WO2011024441A1 - Ercc6l comme gènes cibles pour le diagnostic et la thérapie du cancer - Google Patents

Ercc6l comme gènes cibles pour le diagnostic et la thérapie du cancer Download PDF

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WO2011024441A1
WO2011024441A1 PCT/JP2010/005206 JP2010005206W WO2011024441A1 WO 2011024441 A1 WO2011024441 A1 WO 2011024441A1 JP 2010005206 W JP2010005206 W JP 2010005206W WO 2011024441 A1 WO2011024441 A1 WO 2011024441A1
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ercc6l
double
cancer
gene
stranded molecule
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PCT/JP2010/005206
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Yataro Daigo
Yusuke Nakamura
Takuya Tsunoda
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Oncotherapy Science, Inc.
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Priority to CN201080048217XA priority Critical patent/CN102597236A/zh
Priority to JP2012510472A priority patent/JP2013502901A/ja
Priority to EP10811499A priority patent/EP2470652A1/fr
Publication of WO2011024441A1 publication Critical patent/WO2011024441A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to methods of detecting and diagnosing cancer as well as methods of treating and preventing cancer, particularly cancers associated with the overexpression of ERCC6L such as lung cancer.
  • the present invention also relates to methods of screening for a candidate substance for treating and preventing an ERCC6L-associated cancer.
  • the present invention relates to double-stranded molecules that reduce ERCC6L gene expression and uses thereof.
  • Lung cancer is the most common form of cancer, accounting for 1.35 million of the 10.9 million new cases of cancer per year. It is also the leading cause of death from cancer-associated disease, accounting for 1.18 million of the 6.7 million cancer-related deaths worldwide (NPL1).
  • NPL1 cancer-related deaths worldwide
  • cytotoxic agents including paclitaxel, docetaxel, gemcitabine, and vinorelbine have emerged to offer multiple therapeutic choices for patients with advanced NSCLC (non-small cell lung cancer); however, each of the new regimens can provide only modest survival benefits as compared with cisplatin-based therapies (NPL 2).
  • molecular-targeted agents including anti-EGFR or anti-VEGF monoclonal antibody, cetuximab (Erbitux) or Bevacizumab (Avastin), and small molecule inhibitors of EGFR tyrosine kinase, such as gefitinib (Iressa) and erlotinib (Tarceva), have been examined and/or approved for clinical use (NPL 3, 4).
  • These agents display activity against recurrent NSCLC to a certain extent, but the number of patients who could receive a survival benefit is still limited. Meanwhile patients with SCLC (small cell lung cancer) respond favorably to the 1 st line multi-agent chemotherapy, though they often relapse in a short time.
  • ERCC6L excision repair cross-complementing rodent repair deficiency, complementation group 6-like; also referred as to "PICH" is of particular note.
  • ERCC6L has been identified as a member of the SNF2 ATPase family, which is an interaction partner and substrate of Plk1 (NPL10).
  • NPL10 Plk1
  • a recent report demonstrates that ERCC6L is an essential component of checkpoint signaling, and binds to catenated centromere-related DNA to monitor tension developing between sister kinetochores (NPL10).
  • NPL10 sister kinetochores
  • NPL 1 Jemal A, Siegel R, Ward E, et al. CA Cancer J Clin. 2008;58:71-96.
  • NPL 2 Schiller JH, Harrington D, Belani CP, et al. Eastern Cooperative Oncology Group. N Engl J Med 2002;346:92-8.
  • NPL 3 Dowell J, Minna JD, Kirkpatrick P. Nat Rev Drug Discov 2005;4:13-4.
  • NPL 4 Pal SK, Pegram M. Anticancer Drugs 2005;16:483-94.
  • NPL 5 Chute JP, Chen T, Feigal E, Simon R, Johnson BE: J Clin Oncol 1999;17:1794-801.
  • NPL6 Daigo Y, Nakamura Y.
  • the present invention relates to the discovery, through microarray analysis and RT-PCR, that ERCC6L is overexpressed in clinical lung cancer tissues.
  • functional knockdown of endogenous ERCC6L by siRNA in cancer cell lines results in drastic suppression of cancer cell growth, suggesting its essential role in maintaining viability of cancer cells. Since it is only scarcely expressed in adult normal organs, ERCC6L appears to be an appropriate and promising molecular target for a novel therapeutic approach with minimal adverse effect.
  • An increase in the level of expression of ERCC6L as compared to a normal control level indicates that the subject suffers from or is at risk of developing cancer, particularly lung cancer.
  • the ERCC6L gene can be detected by appropriate probes or primer sets or, alternatively, the ERCC6L protein can be detected by anti-ERCC6L antibody.
  • the methods of the present invention can be carried out in vitro or in vivo and use as an index the binding activity to an ERCC6L polypeptide, an expression level of an ERCC6L gene, a biological activity of an ERCC6L polypeptide, or an expression level of a reporter gene or an activity of a reporter gene controlled under a transcriptional regulatory region of the gene product.
  • Substances that bind to an ERCC6L polypeptide, or suppress an ERCC6L expression or activity, or a reporter gene expression or activity can be identified as candidate substances for treating and/or preventing cancer, or inhibiting cancer cell growth.
  • the biological activity of the ERCC6L polypeptide to be detected is preferably cell proliferative activity (cell proliferation enhancing activity).
  • a decrease in the biological activity of the ERCC6L polypeptide as compared to a control level in the absence of the test substance indicates that the test substance may be used to reduce symptoms of lung cancer, or treating and/or preventing lung cancer cancer.
  • the agent is an inhibitory nucleic acid (e.g., an antisense, ribozyme, double-stranded molecule, aptamer).
  • the agent may be a nucleic acid molecule or vector for providing double-stranded molecule. Expression of the gene may be inhibited by introduction of a double-stranded molecule into the target cell in an amount sufficient to inhibit expression of the ERCC6L gene.
  • the method includes the step of administering to a subject a pharmaceutically effective amount of double-stranded molecule against an ERCC6L gene or a vector encoding such a molecule, wherein the double-stranded molecule inhibits an expression of an ERCC6L gene as well as cell proliferation when introduced into a cell expression an ERCC6L gene.
  • a pharmaceutical composition suitable for the treatment and/or prevention of an ERCC6L-associated cancer that includes a pharmaceutically acceptable carrier and an active agent including one or more double-stranded molecules against an ERCC6L gene or a vector encoding such a molecule.
  • a double-stranded molecule against ERCC6L is capable of inhibiting the expression of an ERCC6L gene as well as inhibiting the cell proliferation induced thereby when introduced into a cell expressing an ERCC6L gene.
  • the cancer being treated and/or prevented is lung cancer, including NSCLCs and SCLCs. Examples of NSCLCs include lung adenocarcinoma (ADC), and lung squamous cell carcinoma (SCC).
  • the double-stranded molecules of the present invention are preferably composed of a sense strand and an antisense strand, wherein the sense strand includes a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 7 and 8 and the antisense strand includes a sequence which is complementary to the sense strand.
  • the sense and the antisense strands of the molecule hybridize to each other to form a double-stranded molecule.
  • the double-stranded molecule of the present invention inhibits an expression of the ERCC6L gene as well as cell proliferation.
  • the methods and materials of the present invention are capable of identifying cancer prior to detection of overt clinical symptoms thereof and may be used in the context of cancer therapy without adverse effect.
  • Figure 1 demonstrates the ERCC6L expression in lung cancers and normal tissues.
  • Part A depicts the results of semi quantitative RT-PCR validating the over-expression of ERCC6L in clinical samples of NSCLC (ADC and SCC) and SCLC as compared to normal lung tissues. Appropriate dilutions of each single-stranded cDNA prepared from mRNAs of lung cancer samples were prepared, using the level of beta-actin (ACTB) expression as a quantitative control.
  • Part B depicts the results of semiquantitative RT-PCR validating the expression of ERCC6L in lung cancer cell lines.
  • Part C depicts the results of Northern blot analysis for ERCC6L, demonstrating that several lung cancer cell lines strongly express ERCC6: while most normal human tissues do not.
  • Part D depicts the subcellular localization of exogenous ERCC6L protein in COS-7 cells detected by anti-myc, which were co-stained with DAPI.
  • Figure 2 demonstrates the effect of siRNA against ERCC6L on the growth of lung cancer cells that overexpress ERCC6L.
  • Part A depicts the results of semiquantitative RT-PCR analysis confirming the knockdown effect on ERCC6L expression in SBC-5 cells in response to si-ERCC6L (si-#A or si-#B) but not control siRNAs (LUC or EGFP).
  • Part B depicts the results of colony formation assays of SBC-5 cells transfected with specific siRNAs or control plasmids.
  • Part C depicts the results of MTT assays of SBC-5 cells in response to si-ERCC6L (si-#A or si-#B), si-LUC, or si-EGFP. All assays were performed three times, and in triplicate wells.
  • Figure 3 demonstrates the enhancement of cell growth by ERCC6L introduction into COS-7 cells.
  • Part A depicts the results of Western blot analysis confirming the transient expression of ERCC6L in COS-7 cells.
  • Part B depicts the growth promoting effect in transient expression of ERCC6L in COS-7 cells. Assays were performed three times and in triplicate wells.
  • an isolated or purified antibody refers to an antibody that is substantially free of cellular material such as carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • heterologous protein also referred to herein as a "contaminating protein”
  • the polypeptide is recombinantly produced, it is also preferably substantially free of culture medium, which includes preparations of polypeptide with culture medium less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • the polypeptide When the polypeptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, which includes preparations of polypeptide with chemical precursors or other chemicals involved in the synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation. That a particular protein preparation contains an isolated or purified polypeptide can be shown, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining or the like of the gel.
  • SDS sodium dodecyl sulfate
  • antibodies of the present invention are isolated or purified.
  • An “isolated” or “purified” nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • polypeptide polypeptide
  • peptide protein
  • protein polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly functions to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine).
  • amino acid analog refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium).
  • modified R group or modified backbones e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium.
  • amino acid mimetic refers to chemical compounds that have different structures but similar functions to general amino acids. Amino acids may be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • nucleic acid and nucleic acid molecule
  • gene refers to any genetic material.
  • polynucleotide refers to any polynucleotide.
  • oligonucleotide refers to any polynucleotide.
  • nucleic acid refers to any combination thereof.
  • nucleic acid molecule may be composed of DNA, RNA or a combination thereof.
  • cancer refers to cancer over-expressing the ERCC6L gene.
  • examples of cancers over-expressing ERCC6L include, but are not limited to, lung cancers including SCLC and NSCLC.
  • NSCLC includes, but are not limited to, adenocarcinoma (ADC) and squamous-cell carcinoma (SCC).
  • ADC adenocarcinoma
  • SCC squamous-cell carcinoma
  • double-stranded molecule refers to a nucleic acid molecule that inhibits expression of a target gene, including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)).
  • siRNA short interfering RNA
  • dsRNA double-stranded ribonucleic acid
  • shRNA small hairpin RNA
  • siD/R-NA short interfering DNA/RNA
  • double-stranded molecule is also referred to as “double-stranded nucleic acid”, “double-stranded nucleic acid molecule”, “double-stranded polynucleotide” and “double-stranded polynucleotide molecule”.
  • the ERCC6L Gene and ERCC6L Protein The present invention is based in part on the discovery that the gene encoding ERCC6L is over-expressed in cancer as compared to non-cancerous tissue.
  • Nucleotide sequences of ERCC6L polynucleotide and amino acid sequences of ERCC6L polypeptide are known to those skilled in the art, and obtained, for example, from gene databases on the web site such as GenBank TM .
  • An exemplified nucleotide sequence of ERCC6L polynucleotide is shown in SEQ ID NO: 9
  • an exemplified amino acid sequence of ERCC6L polypeptide is shown in SEQ ID NO: 10.
  • sequence data are also available, for example, via GenBank accession No. BC11486 or No. NM_017669.
  • ERCC6L sequences need not be limited to these sequences and that variants (e.g., functional equivalents and allelic variants) can be used in the present invention as described below.
  • ERCC6L polypeptides are also considered to be "ERCC6L polypeptides".
  • a “functional equivalent” of a protein e.g., an ERCC6L polypeptide
  • a polypeptide that has a biological activity equivalent to the protein e.g., any polypeptide that retains the biological ability of the ERCC6L protein may be used as such a functional equivalent in the present invention.
  • Such functional equivalents include those wherein one or more amino acids are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the ERCC6L protein.
  • the polypeptide may be composed an amino acid sequence having at least about 80% homology (also referred to as sequence identity) to the sequence of the respective protein, more preferably at least about 90% to 95% homology, often about 96%, 97%, 98% or 99% homology.
