JP2013502901A - ERCC6L as a target gene for cancer treatment and diagnosis - Google Patents

Abstract

Described herein are methods aimed at diagnosing a predisposition to developing cancer, particularly lung cancer. The present invention provides a diagnostic method using the expression level of ERCC6L as an indicator of cancer. The present invention further provides methods of screening for therapeutic agents useful in the treatment of ERCC6L-related diseases, such as cancer, eg, lung cancer. The invention further provides a method of inhibiting cell proliferation and treating or alleviating one or more symptoms of an ERCC6L-related disease. The invention also features double-stranded molecules, and vectors and compositions containing them.

Description

Priority This application claims the benefit of U.S. Provisional Patent Application Serial No. 61 / 275,198 on August 25, 2009, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD The present invention relates to methods for detecting and diagnosing cancers, particularly cancers associated with overexpression of ERCC6L, such as lung cancer, and methods for treating and preventing such cancers. The present invention also relates to a method of screening candidate substances for treating and preventing ERCC6L-related cancer. The invention further relates to double-stranded molecules that reduce ERCC6L gene expression and uses thereof.

  Lung cancer is the most common cancer, accounting for 1.35 million out of 10.9 million new cases of cancer per year. Lung cancer is also the top cause of death due to cancer-related diseases, and accounts for 1.18 million cases out of 6.7 million cases of cancer-related deaths worldwide (Non-patent Document 1). Over the last decade, newly developed cytotoxic agents, including paclitaxel, docetaxel, gemcitabine, and vinorelbine, will appear to provide multiple treatment options for advanced NSCLC (non-small cell lung cancer) patients However, each new dosing regimen can only provide a slight life-prolonging effect compared to cisplatin-based therapies (Non-Patent Document 2).

  More recently, molecular targets including small molecule inhibitors of EGFR tyrosine kinases such as anti-EGFR monoclonal antibodies or anti-VEGF monoclonal antibodies Erbitux or Bevacizumab (Avastin), and gefitinib (Iressa) and erlotinib (Tarceva) The agent has been investigated and / or approved for clinical use (3, 4). Although these agents show some activity against recurrent NSCLC, there is still a limit to the number of patients who can experience a life-prolonging effect. SCLC (small cell lung cancer) patients respond favorably to first-line combination chemotherapy, but often relapse within a short period of time. Only 20% of patients with limited-stage disease (LD) can be cured with multidisciplinary treatment, and less than 5% of patients with advanced-stage disease (ED) can achieve 5-year survival after initial diagnosis Not possible (Non-patent Document 5). Therefore, new therapeutic strategies such as the development of more selective and effective molecular targeting agents for lung cancer are awaited.

  In the process of screening novel molecular targets for diagnosing, treating, and preventing human cancer, we include 27,648 genes or expressed sequence tags (ESTs) along with laser microdissection. Using cDNA microarray, comprehensive genomic expression profiling of 101 cases of lung cancer was performed and several candidate molecular targets and biomarkers for lung cancer treatment were found (Patent Documents 1 and 2, Non-Patent Documents 6 to 9) . Of these, ERCC6L (exclusion repair cross-complementing rodent repair defect, complementation group 6-complementation, complementation group 6-like), also referred to as “PICH”, is particularly noteworthy. .

  ERCC6L was identified as a member of the SNF2 ATPase family that is an interaction partner and substrate of Plk1 (Non-patent Document 10). Recent reports demonstrate that ERCC6L is an essential component of checkpoint signaling and binds to linked centromere-related DNA to monitor the tension generated between sister kinetocores (Non-Patent Document 10). ). However, so far, the relationship between ERCC6L and carcinogenesis has not been established.

Citation list
WO2004 / 031413 WO2007 / 013665

Citation list
Jemal A, Siegel R, Ward E, et al. CA Cancer J Clin. 2008; 58: 71-96. Schiller JH, Harrington D, Belani CP, et al. Eastern Cooperative Oncology Group.N Engl J Med 2002; 346: 92-8. Dowell J, Minna JD, Kirkpatrick P. Nat Rev Drug Discov 2005; 4: 13-4. Pal SK, Pegram M. Anticancer Drugs 2005; 16: 483-94. Chute JP, Chen T, Feigal E, Simon R, Johnson BE: J Clin Oncol 1999; 17: 1794-801. Daigo Y, Nakamura Y. Gen Thorac Cardiovasc Surg 2008; 56: 43-53. Kikuchi T, Daigo Y, Katagiri T, et al. Oncogene 2003; 22: 2192-205. Kakiuchi S, Daigo Y, Tsunoda T, Yano S, Sone S, Nakamura Y. Mol Cancer Res 2003; 1: 485-99. Taniwaki M, Daigo Y, Ishikawa N, et al. Int J Oncol 2006; 29: 567-75. Baumann C, et al. Cell 2007; 128: 101-114.

  The present invention relates to the discovery by microarray analysis and RT-PCR that ERCC6L is overexpressed in clinical lung cancer tissues. As demonstrated herein, the functional knockdown of endogenous ERCC6L by siRNA in cancer cell lines results in dramatic suppression of cancer cell proliferation, and thus the viability of cancer cells Suggests its essential role in the maintenance of Since ERCC6L is rarely expressed in adult normal organs, ERCC6L appears to be an appropriate and promising molecular target for new therapeutic approaches with minimal adverse effects.

  Accordingly, providing a method of diagnosing cancer, particularly lung cancer in a subject or determining a predisposition for such cancer, by measuring the expression level of ERCC6L in a subject-derived biological sample One object of the invention. An increased expression level of ERCC6L compared to the normal control level indicates that the subject is suffering from or at risk of developing such cancer, particularly lung cancer. In the method of the present invention, the ERCC6L gene can be detected with an appropriate probe or primer set, or the ERCC6L protein can be detected with an anti-ERCC6L antibody.

It is a further object of the present invention to provide a kit comprising reagents for detecting the transcript or translation product of the ERCC6L gene.
It is a still further object of the present invention to provide a reagent for diagnosing or detecting cancer comprising a nucleic acid that binds to a transcript of the ERCC6L gene or an antibody that binds to a translation product of the ERCC6L gene.
It is still a further object of the present invention to provide the use of a nucleic acid that binds to a transcript of the ERCC6L gene or an antibody that binds to a translation product of the ERCC6L gene for the manufacture of a reagent for diagnosing or detecting cancer. It is.

  It is a still further object of the present invention to provide a method for identifying candidate substances that inhibit the growth of cells that overexpress the ERCC6L gene, such substances may be used to treat ERCC6L-related diseases such as cancer and / or Or useful in prevention. The methods of the invention can be performed in vitro or in vivo and are controlled under the binding activity to ERCC6L polypeptide, the expression level of ERCC6L gene, the biological activity of ERCC6L polypeptide, or the transcriptional regulatory region of the gene product The expression level of the reporter gene or the activity of the reporter gene can be used as an indicator. A substance that binds to ERCC6L polypeptide or suppresses ERCC6L expression or activity, or reporter gene expression or activity, for treating and / or preventing cancer or inhibiting cancer cell proliferation Can be identified as a candidate substance. The biological activity of the ERCC6L polypeptide to be detected is preferably cell proliferation activity (cell proliferation enhancing activity). By reducing the biological activity of the ERCC6L polypeptide compared to a control level in the absence of the test substance, the test substance is used to reduce symptoms of lung cancer or to treat and / or prevent lung cancer. Shown to get.

  Method for treating and / or preventing cancer by administering an agent that inhibits expression of ERCC6L gene and / or function of ERCC6L protein, or inhibition of proliferation of cancerous cells overexpressing ERCC6L It is a still further object of the present invention to provide Preferably, the agent is an inhibitory nucleic acid (eg, antisense, ribozyme, double stranded molecule, aptamer). The agent may be a nucleic acid molecule or vector to provide a double stranded molecule. By introducing a double-stranded molecule into the target cell in an amount sufficient to inhibit the expression of the ERCC6L gene, the expression of the gene can be inhibited. Thus, in a particularly preferred embodiment of the invention, the method comprises administering to a subject a pharmaceutically effective amount of a double-stranded molecule against the ERCC6L gene, or a vector encoding such a molecule, Inhibits ERCC6L gene expression and cell proliferation when introduced into cells that express the ERCC6L gene.

  Suitable for the treatment and / or prevention of ERCC6L-related cancers comprising a pharmaceutically acceptable carrier and an active agent comprising one or more double-stranded molecules for the ERCC6L gene or a vector encoding such a molecule It is a still further object of the present invention to provide a pharmaceutical composition. In the context of the present invention, a double-stranded molecule against ERCC6L can inhibit the expression of the ERCC6L gene when introduced into a cell that expresses the ERCC6L gene and also inhibit the cell proliferation induced thereby. it can. In a preferred embodiment, the cancer undergoing treatment and / or prevention is lung cancer, including NSCLC and SCLC. Examples of NSCLC include lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SCC).

The double-stranded molecule of the present invention is preferably composed of a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 7 and 8. The antisense strand includes a sequence that is complementary to the sense strand. The sense strand and antisense strand of the molecule hybridize to each other to form a double-stranded molecule. When introduced into a cell that expresses the ERCC6L gene, the double-stranded molecule of the present invention inhibits the expression and cell proliferation of the ERCC6L gene.
The methods and materials of the present invention can be used to identify cancers before obvious clinical symptoms of cancer are detected and can be used in the context of cancer treatments that have no adverse effects.

  It will be appreciated by one skilled in the art that one or more aspects of the present invention can meet a particular purpose, while one or more other aspects can meet a particular other purpose. Each objective may not apply equally in all respects to all aspects of the invention. Accordingly, the foregoing objects can alternatively be considered with respect to any one aspect of the present invention. These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings and examples. However, it is to be understood that both the foregoing summary of the invention and the following detailed description are of preferred embodiments and are not intended to limit the invention and other alternative embodiments of the invention. .

  Various aspects and applications of the present invention will become apparent to those skilled in the art from consideration of the following brief description of the drawings, and detailed description of the invention and preferred embodiments thereof.

FIG. 1 shows ERCC6L expression in lung cancer and normal tissues. Part A shows semi-quantitative RT-PCR results demonstrating ERCC6L overexpression in NSCLC (ADC and SCC) and SCLC clinical samples compared to normal lung tissue. Appropriate dilutions of each single-stranded cDNA prepared from mRNA of lung cancer samples were prepared using the level of β-actin (ACTB) expression as a quantity control. Part B shows semi-quantitative RT-PCR results that demonstrate the expression of ERCC6L in lung cancer cell lines. Part C shows the results of Northern blot analysis of ERCC6L, demonstrating that some lung cancer cell lines strongly express ERCC6, but not most normal human tissues. Part D shows the subcellular localization of exogenous ERCC6L protein detected by anti-myc in COS-7 cells co-stained with DAPI. FIG. 2 shows the effect of siRNA against ERCC6L on the growth of lung cancer cells overexpressing ERCC6L. Part A confirms the knockdown effect on ERCC6L expression in SBC-5 cells that responds to si-ERCC6L (si- # A or si- # B) but not to control siRNA (LUC or EGFP). The result of a semi-quantitative RT-PCR analysis is shown. Part B shows the results of a colony formation assay of SBC-5 cells transfected with specific siRNA or control plasmid. Part C shows the results of MTT assay of SBC-5 cells in response to si-ERCC6L (si- # A or si- # B), si-LUC, or si-EGFP. All assays were performed in triplicate wells in triplicate. FIG. 3 shows the enhancement of cell proliferation by introduction of ERCC6L into COS-7 cells. Part A shows the results of Western blot analysis confirming transient expression of ERCC6L in COS-7 cells. Part B shows the growth-promoting effect on transient expression of ERCC6L in COS-7 cells. The assay was performed 3 times in triplicate wells.

