of available receptor by the amount of internalized LDL. To this end, 48h after transfection the supernatant was replaced by pre- warmed fresh Lipoprotein deficient RPMI medium containing 2% LPDS and 3ug/ml LDL, labelled with the lipid dye DiI (LDL-DiI), supplemented with lug/ml Hoechst for staining of cell nuclei. After an incubation period of 60min at 37'C with this staining solution, cells were washed with phosphate buffered saline containing MgCL2 and CaCL2 (PBS+) and fixed with 4%PFA for 30min at RT.

Image acquisition:

Cells were imaged using a fully automated fluorescence microscope from MDC (Molecular Devices Corporation, CA, USA). Per experimental well 6 fields with a dimension of approx. 2 x 1.5 mm were acquired using excitation/emission conditions, optimized to the spectral properties of the two chromophores, DiI and Hoechst.

Image analysis:

To quantify the degree of LDL-DiI uptake, each image acquired in the DiI channel was subjected to an automated image analysis algorithm, programmed using the MetaMorph image analysis software (Universal Imaging/MDC). hi this algorithm, an adaptive intensity threshold was used to define and measure the area covered by LDL-DiI labelled objects.

For each image, this area was normalized to the fraction of total image area covered by cells (cell density).

The normalized LDL-DiI measurements and the cell density values derived from each of the 6 fields for a given well were averaged to obtain two data points (LDL-DiI and cell density) per experimental well. All experimental data points were . normalized to the corresponding control data points taken from wells treated with non-template siRNA on the same plate. Finally, the plate-normalized LDL-DiI and cell density data points from corresponding wells on the 3 replicate plates were averaged to genearate a single mean value and standard deviation. ^■ .

Validation of screening method by confirming positive control genes

As an internal control and for intra plate normalization each 96well screening plate, transfected with 88 different sample siRNAs contained the following 8 control wells: 2 wells with siRNAs directed against FfMGCR, 2 wells against SQLE, 3 wells with unspecific control siRNAs sharing no complete sequence homology with any coding sequence in the human transcriptome and 1 well without any siRNA. In addition to its function as positive control gene, SQLE was also part of the screened library, targeted by 3 different siRNAs. 2 of these 3 siRNAs, one of them being identical to the SQLE positive control siRNA, were confirmed as positive in the screen showing LDL- DiI uptake values of 348% and 522% of the corresponding unspecific control value. SiRNAs targeting HMGCR were not present in the screened siRNA library.

EXAMPLE 4: 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-Ul 33 Plus 2" (Affymetrix) were used for signal detection. Signals were analyzed primarily using GCOS software (Affymetrix) and subsequently with GeneData software.

Expresssion level data are shown in table 4.

EXAMPLE 5: Screening for compounds useful in the treatment and/ύr 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, Briiii M ₅ Pozzan T.; Nature 358 (1992) 325-327). Additionally, after transfection with a recombinant expression plasmid, containing the full length cDNA for human CysLTR2, the screening cell line is stably expressing the CysLTR2 protein (Heise et.al., JBC 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 Cat.# 61965-026; 10% Fetal Calf Serum,

Gibco Cat.# 10270-106; 1,4 mM Natriumpyruvat, Gibco Cat.# 11360-039; 1,8 mM Natriumbicarbonate, Gibco Cat.# 25080-060; 10 mM HEPES ₅ Gibco Cat.# 15290-026) under standard cell culture conditions (96% humidity, 5% v/v CO ₂ , 37 ⁰ C). Culture medium is replaced by Tyrode buffer (containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl ₂ , 2 mM CaCl ₂ , 20 mM Glucose, 20 mM HEPES) plus Coelenterazin (50 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 ^'8 M) stimulus.

EXAMPLE 6: 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. 199 _. 3 129, 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 NaCl, 1 mM MgC12, 1.5 mM EDTA,

10% Glycerin, 20 μL protease inhibitor cocktail set III [CalBiochem, La Jolla, CA USA]).

The cells were disrupted by sonication at 4 ⁰ C and cell debris were removed by centrifugation at 15,000 x g at 4°C for 30 minutes. The supernatant is designated PDE4B cell extract and is, stored at -80 ⁰ C. ' .

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:

Table 3:

Target siRNA SEQ ID NO

No. ID siRNAsense sequence (21-mer)