  • the polypeptide can be encoded by a polynucleotide that hybridizes under stringent conditions to the natural occurring nucleotide sequence of the ERCC6L gene.
  • a polypeptide of the present invention may have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has a function equivalent to that of the human ERCC6L protein of the present invention, it is within the scope of the present invention.
  • stringent (hybridization) conditions refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10 degrees C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm thermal melting point
  • the Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times of background, preferably 10 times of background hybridization.
  • Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 degrees C, or, 5x SSC, 1% SDS, incubating at 65 degrees C, with wash in 0.2x SSC, and 0.1% SDS at 50 degrees C.
  • a condition of hybridization for isolating a DNA encoding a polypeptide functionally equivalent to the human ERCC6L protein can be routinely selected by a person skilled in the art.
  • hybridization may be performed by conducting pre-hybridization at 68 degrees C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 degrees C for 1 hour or longer.
  • the following washing step can be conducted, for example, in a low stringent condition.
  • An exemplary low stringent condition may include 42 degrees C, 2x SSC, 0.1% SDS, preferably 50 degrees C, 2x SSC, 0.1% SDS.
  • High stringency conditions are often preferably used.
  • An exemplary high stringency condition may include washing 3 times in 2x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37 degrees C for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50 degrees C for 20 min.
  • factors such as temperature and salt concentration, can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.
  • modifications of one, two or more amino acids in a protein will not influence the function of the protein.
  • mutated or modified proteins i.e., peptides composed of an amino acid sequence in which one, two, or several amino acid residues have been modified through substitution, deletion, insertion and/or addition
  • mutated or modified proteins have been known to retain the original biological activity (Mark et al., Proc Natl Acad Sci USA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10:6487-500 (1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13 (1982)).
  • the peptides of the present invention may have an amino acid sequence wherein one, two or even more amino acids are added, inserted, deleted, and/or substituted in the ERCC6L sequence.
  • the number of amino acid mutations is not particularly limited. However, it is generally preferred to alter 5% or less of the amino acid sequence. Accordingly, in a preferred embodiment, the number of amino acids to be mutated in such a mutant is generally 30 amino acids or fewer, preferably 20 amino acids or fewer, more preferably 10 amino acids or fewer, more preferably 5 or 6 amino acids or fewer, and even more preferably 3 or 4 amino acids or fewer.
  • An amino acid residue to be mutated is preferably mutated into a different amino acid in which the properties of the amino acid side-chain are conserved (a process known as conservative amino acid substitution).
  • properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W).
  • A, I, L, M, F, P, W, Y, V hydrophilic
  • Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Aspargine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cystein (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).
  • Such conservatively modified polypeptides are included in the present ERCC6L protein.
  • the present invention is not restricted thereto and the ERCC6L protein includes non-conservative modifications, so long as at least one biological activity of the ERCC6L protein is retained.
  • the modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.
  • the ERCC6L gene of the present invention encompasses polynucleotides that encode such functional equivalents of the ERCC6L protein.
  • a gene amplification method for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a polynucleotide encoding a polypeptide functionally equivalent to the ERCC6L protein, using a primer synthesized based on the sequence information of the protein encoding DNA (SEQ ID NO: 9).
  • PCR polymerase chain reaction
  • High homology typically refers to a homology of 40% or higher, preferably 60% or higher, more preferably 80% or higher, even more preferably 90% to 95% or higher, even more preferably 96%, 97%, 98%, 99% or higher.
  • the homology of a particular polynucleotide or polypeptide can be determined by following the algorithm in "Wilbur and Lipman, Proc Natl Acad Sci USA 80: 726-30 (1983)".
  • ERCC6L gene was found to be specifically elevated in lung cancer (Fig. 1A, B). Northern-blot analysis showed that ERCC6L was not expressed in normal tissues, but strongly expressed in lung cancer cell lines (Fig. 1C). Accordingly, the ERCC6L genes identified herein as well as their transcription and translation products find diagnostic utility as a marker for cancers such as lung cancer, and by measuring the expression of ERCC6L in a sample. Cancer can be diagnosed or detected by comparing the expression level of ERCC6L gene between a subject-derived sample with a normal sample. More particularly, the present invention provides a method for detecting, diagnosing and/or determining the presence of or a predisposition for developing cancer, more particularly ERCC6L-associated cancer, by determining the expression level of ERCC6L in the subject.
  • the present invention provides a method for detecting or diagnosing cancer in a subject, such method including the step of determining an expression level of an ERCC6L gene in a subject-derived biological sample, wherein an increase of the level as compared to a normal control level of the gene indicates the presence or suspicion of cancer cells in the sample, which, in turn, suggests that the subject suffers from or is at risk of developing cancer.
  • the expression level of the ERCC6L gene may be determined by any known method, examples of which include: (a) detecting the mRNA of an ERCC6L gene; (b) detecting the protein encoded by an ERCC6L gene; and (c) detecting the biological activity of the protein encoded by an ERCC6L gene.
  • cancers to be diagnosed by the present method are lung cancers, including NSCLCs and SCLCs.
  • NSCLCs include lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SCC).
  • ADC lung adenocarcinoma
  • SCC lung squamous cell carcinoma
  • an intermediate result for examining the condition of a subject may be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose that a subject suffers from the disease.
  • the present invention contemplates the use of ERCC6L as a diagnostic marker for cancer.
  • the present invention may be used to detect or identify cancerous cells in a subject-derived tissue, such cells being characterized by an increase in said expression level as compared to a normal control level of said gene indicates the presence or suspicion of cancer cells in the tissue.
  • ERCC6L expression results may be combined with additional information to assist a doctor, nurse, or other healthcare practitioner in diagnosing a subject as afflicted with the disease.
  • the present invention may provide a doctor with useful information to diagnose a subject as afflicted with the disease.
  • the outcome of the gene expression analysis serves as an intermediate result for further diagnosis of a subject's disease state.
  • [1] A method of detecting or diagnosing cancer in a subject, including determining an expression level of ERCC6L gene in a subject derived biological sample, wherein an increase of said level compared to a normal control level of said gene indicates that said subject suffers from or is at risk of developing cancer.
  • [2] The method of [1], wherein the expression level is at least 10% greater than the normal control level.
  • the method of [1], wherein the cancer is lung cancer.
  • the expression level is determined by detecting hybridization of a probe to a gene transcript of the gene.
  • a subject to be diagnosed by the present method is preferably a mammal.
  • exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • a biological sample from the subject to be diagnosed to perform the diagnosis.
  • Any biological material can be used as the biological sample for the determination so long as it includes the objective transcription or translation product of ERCC6L.
  • the biological samples include, but are not limited to, bodily tissues which are desired for diagnosing or are suspicion of suffering from cancer, and fluids, such as biopsy sample, blood, sputum and urine.
  • the biological sample contains a cell population including an epithelial cell, more preferably a cancerous epithelial cell or an epithelial cell derived from tissue suspected to be cancerous. Further, if necessary, the cell may be purified from the obtained bodily tissues or fluids, and then used as the biological sample.
  • a biological sample may include lung cells or a lung tissue obtained from a subject to be diagnosed.
  • the expression level of ERCC6L in the subject-derived biological sample is determined.
  • the expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art.
  • the mRNA of ERCC6L may be quantified using probes by hybridization methods (e.g., Northern hybridization).
  • the detection may be carried out on a chip or an array.
  • the use of an array is preferable for detecting the expression level of a plurality of genes (e.g., various cancer specific genes) including ERCC6L.
  • Those skilled in the art can prepare such probes utilizing the sequence information of ERCC6L.
  • the cDNA of ERCC6L may be used as the probes.
  • the probe may be labeled with a suitable label, such as dyes, fluorescent and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.
  • the transcription product of ERCC6L may be quantified using primers by amplification-based detection methods (e.g., RT-PCR).
  • primers can also be prepared based on the available sequence information of the gene.
  • the primers used in the Example (SEQ ID NO 1 and 2) may be employed for the detection by RT-PCR or Northern blot, but the present invention is not restricted thereto.
  • a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of ERCC6L.
  • stringent (hybridization) conditions refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degree Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degree Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degree Centigrade for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • the translation product may be detected for the diagnosis of the present invention.
  • the quantity of ERCC6L protein may be determined.
  • a method for determining the quantity of the protein as the translation product includes immunoassay methods that use an antibody specifically recognizing the protein.
  • the antibody may be monoclonal or polyclonal.
  • any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used for the detection, so long as the fragment retains the binding ability to ERCC6L protein.
  • Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the intensity of staining may be observed via immunohistochemical analysis using an antibody against ERCC6L protein. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of ERCC6L gene.
  • the expression level of other cancer-associated genes may also be determined to improve the accuracy of the diagnosis.
  • the expression level of cancer marker gene including ERCC6L gene in a biological sample can be considered to be increased if it increases from the control level of the corresponding cancer marker gene by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
  • the control level may be determined at the same time with the test biological sample by using a sample(s) previously collected and stored from a subject/subjects whose disease state (cancerous or non-cancerous) is/are known.
  • the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of ERCC6L gene in samples from subjects whose disease state are known.
  • the control level can be a database of expression patterns from previously tested cells.
  • the expression level of ERCC6L gene in a biological sample may be compared to multiple control levels, which control levels are determined from multiple reference samples.
  • control level determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample It is preferred, to use the standard value of the expression levels of ERCC6L gene in a population with a known disease state.
  • the standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as standard value.
  • a control level determined from a biological sample that is known to be non-cancerous is referred to as a "normal control level”.
  • control level is determined from a cancerous biological sample, it is referred to as a "cancerous control level”.
  • the subject When the expression level of ERCC6L gene is increased as compared to the normal control level or is similar to the cancerous control level, the subject may be diagnosed to be suffering from or at a risk of developing cancer. Furthermore, in the case where the expression levels of multiple cancer-related genes are compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing cancer.
  • control nucleic acids e.g., housekeeping genes, whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell.
  • control genes include, but are not limited to, beta-actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.
  • ERCC6L is not only a useful diagnostic marker, but is also suitable target for cancer therapy. Therefore, cancer treatment targeting ERCC6L can be achieved by the present invention.
  • the cancer treatment targeting ERCC6L refers to suppression or inhibition of ERCC6L activity and/or expression in the cancer cells.
  • Any anti-ERCC6L agents may be used for the cancer treatment targeting ERCC6L.
  • the anti-ERCC6L agents include following substance or active ingredient: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof.
  • the present invention provides a method of (i) diagnosing whether a subject has the cancer to be treated with anti- ERCC6L agent, and/or (ii) selecting a subject for cancer treatment targeting ERCC6L, which method includes the steps of: a) determining the expression level of ERCC6L in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated; b) comparing the expression level of ERCC6L with a normal control level; c) diagnosing the subject as having the cancer to be treated, if the expression level of ERCC6L is increased as compared to the normal control level; and d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
  • such a method includes the steps of: a) determining the expression level of ERCC6L in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated; b) comparing the expression level of ERCC6L with a cancerous control level; c) diagnosing the subject as having the cancer to be treated, if the expression level of ERCC6L is similar or equivalent to the cancerous control level; and d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
  • kits for Diagnosing Cancer The present invention also provides a kit for diagnosing cancer, which may also be useful in assessing and/or monitoring the efficacy of a cancer therapy.
  • the present invention also provides a kit for determining a subject suffering from cancer that can be treated with the double-stranded molecule of the present invention or vector encoding thereof, which may also be useful in assessing and/or monitoring the efficacy of such cancer treatment.
  • the cancer to be diagnosed by the present kit is lung cancer, including NSCLC and SCLC.
  • the kit includes at least one reagent for detecting the expression level of the ERCC6L gene in a subject-derived biological sample, which reagent may be selected from the group consisting of: (a) a reagent for detecting mRNA of the ERCC6L gene; (b) a reagent for detecting the ERCC6L protein; and (c) a reagent for detecting the biological activity of the ERCC6L protein.
  • Suitable reagents for detecting mRNA of the ERCC6L gene include nucleic acids that specifically bind to or identify the ERCC6L mRNA, such as oligonucleotides which have a complementary sequence to a part of the ERCC6L mRNA. These kinds of oligonucleotides are exemplified by primers and probes that are specific to the ERCC6L mRNA. These kinds of oligonucleotides may be prepared based on methods well known in the art. If needed, the reagent for detecting the ERCC6L mRNA may be immobilized on a solid matrix. Moreover, more than one reagent for detecting the ERCC6L mRNA may be included in the kit.
  • suitable reagents for detecting the ERCC6L protein include antibodies to the ERCC6L protein.
  • the antibody may be monoclonal or polyclonal.