DESCRIPTION OF EMBODIMENTS Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are described below. Describe. However, before describing the materials and methods of the present invention, the specific sizes, shapes, dimensions, materials, methodologies, protocols, etc. described herein can be altered according to routine experimentation and optimization. It should be understood that the present invention is not limited thereto. The terminology used herein is for the purpose of describing particular types or embodiments only and is not intended to limit the scope of the invention which is limited only by the appended claims. It should also be understood.

The disclosure of each publication, patent, or patent application mentioned in this specification is expressly incorporated by reference in its entirety. However, nothing in this specification should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Definition:
As used herein, the words “a”, “an”, and “that” mean “at least one” unless specifically indicated otherwise.
The terms “isolated” and “purified” as used herein in relation to a substance (eg, polypeptide, antibody, polynucleotide, etc.) are those that the substance is otherwise included in natural sources. Indicating that it is substantially free of at least one substance that may be present. Thus, an isolated or purified antibody substantially includes cellular material such as carbohydrates, lipids, or other contaminating proteins derived from the cell or tissue source from which the protein (antibody) is derived. When not chemically or chemically synthesized, it refers to an antibody that is substantially free of chemical precursors or other chemicals. The term “substantially free of cellular material” refers to a preparation of a polypeptide in which the polypeptide is isolated from cellular components of cells from which the polypeptide is isolated or recombinantly produced. Including. Thus, a polypeptide that is substantially free of cellular material is less than about 30%, 20%, 10%, or 5% heterologous protein (also referred to herein as “contaminating protein”) (by dry weight). A preparation of a polypeptide having Where the polypeptide is produced recombinantly, the polypeptide is also preferably substantially free of culture medium, and the culture medium is less than about 20%, 10%, or 5% of the volume of the protein preparation. Peptide preparations are included. If the polypeptide is made by chemical synthesis, the polypeptide is preferably substantially free of chemical precursors or other chemicals, and the chemical precursors or other chemicals involved in protein synthesis are protein preparations. Polypeptide preparations that are less than about 30%, 20%, 10%, 5% (by dry weight) of the product volume. The inclusion of a specific protein preparation with an isolated or purified polypeptide can be achieved, for example, after sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis of the protein preparation and Coomassie brilliant blue staining of the gel. It can be shown by the appearance of a single band. In preferred embodiments, the antibodies of the invention are isolated or purified.

  Nucleic acid molecules, such as “isolated” or “purified” cDNA molecules, can be substantially free of other cellular material or culture medium when made by recombinant techniques, or chemically synthesized. If present, it may be substantially free of chemical precursors or other chemicals.

  The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein and refer to a polymer of amino acid residues. The term refers to, in addition to natural amino acid polymers, amino acids in which one or more amino acid residues are modified residues or are non-natural residues, eg, artificial chemical mimetics of the corresponding natural amino acids. Applied to polymer.

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Natural amino acids are natural amino acids encoded by the genetic code and natural amino acids that are post-translationally modified in cells (eg, hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine). The phrase “amino acid analog” is a compound having the same basic chemical structure as a natural amino acid (hydrogen, carboxy group, amino group, and α-carbon attached to the R group), but having a modified R group or a modified backbone (eg, Homoserine, norleucine, methionine, sulfoxide, methionine methylsulfonium). The phrase “amino acid mimetic” refers to a chemical substance that has a different structure from a common amino acid but has a similar function.
Amino acids may be referred to herein by their known three-letter or single-letter code as recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

  The terms “gene”, “polynucleotide”, “oligonucleotide”, “nucleic acid”, and “nucleic acid molecule” are used interchangeably unless specifically indicated otherwise, and are generally accepted as well as amino acids. Referenced by the one-letter code. Like amino acids, they include both natural and non-natural nucleic acid polymers. A polynucleotide, oligonucleotide, nucleic acid, or nucleic acid molecule can be composed of DNA, RNA, or a combination thereof.

  Unless otherwise specified, the term “cancer” refers to a cancer that overexpresses the ERCC6L gene. Examples of cancers that overexpress ERCC6L include, but are not limited to, lung cancers including SCLC and NSCLC. NSCLC includes, but is not limited to, adenocarcinoma (ADC) and squamous cell carcinoma (SCC).

  As used herein, the term “double-stranded molecule” refers to, for example, short interfering RNA (siRNA; eg, double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)), and short interfering DNA / Expression of target genes, including RNA (siD / R-NA; eg, DNA and RNA double-stranded chimeras (dsD / R-NA) or DNA and RNA small hairpin chimeras (shD / R-NA)) Refers to a nucleic acid molecule that inhibits. In the present specification, the “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”.

ERCC6L gene and ERCC6L protein:
The present invention is based in part on the discovery that the gene encoding ERCC6L is overexpressed in cancer compared to non-cancerous tissue.
The nucleotide sequence of the ERCC6L polynucleotide and the amino acid sequence of the ERCC6L polypeptide are known to those skilled in the art and can be obtained from a genetic database on a website such as GenBank ™. An exemplary nucleotide sequence of an ERCC6L polynucleotide is shown in SEQ ID NO: 9, and an exemplary amino acid sequence of an ERCC6L polypeptide is shown in SEQ ID NO: 10. Sequence data is also available via, for example, GenBank accession number BC11486 or NM_017669. One skilled in the art recognizes that ERCC6L sequences need not be limited to these sequences and that variants (eg, functional equivalents and allelic variants) can be used in the present invention as described below. Will.

  According to one aspect of the invention, functional equivalents are also considered “ERCC6L polypeptides”. As used herein, a “functional equivalent” of a protein (eg, an ERCC6L polypeptide) is a polypeptide having a biological activity equivalent to that protein. That is, 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 in which one or more amino acids are substituted, deleted, added, or inserted into the natural amino acid sequence of the ERCC6L protein. Alternatively, the polypeptide has at least about 80% homology (also referred to as sequence identity) with each protein sequence, more preferably at least about 90% -95% homology, often about 96% , 97%, 98%, or 99% homologous amino acid sequences. In other embodiments, the polypeptide can be encoded by a polynucleotide that hybridizes under stringent conditions to the native nucleotide sequence of the ERCC6L gene.

  The polypeptide of the present invention may have different amino acid sequence, molecular weight, isoelectric point, presence or absence of sugar chain, or form depending on the cell or host used for production or the purification method used. Nevertheless, so long as it has a function equivalent to the human ERCC6L protein of the present invention, this is within the scope of the present invention.

  The phrase “stringent conditions” typically refers to a nucleic acid molecule that hybridizes to its target sequence in a complex mixture of nucleic acids but to a detectable extent to other sequences. This refers to the condition where soybeans are not used. Stringent conditions are sequence-dependent and can be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Extensive guidance on nucleic acid hybridization can be 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 ° C. lower than the thermal melting temperature (Tm) for the specific sequence at a defined ionic strength and pH. Tm is the temperature (at a given ionic strength, pH, and nucleic acid concentration) at which 50% of the probe complementary to the target hybridizes in equilibrium with the target sequence (because the target sequence is present in excess) 50% of the probe is occupied in equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least twice background, preferably 10 times background hybridization. Examples of stringent hybridization conditions include: 50% formamide, 5 × SSC, and 1% SDS, 42 ° C. incubation, or 5 × SSC, 1% SDS, 65 ° C., plus 0 Wash at 50 ° C. in 2 × SSC and 0.1% SDS.

  In the context of the present invention, hybridization conditions for isolating DNA encoding a polypeptide functionally equivalent to the human ERCC6L protein can be routinely selected by those skilled in the art. For example, by using “Rapid-hyb buffer” (Amersham LIFE SCIENCE), prehybridization is performed at 68 ° C. for 30 minutes or longer, a labeled probe is added, and the mixture is heated at 68 ° C. for 1 hour or longer. Hybridization may be performed. Subsequent washing steps can be performed, for example, under low stringent conditions. Exemplary low stringency conditions include 42 ° C., 2 × SSC, 0.1% SDS, preferably 50 ° C., 2 × SSC, 0.1% SDS. Often high stringency conditions are preferably used. Exemplary high stringency conditions include three 20 minute washes in 2 × SSC, 0.01% SDS at room temperature followed by a 20 minute wash in 1 × SSC, 0.1% SDS at 37 ° C. And washing twice for 20 minutes in 1 × SSC and 0.1% SDS at 50 ° C. is performed. However, since several factors such as temperature and salt concentration can affect the stringency of hybridization, one skilled in the art can appropriately select these factors to achieve the required stringency.

  In general, modification of one, two, or more amino acids in a protein does not affect the function of the protein. Indeed, a mutated or modified protein (ie, a peptide composed of an amino acid sequence in which one, two, or several amino acid residues have been modified by substitution, deletion, insertion, and / or addition) It is known to retain its 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)). Thus, those skilled in the art will recognize that a single amino acid or a small percentage of amino acids may be altered by individual additions, deletions, insertions or substitutions to the amino acid sequence, or protein changes resulting in proteins with similar functions. It will be appreciated that what is considered “modified” is permissible in the context of the present invention. Thus, in one embodiment, the peptides of the invention may have an amino acid sequence that is 1, 2, or even more amino acids added, inserted, deleted, and / or substituted in the ERCC6L sequence. .

  As long as the activity of the protein is maintained, the number of amino acid mutations is not particularly limited. However, it is generally preferred to change 5% or less of the amino acid sequence. Thus, in a preferred embodiment, the number of amino acids to be mutated in such variants is generally 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, more preferably 5 Or 6 amino acids or less, even more preferably 3 or 4 amino acids or less.

The amino acid residue to be mutated is preferably mutated to another amino acid that preserves the properties of the amino acid side chain (a process known as conservative amino acid substitution). Examples of amino acid side chain properties include 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: aliphatic side chains (G, A, V, L, I, P); hydroxyl group-containing side Chains (S, T, Y); sulfur atom-containing side chains (C, M); carboxylic acid and amide-containing side chains (D, N, E, Q); base-containing side chains (R, K, H); Aromatic-containing side chains (H, F, Y, W). 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 each other:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), glutamic acid (E);
3) Asparagine (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) Cysteine (C), Methionine (M) (see, eg, Creighton, Proteins 1984).

  Such conservatively modified polypeptides are included in the ERCC6L protein of the present invention. However, the present invention is not limited thereto, and the ERCC6L protein includes non-conservative modifications as long as at least one biological activity of the ERCC6L protein is retained. Furthermore, modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.

  Furthermore, the ERCC6L gene of the present invention encompasses polynucleotides that encode such functional equivalents of the ERCC6L protein. In order to isolate a polynucleotide encoding a polypeptide functionally equivalent to the ERCC6L protein, in addition to hybridization, it was synthesized based on the sequence information of DNA encoding the protein (SEQ ID NO: 9). A gene amplification method using a primer, for example, a polymerase chain reaction (PCR) method can be used. Polynucleotides and polypeptides that are functionally equivalent to the human ERCC6L gene and protein, respectively, usually have a high homology to the original nucleotide or amino acid sequence. “High homology” typically is 40% or more, preferably 60% or more, more preferably 80% or more, even more preferably 90% to 95% or more, even more preferably Refers to 96%, 97%, 98%, 99% or more homology. 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)”.

How to diagnose cancer:
The expression of ERCC6L gene was found to be specifically elevated in lung cancer (FIGS. 1A, B). Northern blot analysis showed that ERCC6L is not expressed in normal tissues but strongly expressed in lung cancer cell lines (FIG. 1C). Therefore, the ERCC6L genes identified herein and their transcripts and translation products are diagnostically useful as markers for cancers such as lung cancer by measuring the expression of ERCC6L in a sample. Cancer can be diagnosed or detected by comparing the expression level of the ERCC6L gene between a subject-derived sample and a normal sample. More specifically, the present invention detects, diagnoses, diagnoses the presence of cancer, more particularly the presence of ERCC6L-related cancer, or a predisposition to develop such cancer by measuring the expression level of ERCC6L in a subject. A method of determining and / or determining is provided.