1 113613 1 CCAAGCACGAUGUAUACAGTT

1 105742 2 GCGCAUGGAGCUUUUGGAATT

1 105202 3 GGAGAAUAUCGUGCGCAUCTT

2 117853 4 CGAAAGUUAACAAAACUCCTT

2 117852 5 GGACGGUAUGGAGCAUGUUTT

2 117851 6 GCCUGAACCUUCUCUUGAGTT

3 117417 7 CGUAΆACCUUCCUGAAAGATT

3 117416 8 CCAUUCGUUUAUUAGAAACTT

3 117418 9 CCUUUAAUUACCUUCCUAGTT

4 42711 10 GAAUAUGCCUGUUCCUGUGTT

4 42626 11 GACUCAUUGAACAUAUGCATT

5 10418 12 GGAUUAUGACAGAUUACGATT

5 10505 13 GGCUGUCAAGUAUGUGGAGTT

6 118135 14 GCAGACAAUGGUGUGUACATT

6 116839 IS GCAGGUAUUCGUUGGUUUUTT

7 105039 16 GGCCCAUAUAUUGCAUUUATT

7 105041 17 GGAGGCAGAUAAUGCUGGUTT

8 1147 18 GGCAGACAAGUCAACCGUGTT

8 1243 19 GGCAUGGCCACUACUUUGUTT

9 8225 20 GGAAUGCAUGUAUGCUGUGTT

9 117844 21 CCAAUCCUACUAAUAAACCTT

10 106087 22 GGAAUAUAUGGCAACUUUGTT

10 106089 23 GGGCACACUACACUAUUAATT

11 121011 24 CGAGUUACCUCGUCCGAGUTT

11 121012 25 GCAUGCUAUGGUGUUCUUCTT

12 1766 26 GGUGCACAUCUUCUCUCUGTT

12 1947 27 GGUACCUAUCAGUUUGUAUTT

13 142836 28 CCGAUUACUUAAGUUUCCGTT

13 142834 29 CGACGACAUUUUGUGAAUATT

14 1547 30 GGUUAUUCACAUGGAGCUGTT

14 1452 31 GGGAUUGUUUGUGCUGCAUTT

15 1699 32 GGUUAAGCUGUGUGAUUUUTT

15 118252 33 GCCCUCCAAUAUGCUAGUATT

16 43916 34 GAGAAGGCAUGUCUUUGGGTT

16 43820 35 GAGAGAAGGCAUGUCUUUGTT

17 402 36 GGAGGCUUUGGCUGUAUAUTT

17 401 37 GGAAUGGAAAGUAGGAUUATT

18 117695 38 CCAUCCUGAGAGAUGUGAUTT

18 117693 39 GCCAGAAUACAAGUAUGUATT

19 118261 40 CCCAAGCACUUUAUGCAUATT

19 118260 41 GCGAAUUUUGUGUGAUΫUCTT

20 5146 42 GGCAUGUCUGAGAGACUUATT

20 5237 43 GGAACCUUCUGGCAUAUΫUTT

21 828 44 GGUAUACAAUGCCGUCCUCTT

21 829 45 GGACUUUGGAACUGUGAAUTT

22 19104 46 GGCCAAGCAAUAUCUGUCUTT

22 19196 47 GGGUCUUCAGGAAAGUCUCTT

23 103354 48 GGACCAGCAAAUCACUGCCTT

23 978 49 GGUCUGAACCAGUGGAUGUTT

24 2240 50 GGAUUUGACCUCACAUUCATT

24 2061 51 GGGUAUAGAGUCAGGCAUCTT

25 20115 52 GGUUUUCCAUGGUUAAGUUTT

Target siRNA SEQ ID NO

No. ID siRNA sense sequence (21-mer)