  • any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used as the reagent, so long as the fragment retains the binding ability to the ERCC6L protein.
  • Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the antibody may be labeled with signal generating molecules via direct linkage or an indirect labeling technique. Labels and methods for labeling antibodies and detecting the binding of antibodies to their targets are well known in the art and any labels and methods may be employed for the present invention.
  • more than one reagent for detecting the ERCC6L protein may be included in the kit.
  • the biological activity can be determined by, for example, measuring the cell proliferating activity due to the expressed ERCC6L protein in the biological sample.
  • the cell is cultured in the presence of a subject-derived biological sample, and then by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability the cell proliferating activity of the biological sample can be determined.
  • the reagent for detecting the ERCC6L mRNA may be immobilized on a solid matrix.
  • more than one reagent for detecting the biological activity of the ERCC6L protein may be included in the kit.
  • the kit may contain more than one of the aforementioned reagents.
  • the kit may include a solid matrix and reagent for binding a probe against the ERCC6L gene or antibody against the ERCC6L protein, a medium and container for culturing cells, positive and negative control reagents, and a secondary antibody for detecting an antibody against the ERCC6L protein.
  • tissue samples obtained from subject suffering from cancer or not may serve as useful control reagents.
  • a kit of the present invention may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts (e.g., written, tape, CD-ROM, URL etc.) with instructions for use.
  • These reagents and such may be provided in a container with a label.
  • Suitable containers include bottles, vials, and test tubes.
  • the containers may be formed from a variety of materials, such as glass or plastic.
  • the reagent when the reagent is a probe against the ERCC6L mRNA, the reagent may be immobilized on a solid matrix, such as a porous strip, to form at least one detection site.
  • the measurement or detection region of the porous strip may include a plurality of sites, each containing a nucleic acid (probe).
  • a test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a strip separated from the test strip.
  • the different detection sites may contain different amounts of immobilized nucleic acids, i.e., a higher amount in the first detection site and lesser amounts in subsequent sites.
  • the number of sites displaying a detectable signal provides a quantitative indication of the amount of ERCC6L mRNA present in the sample.
  • the detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
  • the kit of the present invention may further include positive and/or negative controls sample, and/or an ERCC6L standard sample.
  • the positive control sample of the present invention may be prepared by collecting ERCC6L positive samples.
  • ERCC6L positive samples may be obtained, for example, from established lung cancer cell lines, including lung adenocarcinoma cell (ADC) lines such as A427, NCI-H1781, A549, LC319 and the like; lung squamous cell carcinoma (SCC) cell lines such as NCI-H26, EBC-1, NCI-H520, NCI-H2170 and the like; and SCLC cell lines such as DMS114, DMS273, SBC-3, SBC-5, H196, H446 and the like.
  • ADC lung adenocarcinoma cell
  • SCC lung squamous cell carcinoma
  • the ERCC6L positive samples may be obtained from clinical lung cancer tissues, including lung adenocarcinoma tissues, lung squamous cell carcinoma tissues and SCLC tissues.
  • positive control samples may be prepared by determined a cut-off value and preparing a sample containing an amount of an ERCC6L mRNA or protein more than the cut-off value.
  • the phrase "cut-off value" refers to the value dividing between a normal range and a cancerous range.
  • ROC receiver operating characteristic
  • the present kit may include an ERCC6L standard sample containing a cut-off value amount of an ERCC6L mRNA or polypeptide.
  • negative control samples may be prepared from non-cancerous cell lines or non-cancerous tissues such as normal lung tissues, or may be prepared by preparing a sample containing an ERCC6L mRNA or protein less than cut-off value.
  • the present invention provides use of a reagent for preparing a diagnostic reagent for diagnosing cancer.
  • the reagent can be selected from the group consisting of: (a) a reagent for detecting mRNA of the ERCC6L gene; (b) a reagent for detecting the ERCC6L protein; and (c) a reagent for detecting the biological activity of the ERCC6L protein.
  • such reagent is an oligonucleotide that hybridizes to the ERCC6L polynucleotide, or an antibody that binds to the ERCC6L polypeptide.
  • ERCC6L is involved in cancer cell growth. Accordingly, substances that suppress an expression level of ERCC6L gene and/or a biological activity of ERCC6L polypeptide are expected to be useful for treating and/or preventing cancer. Such substances can be screened using an ERCC6L gene, polypeptides encoded by the gene, or transcriptional regulatory region of the gene. Thus, the present invention also provides a method of screening for a candidate substance for treating and/or preventing cancer using ERCC6L gene, ERCC6L polypeptide, or transcriptional regulatory region of the gene.
  • substances to be identified through the present screening methods may be any compound or composition including several compounds.
  • the test substance exposed to a cell or protein according to the screening methods of the present invention may be a single substance or a combination of substances.
  • the substances may be contacted sequentially or simultaneously.
  • the substances screened by the present screening method may be suitable candidate substances for treating and/or preventing cancer, and/or inhibiting cancer cell growth.
  • the cancer is preferably characterized by an association with ERCC6L overexpression.
  • the screened substances may be preferably applied to the cancers correlated or associated with ERCC6L overexpression.
  • the cancers correlated or associated with ERCC6L overexpression are lung cancer, including NSCLCs and SCLCs.
  • NSCLCs include, but are not limited to, lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SCC).
  • test substance for example, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds (including nucleic acid constructs, such as antisense RNA, siRNA, Ribozymes, and aptamer etc.) and natural compounds can be used in the screening methods of the present invention.
  • test substance for example, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds (including nucleic acid constructs, such as antisense RNA, siRNA, Ribozymes, and aptamer etc.) and natural compounds can be used in the screening methods of the present invention.
  • test substance of the present invention can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including (1) biological libraries, (2) spatially addressable parallel solid phase or solution phase libraries, (3) synthetic library methods requiring deconvolution, (4) the "one-bead one-compound” library method and (5) synthetic library methods using affinity chromatography selection.
  • biological libraries using affinity chromatography selection is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des 1997, 12: 145-67).
  • a candidate substance obtained by the present screening method is a proteins
  • for obtaining a DNA encoding the protein either the whole amino acid sequence of the protein may be determined to deduce the nucleic acid sequence coding for the protein, or partial amino acid sequence of the obtained protein may be analyzed to prepare an oligo DNA as a probe based on the sequence, and screen cDNA libraries with the probe to obtain a DNA encoding the protein.
  • the obtained DNA may be confirmed it's usefulness in preparing the candidate substance for treating or preventing cancer.
  • Test substances used in the screenings described herein may also be antibodies that specifically bind to an ERCC6L protein or partial peptides thereof that lack the biological activity of the original proteins in vivo.
  • test substance libraries are well known in the art, herein below, additional guidance in identifying test substances and construction libraries of such substances for the present screening methods are provided.
  • One approach to preliminary screening of test substances suitable for further evaluation utilizes computer modeling of the interaction between the test substance and its target.
  • Computer modeling technology allows for the visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new substances that will interact with the molecule.
  • the three-dimensional construct typically depends on data from x-ray crystallographic analysis or NMR imaging of the selected molecule.
  • the molecular dynamics require force field data.
  • the computer graphics systems enable prediction of how a new substance will link to the target molecule and allow experimental manipulation of the structures of the substance and target molecule to perfect binding specificity. Prediction of what the molecule-substance interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user
  • CHARMm performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
  • test substances may be screened using the methods of the present invention to identify test substances suited to the treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence of cancer, particularly wherein the cancer is lung cancer.
  • Combinatorial chemical synthesis Combinatorial libraries of test substances may be produced as part of a rational drug design program involving knowledge of core structures existing in known inhibitors. This approach allows the library to be maintained at a reasonable size, facilitating high throughput screening.
  • simple, particularly short, polymeric molecular libraries may be constructed by simply synthesizing all permutations of the molecular family making up the library.
  • An example of this latter approach would be a library of all peptides six amino acids in length. Such a peptide library could include every 6 amino acid sequence permutation. This type of library is termed a linear combinatorial chemical library.
  • Combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6).
  • peptide libraries see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptides (e.g., PCT Publication No.
  • WO 91/19735 encoded peptides (e.g., WO 93/20242), random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g., US Patent 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90:6909-13), vinylogous polypeptides (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J Amer Chem Soc 1992, 114: 9217-8), analogous organic syntheses of small compound libraries (Chen et al., J.
  • a second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 1986, 23: 709-15; Geysen et al., J Immunologic Method 1987, 102: 259-74); and the method of Fodor et al. (Science 1991, 251: 767-73) are examples.
  • Furka et al. 14th International Congress of Biochemistry 1988, Volume #5, Abstract FR:013; Furka, Int J Peptide Protein Res 1991, 37: 487-93
  • Houghten US Patent 4,631,211
  • Rutter et al. US Patent 5,010,175) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.
  • Aptamers are macromolecules composed of nucleic acid that bind tightly to a specific molecular target.
  • Tuerk and Gold discloses SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method for selection of aptamers.
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • a large library of nucleic acid molecules ⁇ e.g., 10 15 different molecules) can be used for screening.
  • the present invention provides methods of screening for a candidate substance applicable to the treatment and/or prevention of cancer using an ERCC6L polypeptide.
  • the ERCC6L polypeptide to be used may be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides.
  • the ERCC6L polypeptide may be a recombinant polypeptide, a protein derived from the nature or a partial peptide thereof.
  • ERCC6L polypeptides may be included in ERCC6L polypeptides used for the present screening so long as the modified peptide retains at least one biological activity of the original polypeptide.
  • Examles of the biological activity of the ERCC6L polypeptide include, but are not limited to, cell proliferative activity, helicase activity, ATPase activity and Plk1-binding activity.
  • Preferred examples of such functional equivalents are described above in the section entitled "The ERCC6L gene and ERCC6L protein".
  • a preferred example of such functional equivalents includes a polypeptide containing a helicase domain of ERCC6L polypeptide (e.g., 61 to 631 of SEQ ID NO: 10).
  • the polypeptides may be further linked to other substances, so long as the linking process and linked substance do not interfere with the biological activity of the original polypeptide and/or fragment.
  • Usable substances include, for example: peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc. These kinds of modifications may be performed to confer additional functions or to stabilize the polypeptide and fragments.
  • the polypeptides used for the present method may be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on a selected amino acid sequence. For example, conventional peptide synthesis methods that can be adopted for the synthesis include: 1) Peptide Synthesis, Interscience, New York, 1966; 2) The Proteins, Vol.
  • polypeptides may be obtained by adapting any known genetic engineering methods to the production of the instant polypeptides (e.g., Morrison J., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62).
  • a suitable vector including a polynucleotide encoding the objective protein in an expressible form e.g., downstream of a regulatory sequence including a promoter
  • the host cell is cultured to produce the protein.
  • a gene encoding an ERCC6L polypeptide are expressed in host (e.g., animal) cells and such by inserting the gene into a vector for expressing foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8.
  • a promoter may be used for the expression. Any commonly used promoters may be employed, including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3.
  • the EF-alpha promoter (Kim et al., Gene 1990, 91:217-23), the CAG promoter (Niwa et al., Gene 1991, 108:193), the RSV LTR promoter (Cullen, Methods in Enzymology 1987, 152:684-704), the SR-alpha promoter (Takebe et al., Mol Cell Biol 1988, 8:466), the CMV immediate early promoter (Seed et al., Proc Natl Acad Sci USA 1987, 84:3365-9), the SV40 late promoter (Gheysen et al., J Mol Appl Genet 1982, 1:385-94), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 1989, 9:946), the HSV TK promoter, and such.
  • the introduction of the vector into host cells to express an ERCC6L polypeptide may be performed according to any conventional methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 1987, 15:1311-26), the calcium phosphate method (Chen et al., Mol Cell Biol 1987, 7:2745-52), the DEAE dextran method (Lopata et al., Nucleic Acids Res 1984, 12:5707-17; Sussman et al., Mol Cell Biol 1985, 4:1641-3), the Lipofectin method (Derijard B, Cell 1994, 7:1025-37; Lamb et al., Nature Genetics 1993, 5:22-30; Rabindran et al., Science 1993, 259:230-4), and such.
  • ERCC6L polypeptides may also be produced in vitro using a conventional in vitro translation system.
  • ERCC6L binding Substance In the context of the present invention, an over-expression of ERCC6L gene was detected in lung cancer, in spite of no expression in normal organs (Fig. 1). Accordingly, using the ERCC6L gene and proteins encoded thereby, the present invention provides a method of screening for a substance that binds to ERCC6L polypeptide. Due to the expression of ERCC6L in cancer, a substance binds to ERCC6L polypeptide is expected to suppress the proliferation of cancer cells, and thus be useful for treating and/or preventing cancer.