Accordingly, the present invention provides a method of detecting or diagnosing cancer in a subject, the method comprising measuring the expression level of the ERCC6L gene in a biological sample from the subject, compared to the normal control level of the gene An increase in the level indicates that cancer cells are present or suspected in the sample, and then the subject is suffering from or develops cancer. It is suggested that there is a risk to
The expression level of the ERCC6L gene can be measured by any known method.
(A) detecting mRNA of the ERCC6L gene;
(B) detecting the protein encoded by the ERCC6L gene; and (c) detecting the biological activity of the protein encoded by the ERCC6L gene.

In a preferred embodiment, the cancer diagnosed by the methods of the present invention is lung cancer, including NSCLC and SCLC. NSCLC includes lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SCC).
In accordance with the present invention, intermediate results for examining a subject's condition may be provided. Such intermediate results can be combined with additional information to assist doctors, nurses, or other interested parties in diagnosing a subject as suffering from a disease. The present invention therefore contemplates the use of ERCC6L as a diagnostic marker for cancer.

  Alternatively, the present invention can be used to detect or identify cancerous cells in a tissue derived from a subject, wherein the expression level is increased compared to a normal control level of the gene. Such cells that indicate that cancer cells are present or suspected in the tissue. The results of ERCC6L expression can be combined with additional information to assist doctors, nurses, or other medical personnel in diagnosing a subject as suffering from a disease. In other words, the present invention may provide doctors with information useful for diagnosing a subject as suffering from a disease. For example, in accordance with the present invention, in the case of doubt about the presence of cancer cells in tissue taken from a subject, in addition to the expression level of the ERCC6L gene, histopathology, the level of a known tumor marker in the blood, Considering various aspects of the disease, including the clinical course of the subject and the like, a clinical decision can be reached. For example, some well-known diagnostic lung tumor markers in blood are IAP, ACT, BFP, CA19-9, CA50, CA72-4, CA130, CEA, KMO-1, NSE, SCC, SP1, Span-1 , TPA, CSLEX, SLX, STN, and CYFRA. That is, in this particular embodiment of the invention, the results of gene expression analysis serve as intermediate results for further diagnosis of the disease state of interest.

The following methods [1] to [10] are of particular interest for the present invention:
[1] A method for detecting or diagnosing cancer in a subject, comprising measuring the expression level of an ERCC6L gene in a biological sample derived from the subject, wherein the level is increased compared to a normal control level of the gene A method wherein the subject is shown to be suffering from or at risk of developing cancer.
[2] The method of [1], wherein the expression level is at least 10% higher than the normal control level.
[3] The expression level is
(A) detecting ERCC6L gene mRNA;
Detected by a method selected from (b) detecting a protein encoded by the ERCC6L gene, and (c) detecting a biological activity of the protein encoded by the ERCC6L gene [1] The method described.
[4] The method according to [1], wherein the cancer is lung cancer.
[5] The method according to [3], wherein the expression level is measured by detecting hybridization of a probe to a gene transcript of the gene.
[6] The method according to [3], wherein the expression level is measured as the expression level of the gene by detecting the binding of the antibody to the protein encoded by the gene.
[7] The method according to [1], wherein the biological sample includes a biopsy sample, sputum, or blood.
[8] The method according to [1], wherein the subject-derived biological sample contains epithelial cells.
[9] The method according to [1], wherein the subject-derived biological sample contains cancer cells.
[10] The method according to [1], wherein the subject-derived biological sample contains cancerous epithelial cells.

The method for diagnosing cancer of the present invention will be described in more detail below.
The subject to be diagnosed by the method of the present invention is preferably a mammal. Exemplary mammals include, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, and cows.

  It is preferable to make a diagnosis by collecting a biological sample from a subject to be diagnosed. Any biological material can be used as a biological sample for measurement as long as it contains the desired transcript or translation product of ERCC6L. Biological samples include body tissues where it is desired or suspected to be diagnosed with cancer, and body fluids such as biopsy samples, blood, sputum, and urine. It is not limited to. Preferably, the biological sample contains a cell population comprising epithelial cells, more preferably cancerous epithelial cells, or epithelial cells derived from a tissue suspected of being cancerous. Furthermore, if necessary, cells may be purified from the collected body tissue or body fluid, and then used as a biological sample. In preferred embodiments, the biological sample may comprise lung cells or lung tissue collected from a subject to be diagnosed.

  According to the present invention, the expression level of ERCC6L in a subject-derived biological sample is measured. Expression levels can be measured at the transcription (nucleic acid) product level using methods known in the art. For example, ERCC6L mRNA may be quantified using a probe by a hybridization method (eg, Northern hybridization). Detection can be performed on a chip or array. The use of an array is preferred for detecting the expression level of multiple genes (eg, various cancer specific genes) including ERCC6L. One skilled in the art can prepare such probes using the sequence information of ERCC6L. For example, ERCC6L cDNA may be used as a probe. If necessary, the probe may be labeled with an appropriate label such as a dye, a fluorescent substance, and an isotope, and the expression level of the gene may be detected as the intensity of the hybridized label.

  Furthermore, the transcription product of ERCC6L may be quantified using a primer by a detection method based on amplification (for example, RT-PCR). Such primers can also be prepared based on available sequence information of the gene. For example, the primers (SEQ ID NOs 1 and 2) used in the “Examples” may be used for detection by RT-PCR or Northern blot, but the present invention is not limited thereto.

  Specifically, the probe or primer used in the method of the present invention hybridizes with ERCC6L mRNA under stringent, moderately stringent, or low stringent conditions. As used herein, the phrase “stringent (hybridization) conditions” refers to conditions under which a probe or primer hybridizes to its target sequence but not to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of stringent conditions is selected to be about 5 ° C. lower than the melting temperature (Tm) of the specific sequence at a given ionic strength and pH. Tm is the temperature (at a given ionic strength, pH, and nucleic acid concentration) at which 50% of the probe complementary to the target sequence hybridizes with the target sequence at equilibrium. Since target sequences are generally present in excess, at Tm, 50% of the probe is occupied at equilibrium. Typically, stringent conditions include a salt concentration of less than about 1.0 M sodium ions at pH 7.0-8.3, typically about 0.01-1. 0 M, conditions for short probes or primers (eg, 10-50 nucleotides) at a temperature of at least about 30 ° C and for longer probes or primers at least about 60 ° C. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

  Alternatively, the translation product can be detected for the diagnosis of the present invention. For example, the amount of ERCC6L protein may be measured. The method for measuring the amount of the protein as a translation product includes an immunoassay using an antibody that specifically recognizes the protein. The antibody may be monoclonal or polyclonal. Furthermore, any fragment or modification of an antibody (eg, chimeric antibody, scFv, Fab, F (ab ′) 2, Fv, etc.) can be used for detection as long as the fragment retains the ability to bind to the ERCC6L protein. . Methods for preparing such types of antibodies for detecting proteins are well known in the art, and any method in the present invention can be used to prepare such antibodies and their equivalents .

  As another method for detecting the expression level of the ERCC6L gene based on its translation product, the staining intensity may be observed by immunohistochemical analysis using an antibody against the ERCC6L protein. That is, the observation of intense staining indicates an increase in the presence of the protein and at the same time a high expression level of the ERCC6L gene.

Furthermore, in order to improve the accuracy of diagnosis, in addition to the expression level of the ERCC6L gene, the expression level of other cancer-related genes such as genes that are known to be differentially expressed in cancer is measured. May be.
The expression level of a cancer marker gene comprising an ERCC6L gene in a biological sample is increased by, for example, 10%, 25%, or 50% from the control level of the corresponding cancer marker gene; or more than 1.1 fold , Over 1.5 times, over 2.0 times, over 5.0 times, over 10.0 times, or more, may be considered as rising.

The control level is measured at the same time as the test biological sample by using a sample previously collected and stored from one or more subjects with known disease state (cancerous or non-cancerous). May be. Alternatively, the control level may be determined by statistical methods based on results obtained by analyzing the pre-measured expression level of the ERCC6L gene in a sample from a subject whose disease state is known. . In addition, the control level may be a database of expression patterns from previously tested cells. Furthermore, according to one aspect of the present invention, the expression level of the ERCC6L gene in a biological sample may be compared to a plurality of control levels measured from a plurality of reference samples. It is preferred to use a control level measured from a reference sample derived from a tissue type similar to the tissue type of the subject-derived biological sample. Furthermore, it is preferable to use a reference value for the expression level of the ERCC6L gene in a population whose disease state is known. The reference value may be obtained by any method known in the art. For example, the average value +/− 2 S.I. D. Or mean +/- 3 S. D. Can be used as a reference value.
In the context of the present invention, a control level measured from a biological sample that has been found not to be cancerous is referred to as a “normal control level”. On the other hand, if the control level is measured from a cancerous biological sample, this is referred to as the “cancerous control level”.

  If the expression level of the ERCC6L gene is elevated compared to the normal control level or similar to the cancerous control level, the subject has cancer or is at risk of developing cancer Can be diagnosed. In addition, when comparing the expression levels of multiple cancer-related genes, because the gene expression pattern between the sample and the cancerous reference is similar, the subject is suffering from cancer, or It is indicated that there is a risk of developing cancer.

  The difference between the expression level of the test biological sample and the control level is the expression level of a control nucleic acid, such as a housekeeping gene, whose expression level is known not to vary depending on the cancerous or non-cancerous state of the cell. Can be normalized to. Exemplary control genes include, but are not limited to, β-actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1.

In the present invention, it is revealed that ERCC6L is not only a useful diagnostic marker but also a suitable target for cancer treatment. Therefore, cancer treatment targeting ERCC6L can be achieved by the present invention. In the present invention, cancer treatment targeting ERCC6L refers to suppression or inhibition of ERCC6L activity and / or expression in cancer cells. Any anti-ERCC6L agent can be used for cancer treatment targeting ERCC6L. In the present invention, the anti-ERCC6L agent includes the following substances or active ingredients:
(A) the double-stranded molecule of the present invention,
(B) DNA encoding it, and (c) a vector encoding it.

Accordingly, in a preferred embodiment, the present invention comprises (i) diagnosing whether a subject has a cancer to be treated with an anti-ERCC6L agent and / or (ii) subjecting the subject for cancer treatment targeting ERCC6L. Providing a method of selecting, the method comprising:
a) measuring the expression level of ERCC6L in cancer cells or tissues taken from a subject suspected of having a cancer to be treated;
b) comparing the expression level of ERCC6L with a normal control level;
c) diagnosing that the subject has a cancer to be treated when the expression level of ERCC6L is elevated compared to a normal control level; and d) the cancer to be treated by the subject in step c) Selecting the subject for cancer treatment.

Alternatively, such a method is
a) measuring the expression level of ERCC6L in cancer cells or tissues taken from a subject suspected of having a cancer to be treated;
b) comparing the expression level of ERCC6L with a cancerous control level;
c) diagnosing the subject as having a cancer to be treated if the expression level of ERCC6L is similar or equivalent to a cancerous control level; and d) treating the subject in step c) Selecting a subject for cancer treatment when diagnosed with having a cancer.

Kit for diagnosing cancer:
The present invention also provides a kit for diagnosing cancer, which may also be useful for evaluating and / or monitoring the effectiveness of cancer treatment. 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 a vector encoding the same, the kit also comprising such a cancer. It can also be useful to evaluate and / or monitor the effectiveness of a treatment. Preferably, the cancer diagnosed by the kit of the present invention is lung cancer including NSCLC and SCLC. More preferably, the kit comprises at least one reagent for detecting the expression level of the ERCC6L gene in a subject-derived biological sample, the reagent comprising:
(A) a reagent for detecting mRNA of the ERCC6L gene;
(B) a reagent for detecting ERCC6L protein; and (c) a reagent for detecting biological activity of ERCC6L protein.