25 20024 53 GGAAGUCUUGUCCUGGUCUTT

26 118397 54 GGAGCAGACUAGGCAUAUCTT

26 118398 55 GCAGACUAGGCAUAUCUUUTT

27 104835 56 GGCGAUAUGGAGGUGUACATT

27 104830 57 GGAGGCCAUUUGGUCUUCATT

28 119221 58 CGGUUUCAUGCCGACUUCATT

29 105141 59 GGCAGCCAGACUGCAUUUATT

30 122236 SO GUUGUCUGCUUACCUUAACTT

31 43781 61 GCUCUAAGGAAGACUUCAATT

32 103636 52 GGCUGGAUGGAUGCAUUUATT

33 45922 63 GCUACAUCCGCUCCAAGAATT

34 117371 64 CCAUUCUAGAUUGCAUUUATT

35 111157 G5 GCGUGGCAAAGUCCAAGAUTT

36 38979 66 GGUGAUCAAGAAGGUAAAGTT

37 121933 67 GGAAUCAUUGCAUGCUUAATT

38 119829 58 GCCUUGAUAUAAGGUGUAUTT

39 19471 69 GGAAAUAAUAAGGGAGUAATT

40 120442 70 GGCAUUAAUUCAUGGACUATT

41 105154 71 GGCAGUCUCUGGUAUACACTT

42 6196 72 GGUCUCAUAGGGAAUAUAUTT

43 105753 73 GUGGCAGUGUGACCAAGAATT

44 21895 74 GGAAAGUAAGUGCUGGUCUTT

45 120445 75 GGCUCUGCUAGACCAAGAATT

46 111807 76 GGUGGUUCGAAAGACUUCATT

47 1721 77 GGUCAUCAGCAUGGAGUACTT

48 1774 78 GGACAGUGACGAACUAUUUTT

49 117712 79 GCGAGUUUUACCUGAUAAATT

50 1795 80 GGGAAGACUUUCGCUUCUGTT

51 117084 81 GCUCCUGAUCAUGGUGUAUTT

52 119926 82 GGAGAAAAGGAGACUUCAATT

53 9067 83 GGCUAUUUGCCAGCAUAUATT

54 121179 84 CGGACGACUACGUCUUUUATT

55 38327 85 GGGAAGUCUUAUUUUGUCATT

56 111813 86 CCCUAAGCAAUGUGCAUACTT

57 107569 87 GGUCUAGGGACAAUAAGUATT

58 10560 88 GGCAUCCUGAUAUGCUUACTT

59 10235 89 GGUGCUAGAGUGUGGAGUUTT

60 104127 90 GGACAAAGUGUUUGCUUGATT

61 112077 91 CGGAGUGUACACUGUUCACTT

62 105010 92 GGAAGAGAGUUGUAUGUACTT

63 4154 93 GGCUGAUUGUUGUGACAUUTT

64 40980 94 GGAAAGACCAUGUUUGUAGTT

65 120756 95 CCUGGUGGUUUGUGUAUGATT

66 1627 96 GGAAGUϋUACUGGAUUUCUTT

67 122128 97 CCAAAGUGGGCAUGCAUUGTT

68 2207 98 GGCAUCAUUAAACAAGCCATT

69 4633 99 GGUACAUAGCAAUCUGUCATT

70 119952 100 CCGUGGUGGAGUUGCUUAATT

71 105325 101 GAAAAAAUCGUUCCAAGAATT

72 112330 102 GGAUGUGCUGCCUAAGUAUTT

73 121576 103 CGUGUAAAUAGUGGUAAAUTT

74 117799 104 CGAUUCUCUACACAGGAGUTT

75 104458 105 GGAUCAAUUUAUCAGAAAGTT

76 112453 106 GCAUUAUCGACAUGCAUUGTT

77 121337 107 CCAAUCAUCGAUUCUGCCATT

78 110844 108 GGAUCCAACGACCAAGAACTT

79 119422 109 CCGAUCACAAAGCCUUCUATT

80 119276 110 GGUAUACCUUAAGCCGUACTT

Target siRNA SEQIDNO

No. ID siRNA sense sequence (21-mer)