  • the present invention also provides a method of screening for a candidate substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing cancer using the ERCC6L polypeptide.
  • an embodiment of this screening method includes the steps of: (a) contacting a test substance with an ERCC6L polypeptide ; (b) detecting the binding activity between the polypeptide and the test substance; and (c) selecting the test substance that binds to the polypeptide.
  • the potential therapeutic effect of a test substance for treating and/or preventing cancer can also be evaluated or estimated.
  • the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance for treating and/or preventing cancer and/or inhibiting cancer associated with over-expression of ERCC6L, the method including steps of: (a) contacting a test substance with a polypeptide encoded by a polynucleotide of ERCC6L; (b) detecting the binding activity between the polypeptide and the test substance; and (c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance binds to the polypeptide.
  • the therapeutic effect may be correlated with the binding level of the test substance and ERCC6L protein(s).
  • the test substance when the test substance binds to an ERCC6L protein, the test substance may identified or selected as a candidate substance having the requisite therapeutic effect.
  • the test substance when the test substance does not bind to an ERCC6L protein, the test substance may characterized as having no significant therapeutic effect. The method of the present invention will be described in more detail below.
  • the ERCC6L polypeptide to be used for screening may be a recombinant polypeptide or a protein derived from the nature or a partial peptide thereof.
  • the polypeptide to be contacted with a test substance may be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides.
  • test substances used by the present invention may be proteins such as antibodies or synthetic chemical compounds.
  • a method of screening substances that bind to an ERCC6L polypeptide many methods well known by a person skilled in the art may be used. Such a screening may be conducted by, for example, immunoprecipitation method.
  • an ERCC6L polypeptide contains an antibody recognition site.
  • ERCC6L polypeptides to be used for the present screening method may be prepared as described above.
  • the polypeptide encoded by ERCC6L gene can be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C- terminus of the polypeptide.
  • a commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (1995)).
  • Vectors which can express a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescence protein (GFP) and so on by the use of its multiple cloning sites are commercially available. Also, a fusion protein prepared by introducing only small epitopes consisting of several to a dozen amino acids so as not to change the property of the ERCC6L polypeptide by the fusion is also reported.
  • a fusion protein prepared by introducing only small epitopes consisting of several to a dozen amino acids so as not to change the property of the ERCC6L polypeptide by the fusion is also reported.
  • Epitopes such as polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such, and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the ERCC6L polypeptide (Experimental Medicine 13: 85-90 (1995)).
  • His-tag polyhistidine
  • influenza aggregate HA human c-myc
  • FLAG Vesicular stomatitis virus glycoprotein
  • VSV-GP Vesicular stomatitis virus glycoprotein
  • T7-tag T7 gene 10 protein
  • HSV-tag human simple herpes virus glycoprotein
  • E-tag an epitope on monoclonal phage
  • an immune complex is formed by adding these antibodies to cell lysate prepared using an appropriate detergent.
  • the immune complex consists of the ERCC6L polypeptide, a polypeptide including the binding ability with the polypeptide, and an antibody. Immunoprecipitation can be also conducted using antibodies against the ERCC6L polypeptide, besides using antibodies against the above epitopes, which antibodies can be prepared as described above.
  • An immune complex can be precipitated, for example by Protein A sepharose or Protein G sepharose when the antibody is a mouse IgG antibody.
  • an immune complex can be formed in the same manner as in the use of the antibody against the ERCC6L polypeptide, using a substance specifically binding to these epitopes, such as glutathione-Sepharose 4B.
  • Immunoprecipitation can be performed by following or according to, for example, the methods in the literature (Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)).
  • SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Since the protein bound to the ERCC6L polypeptide is difficult to detect by a common staining method, such as Coomassie staining or silver staining, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, 35 S-methionine or 35 S-cystein, labeling proteins in the cells, and detecting the proteins.
  • the target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.
  • a protein binding to the ERCC6L polypeptide can be obtained by preparing a cDNA library from cultured cells expected to express a protein binding to the ERCC6L polypeptide using a phage vector (e.g., ZAP), expressing the protein on LB-agarose, fixing the protein expressed on a filter, reacting the purified and labeled ERCC6L polypeptide with the above filter, and detecting the plaques expressing proteins bound to the ERCC6L polypeptide according to the label.
  • a phage vector e.g., ZAP
  • the polypeptide of the invention may be labeled by utilizing the binding between biotin and avidin, or by utilizing an antibody that specifically binds to the ERCC6L, or a peptide or polypeptide (for example, GST) that is fused to the ERCC6L polypeptide. Methods using radioisotope or fluorescence and such may be also used.
  • a two-hybrid system utilizing cells may be used ("MATCHMAKER Two-Hybrid system", “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene); the references “Dalton and Treisman, Cell 68: 597-612 (1992)", “Fields and Sternglanz, Trends Genet 10: 286-92 (1994)”).
  • the polypeptide of the invention is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells.
  • a cDNA library is prepared from cells expected to express a protein binding to the polypeptide of the invention, such that the library, when expressed, is fused to the VP16 or GAL4 transcriptional activation region.
  • the cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the polypeptide of the invention is expressed in yeast cells, the binding of the two activates a reporter gene, making positive clones detectable).
  • a protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E. coli and expressing the protein.
  • a reporter gene for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.
  • a substance binding to an ERCC6L polypeptide may also be screened using affinity chromatography.
  • an ERCC6L polypeptide may be immobilized on a carrier of an affinity column, and a composition containing test substances is applied to the column.
  • a composition herein may be, for example, cell extracts, cell lysates, antibody libraries etc. After loading test substances, the column is washed, and substances bound to the ERCC6L polypeptide can be collected.
  • the test substance is a protein
  • the amino acid sequence of the obtained protein is analyzed, an oligo DNA is synthesized based on the sequence, and cDNA libraries are screened using the oligo DNA as a probe to obtain a DNA encoding the protein.
  • a biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound substance in the present invention.
  • a biosensor When such a biosensor is used, the interaction between an ERCC6L polypeptide and a test substance can be observed real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between an ERCC6L polypeptide and a test substance using a biosensor such as BIAcore.
  • the ERCC6L protein is characterized as having the activity of promoting cell proliferation of cancer cells (Fig. 3). Using this biological activity as an index, the present invention provides a method for screening a substance that suppresses the proliferation of cancer cells expressing ERCC6L, and a method of screening for a substance for treating and/or preventing the cancer, particular ERCC6-L associated cancers such as lung cancer.
  • the present invention provides a method of screening for a candidate substance for treating and/or preventing cancer using the polypeptide encoded by ERCC6L gene including the steps as follows: (a) contacting a test substance with a polypeptide encoded by a polynucleotide of ERCC6L (ERCC6L polypeptide) or fragment thereof; (b) detecting the biological activity of the polypeptide or the fragment of step (a); and (c) selecting the test substance that suppresses the biological activity of the polypeptide encoded by the polynucleotide of ERCC6L (ERCC6L polypeptide) or the fragment as compared to the biological activity of said polypeptide detected in the absence of the test substance.
  • the therapeutic effect of the test substance in suppressing the biological activity (e.g., the cell-proliferating activity) of ERCC6L, or a candidate substance for treating and/or preventing cancer may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance that suppresses the biological activity of ERCC6L, or a candidate substance for treating and/or preventing cancer, using the ERCC6L polypeptide or fragments thereof, including the following steps: a) contacting a test substance with the ERCC6L polypeptide or a functional fragment thereof; and b) detecting the biological activity of the polypeptide or fragment of step (a), and c) correlating the biological activity of b) with the therapeutic effect of the test substance.
  • a method of screening for a candidate substance that suppresses the biological activity of ERCC6L, or a candidate substance for treating and/or preventing cancer using the ERCC6L polypeptide or fragments thereof, including the following steps: a) contacting a test substance
  • the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance in the treatment and/or prevention of cancer and/or in the inhibition of the growth of a cancer associated with over-expression of ERCC6L, the method including steps of: (a) contacting a test substance with the ERCC6L polypeptide or a functional fragment thereof; (b) detecting the biological activity of the polypeptide or fragment of step (a); and (c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance suppresses the biological activity of the polypeptide encoded by the polynucleotide of ERCC6L gene as compared to the biological activity of said polypeptide detected in the absence of the test substance.
  • the therapeutic effect may be correlated with the biological activity of the ERCC6L polypeptide or a functional fragment thereof.
  • the test substance when the test substance suppresses or inhibits the biological activity of the ERCC6L polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect.
  • the test substance when the test substance does not suppress or inhibit the biological activity of the ERCC6L polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
  • the method of the present invention will be described in more detail below.
  • Any polypeptides can be used for screening so long as they suppress a biological activity of the ERCC6L protein.
  • biological activity includes cell-proliferating activity (herein, also referred to as “cell proliferative activity”, “cell proliferation enhancing activity” or “cell proliferation promoting activity”), helicase activity, ATPase activity and Plk1-binding activity of the ERCC6L protein.
  • cell proliferative activity cell proliferation enhancing activity
  • cell proliferation promoting activity cell proliferation promoting activity
  • helicase activity helicase activity
  • ATPase activity ATPase activity
  • Plk1-binding activity of the ERCC6L protein Plk1-binding activity of the ERCC6L protein.
  • ERCC6L protein can be used and polypeptides functionally equivalent to these proteins can also be used.
  • Such polypeptides may be expressed endogenously or exogenously by cells.
  • the present invention also provides a screening method following the method described in the above "Screening" section, such method including the steps of: a) contacting a test substance with the ERCC6L polypeptide or a fragment thereof; b) detecting the binding between the polypeptide or fragment and the test substance; c) selecting the test substance that binds to the polypeptide; d) contacting the test substance selected in step c) with the ERCC6L polypeptide or a fragment thereof; e) comparing the biological activity of the polypeptide or fragment with the biological activity detected in the absence of the substance; and f) selecting the substance that suppresses the biological activity of the polypeptide as a candidate substance for treating or preventing lung cancer.
  • the substance isolated by this screening is a candidate for antagonists of the polypeptide encoded by ERCC6L gene.
  • antagonist refers to molecules that inhibit the function of the polypeptide by binding thereto. This term also refers to molecules that reduce or inhibit expression of the gene encoding ERCC6L.
  • a substance isolated by this screening is a candidate for substances which inhibit the in vivo interaction of the ERCC6L polypeptide with molecules (including DNAs and proteins).
  • the biological activity to be detected in the present method is cell proliferation
  • it can be detected, for example, by preparing cells which express the ERCC6L polypeptide, culturing the cells in the presence of a test substance, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by measuring survival cells or the colony forming activity, for example, shown in Fig. 3.
  • the substances that reduce the speed of proliferation of the cells expressed ERCC6L are selected as candidate substance for treating or preventing cancer.
  • the method includes the step of: (a) contacting a test substance with cells overexpressing ERCC6L; (b) measuring cell-proliferating activity; and (c) selecting the test substance that reduces the cell-proliferating activity in the comparison with the cell-proliferating activity in the absence of the test substance.
  • the method of the present invention may further include the steps of: (d) selecting the test substance that have no effect to the cells no or little expressing ERCC6L.
  • suppress the biological activity are preferably at least 10% suppression of the biological activity of ERCC6L in comparison with in absence of the substance, more preferably at least 25%, 50% or 75% suppression and most preferably at 90% suppression.
  • control cells that do not express ERCC6L polypeptide are used.
  • the present invention also provides a method of screening for a candidate substance that inhibits cell growth or a candidate substance for treating and/or preventing an ERCC6L- associated disease, using the ERCC6L polypeptide or fragments thereof including the steps as follows: a) culturing cells which express an ERCC6L polypeptide or a functional fragment thereof, and control cells that do not express an ERCC6L polypeptide or a functional fragment thereof in the presence of the test substance; b) detecting the biological activity of the cells which express the protein and control cells; and c) selecting the test substance that inhibits the biological activity in the cells which express the protein as compared to the proliferation detected in the control cells and in the absence of said test substance.
  • suppressing the biological activity of ERCC6L reduces cell growth.
  • candidate substance that inhibits the biological activity of ERCC6L candidate substance that have the potential to treat and/or prevent cancers can be identified.
  • the potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic substance, compoundes or agent for cancers. For example, when a substance that inhibits the biological activity of an ERCC6L protein also inhibits the activity of a cancer, it may be concluded that such a substance has an ERCC6L specific therapeutic effect.
  • the present invention provides a method of screening for a substance that inhibits the expression of ERCC6L.
  • a substance that inhibits the expression of ERCC6L is expected to suppress the proliferation of cancer cells, and thus is useful for treating or preventing cancer, particularly ERCC6L-associated cancers such as lung cancer. Therefore, the present invention also provides a method for screening a substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing cancer.