  Suitable reagents for detecting ERCC6L gene mRNA include nucleic acids that specifically bind to or identify ERCC6L mRNA, such as oligonucleotides having a complementary sequence to a portion of ERCC6L mRNA. included. This type of oligonucleotide is exemplified by primers and probes that are specific for ERCC6L mRNA. Such types of oligonucleotides can be prepared based on methods well known in the art. If necessary, a reagent for detecting ERCC6L mRNA may be immobilized on a solid substrate. In addition, two or more reagents for detecting ERCC6L mRNA may be included in the kit.

  On the other hand, suitable reagents for detecting ERCC6L protein include antibodies against ERCC6L protein. The antibody may be monoclonal or polyclonal. Furthermore, any fragment or modified antibody (eg, chimeric antibody, scFv, Fab, F (ab ′) 2, Fv, etc.) can be used as a reagent as long as the fragment retains the ability to bind to the ERCC6L protein. . Methods for preparing such types of antibodies for detecting proteins are well known in the art, and any method in the present invention can be used to prepare such antibodies and their equivalents . In addition, the antibody may be labeled with a signal generating molecule by direct linkage or indirect labeling techniques. Labels and methods for labeling antibodies and detecting antibody binding to their targets are well known in the art, and any label and method may be used in the present invention. In addition, two or more reagents for detecting ERCC6L protein may be included in the kit.

  Further, for example, the biological activity can be measured by measuring the cell proliferation activity by the expressed ERCC6L protein in the biological sample. For example, measuring cell proliferation activity of a biological sample by culturing cells in the presence of a biological sample from the subject and then detecting the rate of proliferation or measuring the cell cycle or colony forming ability Can do. If necessary, a reagent for detecting ERCC6L mRNA may be immobilized on a solid substrate. In addition, more than one reagent for detecting the biological activity of the ERCC6L protein may be included in the kit.

  The kit can include two or more of the aforementioned reagents. In addition, the kit detects solid substrates and reagents for binding probes to the ERCC6L gene or antibodies to the ERCC6L protein, media and containers for culturing cells, positive and negative control reagents, and antibodies to the ERCC6L protein. Secondary antibodies. For example, a tissue sample taken from a subject with or without cancer can serve as a useful control reagent. The kits of the present invention comprise commercial and user, including buffers, diluents, filters, needles, syringes, and packaging inclusions (eg, documents, tapes, CD-ROMs, URLs, etc.) with instructions for use. It may further include other materials desirable from the viewpoint of the above. These reagents and the like can be provided in a labeled container. Suitable containers include bottles, vials, and test tubes. The container may be formed from a variety of materials such as glass or plastic.

  As one aspect of the present invention, when the reagent is a probe for ERCC6L mRNA, the reagent may be immobilized on a solid substrate such as a porous strip to form at least one detection site. The measurement or detection region of the porous strip can include multiple sites, each containing a nucleic acid (probe). The test strip can also include sites for negative and / or positive controls. Alternatively, the control site can be located on a separate strip from the test strip. Optionally, the different detection sites can contain different amounts of immobilized nucleic acid, ie, a larger amount of immobilized nucleic acid at the first detection site and a smaller amount of immobilized nucleic acid at subsequent sites. When a test sample is added, the number of sites that exhibit a detectable signal provides a quantitative indicator of the amount of ERCC6L mRNA present in the sample. The detection site may be configured in any shape that can be suitably detected and is typically in the shape of a bar or dot across the width of the test strip.

  The kit of the present invention may further comprise positive and / or negative control samples and / or ERCC6L standard samples. The positive control sample of the present invention can be prepared by collecting ERCC6L positive samples. Such ERCC6L positive samples include, for example, lung adenocarcinoma cell (ADC) strains such as A427, NCI-H1781, A549, LC319; lung squamous epithelium such as NCI-H26, EBC-1, NCI-H520, NCI-H2170 It may be obtained from established lung cancer cell lines, including cancer (SCC) cell lines; and SCLC cell lines such as DMS114, DMS273, SBC-3, SBC-5, H196, H446. Alternatively, ERCC6L positive samples may be obtained from clinical lung cancer tissues including lung line cancer tissue, lung squamous cell carcinoma tissue, and SCLC tissue. Alternatively, a positive control sample may be prepared by determining a cutoff value and preparing a sample containing an amount of ERCC6L mRNA or protein greater than the cutoff value. In this specification, the phrase “cutoff value” refers to a value that divides the normal range and the cancerous range. For example, one skilled in the art can determine a cutoff value using a receiver operating characteristic (ROC) curve. The kit of the present invention may comprise an ERCC6L standard sample comprising an amount of ERCC6L mRNA or polypeptide cutoff value. Conversely, a negative control sample may be prepared from a non-cancerous tissue such as a non-cancerous cell line or normal lung tissue, or by preparing a sample containing ERCC6L mRNA or protein below the cutoff value. May be prepared.

Alternatively, the present invention provides the use of a reagent for preparing a diagnostic reagent for diagnosing cancer. In some embodiments, the reagent is
(A) a reagent for detecting mRNA of the ERCC6L gene;
(B) a reagent for detecting ERCC6L protein; and (c) a reagent for detecting biological activity of ERCC6L protein.
Specifically, such a reagent is an oligonucleotide that hybridizes to an ERCC6L polynucleotide, or an antibody that binds to an ERCC6L polypeptide.

Anti-cancer screening:
Through the present invention, it was demonstrated that ERCC6L is involved in the growth of cancer cells. Therefore, substances that suppress the expression level of the ERCC6L gene and / or the biological activity of the ERCC6L polypeptide are expected to be useful for treating and / or preventing cancer. Such substances can be screened using the ERCC6L gene, a polypeptide encoded by the gene, or the transcriptional regulatory region of the gene. Therefore, the present invention also provides a method of screening candidate substances for treating and / or preventing cancer using the ERCC6L gene, ERCC6L polypeptide, or the transcriptional regulatory region of the gene.

  In the context of the present invention, the substance identified by the screening method of the present invention may be any compound or composition comprising several compounds. Furthermore, the test substance exposed to the cell or protein according to the screening method of the present invention may be a single substance or a combination of substances. When a combination of substances is used in the method, the substances may be contacted sequentially or simultaneously.

  The substance screened by the screening method of the present invention can be a candidate substance suitable for treating and / or preventing cancer and / or inhibiting the growth of cancer cells. In the present invention, the cancer is preferably characterized by its association with ERCC6L overexpression. Therefore, the substance to be screened can preferably be applied to a cancer that correlates or is associated with ERCC6L overexpression. In preferred embodiments, the cancer correlated or associated with ERCC6L overexpression is lung cancer, including NSCLC and SCLC. NSCLC includes, but is not limited to, lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SCC).

  In the screening method of the present invention, any test substance, for example, cell extract, cell culture supernatant, fermented microorganism product, marine organism extract, plant extract, purified protein or crude protein, peptide, non- Peptide compounds, synthetic micromolecular compounds (including nucleic acid constructs such as antisense RNA, siRNA, ribozymes, and aptamers), and natural compounds can be used. The test substance of the present invention comprises (1) a biological library method, (2) a spatially addressable parallel solid phase library method or a solution phase library method, and (3) a synthesis requiring deconvolution. Any of a number of approaches in combinatorial library methods known in the art, including library methods, (4) “one bead one compound” library methods, and (5) synthetic library methods using affinity chromatography selection. Can also be obtained. Biological library methods using affinity chromatography selection are limited to peptide libraries, but the other four approaches apply to peptide libraries of substances, non-peptide oligomer libraries, or small molecule libraries of compounds Yes (Lam, Anticancer Drug Des 1997, 12: 145-67). Examples of methods for synthesizing molecular libraries can be found in the art (DeWitt et al., Proc Natl Acad Sci USA 1993, 90: 6909-13, Erb et al., Proc Natl Acad Sci USA 1994, 91: 11422-6, Zuckermann et al., J Med Chem 37: 2678-85, 1994, Cho et al., Science 1993, 261: 1303-5, Carell et al., Angew Chem Int Ed Engl 1994, 33: 2059, Carell et al., Angew Chem Int Ed Engl 1994, 33: 2061, Gallop et al., J Med Chem 1994, 37: 1233-51). Compound libraries can be in solution (see Houghten, Bio / Techniques 1992, 13: 412-21) or beads (Lam, Nature 1991, 354: 82-4), chips (Fodor, Nature 1993, 364: 555-6), bacteria (US Pat. No. 5,223,409), spores (US Pat. Nos. 5,571,698, 5,403,484, and 5,223,409). ), Plasmid (Cull et al., Proc Natl Acad Sci USA 1992, 89: 1865-9) or phage (Scott and Smith, Science 1990, 249: 386-90, Devlin, Science 1990, 249: 404-6, Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82, Felici, J Mol Biol 1991, 222: 301-10, US Patent Application No. 2002103360).

A compound obtained by converting a part of the structure of a substance screened by any one of the screening methods of the present invention by addition, deletion, and / or substitution is included in the substance obtained by the screening method of the present invention. .
Further, when the candidate substance obtained by the screening method of the present invention is a protein, in order to obtain DNA encoding the protein, the entire amino acid sequence of the protein is determined, and the nucleic acid sequence encoding the protein is determined. Or analyzing a partial amino acid sequence of the obtained protein, preparing an oligo DNA as a probe based on the sequence, screening a cDNA library using the probe, and Encoding DNA may be obtained. The obtained DNA can be confirmed for its usefulness in the preparation of candidate substances for treating or preventing cancer.

The test substance used in the screening described herein may also be an antibody that specifically binds to the ERCC6L protein or a partial peptide thereof that lacks the biological activity of the original protein in vivo.
Although the construction of test substance libraries is well known in the art, further guidance regarding the identification of test substances and the construction of such substance libraries for the present screening methods is provided herein below.

(I) Molecular modeling:
Construction of a test substance library is facilitated by knowing the molecular structure of substances known to have the required properties and / or the molecular structure of the ERCC6L protein. One approach to prescreen test substances suitable for further evaluation is to use computer modeling of the interaction between the test substance and its target.
Computer modeling techniques allow visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new materials that are believed to interact with the molecule. Three-dimensional construction typically depends on X-ray crystallographic or NMR imaging data of selected molecules. Molecular dynamics requires force field data. A computer graphics system makes it possible to predict how a new substance will bind to a target molecule, allowing experimental manipulation of the structure of the substance and the target molecule to improve binding specificity. A user-friendly menu-style interface is usually provided between the molecular design program and the user to predict what will happen to a molecule-substance interaction when a minor change is made to one or both. There is also a need for molecular mechanics software and a computationally intensive computer.

  Examples of molecular modeling systems outlined above include the CHARMm program and the QUANTA program (Polygen Corporation, Waltham, Mass). CHARMm performs the functions of energy minimization and molecular dynamics. QUANTA performs molecular structure building, graphic modeling, and analysis. QUANTA allows interactive construction, modification, visualization, and behavioral analysis of molecules relative to each other.

  Numerous papers have published on computer modeling of drugs that interact with specific proteins, such as Rotivinen et al. Acta Pharmaceutica Fennica 1988, 97: 159-66, Ripka, New Scientist 1988, 54-8, McKinlay &; Rossmann, Annu Rev Pharmacol Toxiciol 1989, 29: 111-22, Perry & Davies, Prog Clin Biol Res 1989, 291: 189-93, Lewis & Dean, Proc R Soc Lond 1989, 236: 125-40, 141 -62, and Askew et al., J Am Chem Soc 1989, 111: 1082-90 for model receptors for nucleic acid components.

  Other computer programs for screening and schemating chemicals are available from BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc (Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario). For example, DesJarlais et al., J Med Chem 1988, 31: 722-9, Meng et al., J Computer Chem 1992, 13: 505-24, Meng et al., Proteins 1993, 17: 266-78, Shoichet et al., Science 1993, 259: 1445-50.

  As described below, once the putative inhibitor has been identified, any number of variants can be constructed based on the chemical structure of the identified putative inhibitor using combinatorial chemistry techniques. The resulting library of putative inhibitors or "test substances" is suitable for testing if the cancer is lung cancer, treating and / or preventing cancer and / or preventing postoperative recurrence of cancer Screening may be done using the methods of the invention for identifying substances.