81 118817 111 GCCAAAAAACUGGGUGUACTT

82 118114 112 CGAUGAACUAUAGCAUAUUTT

83 118462 113 GCCGAAGGUGUUUGCUUAUTT

84 46510 114 GACUCCAGAACCϋϋCUCUCTT

85 119129 115 CGAUGACCAAUCCCUUUAUTT

86 119399 116 GCAAGGUCAUAGAACUCGGTT

87 122166 117 GAAAGGAUUAGAUUGGGUATT

88 45063 118 GCAUCCUGACGGUUGAUGUTT

89 117601 119 GCUUCACUUGUGACAGGACTT

90 118446 120 GCAACUUGUUUUGCAUAUGTT

91 18240 121 GGAAGUUCCUCAUUGCUUUTT

92 104559 122 GGAGUCCUAUGACUAUCAGTT

93 1407 123 GGUGGAUGUAUAGCAUUUUTT

94 119077 124 GGAGAUGAGGUUGAUUUCATT

95 1371 125 GGGCAGGCAUAUGGAGUAUTT

96 106537 12S GGACCUGUUGAUGCUAGUUTT

97 120500 127 GCAGAUACGAΆAUGGUGUUTT

98 111654 128 CGAAUGUUCGUGCACAUUUTT

99 118718 129 CCCUUAAAGCUCACUUCAATT

100 120608 130 GCACGAGACGCACUUUUAUTT

101 142253 131 GGAGCUACAGAGUCUUCGATT

102 111081 132 GGCAGACCGAAGAGAAUACTT

103 103786 133 GAACGGCAAAGAUUACUACTT

104 121943 134 CCAUUGUAUUGUUGCUUAGTT

105 121806 135 CGAAUGUCCUAGUGCAUUUTT

106 15527 136 GGAAUACUAAGAUGCUUGGTT

107 143005 137 GCACUGUUUUUAGCAUUACTT

108 110607 138 CCUGAGCGAGAGACUUUUUTT

109 107834 139 GGGUCUAUAGAGUUUAUGATT

110 121163 140 GGUGCAAUGCGACUAUCACTT

111 118522 141 CCUAUGACUUUGUCUUCGATT

112 120179 142 CGAAUGAGUUUUGUGCUUCTT

113 34999 143 GGUUCUAUAGUCCUUUUAATT

114 6091 144 GGUAAUAGGAGAAUAGGUGTT

115 103829 145 GGAAAUGACUACAGAGCUGTT

116 119396 146 CGUACCUACGGCCAUUGCATT

117 8816 147 GGAGACAAAGUGCAUAUAATT

118 15339 148 GGAAAAGGCAUCCAUGCUUTT

119 29459 149 GGCAAGAGGAUAUUCUUCUTT

120 16059 150 GGAUGAUGCUGACUGUUCGTT

121 104173 151 GGUGGUGGAAUGUGUCUCUTT

122 120611 152 GGUCUUCAUGCCCUAUCACTT

123 142929 153 CCUACAUUCUCGAUUUΫUCTT

124 35220 154 GGCCUUCAAGAAGGAGCUGTT

125 119469 155 GCCUUGGGAAACAACUUUUTT

126 121471 156 GCAUACUUCUAGGAUAGCUTT

127 122003 157 GGAAGUCAUUCUUAAGGACTT

128 114058 158 GGCAGUUAUCCAGCAUUUCTT

129 117031 159 GCAUAGUUGGUCUUGGUGUTT

130 110906 160 GCAUCUAUCAGAUUAAGUUTT

131 119517 161 CGACAGUCUAAUGGAGCUUTT

132 114048 162 CCAAACAGGGUAAUCUUGATT

133 809 163 GGGAAGAAGCCAAGCCUUATT

134 137195 164 CCAUCGGUAUAGUGGAAGCTT

135 118341 165 CGAGACAAAGCACUUCAUCTT

136 46319 166 GCAAAGACCUGUGAAGCUATT

137 7204 167 GGAACUGGCAUUGGACUCATT

138 119081 168 CCGUGGACCAGUACUUUUATT

Target SiRNA SEQIDNO

No. ID siRNA sense sequence (21-mer)

139 745 169 GGUUUACAAGGCCAAGAAUTT

140 119216 170 GCUAUGCCUGGUUGCUUAGTT

141 117277 171 GCAUCAUGUCGGCCUUCUUTT

142 14775 172 GGGϋUGCUCAUUUUGGGUATT

143 119182 173 CCUCCUGAUCAAUAAGUAUTT

144 1286 174 GGUCAACAAUCACCUUUUCTT

145 1961 175 GGA&CUCAGAAACCAAGAATT

146 119782 17S GGACUCAAGUAUGACUUCCTT

147 135366 177 CCAAGGUCCUGGAAUGUCUTT

148 42362 178 GCAUUGUGAACCAGGUCUCTT

149 12155 179 GGAUGUGCGAGUUCAAGUGTT

150 11 180 GGAGCACCUGAUUCCUUUCTT

151 7881 181 GGAACAGCAACAAUUCCGGTT

152 10428 182 GGAUUUGCCAAAUGCUAUGTT

153 16123 183 GGGAAUUGAGGAAAGCCUUTT

154 1049 184 GGAAAAGAUCCAGGAGUAUTT

155 919 185 GGUACUGGUGAUGGAGUACTT

156 121066 186 CCAAAGACGGUGGAUAUAGTT

157 105169 187 GGAAAUCUAGUGGUGACUATT

158 36311 188 GGCϋUUUACCAGAUUUCUUTT

159 5884 189 GGUAUGAUGUUUGUGAAUATT

160 44940 190 GUGGAUCCGAGACAUGUACTT

161 1398 191 GGCAAUCAGGAUGUAAAGUTT

162 104626 192 GGAAAGACGUAACAGGAGATT

163 15563 193 GGAACGACAGUGGGUAGAUTT

164 136772 194 CCCACAUAUCUGCUCUGUATT

165 46183 195 GAUACCAAUGGUGCUGUUGTT

166 5377 196 GGCCUUCUCCAAGCACAUCTT

167 103491 197 GGUGGGUAUGUACAAGGUCTT

168 117929 198 GGUCACAAUUUCUCUUGAUTT

169 110591 199 CCUCAUUAUCGCCAAGGAUTT

170 103534 200 GGACAGUACAAUUUUGACCTT

171 119066 201 GCGAGUGUUUACGGGUGUUTT

172 106403 202 GGUCUACUUGUACUAUCAATT

173 121059 203 GCGAGAAUAUUGCCUUCUUTT

174 121219 204 GCAUGAUCAGUCCUGAUUGTT

175 117366 205 CCUUUGCACUAUAUACAUCTT

176 105936 206 GGCCAAAGGACUUCUUUUGTT

177 105919 207 GCCCUAGUUCUCGCAUUGATT

178 103932 208 GGAAGCAUCCAUCAGCUGGTT

179 43139 209 GUCUCCUGCUUUGUAUCCATT

180 9108 210 GGCACAGGUGUAAAUAUAGTT

181 111592 211 GGUUAUUUUACAUCAUUCCTT

182 104388 212 GGAUGGCUAGUAGUAACACTT

183 115931 213 GCCCAAGUGUUUCAGUGACTT

184 30832 214 GGCGGCUUCCUUGGAGUAUTT

185 105076 215 GGAUGAACUGAUCGCUUAUTT

186 44885 216 GUUCACUGUUGGCAAGGAATT

187 103580 217 GGAGCUUAUGGUUCUGUCATT

188 1555 218 GGUCAGAGAUGUAGGACCUTT

189 105163 219 GGUAAUCAUGUUACUAACCTT

190 105773 220 GCAGCCUUGUGUUUGGGUATT

191 37190 221 GGUGUUUCUGGAUGCAUAUTT

192 121953 222 GCUCUCAGCGUGCUAGUAATT

193 9040 223 GGACAUUGUGUUCCUGAUCTT

194 119716 224 GGAAAUCAAGCCCUGCCAATT

195 1794 225 GGUGCAGAACAUCAAGUUCTT

196 115136 22S CCCUAUAUCCAGCAUGCGATT

Target siRNA SEQIDNO

No. ID siRNAsensesequence (21-mer)