  • such screening may include, for example, the following steps: (a) contacting a test substance with a cell expressing ERCC6L; and (b) selecting the test substance that reduces the expression level of ERCC6L as compared to a control.
  • such screening may include, for example, the following steps: a) contacting a test substance with a cell expressing the ERCC6L gene; b) detecting the expression level of the ERCC6L gene; and c) correlating the expression level of b) with the therapeutic effect of the test substance.
  • the therapeutic effect may be correlated with the expression level of the ERCC6L gene.
  • the test substance when the test substance reduces the expression level of the ERCC6L gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect.
  • the test substance when the test substance does not reduce the expression level of the ERCC6L gene as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
  • the method of the present invention will be described in more detail below.
  • Cells expressing the ERCC6L include, for example, cell lines established from lung cancer or cell lines transfected with ERCC6L expression vectors; any of such cells can be used for the above screening of the present invention.
  • the expression level can be estimated by methods well known to one skilled in the art, for example, RT-PCR, Northern blot assay, Western blot assay, immunostaining and flow cytometry analysis.
  • the phrase "reduce the expression level" as defined herein are preferably at least 10% reduction of expression level of ERCC6L in comparison to the expression level in absence of the substance, more preferably at least 25%, 50% or 75% reduced level and most preferably at least 95% reduced level.
  • the substance herein includes chemical compounds, double-strand nucleotides, and so on. The preparation of the double-strand nucleotides will be described bellow.
  • a substance that reduces the expression level of ERCC6L can be selected as candidate substances to be used for the treatment or prevention of cancer.
  • the screening method of the present invention may include the following steps: (a) contacting a test substance with a cell into which a vector, including the transcriptional regulatory region of ERCC6L and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; (b) measuring the expression or activity of said reporter gene; and (c) selecting the test substance that reduces the expression or activity of said reporter gene.
  • reporter genes and host cells are well known in the art.
  • Illustrative reporter genes include, but are not limited to, luciferase, green fluorescence protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed), Chrolamphenicol Acetyltransferase (CAT), lacZ and beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa and so on.
  • the reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of ERCC6L gene.
  • the transcriptional regulatory region of ERCC6L gene herein is the region from transcription stat site to at least 500bp upstream, preferably 1000bp, more preferably 5000 or 10000bp upstream.
  • a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR.
  • the reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of the gene. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).
  • the vector containing the said reporter construct is introduced into host cells and the expression or activity of the reporter gene is detected by methods well known in the art (e.g., using luminometer, absorption spectrometer, flow cytometer and so on).
  • "Reduces the expression or activity” as defined herein are preferably at least 10% reduction of the expression or activity of the reporter gene in comparison with in absence of the test substance, more preferably at least 25%, 50% or 75% reduction and most preferably at least 95% reduction.
  • the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of ERCC6L, the method including steps of: (a) contacting a test substance with a cell into which a vector, including the transcriptional regulatory region of ERCC6L and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; (b) measuring the expression level or activity of said reporter gene; and (c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance reduces the expression level or activity of said reporter gene.
  • the therapeutic effect of the test substance on inhibiting the cell growth or a candidate substance for treating or preventing ERCC6L associating disease may be evaluated. Therefore, the present invention also provides a method for screening a candidate substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing ERCC6L associating disease.
  • the present invention provides a method which includes the following steps of: (a) contacting a test substance with a cell into which a vector, composed of the transcriptional regulatory region of the ERCC6L gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; (b) detecting the expression level or activity of said reporter gene; and (c) correlating the expression level of (b) with the therapeutic effect of the test substance.
  • the therapeutic effect may be correlated with the expression level or activity of said reporter gene.
  • the test substance when the test substance reduces the expression level or activity of said reporter gene as compared to a level detected in the absence of the test substance, the test substance may be identified or selected as the candidate substance having the therapeutic effect.
  • the test substance when the test substance does not reduce the expression level or activity of said reporter gene as compared to a level detected in the absence of the test substance, the test substance may be identified as the substance having no significant therapeutic effect.
  • candidate substances that have the potential to treat or prevent cancers e.g., lung cancer, ) can be identified.
  • the therapeutic potential of these candidate substances may be evaluated by second and/or further screening to identify therapeutic substance for cancers. For example, when a substance that binds to the ERCC6L polypeptide inhibits the above-described activities of cancer, it may be concluded that such a substance has the ERCC6L-specific therapeutic effect.
  • the term "isolated double-stranded molecule” refers to a nucleic acid molecule that inhibits expression of a target gene and includes, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g. double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)).
  • siRNA short interfering RNA
  • dsRNA double-stranded ribonucleic acid
  • shRNA small hairpin RNA
  • siD/R-NA short interfering DNA/RNA
  • a target sequence is a nucleotide sequence within mRNA or cDNA sequence of a gene, which will result in suppress of translation of the whole mRNA if a double-stranded nucleic acid molecule of the invention was introduced within a cell expressing the gene.
  • a nucleotide sequence within mRNA or cDNA sequence of a gene can be determined to be a target sequence when a double-stranded polynucleotide including a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene.
  • the double stranded polynucleotide by which suppresses the gene expression may consists of the target sequence and 3'overhang having 2 to 5 nucleotides in length (e.g., uu).
  • a sense strand sequence of a double-stranded cDNA i.e., a sequence that mRNA sequence is converted into DNA sequence
  • a double-stranded molecule is composed of a sense strand that has a sequence corresponding to a target sequence and an antisense strand that has a complementary sequence to the target sequence, and the antisense strand hybridizes with the sense strand at the complementary sequence to form a double-stranded molecule.
  • the phrase "corresponding to" means converting a target sequence according to the kind of nucleic acid that constitutes a sense strand of a double-stranded molecule.
  • the target sequences are mainly shown in DNA.
  • the present invention also provides a double-stranded molecule whose target sequence includes or is limited to SEQ ID NO: 7 or SEQ ID NO: 8 which is shown in DNA but can be replaced with RNA..
  • a complementary sequence to a target sequence for an antisense strand of a double-stranded molecule can be defined according to the kind of nucleic acid that constitutes the antisense strand.
  • a double-stranded molecule may have one or two 3'overhangs having 2 to 5 nucleotides in length (e.g., uu) and/or a loop sequence that links a sense strand and an antisense strand to form hairpin structure, in addition to a sequence corresponding to a target sequence and complementary sequence thereto.
  • siRNA refers to a double-stranded RNA molecule that prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed.
  • the siRNA includes an ERCC6L sense nucleic acid sequence (also referred to as “sense strand”), an ERCC6L antisense nucleic acid sequence (also referred to as “antisense strand”) or both.
  • the siRNA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, e.g., a hairpin.
  • the siRNA may either be a dsRNA or shRNA.
  • dsRNA refers to a construct of two RNA molecules composed of complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded RNA molecule.
  • the nucleotide sequence of two strands may include not only the "sense” or "antisense” RNAs selected from a protein coding sequence of target gene sequence, but also RNA molecule having a nucleotide sequence selected from non-coding region of the target gene.
  • shRNA refers to an siRNA having a stem-loop structure, composed of first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions are sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
  • siD/R-NA refers to a double-stranded polynucleotide molecule which is composed of both RNA and DNA, and includes hybrids and chimeras of RNA and DNA and prevents translation of a target mRNA.
  • a hybrid indicates a molecule wherein a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize to each other to form the double-stranded molecule; whereas a chimera indicates that one or both of the strands composing the double stranded molecule may contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used.
  • the siD/R-NA includes an ERCC6L sense nucleic acid sequence (also referred to as “sense strand”), an ERCC6L antisense nucleic acid sequence (also referred to as “antisense strand”) or both.
  • the siD/R-NA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences from the target gene, e.g., a hairpin.
  • the siD/R-NA may either be a dsD/R-NA or shD/R-NA.
  • the term "dsD/R-NA” refers to a construct of two molecules composed of complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded polynucleotide molecule.
  • the nucleotide sequence of two strands may include not only the "sense” or "antisense” polynucleotides sequence selected from a protein coding sequence of target gene sequence, but also polynucleotide having a nucleotide sequence selected from non-coding region of the target gene.
  • One or both of the two molecules constructing the dsD/R-NA are composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules is composed of RNA and the other is composed of DNA (hybrid double-strand).
  • shD/R-NA refers to an siD/R-NA having a stem-loop structure, composed of a first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions are sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the loop region of an shD/R-NA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
  • an "isolated nucleic acid” is a nucleic acid removed from its original environment (e.g., the natural environment if naturally occurring) and thus, synthetically altered from its natural state.
  • examples of isolated nucleic acid includes DNA, RNA, and derivatives thereof.
  • dsRNA Fig. 2
  • the present invention provides isolated double-stranded molecules that are capable of inhibiting the expression of an ERCC6L gene when introduced into a cell expressing the gene.
  • the target sequence of double-stranded molecule may be designed by an siRNA design algorithm such as that mentioned below.
  • Examples of ERCC6L target sequences include nucleotides such as: SEQ ID NO: 7 or 8.
  • the double-stranded molecule of the present invention will be described in more detail below.
  • Methods for designing double-stranded molecules having the ability to inhibit target gene expression in cells are known. (See, for example, US Patent No. 6,506,559, herein incorporated by reference in its entirety).
  • a computer program for designing siRNAs is available from the Ambion website (http://www.ambion.com/techlib/misc/siRNA_finder.html). The computer program selects target nucleotide sequences for double-stranded molecules based on the following protocol.
  • Target Sites 1. Beginning with the AUG start codon of the transcript, scan downstream for AA di-nucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al. don't recommend designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these may be richer in regulatory protein binding sites, and UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex. 2. Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences.
  • BLAST which can be found on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/, is used (Altschul SF et al., Nucleic Acids Res 1997 Sep 1, 25(17): 3389-402). 3. Select qualifying target sequences for synthesis. Selecting several target sequences along the length of the gene to evaluate is typical.
  • the target sequence of the isolated double-stranded molecules of the present invention were designed as SEQ ID NO: 7 and 8 for ERCC6L gene.
  • Double-stranded molecules targeting the above-mentioned target sequences were respectively examined for their ability to suppress the growth of cells expressing the target genes. Therefore, the present invention provides double-stranded molecule targeting the sequences of SEQ ID NO: 7 and 8 for ERCC6L gene.
  • the double-stranded molecule of the present invention may be directed to a single target ERCC6L gene sequence or may be directed to a plurality of target ERCC6L gene sequences.
  • a double-stranded molecule of the present invention targeting the above-mentioned targeting sequence of ERCC6L gene include isolated polynucleotide that contain the nucleic acid sequences of target sequences and/or complementary sequences to the target sequence.
  • Example of polynucleotide targeting ERCC6L gene includes that containing the sequence of SEQ ID NO: 7 or 8 and/or complementary sequences to these nucleotides;
  • the present invention is not limited to this example, and minor modifications in the aforementioned nucleic acid sequences are acceptable so long as the modified molecule retains the ability to suppress the expression of ERCC6L gene.
  • the phrase "minor modification" as used in connection with a nucleic acid sequence indicates one, two or several substitution, deletion, addition or insertion of nucleic acids to the sequence.
  • a double-stranded molecule is composed of two polynucleotides, one polynucleotide has a sequence corresponding to a target sequence, i.e., sense strand, and another polypeptide has a complementary sequence to the target sequence, i.e., antisense strand.
  • the sense strand polynucleotide and the antisense strand polynucleotide hybridize to each other to form double-stranded molecule.
  • double-stranded molecules include dsRNA and dsD/R-NA.
  • a double-stranded molecule is composed of a polynucleotide that has both a sequence corresponding to a target sequence, i.e., sense strand, and a complementary sequence to the target sequence, i.e., antisense strand.
  • the sense strand and the antisense strand are linked by a intervening strand, and hybridize to each other to form a hairpin loop structure.
  • Examples of such double-stranded molecule include shRNA and shD/R-NA.
  • a double-stranded molecule of the present invention is composed a sense strand polynucleotide having a nucleotide sequence of the target sequence and anti-sense strand polynucleotide having a nucleotide sequence complementary to the target sequence, and both of polynucleotides hybridize to each other to form the double-stranded molecule.
  • a part of the polynucleotide of either or both of the strands may be RNA, and when the target sequence is defined with a DNA sequence, the nucleotide "t" within the target sequence and complementary sequence thereto is replaced with "u".
  • such a double-stranded molecule of the present invention includes a stem-loop structure, composed of the sense and antisense strands.
  • the sense and antisense strands may be joined by a loop.