(Ii) Combinatorial chemical synthesis:
A combinatorial library of test substances can be created as part of a rational drug design program that includes knowledge of the core structure present in known inhibitors. This approach makes it possible to maintain the library at an appropriate size, which facilitates high throughput screening. Alternatively, a simple, particularly short polymer molecular library may be constructed by simply synthesizing all the permutations of the molecular families that make up the library. An example of this latter approach is a library where all peptides are 6 amino acids long. Such a peptide library may contain a sequence permutation every 6 amino acids. This type of library is referred to as a linear combinatorial chemical library.

  The preparation of combinatorial chemical libraries is well known to those skilled in the art and can be made by either chemical synthesis or biological synthesis. Combinatorial chemical libraries include peptide libraries (eg, US Pat. No. 5,010,175, Furka, Int J Pept Prot Res 1991, 37: 487-93, Houghten et al., Nature 1991, 354: 84-6 Is included), but is not limited to. Other chemistries for creating chemical diversity libraries can also be used. Such chemistry includes peptides (eg, WO 91/19735), encoded peptides (eg, WO 93/20242), random bio-oligomers (eg, WO 92/00091), benzodiazepines (eg US Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines, and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90: 6909- 13), vinylogous polypeptide (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with glucose backbone (Hirschmann et al., J Amer Chem Soc 1992) , 114: 9217-8), similarity of small molecule library Organic synthesis (Chen et al., J. Amer Chem Soc 1994, 116: 2661), oligocarbamate (Cho et al., Science 1993, 261: 1303), and / or peptidylphosphonate (Campbell et al. al., J Org Chem 1994, 59: 658), nucleic acid library (Ausubel, Current Protocols in Molecular Biology 1995 supplement; Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory, New York, USA ), Peptide nucleic acid libraries (see, eg, US Pat. No. 5,539,083), antibody libraries (eg, Vaughan et al., Nature Biotechnology 1996, 14 (3): 309-14 , And International Application PCT / US96 / 10287), carbohydrate libraries (eg, Liang et al., Science 1996, 274: 1520-22, US Pat. No. 5,59). 853), as well as small organic libraries such as benzodiazepines (Gordon EM. Curr Opin Biotechnol. 1995 Dec 1; 6 (6): 624-31.), Isoprenoids (US Pat. No. 5,569). 588), thiazolidinone and metathiazoneone (US Pat. Nos. 5,549,974), pyrrolidine (US Pat. Nos. 5,525,735 and 5,519,134), morpholino compounds (US patents) No. 5,506,337), benzodiazepines (see US Pat. No. 5,288,514) and the like, but are not limited thereto.

  Equipment for the preparation of combinatorial libraries is commercially available (eg, 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster Cit, CA 90 (See Millipore, Bedford, MA). In addition, numerous combinatorial libraries are commercially available on their own (eg, ComGenex, Princeton, NJ, Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia). , MD etc.)

(Iii) Other candidates:
Another approach uses recombinant bacteriophage to create a library. This “phage method” (Scott & Smith, Science 1990, 249: 386-90, Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82, Devlin et al., Science 1990, 249: 404- 6) can be used to build very large libraries (eg 106-108 chemicals). The second approach uses primarily chemical methods, but is not limited to Geysen's 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 Pat. No. 4,631,211), And Rutter et al. (US Pat. No. 5,010,175) describe a method for producing a mixture of peptides that can be tested as agonists or antagonists.

Aptamers are macromolecules composed of nucleic acids that bind strongly to specific molecular targets. Tuerk and Gold (Science. 249: 505-510 (1990)) disclose a SELEX (systematic evolution of ligands by exponential enrichment) method for aptamer selection. In the SELEX method, a large library of nucleic acid molecules (eg, 10 ¹⁵ different molecules) can be used for screening.

I. Protein-Based Screening Method The present invention provides a method of screening candidate substances applicable to the treatment and / or prevention of cancer using ERCC6L polypeptide. In the context of the screening method of the present invention, the ERCC6L polypeptide used may be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier, or a fusion protein fused to another polypeptide. Furthermore, the ERCC6L polypeptide may be a recombinant polypeptide, a naturally derived protein, or a partial peptide thereof.

  As long as the modified peptide retains at least one biological activity of the original polypeptide, in addition to the native ERCC6L polypeptide, a functional equivalent of the polypeptide is also included in the ERCC6L polypeptide used in the screening of the present invention. May be included. Examples of biological activities of ERCC6L polypeptides include, but are not limited to, cell proliferation activity, helicase activity, ATPase activity, and Plk1 binding activity. Preferred examples of such functional equivalents are described above in the section entitled “ERCC6L gene and ERCC6L protein”. For example, preferred examples of such functional equivalents include polypeptides comprising the helicase domain of ERCC6L polypeptide (eg, 61-631 of SEQ ID NO: 10).

The polypeptide may be further linked to other substances as long as the ligation process and the linked substance do not interfere with the biological activity of the original polypeptide and / or fragment. Usable materials include, for example, peptides, lipids, sugars and sugar chains, acetyl groups, natural and synthetic polymers, and the like. Such types of modifications can be made to confer additional functions or to stabilize the polypeptides and fragments. Polypeptides used in the methods of the invention may be obtained from nature as natural proteins by conventional purification methods or through chemical synthesis based on selected amino acid sequences. For example, conventional peptide synthesis methods that can be employed in this synthesis include the following:
1) Peptide Synthesis, Interscience, New York, 1966;
2) The Proteins, Vol. 2, Academic Press, New York, 1976;
3) Peptide synthesis, Maruzen, 1975;
4) Fundamentals and experiments of peptide synthesis, Maruzen, 1985;
5) Development of pharmaceuticals (continued 14) Peptide synthesis, Hirokawa Shoten, 1991;
6) International Publication No. 99/67288; and 7) Barany G. & Merrifield RB, Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.

Alternatively, any known genetic engineering method for producing a polypeptide of interest may be adapted to obtain the protein (eg, Morrison J., J Bacteriology 1977, 132: 349-51; Clark -Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62). For example, first, an appropriate vector containing a polynucleotide encoding the protein of interest in a form that allows expression of the protein of interest (eg, downstream of a regulatory sequence containing a promoter) is prepared, transformed into an appropriate host cell, and then Host cells are cultured to produce the protein. More specifically, by inserting a gene encoding an ERCC6L polypeptide into a vector for expressing a foreign gene, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8, For example, it is expressed in animal cells. A promoter can be used for expression. For example, SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 1982, 83-141), EF-α promoter (Kim et al., Gene 1990, 91: 217-23 ), CAG promoter (Niwa et al., Gene 1991, 108: 193), RSV LTR promoter (Cullen, Methods in Enzymology 1987, 152: 684-704), SRα promoter (Takebe et al., Mol Cell Biol 1988, 8 : 466), CMV early promoter (Seed et al., Proc Natl Acad Sci USA 1987, 84: 3365-9), SV40 late promoter (Gheysen et al., J Mol Appl Genet 1982, 1: 385-94), Any commonly used promoter can be used, including adenovirus late promoter (Kaufman et al., Mol Cell Biol 1989, 9: 946), HSV TK promoter, and the like. Introduction of the vector into the host cell for expression of the ERCC6L polypeptide can be accomplished by any conventional method, such as electroporation (Chu et al., Nucleic Acids Res 1987, 15: 1311-26), calcium phosphate method (Chen et al., Mol Cell Biol 1987, 7: 2745-52), DEAE dextran method (Lopata et al., Nucleic Acids Res 1984, 12: 5707-17; Sussman et al., Mol Cell Biol 1985, 4: 1641- 3), 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) be able to.
ERCC6L polypeptides may also be produced in vitro using conventional in vitro translation systems.

(I) Screening for ERCC6L binding substance:
In the context of the present invention, overexpression of the ERCC6L gene was detected in lung cancer even though the ERCC6L gene was not expressed in normal organs (FIG. 1). Therefore, using the ERCC6L gene and the protein encoded thereby, the present invention provides a method of screening for a substance that binds to the ERCC6L polypeptide. Since ERCC6L is expressed in cancer, a substance that binds to ERCC6L polypeptide is expected to suppress the growth of cancer cells and thus be useful for treating and / or preventing cancer. Therefore, the present invention also provides a method for screening a candidate substance that suppresses the growth of cancer cells using the ERCC6L polypeptide, and a method for screening a candidate substance for treating or preventing cancer. In particular, one aspect of this screening method is:
(A) contacting a test substance with an ERCC6L polypeptide;
(B) detecting a binding activity between the polypeptide and the test substance; and (c) selecting a test substance that binds to the polypeptide.

Alternatively, according to the present invention, the potential therapeutic effect of a test substance for treating and / or preventing cancer can also be evaluated or estimated. In certain embodiments, the present invention provides methods for assessing or estimating the therapeutic effect of a test substance to treat and / or prevent cancer and / or inhibit cancer associated with overexpression of ERCC6L; The method
(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 test substance with a potential therapeutic effect exhibited when the substance binds to the polypeptide. Including.

In the context of the present invention, the therapeutic effect can be correlated with the binding level of the test substance and the ERCC6L protein. For example, when the test substance binds to the ERCC6L protein, the test substance can be identified or selected as a candidate substance having the required therapeutic effect. Alternatively, if the test substance does not bind to the ERCC6L protein, the test substance can be characterized as not having a significant therapeutic effect.
The method of the present invention will be described in more detail below.

The ERCC6L polypeptide used for screening may be a recombinant polypeptide or a naturally derived protein, or a partial peptide thereof. The polypeptide to be contacted with the test substance may be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier, or a fusion protein fused with another polypeptide.
In a preferred embodiment, the test substance used according to the present invention may be a protein such as an antibody or a synthetic compound. As a method for screening for a substance that binds to the ERCC6L polypeptide, many methods well known to those skilled in the art may be used. Such screening can be performed, for example, by immunoprecipitation.

When using immunoprecipitation, the ERCC6L polypeptide preferably contains an antibody recognition site. The ERCC6L polypeptide used in the screening method of the present invention can be prepared as described above.
Alternatively, the polypeptide encoded by the ERCC6L gene can be obtained by introducing a monoclonal antibody recognition site (epitope) whose specificity has been clarified into the N-terminus or C-terminus of the polypeptide. It can be expressed as a fusion protein containing. A commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (1995)). Vectors that can express a fusion protein with, for example, β-galactosidase, maltose-binding protein, glutathione S-transferase, green fluorescent protein (GFP) and the like by using the plurality of cloning sites are commercially available. In addition, a fusion protein prepared by introducing only a small epitope consisting of several amino acids to 12 amino acids so that the characteristics of the ERCC6L polypeptide does not change by fusion has also been reported. Epitopes such as polyhistidine (His tag), influenza agglutinin HA, human c-myc, FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7 tag), human herpes simplex virus glycoprotein ( HSV tags), E tags (epitopes on monoclonal phage), etc., and monoclonal antibodies that recognize them can be used as an epitope-antibody system for screening proteins that bind to ERCC6L polypeptide (Experimental Medicine 13 : 85-90 (1995)).

  In immunoprecipitation, immune complexes are formed by adding these antibodies to cell lysates prepared using an appropriate detergent. The immune complex consists of an ERCC6L polypeptide, a polypeptide having the ability to bind to the polypeptide, and an antibody. In addition to the use of antibodies against the above epitopes, immunoprecipitation can also be performed using antibodies against ERCC6L polypeptides, which can be prepared as described above. When the antibody is a mouse IgG antibody, the immune complex can be precipitated, for example, with protein A sepharose or protein G sepharose. When the polypeptides encoded by the ERCC6L gene are prepared as fusion proteins with epitopes such as GST, the immune complex is a substance that specifically binds to these epitopes, similar to the use of antibodies against ERCC6L polypeptides. For example, it can be formed using glutathione-Sepharose 4B.