197 116921 227 CCGAAAUGGAUGCAUUTJCATT

198 117213 228 GCUGUGUCCCUUAGCCUUUTT

199 10426 229 GGGUCCGAUUUGCCAUCGATT

200 657 230 GGCUCACCAUGAUGAUUAATT

201 46002 231 GCUCCAAGSAGGAGAAUAGTT

202 105830 232 GCCUAUCCUACUAGAACUUTT

203 104815 233 GGCUCCUCUUACCUCCCAATT

204 142280 234 GCUUUAUUGAGUCACUGCCTT

205 1940 235 GGUGAUGAAUUUACCAUCATT

206 103397 23S GGGUGUGGUCUGAAUUACCTT

207 117187 237 GCAGCUCAAGUUAGCAUUUTT

208 119405 238 CCAGAUGAGCUUUCAAGUUTT

209 111208 239 CCAAUUGGAUAGAGGGUACTT

210 118568 240 CCAUCAAGGGCUAUGCAUGTT

211 383 241 GGUUGUUCAUGAUGCUAACTT

212 118038 242 CCAAAGAUAUCCUGAUUAATT

213 42454 243 GGCUUUUCUGUCAUGCUUGTT

214 118423 244 CCUGGAGAGCAUCUUUUCATT

215 104231 245 GGAGGUGGUCUGUAAAAGGTT

216 121036 246 CCAGCAAGUGCGACUGUAUTT

217 5834 247 GGAAAUUUGGAAAUCCUAGTT

218 114204 248 CCCUAACAAGCUGGUGUUATT

219 119258 249 CCUAAGCUUCCAUGUAGCCTT

220 111728 250 GGAAAAACCCUCAGGUGUGTT

221 119072 251 GCAAGAUUGUCGGCAAGUGTT

222 121053 252 CGAGGGUUGGGUUUGCUUUTT

223 142803 253 CCUUUGACUAAUAGGAGUUTT

224 117248 254 GCAAUCACAUUGCUCUGUATT

225 111369 255 CCACUGACCCACAGACUCUTT

226 118232 25S GCACUGACCUCUGUGACUUTT

227 10238 257 GGAAAGACCACCAUCCUAUTT

228 118663 258 GCUUCCGGGAAGAAUAUAUTT

229 13014 259 GGAACAGCGAGCUGUUUGATT

230 118922 260 GGCAUAAAUUCCUUCΫCAGTT

231 119792 261 GGCCGAGACAAAGAAGCUUTT

232 103447 262 GGUCUUGUCGGCAGUGAAATT

233 23376 263 GGGAAGACUCGAACCUUΫUTT

234 17099 2S4 GGUUGUGAUGUGGUAGUUATT

235 138240 265 CCUCCUACGGGCCUUUUAUTT

236 9004 266 GGUGUUUAUAGUGGUGUUUTT

237 1785 267 GGAUGUGCAGAAAGUGCUGTT

238 107446 268 GGACAAUCGCACUUUAUACTT

239 108267 269 GGUAUUDUCUACGGGUGΫUTT

240 122036 270 CCAAACUGUACAGACUCUCTT

241 118553 271 GCCAAGCAGGAGAUUAACATT

242 119612 272 CCGAUGUGAAAGGCUGUAGTT

243 121775 273 CGAAAGACGGUGACUCUUGTT

244 110854 274 GGAAUGUGUAGAAAGAUCGTγ

245 126062 275 CCAGUUUUAGAUGACUCUUTT

246 118792 276 CCUUGGAUUACAUAAGGAUTT

247 120597 277 CGGGCCAGUAUGGUGCAUUTT

248 119183 278 CCGCAUGGGAGAUGUUCUUTT

249 121277 279 GGGCCCAAAGAAAGAGCUATT

250 117497 280 GCAGGGUUCAUGCAUAUGATT

251 1704 281 GGUGUCAGAUAGUAAUAUCTT

252 119905 282 GCAGUUCUUACUGGACUCATT

253 121507 283 GCAUCUGGCUAUUAAGUGCTT

254 121103 284 GGUCAACϋϋUCAAUUACUGTT

Target SiRNA SEQID NO

No. ID siRNA sense sequence (21-mer)