  • the present invention also provides the double-stranded molecule composed of a single polynucleotide containing both the sense strand and the antisense strand linked or flanked by an intervening single-strand.
  • double-stranded molecules targeting the ERCC6L gene may have a sequence selected from among SEQ ID NOs: 7 and 8 as a target sequence.
  • preferable examples of the double-stranded molecule of the present invention include polynucleotide and a complementary sequence thereto, and a polynucleotide that has a sequence corresponding to SEQ ID NO: 7 and 8 and a complementary sequence thereto.
  • the term "several" as applies to nucleic acid substitutions, deletions, additions and/or insertions may mean 3-7, preferably 3-5, more preferably 3-4, even more preferably 3 nucleic acid residues.
  • a double-stranded molecule of the present invention can be tested for its ability using the methods utilized in the Examples.
  • double-stranded molecules composed of sense strands of various portions of mRNA of ERCC6L genes or antisense strands complementary thereto were tested in vitro for their ability to decrease production of ERCC6L gene product in cancer cell lines according to standard methods.
  • reduction in ERCC6L gene product in cells contacted with the candidate double-stranded molecule compared to cells cultured in the absence of the candidate molecule can be detected by, e.g.
  • RT-PCR using primers for the ERCC6L mRNA mentioned under Example 1 item "Semiquantitative reverse transcription RT-PCR”. Sequences that decrease the production of an ERCC6L gene product in in vitro cell-based assays can then be tested for their inhibitory effects on cell growth. Sequences that inhibit cell growth in in vitro cell-based assay can then be tested for their in vivo ability using animals with cancer, e.g. nude mouse xenograft models, to confirm decreased production of an ERCC6L gene product and decreased cancer cell growth.
  • cancer e.g. nude mouse xenograft models
  • the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a polynucleotide
  • binding means the physical or chemical interaction between two polynucleotides.
  • the polynucleotide includes modified nucleotides and/or non-phosphodiester linkages, these polynucleotides may also bind each other as same manner.
  • complementary polynucleotide sequences hybridize under appropriate conditions to form stable duplexes containing few or no mismatches.
  • the sense strand and antisense strand of the isolated polynucleotide of the present invention can form double-stranded molecule or hairpin loop structure by the hybridization.
  • such duplexes contain no more than 1 mismatch for every 10 matches.
  • such duplexes contain no mismatches.
  • the polynucleotide is preferably less than 7006 nucleotides in length for ERCC6L.
  • the polynucleotide is less than 500, 200, 100, 75, 50, or 25 nucleotides in length for ERCC6L.
  • the isolated polynucleotides of the present invention are useful for forming double-stranded molecules against ERCC6L gene or preparing template DNAs encoding the double-stranded molecules.
  • the polynucleotides may be longer than 19 nucleotides, preferably longer than 21 nucleotides, and more preferably has a length of between about 19 and 25 nucleotides.
  • the present invention provides a double-stranded molecule composed of a sense strand and an antisense strand, wherein the sense strand is a nucleotide sequence corresponding to a target sequence.
  • the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.
  • the double-stranded molecule serves as a guide for identifying homologous sequences in mRNA for the RISC complex, when the double-stranded molecule is introduced into cells.
  • the identified target RNA is cleaved and degraded by the nuclease activity of Dicer, through which the double-stranded molecule eventually decreases or inhibits production (expression) of the polypeptide encoded by the RNA.
  • a double-stranded molecule of the invention can be defined by its ability to generate a single-strand that specifically hybridizes to the mRNA of the ERCC6L gene under stringent conditions.
  • target sequence or “target nucleic acid” or “target nucleotide”.
  • target nucleic acid or “target nucleotide”.
  • nucleotide sequence of the "target sequence” can be shown using not only the RNA sequence of the mRNA, but also the DNA sequence of cDNA synthesized from the mRNA.
  • the double-stranded molecules of the invention may contain one or more modified nucleotides and/or non-phosphodiester linkages.
  • Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule.
  • the skilled person will be aware of other types of chemical modification which may be incorporated into the present molecules (WO03/070744; WO2005/045037).
  • modifications can be used to provide improved resistance to degradation or improved uptake.
  • modifications include, but are not limited to, phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base” nucleotides, 5'-C- methyl nucleotides, and inverted deoxybasic residue incorporation (US20060122137).
  • modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule.
  • modifications include, but are not limited to, chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3' or 5' terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2 -fluoro modified ribonucleotides and 2'-deoxy ribonucleotides (WO2004/029212).
  • modifications can be used to increased or decreased affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (WO2005/044976).
  • an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine.
  • an unmodified purine can be substituted with a 7-deaza, 7-alkyl, or 7-alkenyl purine.
  • the double-stranded molecule is a double-stranded molecule with a 3' overhang
  • the 3'- terminal nucleotide overhanging nucleotides may be replaced by deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200).
  • published documents such as US20060234970 are available.
  • the present invention is not limited to these examples and any known chemical modifications may be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene.
  • the double-stranded molecules of the present invention may include both DNA and RNA, e.g., dsD/R-NA or shD/R-NA.
  • RNA e.g., dsD/R-NA or shD/R-NA.
  • a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability.
  • DNA and RNA i.e., a hybrid type double-stranded molecule composed of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule containing both DNA and RNA on any or both of the single strands (polynucleotides), or the like may be formed for enhancing stability of the double-stranded molecule.
  • the hybrid of a DNA strand and an RNA strand may be either where the sense strand is DNA and the antisense strand is RNA, or vice versa so long as it can inhibit expression of the target gene when introduced into a cell expressing the gene.
  • the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA.
  • the chimera type double-stranded molecule may be either where both of the sense and antisense strands are composed of DNA and RNA, or where any one of the sense and antisense strands is composed of DNA and RNA so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
  • the molecule preferably contains as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule is required to be RNA within a range to induce sufficient inhibition of the expression.
  • an upstream partial region i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands
  • the upstream partial region indicates the 5' side (5'-end) of the sense strand and the 3' side (3'-end) of the antisense strand.
  • regions flanking to 5'-end of sense strand and/or 3'-end of antisense strand are referred to upstream partial region.
  • a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand are composed of RNA.
  • the chimera or hybrid type double-stranded molecule of the present invention include following combinations.
  • sense strand 5'-[---DNA---]-3' 3'-(RNA)-[DNA]-5' :antisense strand
  • sense strand 5'-(RNA)-[DNA]-3' 3'-(RNA)-[DNA]-5' :antisense strand
  • sense strand 5'-(RNA)-[DNA]-3' 3'-(---RNA---)-5' :antisense strand
  • the upstream partial region preferably is a domain composed of 9 to 13 nucleotides counted from the terminus of the target sequence or complementary sequence thereto within the sense or antisense strands of the double-stranded molecules.
  • preferred examples of such chimera type double-stranded molecules include those having a strand length of 19 to 21 nucleotides in which at least the upstream half region (5' side region for the sense strand and 3' side region for the antisense strand) of the polynucleotide is RNA and the other half is DNA. In such a chimera type double-stranded molecule, the effect to inhibit expression of the target gene is much higher when the entire antisense strand is RNA (US20050004064).
  • the double-stranded molecule may form a hairpin, such as a short hairpin RNA (shRNA) and short hairpin consisting of DNA and RNA (shD/R-NA).
  • shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • the shRNA or shD/R-NA includes the sense target sequence and the antisense target sequence on a single strand wherein the sequences are separated by a loop sequence.
  • the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which is then bound to the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • a loop sequence composed of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form the hairpin loop structure.
  • the present invention also provides a double-stranded molecule having the general formula 5'-[A]-[B]-[A']-3', wherein [A] is the sense strand containing a nucleotide sequence corresponding to a target sequence, [B] is an intervening single-strand and [A'] is the antisense strand containing a complementary sequence to the target sequence.
  • the target sequence may be selected from among, for example, nucleotide sequences of SEQ ID NO: 7 and 8 for ERCC6L.
  • the present invention is not limited to these examples, and the target sequence in [A] may be modified sequences from these examples so long as the double-stranded molecule retains the ability to suppress the expression of the targeted ERCC6L gene.
  • the region [A] hybridizes to [A'] to form a loop composed of the region [B].
  • the intervening single-stranded portion [B], i.e., loop sequence may be preferably 3 to 23 nucleotides in length.
  • the loop sequence for example, can be selected from among the following sequences (http://www.ambion.com/techlib/tb/tb_506.html).
  • loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26): CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26; UUCG: Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003 Feb 18, 100(4): 1639-44, Epub 2003 Feb 10; and UUCAAGAGA: Dykxhoorn DM et al., Nat Rev Mol Cell Biol 2003 Jun, 4(6): 457-67.
  • the loop sequence can be selected from among AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA; however, the present invention is not limited thereto: CUGCACAACCAACUCUUUG-[B]- CAAAGAGUUGGUUGUGCAG (for target sequence SEQ ID NO: 7). AUGUUAAUCAAUAACUGGC-[B]- GCCAGUUAUUGAUUAACAU (for target sequence SEQ ID NO: 8).
  • nucleotides can be added to 3'end of the sense strand and/or antisense strand of the target sequence, as 3' overhangs.
  • the preferred examples of nucleotides constituting a 3' overhang include "t" and "u", but are not limited to.
  • the number of nucleotides to be added is at least 2, generally 2 to 10, preferably 2 to 5.
  • the added nucleotides form single strand at the 3'end of the antisense strand of the double-stranded molecule.
  • a 3' overhang sequence may be added to the 3' end of the single polynucleotide.
  • the method for preparing the double-stranded molecule is not particularly limited though it is preferable to use a chemical synthetic method known in the art.
  • sense and antisense single-stranded polynucleotides are separately synthesized and then annealed together via an appropriate method to obtain a double-stranded molecule.
  • Specific example for the annealing includes wherein the synthesized single-stranded polynucleotides are mixed in a molar ratio of preferably at least about 3:7, more preferably about 4:6, and most preferably substantially equimolar amount (i.e., a molar ratio of about 5:5).
  • the mixture is heated to a temperature at which double-stranded molecules dissociate and then is gradually cooled down.
  • the annealed double-stranded polynucleotide can be purified by usually employed methods known in the art.
  • Example of purification methods include methods utilizing agarose gel electrophoresis or wherein remaining single-stranded polynucleotides are optionally removed by, e.g., degradation with appropriate enzyme.
  • the regulatory sequences flanking ERCC6L sequences may be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner.
  • the double-stranded molecules can be transcribed intracellularly by cloning ERCC6L gene templates into a vector containing, e.g., a RNA pol III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.
  • snRNA small nuclear RNA
  • the double-stranded molecules may be transcribed intracellularly by cloning its coding sequence into a vector containing a regulatory sequence that directs the expression of the double-stranded molecule in an adequate cell (e.g., a RNA poly III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter) adjacent to the coding sequence.
  • a regulatory sequence that directs the expression of the double-stranded molecule in an adequate cell e.g., a RNA poly III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter
  • the regulatory sequences flanking the coding sequences of double-stranded molecule may be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. Details of vectors which are capable of producing the double-stranded molecules are described below.
  • Vector containing a double-stranded molecule of the present invention contemplates vectors containing one or more of the double-stranded molecules described herein, and a cell containing such a vector. Of particular interest to the present invention are the following vector of [1] to [10].
  • a vector encoding a double-stranded molecule that, when introduced into a cell, inhibits in vivo expression of ERCC6L and cell proliferation, such molecules composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule;
  • the vector of [1], encoding the double-stranded molecule acts on mRNA, matching a target sequence of SEQ ID NO: 7 or 8;
  • the vector of [1], wherein the sense strand contains a sequence corresponding to a target sequence of SEQ ID NO: 7 or 8;
  • a vector of the present invention preferably encodes a double-stranded molecule of the present invention in an expressible form.
  • the phrase "in an expressible form” indicates that the vector, when introduced into a cell, will express the molecule.
  • the vector includes regulatory elements necessary for expression of the double-stranded molecule.
  • Such vectors of the present invention may be used for producing the present double-stranded molecules, or directly as an active ingredient for treating cancer.
  • Vectors of the present invention can be produced, for example, by cloning ERCC6L sequence into an expression vector so that regulatory sequences are operatively-linked to ERCC6L sequence in a manner to allow expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5).
  • RNA molecule that is the antisense to mRNA is transcribed by a first promoter (e.g., a promoter sequence flanking to the 3' end of the cloned DNA) and RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA).
  • a first promoter e.g., a promoter sequence flanking to the 3' end of the cloned DNA
  • RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA).
  • the sense and antisense strands hybridize in vivo to generate a double-stranded molecule constructs for silencing of the gene.