Immunoprecipitation can be performed, for example, according to or according to the method of 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 an appropriate concentration of gel. Since proteins bound to ERCC6L polypeptide are difficult to detect by common staining methods such as Coomassie or silver staining, cells in culture media containing a radioisotope, ³⁵ S-methionine or ³⁵ S-cysteine are used. The detection sensitivity for the protein can be improved by culturing the protein, labeling the protein in the cell, and detecting the protein. If the molecular weight of the protein is known, the target protein can be purified directly from an SDS-polyacrylamide gel and its sequence can be determined.

  As a method for screening a protein binding to the polypeptide using the ERCC6L polypeptide, West-Western blot analysis (Skolnik et al., Cell 65: 83-90 (1991)) can be used. Specifically, a phage vector (eg, ZAP) is used to prepare a cDNA library from cultured cells that are predicted to express a protein that binds to an ERCC6L polypeptide, and the protein is expressed on LB-agarose. By immobilizing the expressed protein on a filter, reacting the purified and labeled ERCC6L polypeptide with the above filter, and detecting plaques expressing the protein bound to the ERCC6L polypeptide according to the label, the ERCC6L polypeptide Proteins that bind can be obtained. The polypeptide of the present invention utilizes an antibody that specifically binds to a peptide or polypeptide (for example, GST) fused to ERCC6L or ERCC6L polypeptide by utilizing the binding between biotin and avidin. Can be labeled. A method using a radioisotope or fluorescence may also be used.

  Alternatively, in another aspect of the screening method of the present invention, a cell-based two-hybrid system may be used (“MATCHMAKER two-hybrid system”, “mammalian MATCHMAKER two-hybrid assay kit”, “MATCHMAKER one-hybrid system” ( Clontech); “HybriZAP two-hybrid vector system” (Stratagene); references “Dalton and Treisman, Cell 68: 597-612 (1992)”, “Fields and Sternglanz, Trends Genet 10: 286-92 (1994)”).

  In the two-hybrid system, the polypeptide of the present invention is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells. A cDNA library is prepared from cells predicted to express a protein that binds to the polypeptide of the present invention so that when the library is expressed, it fuses with the VP16 or GAL4 transcriptional activation region. Next, this cDNA library is introduced into the above yeast cell, and the cDNA derived from the library is isolated from the detected positive clone (when the protein that binds to the polypeptide of the present invention is expressed in the yeast cell, The combination of the two activates the reporter gene and positive clones are detected). The protein encoded by the cDNA can be prepared by introducing the cDNA isolated as described above into E. coli and expressing the protein. As the reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and the like can be used in addition to the HIS3 gene.

  Substances that bind to the ERCC6L polypeptide may be screened using affinity chromatography. For example, the ERCC6L polypeptide can be immobilized on an affinity column carrier and a composition containing the test substance can be applied to the column. The composition herein may be, for example, a cell extract, cell lysate, antibody library, and the like. After loading the test substance, the column can be washed and the substance bound to the ERCC6L polypeptide can be recovered. When 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, a cDNA library is screened using the oligo DNA as a probe, Obtain DNA encoding the protein.

  A biosensor using the surface plasmon resonance phenomenon may be used in the present invention as a means for detecting or quantifying the bound substance. When such a biosensor is used, the interaction between the ERCC6L polypeptide and the test substance can be observed in real time as a surface plasmon resonance signal using only a very small amount of polypeptide and without labeling. (For example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the ERCC6L polypeptide and the test substance using a biosensor such as BIAcore.

  Methods for screening molecules that bind when an immobilized ERCC6L polypeptide is exposed to synthetic chemicals, natural material banks, or random phage peptide display libraries, and chemicals as well as proteins that bind to ERCC6L protein High-throughput screening methods based on combinatorial chemistry techniques to isolate (including agonists and antagonists) (Wrighton et al., Science 273: 458-64 (1996); Verdine, Nature 384: 11-13 (1996); Hogan, Nature 384: 17-9 (1996)) are well known to those skilled in the art.

(Ii) Screening for substances that inhibit the biological activity of ERCC6L:
In the context of the present invention, the ERCC6L protein is characterized as having activity that promotes cell proliferation of cancer cells (FIG. 3). Using this biological activity as an indicator, the present invention provides a method for screening a substance that suppresses the growth of cancer cells that express ERCC6L, as well as treating ERCC6-L-related cancers such as cancer, particularly lung cancer, and A method of screening for a substance for prevention is provided. Accordingly, the present invention provides a method for screening a candidate substance for treating and / or preventing cancer using a polypeptide encoded by the ERCC6L gene, comprising the following steps:
(A) contacting a test substance with a polypeptide encoded by a polynucleotide of ERCC6L (ERCC6L polypeptide) or a fragment thereof;
(B) detecting the biological activity of said polypeptide or fragment of step (a); and (c) a polypeptide encoded by a polynucleotide of ERCC6L detected in the absence of the test substance ( Selecting a test substance that inhibits the biological activity of the polypeptide or fragment as compared to the biological activity of the ERCC6L polypeptide).

According to the present invention, the therapeutic effect of a test substance in suppressing the biological activity (eg, cell proliferation activity) of ERCC6L, or the therapeutic effect of a candidate substance for treating and / or preventing cancer can be evaluated. Therefore, the present invention also provides a method for screening a candidate substance that suppresses the biological activity of ERCC6L or a candidate substance for treating and / or preventing cancer using ERCC6L polypeptide or a fragment thereof, The method includes the following steps:
a) contacting a test substance with an ERCC6L polypeptide or functional fragment thereof; and b) detecting the biological activity of said polypeptide or fragment of step (a); and c) biological of b) Correlating activity with the therapeutic effect of the test substance.

Alternatively, in certain embodiments, the present invention provides methods for assessing or estimating the therapeutic effect of a test substance in treating and / or preventing cancer and / or inhibiting cancer growth associated with ERCC6L overexpression. The method
(A) contacting the test substance with an ERCC6L polypeptide or a functional fragment thereof;
(B) detecting the biological activity of the polypeptide or fragment of step (a); and (c) the substance is encoded by a polynucleotide of the ERCC6L gene detected in the absence of the test substance. Correlating the test substance with a potential therapeutic effect shown when inhibiting the biological activity of the polypeptide as compared to the biological activity of the polypeptide.

Such cancers include lung cancer.
In the context of the present invention, the therapeutic effect can be correlated with the biological activity of the ERCC6L polypeptide or functional fragment thereof. For example, if the test substance suppresses or inhibits the biological activity of the ERCC6L polypeptide or functional fragment thereof as compared to the level detected in the absence of the test substance, the test substance is Can be identified or selected as a candidate substance having a therapeutic effect. Alternatively, if the test substance does not suppress or inhibit the biological activity of the ERCC6L polypeptide or functional fragment thereof as compared to the level detected in the absence of the test substance, the test substance is It can be identified as a substance that has no significant therapeutic effect.
The method of the present invention will be described in more detail below.

  Any polypeptide can be used for screening as long as it suppresses the biological activity of the ERCC6L protein. Such biological activities include cell-proliferating activity of ERCC6L protein (herein, “cell proliferative activity”, “cell proliferation enhancing activity”), Or “cell proliferation promoting activity”), helicase activity, ATPase activity, and Plk1 binding activity. For example, ERCC6L protein can be used, and polypeptides functionally equivalent to these proteins can also be used. Such polypeptides can be expressed endogenously or exogenously by the cell.

In another aspect, the present invention also provides a screening method following the method described in the “Screening” section above, the method comprising:
a) contacting a test substance with an ERCC6L polypeptide or fragment thereof;
b) detecting binding between the polypeptide or fragment and the test substance;
c) selecting a test substance that binds to the polypeptide;
d) contacting the test substance selected in step c) with an ERCC6L polypeptide or 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) a test substance that inhibits the biological activity of the polypeptide. Selecting 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 the ERCC6L gene. The term “antagonist” refers to a molecule that inhibits its function by binding to a polypeptide. The term also refers to a molecule that decreases or inhibits the expression of the gene encoding ERCC6L. In addition, substances isolated by this screening are candidates for substances that inhibit the in vivo interaction of ERCC6L polypeptides and molecules (including DNA and proteins).

  When the biological activity detected in the method of the present invention is cell proliferation, for example, a cell expressing ERCC6L polypeptide is prepared, the cell is cultured in the presence of a test substance, and the rate of cell proliferation It can be detected by determining the cell cycle, etc., and by measuring the viable cell or colony forming activity shown, for example, in FIG. A substance that decreases the growth rate of cells expressing ERCC6L is selected as a candidate substance for treating or preventing cancer.

More specifically, the method comprises
(A) contacting the test substance with a cell overexpressing ERCC6L;
(B) measuring cell proliferation activity; and (c) selecting a test substance that reduces cell proliferation activity compared to cell proliferation activity in the absence of the test substance.
In a preferred embodiment, the method of the invention comprises:
(D) The method may further comprise selecting a test substance that does not affect cells that do not express or hardly express ERCC6L.

  The phrase “suppressing biological activity” as defined herein preferably means at least 10% inhibition, more preferably at least 25%, of the biological activity of ERCC6L compared to the absence of the substance. %, 50%, or 75% inhibition, and most preferably 90% inhibition.

In a preferred embodiment, control cells that do not express ERCC6L polypeptide are used. Therefore, the present invention also provides a method for screening a candidate substance that inhibits cell proliferation or a candidate substance for treating and / or preventing ERCC6L-related diseases using ERCC6L polypeptide or a fragment thereof, as described below. Including the following stages:
a) culturing cells expressing an ERCC6L polypeptide or functional fragment thereof and control cells not expressing the ERCC6L polypeptide or functional fragment thereof in the presence of a test substance;
b) detecting the biological activity of cells expressing the protein and control cells; and c) expressing the protein in control cells and compared to growth detected in the absence of the test substance. Selecting a test substance that inhibits biological activity in the cell.
As demonstrated herein, inhibiting ERCC6L biological activity reduces cell proliferation. Therefore, by screening candidate substances that inhibit the biological activity of ERCC6L, it is possible to identify candidate substances that have the potential to treat and / or prevent cancer. The likelihood that these candidate substances will treat or prevent cancer can be assessed by secondary and / or further screening to identify therapeutic substances, therapeutic compounds, or therapeutic agents for cancer. For example, if a substance that inhibits the biological activity of ERCC6L protein also inhibits the activity of cancer, it can be concluded that such substance has an ERCC6L-specific therapeutic effect.

II. Screening for substances that alter ERCC6L expression:
In the context of the present invention, reduction of ERCC6L expression by siRNA causes inhibition of cancer cell growth (FIG. 2ABC). Therefore, the present invention provides a method for screening for a substance that inhibits the expression of ERCC6L. Substances that inhibit the expression of ERCC6L are expected to suppress the growth of cancer cells and are therefore useful for treating or preventing cancer, particularly ERCC6L-related cancers such as lung cancer. Therefore, the present invention also provides a method for screening a substance that suppresses the growth of cancer cells, and a method for screening a candidate substance for treating or preventing cancer. In the context of the present invention, such screening may include, for example, the following steps:
(A) contacting a test substance with a cell expressing ERCC6L; and (b) selecting a test substance that reduces the expression level of ERCC6L compared to a control.

In the context of the present invention, such screening may include, for example, the following steps:
a) contacting the 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.

In the context of the present invention, the therapeutic effect can be correlated with the expression level of the ERCC6L gene. For example, if the test substance reduces the expression level of the ERCC6L gene compared to the level detected in the absence of the test substance, the test substance is identified or selected as a candidate substance having a therapeutic effect Can do. Alternatively, if the test substance does not reduce the expression level of the ERCC6L gene compared to the level detected in the absence of the test substance, the test substance is regarded as a substance that does not have a significant therapeutic effect. Can be identified.
The method of the present invention will be described in more detail below.