255 6501 285 GGUGCCCAGAACUCUϋUUCTT

256 43395 286 GAACAAGAACUACAAGGAGTT

257 1541 287 GGACAGAAGCUACAUCUGCTT

258 648 288 GGAGACCUUCAACCUUCUGTT

259 22653 289 GGGAUCUOACAUCUAUCAGTT

260 112041 290 CGACAGAAUAUAACUAACCTT

261 116939 291 GGAGUUCUAUGUCUGCAUCTT

262 111049 292 GGCAUUCCCUAUGGCUUAGTT

263 120730 293 GGACUUCACGGGUAUGGUUTT

264 1776 294 GGAUOAUUACUACCUGUCATT

265 139134 295 CCUCUAAUAGAACUGUCUATT

266 118865 296 GCAAUAUUUAAGCUGUUCUTT

267 119034 297 GCAACACACAUGUGUAUGGTT

268 41802 298 GAUAGAUAGGACAACUUCATT

269 115614 299 GCCUUACAGUUGGCAAGUCTT

270 120142 300 CGUGAUGGCUGCAUAUGGATT

271 129420 301 GGUUAUAGUGUGAGACUUGTT

272 35139 302 GGGAGAAACGCGCCUUAAUTT

273 112237 303 GCCAACGACUUAGCCUUACTT

274 118332 304 GCACUUAUOUACACUUCAGTT

275 202390 305 CCAGGAAUUGUUCUAGUAATT

276 111442 306 GCCUUUCGUGGUAUCUGCATT

277 119734 307 GCAACGUGAUCAAAGGUAUTT

27θ 46555 308 GCUCCUCAUCACACUGUCATT

279 112493 309 GCCUUUACUCUGAGGAUAATT

280 120542 310 CCCAACCAUAAUGGUAAAATT

281 143975 311 CGACUUUCGCGCCAAGAUGTT

282 202509 312 CCGGAAUAUUGCUACAUACTT

283 26615 313 GGCUAAUGUCAUCUGUAUATT

284 2021 314 GGUAAUUUGUUCUUGGUGATT

285 1017 315 GGAAGUUCGUGAACAUGUATT

286 44902 316 GGCUGAGAACGGGAAGCUUTT

287 121821 317 GCACGGAGAUAAUGAGAAUTT

288 3573 318 GGUACUAUUUUGAGGUGGATT

289 111379 319 CGGGAUUUAUUCAGCCUUGTT

290 119725 320 GGCAACCUAUUAGGCAUGATT

291 119012 321 GGCUAUGCCACUGGACUCATT

292 11202 322 GGAUUUCAUUUCAGGUGGATT

293 119352 323 GGACCUGUACAUGCUUCGATT

294 111028 324 GGUCACAAGUUGCCAUCUATT

295 6242 325 GGACCACUGAGAACUUUUGTT

296 6829 326 GGUGAGUACAUGAGCUGCUTT

297 120986 327 CCCUAGUCUUCGAAAUGUUTT

298 282 328 GGCCUGCCAGGAGGUGUUGTT

299 119249 329 GGAACCGGUAUACAGUGAUTT

300 121002 330 GGCAUCUGGGUGGGUUUUATT

301 112098 331 GCGCAUUCCUUUGAUUGGUTT

302 120006 332 GGAGCUAGAUUCAUAUCCUTT

303 9145 333 GGUCUGGUCUGAGGAGCUATT

304 45672 334 GCUGGCAGAUCUGCUUCUATT

305 5997 335 GGUACGUCACCAUCϋUUUATT

306 5922 336 GGCCAUAGUCCUGUUCACCTT

307 112027 337 GGGUUAAAAUGGCUGCCAUTT

308 42079 338 GGUGGACUACUCACGUUUUTT

309 104120 339 GGGAAAGGGAUAUGUAAUATT

310 104465 340 GGCACUGGUUAGGAAGGAUTT

311 118241 341 GGUUGGCAAGAACCUGAUUTT

312 12487 342 GGUCAUGUGGUUGGACUACTT

Target SiRNA SEQIDNO

No. ID siRNA sense sequence (21-mer)