  • two vectors constructs respectively encoding the sense and antisense strands of the double-stranded molecule are utilized to respectively express the sense and anti-sense strands and then forming a double-stranded molecule construct.
  • the cloned sequence may encode a construct having a secondary structure (e.g., hairpin); namely, a single transcript of a vector contains both the sense and complementary antisense sequences of the target gene.
  • the vectors of the present invention may also be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; US Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720.
  • DNA-based delivery technologies include "naked DNA”, facilitated (bupivacaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., US Patent No. 5,922,687).
  • the vectors of the present invention include, for example, viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, such as vaccinia or fowlpox (see, e.g., US Patent No. 4,722,848). This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the double-stranded molecule. Upon introduction into a cell expressing the target gene, the recombinant vaccinia virus expresses the molecule and thereby suppresses the proliferation of the cell.
  • Another example of useable vector includes Bacille Calmette Guerin (BCG). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60.
  • a wide variety of other vectors are useful for therapeutic administration and production of the double-stranded molecules; examples include adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14: 571-85.
  • dsRNAs for ERCC6L were tested for their ability to inhibit cell growth.
  • the dsRNA for ERCC6L (Fig. 2B, C) effectively knocked down the expression of the gene in several cancer cell lines, which coincided with suppression of cell proliferation.
  • the present invention provides methods for inhibiting cancer cell growth by inducing dysfunction of the ERCC6L gene via inhibiting the expression of ERCC6L.
  • ERCC6L gene expression can be inhibited by any of the aforementioned double-stranded molecules of the present invention that specifically target the ERCC6L gene.
  • the present invention provides methods to treat patients with cancer by administering a double-stranded molecule against ERCC6L gene or a vector expressing the molecule without adverse effect because ERCC6L gene was minimally detected in normal organs (Fig. 1C).
  • [1] A method for inhibiting a growth of cancer cell or treating a cancer, wherein the cancer cell or the cancer expresses at least one ERCC6L gene, such method including the step of administering at least one isolated double-stranded molecule inhibiting the expression of ERCC6L gene in a cell over-expressing the gene and the cell proliferation, or vector encoding such double-stranded molecule , wherein the double-stranded molecule is composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule; [2] The method of [1], wherein the double-stranded molecule acts at mRNA which matches a target sequence of SEQ ID NO: 7 or 8; [3] The method of [2], wherein the sense strand contains the sequence corresponding to a target sequence of SEQ ID NO: 7 or 8; [4] The method of [1], wherein the cancer to be treated
  • the growth of cells expressing an ERCC6L gene may be inhibited by contacting the cells with a double-stranded molecule against an ERCC6L gene, a vector expressing the molecule or a composition containing the same.
  • the cell may be further contacted with a transfection agent. Suitable transfection agents are known in the art.
  • the phrase "inhibition of cell growth" indicates that the cell proliferates at a lower rate or has decreased viability as compared to a cell not exposed to the molecule.
  • Cell growth may be measured by methods known in the art, e.g., using the MTT cell proliferation assay.
  • the growth of any kind of cell may be suppressed according to the present method so long as the cell expresses or over-expresses the target gene of the double-stranded molecule of the present invention.
  • Exemplary cells include lung cancer.
  • patients suffering from or at risk of developing disease related to ERCC6L may be treated with the administration of a double-stranded molecule of the present invention, at least one vector expressing the molecule or a composition containing the molecule.
  • patients suffering from cancer may be treated according to the present methods.
  • the type of cancer may be identified by standard methods according to the particular type of tumor to be diagnosed.
  • patients treated by the methods of the present invention are selected by detecting the expression of ERCC6L in a biopsy from the patient by RT-PCR or immunoassay.
  • the biopsy specimen from the subject is confirmed for ERCC6L gene over-expression by methods known in the art, for example, immunohistochemical analysis or RT-PCR.
  • each of the molecules may have different structures but act on mRNA that matches the same target sequence of ERCC6L.
  • plural kinds of the double-stranded molecules may act on mRNA that matches a different target sequence of same gene.
  • the method may utilize double-stranded molecules directed to one, two or more target sequence of ERCC6L.
  • a double-stranded molecule of present invention may be directly introduced into the cells in a form to achieve binding of the molecule with corresponding mRNA transcripts.
  • a DNA encoding the double-stranded molecule may be introduced into cells as a vector.
  • transfection-enhancing agent such as FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical), may be employed.
  • a treatment is deemed “efficacious” if it leads to clinical benefit such as, reduction in expression of ERCC6L gene, or a decrease in size, prevalence, or metastatic potential of the cancer in the subject.
  • “efficacious” means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer. Efficaciousness is determined in association with any known method for diagnosing or treating the particular tumor type.
  • prevention and prophylaxis can occur “at primary, secondary and tertiary prevention levels.” While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g. reducing the proliferation and metastasis of tumors.
  • the treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence thereof include any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis.
  • Effectively treating and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer.
  • reduction or improvement of symptoms constitutes effectively treating and/or the prophylaxis include 10%, 20%, 30% or more reduction, or stable disease.
  • a double-stranded molecule of the present invention degrades ERCC6L mRNA in substoichiometric amounts. Without wishing to be bound by any theory, it is believed that the double-stranded molecule of the invention causes degradation of the target mRNA in a catalytic manner. Thus, as compared to standard cancer therapies, the present invention requires the delivery of significantly less double-stranded molecule at or near the site of cancer in order to exert therapeutic effect.
  • an effective amount of the double-stranded molecule of the present invention can readily determine an effective amount of the double-stranded molecule of the present invention to be administered to a given subject, by taking into account factors such as body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is regional or systemic.
  • an effective amount of the double-stranded molecule of the invention is an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM to about 50 nM, more preferably from about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of the double-stranded molecule can be administered.
  • the precise dosage required for a particular circumstance may be readily and routinely determined by one of skill in the art.
  • the present methods can be used to inhibit the growth or metastasis of cancer expressing ERCC6L gene; for example lung cancer.
  • a double-stranded molecule containing a target sequence of ERCC6L gene e.g., SEQ ID NO: 7 and 8) is particularly preferred for the treatment of cancer.
  • the double-stranded molecule of the present invention can also be administered to a subject in combination with a pharmaceutical composition different from the double-stranded molecule.
  • the double-stranded molecule of the present invention can be administered to a subject in combination with another therapeutic method designed to treat cancer.
  • the double-stranded molecule of the present invention can be administered in combination with therapeutic methods currently employed for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents, such as cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen).
  • the double-stranded molecule can be administered to the subject either as a naked double-stranded molecule, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector which expresses the double-stranded molecule.
  • Suitable delivery reagents for administration in conjunction with the present a double-stranded molecule include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.
  • a preferred delivery reagent is a liposome.
  • Liposomes can aid in the delivery of the double-stranded molecule to a particular tissue, such as lung tumor tissue, and can also increase the blood half-life of the double-stranded molecule.
  • Liposomes suitable for use in the context of the present invention may be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example as described in Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467; and US Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369, the entire disclosures of which are herein incorporated by reference.
  • the liposomes encapsulating the double-stranded molecule of the present invention include a ligand molecule that can deliver the liposome to the cancer site.
  • Ligands which bind to receptors prevalent in tumor or vascular endothelial cells such as monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens, are preferred.
  • the liposomes encapsulating the present double-stranded molecule are modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example, by having opsonization-inhibition moieties bound to the surface of the structure.
  • a liposome of the invention can include both opsonization-inhibition moieties and a ligand.
  • Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane.
  • an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids.
  • These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); e.g., as described in US Pat. No.
  • Liposomes modified with opsonization-inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes.
  • Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature.
  • target tissue characterized by such microvasculature defects for example, solid tumors, will efficiently accumulate these liposomes; see Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53.
  • the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation in liver and spleen.
  • liposomes of the invention that are modified with opsonization-inhibition moieties can deliver the present double-stranded molecule to tumor cells.
  • Opsonization inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons.
  • Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM.sub.1.
  • Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable.
  • the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide.
  • the opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.
  • natural polysaccharides containing amino acids or carboxylic acids e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan
  • aminated polysaccharides or oligosaccharides linear or branched
  • the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof.
  • Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".
  • the opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques. For example, an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane.
  • a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH. sub. 3 and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratio at 60 degrees C.
  • Vectors expressing a double-stranded molecule of the present invention are discussed above. Such vectors expressing at least one double-stranded molecule of the present invention can also be administered directly or in conjunction with a suitable delivery reagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.
  • a suitable delivery reagent including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.
  • the double-stranded molecule of the present invention can be administered to the subject by any means suitable for delivering the double-stranded molecule into cancer sites.
  • the double-stranded molecule can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.
  • Suitable enteral administration routes include oral, rectal, or intranasal delivery.
  • Suitable parenteral administration routes include intravesical and intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a suppository or an implant including a porous, non-porous, or gelatinous material); and inhalation. It is preferred that injections or infusions of the double-stranded molecule or vector be given at or near the site of the cancer.
  • intravesical and intravascular administration e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial in
  • the double-stranded molecule of the present invention can be administered in a single dose or in multiple doses. Where the administration of the double-stranded molecule of the present invention is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. Injection of the double-stranded molecule directly into the tissue is at or near the site of cancer preferred. Multiple injections of the double-stranded molecule into the tissue at or near the site of cancer are particularly preferred.
  • the double-stranded molecule can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site.
  • the double-stranded molecule can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, more preferably from about seven to about ten days.
  • the double-stranded molecule is injected at or near the site of cancer once a day for seven days.
  • the effective amount of a double-stranded molecule administered to the subject can include the total amount of a double-stranded molecule administered over the entire dosage regimen.
  • a cancer overexpressing ERCC6L can be treated with at least one active ingredient selected from the group consisting of: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof.
  • the cancer includes, but is not limited to, lung cancer. Accordingly, prior to the administration of the double-stranded molecule of the present invention as active ingredient, it is preferable to confirm whether the expression level of ERCC6L in the cancer cells or tissues to be treated is enhanced as compared with normal cells of the same organ.
  • the present invention provides a method for treating a cancer (over)expressing ERCC6L, such method including the steps of: i) determining the expression level of ERCC6L in cancer cells or tissue(s) obtained from a subject with the cancer to be treated; ii) comparing the expression level of ERCC6L with normal control; and iii) administrating at least one component selected from the group consisting of (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof, to a subject with a cancer overexpressing ERCC6L compared with normal control.
  • the present invention also provides a pharmaceutical composition containing at least one component selected from the group consisting of: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof, for use in administrating to a subject having a cancer overexpressing ERCC6L.
  • the present invention further provides a method for identifying a subject to be treated with: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, or (c) a vector encoding thereof, which method may include the step of determining an expression level of ERCC6L in subject-derived cancer cells or tissue(s), wherein an increase of the level compared to a normal control level of the gene indicates that the subject has cancer which may be treated with.
  • a subject to be treated by the present method is preferably a mammal.
  • exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • the expression level of ERCC6L in cancer cells or tissues obtained from a subject is determined.
  • the expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art.
  • the mRNA of ERCC6L may be quantified using probes by hybridization methods (e.g., Northern hybridization).
  • the detection may be carried out on a chip or an array.
  • the use of an array is preferable for detecting the expression level of ERCC6L.
  • Those skilled in the art can prepare such probes utilizing the sequence information of ERCC6L.
  • the cDNA of ERCC6L may be used as the probes.
  • the probes may be labeled with a suitable label, such as dyes, fluorescent substances and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.
  • a suitable label such as dyes, fluorescent substances and isotopes
  • the transcription product of ERCC6L may be quantified using primers by amplification-based detection methods (e.g., RT-PCR). Such primers may be prepared based on the available sequence information of the gene.
  • a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of ERCC6L.
  • stringent (hybridization) conditions refers to conditions under which a probe or primer will hybridize to its target sequence, but not to other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degree Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to their target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degree Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degree Centigrade for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • the translation product may be detected for the diagnosis of the present invention.
  • the quantity of observed protein SEQ ID NO: 10.
  • Methods for determining the quantity of the protein as the translation product include immunoassay methods that use an antibody specifically recognizing the protein.
  • the antibody may be monoclonal or polyclonal.
  • any fragment or modification e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.
  • Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the intensity of staining may be measured via immunohistochemical analysis using an antibody against the ERCC6L protein. Namely, in this measurement, strong staining indicates increased presence/level of the protein and, at the same time, high expression level of ERCC6L gene.
  • the expression level of a target gene, e.g., the ERCC6L gene, in cancer cells can be determined to be increased if the level increases from the control level (e.g., the level in normal cells) of the target gene by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
  • the control level may be determined at the same time with the cancer cells by using a sample(s) previously collected and stored from a subject/subjects whose disease state(s) (cancerous or non-cancerous) is/are known.