  Cells expressing ERCC6L include, for example, cell lines established from lung cancer or cell lines transfected with an ERCC6L expression vector; any such cells can be used in the above screening of the present invention. . Expression levels can be assessed by methods well known to those skilled in the art, such as RT-PCR, Northern blot assay, Western blot assay, immunostaining, and flow cytometric analysis. The phrase “decrease expression level” as defined herein is preferably a reduction of at least 10%, more preferably at least 25%, of the expression level of ERCC6L compared to the expression level in the absence of the substance. , 50%, or 75% level reduction, and most preferably at least 95% level reduction. Substances in the present specification include compounds, double-stranded nucleotides and the like. The preparation of double stranded nucleotides is described below. In this screening method, a substance that decreases the expression level of ERCC6L can be selected as a candidate substance used for the treatment or prevention of cancer.

Alternatively, the screening method of the present invention may comprise the following steps:
(A) contacting a test substance with a cell into which a vector containing a transcriptional regulatory region of ERCC6L and a reporter gene expressed under the control of the transcriptional regulatory region is introduced;
(B) measuring the expression or activity of the reporter gene; and (c) selecting a test substance that decreases the expression or activity of the reporter gene.

  Suitable reporter genes and host cells are well known in the art. Exemplary reporter genes include luciferase, green fluorescent protein (GFP), red sea anemone (Discosoma sp.) Red fluorescent protein (DsRed), chloramphenicol acetyltransferase (CAT), lacZ, and β-glucuronidase (GUS) The host cell is COS7, HEK293, HeLa and the like. The reporter construct required for screening can be prepared by linking the reporter gene sequence to the transcriptional regulatory region of the ERCC6L gene. In the present specification, the transcriptional regulatory region of the ERCC6L gene is a region from the transcription start site to at least 500 bp upstream, preferably 1000 bp, more preferably 5000 or 10,000 bp upstream. Nucleotide segments containing transcriptional regulatory regions can be isolated from genomic libraries or can be amplified by PCR. The reporter construct required for screening can be prepared by linking the reporter gene sequence to the transcriptional regulatory region of the ERCC6L gene. Methods for identifying transcriptional regulatory regions and assay protocols are also well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).

  A vector containing the reporter construct is introduced into a host cell, and the expression or activity of the reporter gene is detected by a method well known in the art (for example, using a luminometer, absorptiometer, flow cytometer, etc.). As used herein, “reducing expression or activity” preferably means a decrease of at least 10%, more preferably at least 25%, of the expression or activity of a reporter gene compared to the absence of a test substance. , 50%, or 75% reduction, and most preferably at least 95% reduction.

Alternatively, in certain embodiments, the present invention also provides a method of assessing or estimating the therapeutic effect of a test substance on cancer treatment or prevention, or inhibition of cancer associated with ERCC6L overexpression, the method comprising: ,
(A) contacting a test substance with a cell into which a vector containing a transcriptional regulatory region of ERCC6L and a reporter gene expressed under the control of the transcriptional regulatory region is introduced;
(B) measuring the expression level or activity of the reporter gene; and (c) correlating the test substance with a potential therapeutic effect shown when the test substance decreases the expression level or activity of the reporter gene. Including stages.

  According to the present invention, the therapeutic effect of a test substance on the inhibition of cell proliferation or the therapeutic effect of a candidate substance for treating or preventing an ERCC6L-related disease can be evaluated. Therefore, the present invention also provides a method for screening a candidate substance that suppresses the growth of cancer cells and a method for screening a candidate agent for treating or preventing an ERCC6L-related disease.

According to another aspect, the present invention provides a method comprising the following steps:
(A) contacting a test substance with a cell into which a vector composed of a transcriptional regulatory region of the ERCC6L gene and a reporter gene expressed under the control of the transcriptional regulatory region is introduced;
(B) detecting the expression level or activity of the reporter gene; and (c) correlating the expression level of (b) with the therapeutic effect of the test substance.

  In the present invention, the therapeutic effect can be correlated with the expression level or activity of the reporter gene. For example, if the test substance reduces the expression level or activity of the reporter gene compared to the level detected in the absence of the test substance, the test substance is used as a candidate substance having a therapeutic effect. Can be identified or selected. Alternatively, if the test substance does not reduce the expression level or activity of the reporter gene compared to the level detected in the absence of the test substance, the test substance has a significant therapeutic effect. Can be identified as non-existing substances.

  (I) binds to an ERCC6L polypeptide; (ii) suppresses / reduces biological activity (eg, cell proliferation activity) of the ERCC6L polypeptide; or (iii) a candidate substance that decreases the expression level of the ERCC6L gene. By screening, a candidate substance having a possibility of treating or preventing cancer (for example, lung cancer) can be identified. The therapeutic potential of these candidate substances can be assessed by secondary and / or further screening to identify therapeutic substances for cancer. For example, if a substance that binds to an ERCC6L polypeptide inhibits the above activity of cancer, it can be concluded that such substance has an ERCC6L-specific therapeutic effect.

Double-stranded molecule:
As used herein, the term “isolated double-stranded molecule” refers to a nucleic acid molecule that inhibits expression of a target gene, eg, small interfering RNA (siRNA; eg, double-stranded ribonucleic acid ( dsRNA) or small hairpin RNA (shRNA)) and small interfering DNA / RNA (siD / R-NA; for example, DNA and RNA double stranded chimera (dsD / R-NA) or DNA and RNA small hairpin Chimera (shD / R-NA)).

  As used herein, a “target sequence” is a gene sequence that results in suppression of translation of the entire mRNA when a double-stranded nucleic acid molecule targeting the sequence is introduced into a cell that expresses the gene. A nucleotide sequence within an mRNA or cDNA sequence. When a double-stranded polynucleotide containing a sequence corresponding to the target sequence inhibits the expression of the gene in a cell expressing the gene, the nucleotide sequence in the mRNA or cDNA sequence of the gene is determined to be the target sequence Can be done. A double-stranded polynucleotide that suppresses gene expression may consist of a target sequence and a 3 ′ overhang (eg, uu) 2 to 5 nucleotides in length.

When the target sequence is indicated by a cDNA sequence, a sense strand sequence of double-stranded cDNA, that is, a sequence in which an mRNA sequence is converted into a DNA sequence is used to define the target sequence. The double-stranded molecule consists of a sense strand having a sequence corresponding to the target sequence and an antisense strand having a complementary sequence to the target sequence, and the antisense strand hybridizes in the complementary sequence with the sense strand. Form.
In the present specification, the phrase “corresponding to” means that the target sequence is converted according to the type of nucleic acid constituting the sense strand of the double-stranded molecule. For example, when the target sequence is represented by a DNA sequence and the sense strand of a double-stranded molecule has an RNA region, the base “t” in the RNA region is replaced with the base “u”. On the other hand, when the target sequence is represented by an RNA sequence and the sense strand of the double-stranded molecule has a DNA region, the base “u” in the DNA region is replaced with the base “t”. In the context of the present invention, the target sequence is mainly represented by DNA. In other words, the present invention also includes a double-stranded molecule whose target sequence comprises or is limited to SEQ ID NO: 7 or SEQ ID NO: 8, which is represented by DNA but can be replaced by RNA. provide.

Similarly, with respect to the antisense strand of a double-stranded molecule, a complementary sequence to the target sequence can be defined depending on the type of nucleic acid constituting the antisense strand.
A double-stranded molecule has one or two 3 ′ overhangs (eg, uu) having a length of 2-5 nucleotides and / or a sense strand in addition to the sequence corresponding to the target sequence and its complementary sequence. It may have a loop sequence that links the antisense strand to form a hairpin structure.

  As used herein, the term “siRNA” refers to a double-stranded RNA molecule that prevents translation of a target mRNA. Standard techniques for introducing siRNA into cells are used, including techniques where DNA is the 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 a sense nucleic acid sequence of the target gene and a complementary antisense nucleic acid sequence, such as a hairpin. The siRNA can be either dsRNA or shRNA.

  As used herein, the term “dsRNA” is a construct of two RNA molecules that are composed of complementary sequences to each other and are annealed together through complementary sequences to form a double-stranded RNA molecule. Point to. Double-stranded nucleotide sequences include not only “sense” or “antisense” RNA selected from the protein coding sequence of the target gene sequence, but also RNA molecules having nucleotide sequences selected from non-coding regions of the target gene. May include.

  As used herein, the term “shRNA” refers to a siRNA having a stem-loop structure consisting of a first region and a second region that are complementary to each other, ie, a sense strand and an antisense strand. The degree of region complementarity and directivity is sufficient for base pairing to occur between the regions, the first region and the second region being joined by a loop region, and the nucleotides (or nucleotides) in the loop region. The lack of base pairing between the analogues) results in a loop. The loop region of shRNA is a single-stranded region interposed between the sense strand and the antisense strand, and may also be referred to as “intervening single strand”.

  As used herein, the term “siD / R-NA” refers to a double-stranded polynucleotide molecule consisting of both RNA and DNA, including RNA and DNA hybrids and chimeras that interfere with the translation of the target mRNA. To do. In this specification, a hybrid is a molecule in which a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize with each other to form a double-stranded molecule, while a chimera constitutes a double-stranded molecule. One or both of the strands to be able to contain RNA and DNA. Standard techniques for introducing siD / R-NA into cells 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. SiD / R-NA can be constructed such that a single transcript has both a sense and complementary antisense nucleic acid sequence from the target gene, such as a hairpin. The siD / R-NA can be either dsD / R-NA or shD / R-NA.

  As used herein, the term “dsD / R-NA” refers to two sequences that are complementary to each other and are annealed together through complementary sequences to form a double-stranded polynucleotide molecule. Refers to a molecular construct. The double-stranded nucleotide sequence includes not only a “sense” or “antisense” polynucleotide sequence selected from the protein coding sequence of the target gene sequence, but also a polynucleotide having a nucleotide sequence selected from a non-coding region of the target gene. Nucleotides may also be included. One or both of the two molecules that make up dsD / R-NA consist of both RNA and DNA (chimeric molecules), or alternatively one of the molecules consists of RNA and the other consists of DNA (hybrid) Double stranded).

The term “shD / R-NA” as used herein has a stem-loop structure consisting of a first region and a second region that are complementary to each other, ie, the sense strand and the antisense strand. Refers to siD / R-NA. The degree of region complementarity and directivity is sufficient for base pairing to occur between the regions, the first region and the second region being joined by a loop region, and nucleotides (or nucleotides) within the loop region. The lack of base pairing between the analogues) results in a loop. The loop region of shD / R-NA is a single-stranded region interposed between the sense strand and the antisense strand, and may also be referred to as “intervening single strand”.
As used herein, an “isolated nucleic acid” is a nucleic acid that has been removed from its original environment (eg, the natural environment if it is naturally occurring) and thus artificially altered from its natural state. In the context of the present invention, examples of isolated nucleic acids include DNA, RNA, and derivatives thereof.

A double-stranded molecule against the ERCC6L gene that hybridizes to the target mRNA normally binds to a single-stranded mRNA transcript of the gene, thereby preventing translation of the ERCC6L protein and thus inhibiting expression of the protein, Reduce or inhibit the production of ERCC6L protein encoded by the ERCC6L gene. As described herein, the expression of the ERCC6L gene in several cancer cell lines was inhibited by dsRNA (FIG. 2). Accordingly, the present invention provides an isolated double-stranded molecule that can inhibit the expression of the gene when introduced into a cell that expresses the ERCC6L gene. The target sequence of the double-stranded molecule can be designed by siRNA design algorithms such as those mentioned below.
Examples of ERCC6L target sequences include nucleic acids such as SEQ ID NO: 7 or 8.