313 139155 343 GCUAUUGUAUAAAGGAUGGTT

314 7014 344 GGGAGCCUUUGCCUACAUATT

315 107742 345 GGGAUUUUGGGACUGUUCUTT

316 122070 34S GCUAGGGAAGUDUGCCGUUTT

317 119167 347 CCUAUGACUUUUUUGGGUUTT

318 121082 348 CGAGCCAAUCAUUUCUUCATT

319 34166 349 GGAUGAAGAUGAAGUGCUUTT

320 36798 350 GGUGAUUCUGGACAAGGAUTT

321 6764 351 GGACAUUGCUGAGGUGGAUTT

322 112281 352 GGUGAAAGCAUAACUUUUUTT

323 1359 353 GGGCUAGAAAAGAAUGUAATT

324 120792 354 GAGCAAGAGAUGACCUUAUTT

325 120227 355 GCCAUAGUAUCAUUGGGCCTT

326 117144 356 CGAUAGGAAUAGCUUUUAUTT

327 202463 357 GCUCUGACCAAGUGGAGAUTT

328 326 358 GGAGCUAGAGCUUGAUGAGTT

329 121132 359 GCUAUAACCGAGGAAAUUUTT

330 40762 3SO GGAAAACUGUGAACUUUCUTT

331 106985 361 GGUUAUGGGUAUUGGUGUCTT

332 118634 362 GCGCAGCUGUCUUUAGCCATT

333 1709 363 GGACACUUCAUCGUGAGGATT

334 119230 364 GGUAAUCUACAGUGAGCUGTT

335 111644 365 GGACAACCϋUCCGACϋUUUTT

336 103331 36S GGAUCGAUAUGCCUUGGCATT

337 105105 367 GGCCCAUAAUAGCCAUGCCTT

338 6671 368 GGUCACCAUCCAGAAUGUCTT

339 117749 369 GCUCACAUGCAGUAGACUUTT

340 104678 370 GGUACUAAUGCAUCUGCAUTT

341 4236 371 GGUUCAUCUUUCAUGUGAATT

342 119150 372 CCUUGUUAACAGUGGAGUATT

343 120536 373 GCCUAGAUCUUGCAUUUAATT

344 4084 374 GGCCUUCCUACCACUUCAUTT

345 8584 375 GGAGAAAAAGAUGUCUUUUTT ■

346 118025 376 GGAAGCAUUCAAGCAUUUATT

347 104025 377 GGUUGAUGCGGAGCUGUUUTT

348 142304 378 CCGGAUGUUAACCUUUAACTT

349 119004 379 GCCAUCUUAGCAACUUUCUTT

350 112199 380 GCAGGUCCCUACUAUCAACTT

351 140383 381 CCUCACUUCUAGACUUUCATT

352 43202 382 GCAUUGGGAUAUUAAGUAGTT

353 118558 383 CCUUUACGGCGUAAUCCUGTT

354 122033 384 CGGUCUGUGUGCUGUUUGATT

355 110947 385 GCCUAUGGUAGCUGGACUUTT

356 122374 386 GAAGCAGAGCUGACCUUAGTT

357 121768 387 CCCAUAGUGAAAUGUGCUGTT

358 110667 388 GGAUGUGAAAGAGUCUUUUTT

359 119683 389 GCAAGAAAAACGAACUCUUTT

360 105368 390 GCAUUUGUUGAUCUGUUCATT

361 117476 391 GCUGUGCAAAGAACUAUCCTT

362 8110 392 GGAAGUAUAGAAGAAUGUGTT

363 108254 393 GGUACGCUGUUAAGUAUUATT

364 119254 394 CGUGUCAAAGCUGGUUACGTT

365 120528 395 CCAGAGAUUUGUUUUAUGATT

366 114721 396 GGAAUUAAGACAUGGUGUGTT

367 120404 397 GCCAAGACCGAUGUGAAUGTT

368 121560 398 GCUCCUGUUACAACCUGUGTT

369 8759 399 GGGACACCAAGACCUACAUTT

370 121689 400 GCGAUCAUGUCUACAAGGGTT

Target SiRNA SEQ ID NO

No. ID siRNA sense sequence (21-mer)