  • normal cells obtained from non-cancerous regions of an organ that has the cancer to be treated may be used as normal control.
  • the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of ERCC6L gene in samples from subjects whose disease states are known.
  • the control level can be derived from a database of expression patterns from previously tested cells.
  • the expression level of ERCC6L gene in a biological sample may be compared to multiple control levels, which are determined from multiple reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample. Moreover, it is preferred to use the standard value of the expression levels of ERCC6L gene in a population with a known disease state. The standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as the standard value.
  • a control level determined from a biological sample that is known to be non-cancerous is referred to as a "normal control level”.
  • the control level is determined from a cancerous biological sample, it is referred to as a "cancerous control level”.
  • the expression level of ERCC6L gene is increased as compared to the normal control level, or is similar/equivalent to the cancerous control level, the subject may be diagnosed with cancer to be treated.
  • compositions containing a double-stranded molecule of the present invention are the following compositions [1] to [32]: [1] A composition for inhibiting growth of a cancer cell or treating a cancer, wherein the cancer and the cancer cell express ERCC6L gene, including at least one isolated double-stranded molecule that inhibits the expression of ERCC6L gene and the cell proliferation or vector encoding such doucle-stranded molecule, further wherein the molecule is composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule; [2] The composition of [1], wherein the double-stranded molecule acts at mRNA which matches a target sequence of SEQ ID NO: 7 or 8; [3] The composition of [2], wherein the double-stranded molecule, wherein
  • compositions of the present invention are described in additional detail below.
  • the double-stranded molecule of the present invention is preferably formulated as pharmaceutical compositions prior to administering to a subject, according to techniques known in the art.
  • Pharmaceutical composition of the present invention is characterized as being at least sterile and pyrogen-free.
  • pharmaceutical composition include formulation for human and veterinary use. Methods for preparing pharmaceutical compositions of the present invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
  • the present pharmaceutical composition contains the double-stranded molecule or vector encoding that of the present invention (e.g., 0.1 to 90% by weight), or a pharmaceutically acceptable salt of the molecule, mixed with a pharmaceutically acceptable carrier medium.
  • Preferred physiologically acceptable carrier media are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
  • the composition may contain plural kinds of the double-stranded molecules, each of the molecules may be directed to the same target sequence, or different target sequences of ERCC6L.
  • the composition may contain double-stranded molecules directed to ERCC6L.
  • the composition may contain double-stranded molecules directed to one, two or more target sequences ERCC6L.
  • the present composition may contain a vector coding for one or plural double-stranded molecules.
  • the vector may encode one, two or several kinds of the present double-stranded molecules.
  • the present composition may contain plural kinds of vectors, each of the vectors coding for a different double-stranded molecule.
  • the present double-stranded molecule may be contained as liposomes in the present composition. See the section entitled “Methods of Treating Cancer using the Double-Stranded Molecule" for details of liposomes.
  • compositions of the present invention can also include conventional pharmaceutical excipients and/or additives.
  • Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • Pharmaceutical compositions of the present invention can be packaged for use in liquid form, or can be lyophilized.
  • conventional nontoxic solid carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a solid pharmaceutical composition for oral administration can include any of the carriers and excipients listed above and 10-95%, preferably 25-75%, of one or more double-stranded molecule of the invention.
  • a pharmaceutical composition for aerosol (inhalational) administration can include 0.01-20% by weight, preferably 1-10% by weight, of one or more double-stranded molecule of the present invention encapsulated in a liposome as described above, and propellant.
  • a carrier can also be included as desired; e.g., lecithin for intranasal delivery.
  • the present composition may contain other pharmaceutically active ingredients so long as they do not inhibit the in vivo function of the double-stranded molecules of the present invention.
  • the composition may contain chemotherapeutic agents conventionally used for treating cancers.
  • the present invention provides for the use of the double-stranded nucleic acid molecule of the present invention in manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of ERCC6L gene.
  • the present invention relates to a use of double-stranded nucleic acid molecule inhibiting the expression of an ERCC6L gene in a cell, which molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and target to a sequence of SEQ ID NO: 7 or 8, for manufacturing a pharmaceutical composition for treating cancer expressing ERCC6L gene.
  • the present invention further provides the double-stranded nucleic acid molecules of the present invention for use in treating a cancer expressing the ERCC6L gene.
  • the present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of ERCC6L gene, wherein the method or process includes a step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the expression of ERCC6L gene in a cell, which over-expresses the gene, which molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and target to a sequence of SEQ ID NO: 7 or 8 as active ingredients.
  • the present invention provides a method or process for manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of ERCC6L gene, wherein the method or process includes a step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double-stranded nucleic acid molecule inhibiting the expression of ERCC6L gene in a cell, which over-expresses the gene, which molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NO: 7 or 8.
  • Example 1 General Methods Lung cancer cell lines and tissue samples.
  • the human lung cancer cell lines used in this study were as follows: lung adenocarcinomas A427, NCI-H1781, A549, and LC319; lung squamous cell carcinomas (SCC) NCI-H226, EBC-1, NCI-H520, and NCI-H2170; and SCLCs DMS114, DMS273, SBC-3, SBC-5, H196, and H446. All cells were grown in monolayers in appropriate medium supplemented with 10% FCS and were maintained at 37 degrees C in atmospheres of humidified air with 5% CO 2 .
  • Human small airway epithelial cells (SAEC) were grown in optimized medium purchased from Cambrex Bio Science, Inc.
  • RT-PCR Semiquantitative reverse transcription-PCR (RT-PCR) experiments were carried out with the following synthesized ERCC6L-specific primers or with beta-actin (ACTB)-specific primers as an internal control: ERCC6L, 5'-AAAAATCCAG GAGGCCCTAA -3' (SEQ ID NO:1) and 5'- AGATCTTTCTTGCCTTAAGCCTG-3' (SEQ ID NO:2); ACTB, 5'-GAGGTGATAGCATTGCTTTCG-3' (SEQ ID NO:3) and 5'-CAAGTCAGTGTACAGGTAAGC-3' (SEQ ID NO:4). PCR reactions were optimized for the number of cycles to ensure product intensity within the logarithmic phase of amplification.
  • each specimen was mounted with Vectashield (Vector Laboratories, Inc.) containing 4', 6'-diamidine-2'- phenylindoldihydrochloride (DAPI) and visualized with Spectral Confocal Scanning Systems (TSC SP2 AOBS: Leica Microsystems).
  • DAPI Spectral Confocal Scanning Systems
  • RNA interference assay The present inventors had previously established a vector-based RNA interference system, psiH1BX3.0, that was designed to synthesize small interfering RNAs (siRNA) in mammalian cells (Suzuki C et al. Cancer Res 2003;63:7038-41.). Ten micrograms of siRNA expression vector were transfected using 30 micro liter of LipofectAMINE 2000 (Invitrogen) into lung cancer cell lines SBC-5.
  • siRNA small interfering RNAs
  • the transfected cells were cultured for 7 days in the presence of appropriate concentrations of geneticin (G418); the number of colonies was counted by Giemsa staining; and viability of cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide assay at 7 days after the treatment. Briefly, cell counting kit-8 solution (Dojindo) was added to each dish at a concentration of 1/10 volume, and the plates were incubated at 37 degrees C for additional 2 hours. Absorbance was then measured at 490 nm, and at 630 nm as a reference, with a Microplate Reader 550 (Bio-Rad).
  • RNA interference was carried out with the following synthesized ERCC6L-specific primers according to the standard protocol.
  • the target sequences of the synthetic oligonucleotides for RNA interference were as follows: control 1 (luciferase/LUC: Photinus pyralis luciferase gene), 5'-CGTACGCGGAATACTTCGA-3' (SEQ ID NO:5); control 2 (EGFP: gene, a mutant of Aequorea victoria GFP), 5'-GAAGCAGCACGACTTCTTC-3' (SEQ ID NO:6); siRNA-ERCC6L-#A, 5'-CTGCACAACCAACTCTTTG-3' (SEQ ID NO:7); siRNA-ERCC6L-#B, 5'-ATGTTAATCAATAACTGGC-3' (SEQ ID NO:8).
  • Example 2 ERCC6L expression in lung tumors and normal tissues.
  • genes that showed 3-fold or higher level of expression in more than half of 101 lung cancer samples were first screened by cDNA microarray analysis (Daigo Y, et al. Gen Thorac Cardiovasc Surg 2008;56:43-53.; Kikuchi T, et al. Oncogene 2003;22:2192-205.; Kakiuchi S, et al. Mol Cancer Res 2003;1:485-99.; Kakiuchi S, et al. Hum Mol Genet 2004;13:3029-43.; Kikuchi T, et al.
  • Example 3 Inhibition of growth of lung cancer cells by siRNA against ERCC6L.
  • plasmids were constructed to express siRNAs against ERCC6L (si-ERCC6L-#A and -#B) as well as control plasmids (siRNAs for luciferase (LUC) and EGFP) and transfected them into SBC-5 and A549 cells, which strongly expressed ERCC6L.
  • the ERCC6L-mRNA levels in cells transfected with si-ERCC6L-#A or -#B were significantly decreased in comparison with cells transfected with either control siRNAs (Fig. 2A). Significant decreases in the numbers of viable cells were observed (Figs. 2B and 2C).
  • Example 4 Growth-promoting effect of ERCC6L.
  • plasmids designed to express either ERCC6L (pcDNA3.1/myc-His A vector) or mock vector were prepared.
  • transient transfectants were generated using COS-7 cells which scarcely expressed endogenous ERCC6L.
  • the cells that transiently expressed exogenous ERCC6L revealed significant growth promotion compared with the mock-transfected cells (Figs. 3A and 3B).
  • ERCC6L is likely to be an important growth factor of lung cancer cells, although the molecular mechanisms underlying increased ERCC6L expression levels in many cancer cells remains to be clarified. Because ERCC6L should be classified as one of the cancer specific antigens, selective inhibition of ERCC6L activity by molecular targeted agents could be a promising therapeutic strategy that is expected to have a powerful biological activity against cancer with a minimal risk of adverse events. In summary, activation of ERCC6L has a specific functional role for growth of cancer cells. The data strongly imply the possibility of designing new anticancer drugs to specifically target the oncogenic activity of ERCC6L for treatment of lung cancer patients.
  • the data provided herein add to a comprehensive understanding of cancers, facilitate development of novel diagnostic strategies, and provide clues for identification of molecular targets for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of tumorigenesis, and provide indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of cancers.
  • ERCC6L the gene-expression analysis of cancers described herein using the combination of laser-capture dissection and genome-wide cDNA microarray identify ERCC6L as a gene that is markedly elevated in cancer as compared to normal organs.
  • ERCC6L can be conveniently used as a molecular diagnostic marker for identifying and detecting cancer, in particular, lung cancer.
  • the ERCC6L gene and the proteins encoded thereby find utility in diagnostic kits and assays of cancer.
  • the present invention further demonstrates that the cell growth may be suppressed by a double-stranded nucleic acid molecule that specifically targets the ERCC6L gene.
  • the double-stranded nucleic acid molecule is useful for the development of anti-cancer pharmaceuticals.
  • ERCC6L polypeptide is a useful target for the development of anti-cancer pharmaceuticals.
  • substances that block the expression of ERCC6L protein or prevent its activity may find therapeutic utility as anti-cancer agents, particularly anti-cancer agents for the treatment of lung cancer.

Abstract

L'invention concerne des méthodes de diagnostic d'une prédisposition au développement du cancer, en particulier du cancer du poumon. La présente invention porte sur une méthode de diagnostic qui utilise le niveau d'expression de ERCC6L comme index du cancer. La présente invention concerne en outre des méthodes de criblage pour des substances thérapeutiques utiles dans le traitement de maladies associées à ERCC6L, telles qu'un cancer, par exemple le cancer du poumon. L'invention concerne en outre des méthodes d'inhibition du développement cellulaire et de traitement ou d'atténuation d'un ou plusieurs symptômes de maladies associées à ERCC6L. L'invention concerne en outre des molécules à double brin ainsi que des vecteurs et compositions les contenant.
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WO2008144223A2 (fr) * 2007-05-16 2008-11-27 Eli Lilly And Company Composés triazolyl aminopyrimidine

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DAIGO Y. ET AL: "Understanding of molecular aspects of lung carcinogenesis and new therapeutic target discovery based on comprehensive gene and protein-expression profiles", JAPANESE JOURNAL OF LUNG CANCER, vol. 49, 20 October 2009 (2009-10-20), pages 917 - 927, XP008154155 *
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