371 116959 401 CCUCUUAGGUUGUAUCCUUTT

372 18269 402 GGGCUACCUUGAGACUUUCTT

373 4118 403 GGCAGAUGUGGCAUGUCUUTT

374 120313 404 GGGUGGGAGUUGGUAAAGATT

375 14778 405 GGUGAGAAUUUCAAGGAUUTT

376 1554 4OS GGAAAAUCGGAUGUGGAUCTT

377 120581 407 CCCUCUAUAAGGCUUGGUCTT

378 135789 408 GCCUCAGUAAGAUAGCAUCTT

379 104313 409 GGUAGCAGAUGGACUAACUTT

380 5020 410 GGCACUGAAACUCACACUGTT

381 103787 411 GGCUUUGGCAAGGUGUACATT

382 38222 412 GGCAUCUUGGUUGUAAGUCTT

383 119010 413 GGGCUAUAGCCCUCAUAACTT

384 119464 414 GCUGCCACCAACAAUAUAUTT

385 111967 415 GCAAUAUCCGACUACUGCATT

386 117597 416 CCAACACΫUUUGACCUUGATT

387 616 417 GGUGGACAAUCACCUUUUUTT

388 104271 418 GGAAAGACGGAUUGCAUUATT

389 41648 419 GUACUUUAACAUCGUCGCUTT

390 116760 420 CCUUUUCUGUAUAUAGCCATT

391 103742 421 GGAGCCUCUGCUGAGUAUATT

392 121625 422 GCUAUCAAUGGUGUAAUACTT

393 108793 423 GGAGCUCGAGUAUAUUUAGTT

394 46149 424 GAAAAGAUAUUGGAUGCUCTT

395 121965 425 GCCUAΆUUGAUUUCAUUCUTT

396 104655 42S GGAAAUGCAGCCUAGUCUUTT

397 3025 427 GGAAGAUUAUUCUGAAUACTT

398 23439 428 GGUUGAUGUAACCUUUUAATT

399 31620 429 GGUGGAAAUGAUGUUUAUCTT

400 4069 430 GGACAACUUUGGUGCUGUGTT

401 107669 431 GGCUUUGGGAUAUGCUGUATT

402 9248 432 GGAGUCCAACAAGAAGCACTT

403 106198 433 GGAUCUGUACCCAGUAAUCTT

404 112515 434 CCAUCAAUGUAACUUGUACTT

405 8537 435 GGAAAAUGACUUGUAAGGUTT

406 110610 436 CCAACCAUSGGCAUAUCCUTT

407 43265 437 GACCUGGCCAAGUACAUGATT

408 180 438 GGAAGUGGCAGUGAAGCUATT

409 117634 439 CCUAUCCAUUACUUAAGGATT

410 8947 440 GGUGCAGAAACCUGUGAAGTT

411 10429 441 GGACUGUGGAGACGUAAAUTT

412 107317 442 GGUAUGCGACGGGAAAGUATT

413 121476 443 GCUACUUUGGCCGUAAGGUTT

414 126545 444 GGAGGUUAUGGCCAUUGCATT

415 202300 445 GUUCACCUACAAGCAGAUCTT

416 105208 44£ GGCAAAUGUUCUCACUGCATT

417 109375 447 GGAGUUUCAGACCCUAUCCTT

418 120071 448 GGGAUAUGCAAACCUCAUCTT

419 122067 449 GGACUCAGACACAGGUGAUTT

^' 420 18224 450 GGCGGCAGAACAUUGCUUATT

421 2057 451 GGCAGACAGCUAUACCUUGTT

422 6205 452 GGACAUCAUCACCUUGGAATT

423 103432 453 GGGAUGUUGUAUCUUCAUUTT

424 107085 454 GGUAGCAACAGCGAUUGGATT

425 117546 455 GCUAUGCAGAUGGCGUGUATT

426 41929 456 GCAUUACGGUGUCUUCACCTT

427 112254 457 CCAAUCGAUAAUCACAUUGTT

428 105538 458 GACGGGAACACUCCAAUGATT

Target siRNA SEQ ID NO

No. ID si RNA sense sequence (21-mer)

429 444 459 GGAAAGCAAUCACUUCUCATT

430 6722 460 GGCUUUUUCCAGCCUUACUTT

431 40719 461 GGAGAUCAUUAAGAUUACATT

432 115262 462 GCGGUGUUCAUGAUUUCϋUTT

433 106223 463 GGACAUUCCCACUAUUAUGTT

434 120151 464 GGGAAGGUGAUUUACUUCATT

435 105664 465 GAGAUGACAGCAAUGAGUCTT

436 1020 4S6 GGACAACAAUUUGCAUUAATT

437 112308 467 CGUAAGUCCACAUAUACUUTT

438 120484 468 GGCUAUGAAUUGGACCUUUTT

439 670 4S9 GGAGUUCAAUAAAUGUCUGTT

440 1397 470 GGGAAAACGGCACCUUUUCTT

441 104702 471 GGCAAUCAAGGGUACAUACTT

442 1140 472 GGAUCUGAGGCAGGGUUUUTT

443 112418 473 CCAAGUACGCAAACUUUUCTT

444 104693 474 GGAGUGAUUAGUUCGGGUUTT

445 8943 475 GGAUUCAUAGACUUCAUAGTT

446 107096 476 GGAAAAAUGAAUUGCUUGGTT

447 2531 477 GGUAACUGUGCUGAGGGUCTT

448 118060 478 GGGUCCAAGAGAUAUACUGTT

449 118590 479 GCUCCUUAAGGCUCUUUUGTT

450 118906 480 CCUAUUAGUGUCUCCUACATT

451 106243 481 GGUCACAUUUAUGUGGAAGTT

452 111874 482 CGUGCUUUUUCUCAAGUAUTT

453 8193 483 GGAUGGGCUCAAAUACUACTT

454 2512 484 GGAAAUAAAAGAUCUUGACTT

455 16362 485 GGCUCCUACUACGAGUAUUTT

456 14218 486 GGGUUUAAUACAGCAUGUGTT

457 103493 487 GGCAUAUUCCACUGAOUACTT

458 1176 488 GGUCCACAAGGCAGUGCUGTT

459 111523 489 CGAAGUUAUCAGAGAUGCUTT

460 33700 490 GGAGAAAUGACCUCAUUUUTT

461 2167 491 GGAAUGAUCCAUACUGAAATT

462 105854 492 GACAUCUUAGCCAGGAAAUTT

463 46281 493 GCUGCCUGUGGUGGUAAAATT

464 44894 494 GAAGCUGGAUACAGCAUAUTT

465 38041 495 GGAAAGACAACAAAUACUATT

466 42278 495 GUUAGCUUCAUAAUCUGUϋTT

467 112472 497 CCCUAUCAAGACUUCAUUATT