The following double-stranded molecules [1] to [18] are of particular interest in the present invention:
[1] An isolated double-stranded molecule that inhibits in vivo expression and cell proliferation of ERCC6L when introduced into a cell, and is complementary to a sense strand that hybridizes to each other to form a double-stranded molecule An isolated double-stranded molecule composed of a unique antisense strand;
[2] Double-stranded molecule according to [1], which acts on mRNA corresponding to the target sequence of SEQ ID NO: 7 or 8;
[3] The double-stranded molecule according to [2], wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence of SEQ ID NO: 7 or 8;
[4] The double-stranded molecule according to [3], wherein the sense strand hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of less than about 100 nucleotide pairs;
[5] The double-stranded molecule according to [4], wherein the sense strand hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of less than about 75 nucleotide pairs;
[6] The double-stranded molecule according to [5], wherein the sense strand hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of less than about 50 nucleotide pairs;
[7] The double-stranded molecule according to [6], wherein the sense strand hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of less than about 25 nucleotide pairs;
[8] The double-stranded molecule according to [7], wherein the sense strand hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of about 19 to about 25 nucleotide pairs;
[9] The double-stranded molecule according to [1], composed of a single polynucleotide having both a sense strand and an antisense strand linked by an intervening single strand;
[10] General formula 5 ′-[A]-[B]-[A ′]-3 ′
Wherein [A] is a sense strand comprising a nucleotide sequence corresponding to a target sequence of SEQ ID NO: 7 or 8, is an intervening single strand composed of 3 to 23 nucleotides, and [A ′] is a double-stranded molecule according to [9], which is an antisense strand comprising a sequence complementary to the target sequence;
[11] The double-stranded molecule according to [1], composed of RNA;
[12] The double-stranded molecule according to [1] composed of both DNA and RNA;
[13] The double-stranded molecule according to [12], which is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[14] The double-stranded molecule according to [13], wherein the sense strand and the antisense strand are each composed of DNA and RNA;
[15] The double-stranded molecule according to [12], which is a chimera of DNA and RNA;
[16] The region adjacent to the 3 ′ end of the antisense strand, or both the region adjacent to the 5 ′ end of the sense strand and the region adjacent to the 3 ′ end of the antisense strand are RNA Double-stranded molecule;
[17] The double-stranded molecule according to [16], wherein the adjacent region is composed of 9 to 13 nucleotides; and [18] the double-stranded molecule according to [2], comprising a 3 ′ overhang.

The double-stranded molecule of the present invention is described in more detail below.
Methods for designing double-stranded molecules having the ability to inhibit target gene expression in cells are known. (See, eg, US Pat. No. 6,506,559, which is hereby incorporated by reference in its entirety.) For example, a computer program for designing siRNAs can be found on the Ambion website (http: / /www.ambion.com/techlib/misc/siRNA_finder.html).
The computer program selects the target nucleotide sequence for the double-stranded molecule based on the following protocol.

Target site selection:
1. Search downstream for AA dinucleotide sequences, starting with the AUG start codon of the transcript. Record the occurrence of individual AA and the 3 ′ adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al. 5 'and 3' untranslated regions (UTR) and regions near the start codon (within 75 bases) may have more regulatory protein binding sites, and UTR binding protein and / or translation initiation. SiRNA design against these is not recommended, as the complex can interfere with the binding of the siRNA endonuclease complex.
2. Potential target sites are compared to the appropriate genomic database (human, mouse, rat, etc.) and all target sequences that have significant homology with other coding sequences are excluded from consideration. Basically, BLAST obtained 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. A target sequence that is eligible for synthesis is selected. It is common to select and evaluate several target sequences along the length of the gene.

Using the above protocol, the target sequence of the isolated double-stranded molecule of the present invention was designed as SEQ ID NOs: 7 and 8 for the ERCC6L gene.
Each double-stranded molecule targeting the above target sequence was examined for its ability to inhibit the growth of cells expressing the target gene. Accordingly, the present invention provides double stranded molecules targeting the sequences of SEQ ID NO: 7 and 8 with respect to the 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 multiple target ERCC6L gene sequences.

  The double-stranded molecule of the present invention targeting the above target sequence of the ERCC6L gene comprises an isolated polynucleotide containing the nucleic acid sequence of the target sequence and / or a complementary sequence to the target sequence. Examples of polynucleotides that target the ERCC6L gene include those containing the sequence of SEQ ID NO: 7 or 8, and / or complementary sequences to these nucleotides; however, the invention is limited to this example As long as the modified molecule retains the ability to suppress the expression of the ERCC6L gene, minor modifications in the nucleic acid sequence described above are allowed. As used herein, the phrase “minor modification” as used in reference to a nucleic acid sequence indicates one, two, or several substitutions, deletions, additions, or insertions of the nucleic acid to the sequence.

  In one embodiment, the double-stranded molecule consists of two polynucleotides, one polynucleotide having a sequence corresponding to the target sequence, i.e. the sense strand, and the other polypeptide being a complementary sequence to the target sequence, i.e. Has an antisense strand. The sense strand polynucleotide and the antisense strand polynucleotide hybridize with each other to form a double-stranded molecule. Examples of such double stranded molecules include dsRNA and dsD / R-NA.

In another embodiment, the double-stranded molecule consists of a polynucleotide having both a sequence corresponding to the target sequence, ie the sense strand, and a complementary sequence to the target sequence, ie the antisense strand. Generally, a sense strand and an antisense strand are connected by an intervening strand and hybridize with each other to form a hairpin loop structure. Examples of such double stranded molecules include shRNA and shD / R-NA.
In other words, the double-stranded molecule of the present invention is composed of a sense strand polynucleotide having a nucleotide sequence of a target sequence and an antisense strand polynucleotide having a nucleotide sequence complementary to the target sequence, and both polynucleotides are mutually connected. Hybridizes to form double stranded molecules. In a double-stranded molecule comprising the polynucleotide, part of one or both polynucleotides of the strand may be RNA, and if the target sequence is defined by a DNA sequence, the target sequence and its complement Nucleotide “t” in the sequence is replaced by “u”.

  In one embodiment of the invention, such a double-stranded molecule of the invention comprises a stem loop structure consisting of a sense strand and an antisense strand. The sense strand and the antisense strand can be joined by a loop. Thus, the present invention also provides a double-stranded molecule composed of a single polynucleotide comprising both a sense strand and an antisense strand that are linked by or intervening by an intervening single strand.

In the present invention, the double-stranded molecule targeting the ERCC6L gene may have a sequence selected from SEQ ID NOs: 7 and 8 as the target sequence. Accordingly, preferred examples of the double-stranded molecule of the present invention include polynucleotides and polynucleotides having complementary sequences thereto, and polynucleotides having sequences corresponding to and corresponding to SEQ ID NOs: 7 and 8.
In the context of the present invention, the term “several” as applied to nucleic acid substitutions, deletions, additions and / or insertions means 3-7, preferably 3-5, more preferably 3-5. It may mean 4 and even more preferably 3 nucleic acid residues.

  In accordance with the present invention, a double-stranded molecule of the present invention can be tested for its ability using the methods utilized in the Examples. In the examples described later in this specification, double-stranded molecules comprising sense strands of various parts of mRNA of ERCC6L gene or antisense strands complementary thereto are converted into ERCC6L gene in cancer cell lines according to standard methods. Tested in vitro for the ability to reduce product production. Further, for example, the decrease 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 determined, for example, in Example 1: It can be detected by RT-PCR using primers for ERCC6L mRNA described below. Next, sequences that reduce the production of the ERCC6L gene product in an in vitro cell-based assay can be tested for inhibitory effects on cell proliferation. Subsequently, sequences that inhibit cell proliferation in in vitro cell-based assays were tested for in vivo capacity using animals with cancer, such as nude mouse xenograft models, to reduce ERCC6L gene product production and cancer cells. Reduction of proliferation can be confirmed.

  When the isolated polynucleotide is RNA or a derivative thereof, the base “t” shall be replaced with “u” in the nucleotide sequence. As used herein, the term “complementarity” refers to Watson-Crick or Hoogsteen-type base pairing between nucleotide units of a polynucleotide, and the term “binding” is between two polynucleotides. Means a physical or chemical interaction. If the polynucleotide contains modified nucleotides and / or non-phosphorylated diester linkages, these polynucleotides can also bind to each other in the same way. In general, complementary polynucleotide sequences will hybridize under appropriate conditions to form a stable duplex with little or no mismatch. Furthermore, the sense strand and the antisense strand of the isolated polynucleotide of the present invention can form a double-stranded molecule or a hairpin loop structure by hybridization. In one preferred embodiment, such duplexes contain no more than 1 mismatch per 10 matches. In one particularly preferred embodiment, such duplexes do not contain mismatches when the duplex strands are fully complementary.

  For ERCC6L, the polynucleotide is preferably less than 7006 nucleotides in length. For example, for the ERCC6L gene, the polynucleotide is less than 500 nucleotides in length, less than 200 nucleotides in length, less than 100 nucleotides in length, less than 75 nucleotides in length, less than 50 nucleotides in length, or less than 25 nucleotides in length. The isolated polynucleotide of the present invention is useful for forming a double-stranded molecule against the ERCC6L gene or for preparing a template DNA encoding a double-stranded molecule. When using a polynucleotide to form a double-stranded molecule, the polynucleotide may be longer than 19 nucleotides, preferably longer than 21 nucleotides, more preferably about 19-25 nucleotides in length.

  Accordingly, the present invention provides a double-stranded molecule composed of a sense strand and an antisense strand, and the sense strand is a nucleotide sequence corresponding to a target sequence. In a preferred embodiment, the sense strand hybridizes with the antisense strand at the target sequence to form a double stranded molecule having a length of 19-25 nucleotide pairs.

  The double-stranded molecule serves as a guide for identifying homologous sequences in the mRNA of the RISC complex when the double-stranded molecule is introduced into the cell. The identified target RNA is cleaved and degraded by Dicer's nuclease activity, which ultimately reduces or inhibits production (expression) of the polypeptide encoded by the RNA. Therefore, the double-stranded molecule of the present invention can be defined by the ability to generate a single strand that specifically hybridizes under stringent conditions with the mRNA of the ERCC6L gene. As used herein, the portion of mRNA that hybridizes to a single strand generated from a double stranded molecule is referred to as a “target sequence” or “target nucleic acid” or “target nucleotide”. In the context of the present invention, the nucleotide sequence of the “target sequence” can be indicated not only using the RNA sequence of mRNA, but also using the DNA sequence of cDNA synthesized from the mRNA.

  The double-stranded molecule of the invention may contain one or more modified nucleotides and / or non-phosphorylated diester bonds. Chemical modifications well known in the art can increase the stability, availability and / or cellular uptake of double-stranded molecules. Those skilled in the art will also recognize other types of chemical modifications that can be incorporated into the molecules of the invention (WO 03/070744, WO 2005/045037). In one aspect, modifications may be used to provide improved degradation resistance or improved uptake. Examples of such modifications include phosphorothioate linkages, 2′-O-methyl ribonucleotides (especially on the sense strand of double-stranded molecules), 2′-deoxy-fluoro ribonucleotides, 2′-deoxy ribonucleotides, “universal Including, but not limited to, the incorporation of universal base "nucleotides, 5'-C-methyl nucleotides, and inverted deoxybasic residues (US20060122137).

  In another embodiment, modifications can be used to improve the stability of double-stranded molecules or to increase targeting efficiency. Examples of such modifications include chemical cross-linking between two complementary strands of a double-stranded molecule, chemical modification of the 3 'or 5' end of a strand of a double-stranded molecule, sugar modification, nucleobase modification and / or Alternatively, backbone modifications, 2-fluoro-modified ribonucleotides and 2′-deoxyribonucleotides are included (International Publication No. 2004/029212), but are not limited thereto. In another embodiment, modifications can be used to increase or decrease the affinity for complementary nucleotides in the target mRNA and / or in the complementary double stranded molecular strand (WO 2005/044976). For example, unmodified pyrimidine nucleotides can be replaced with 2-thiopyrimidine, 5-alkynylpyrimidine, 5-methylpyrimidine, or 5-propynylpyrimidine. Furthermore, unmodified purines can be replaced with 7-deazapurines, 7-alkylpurines, or 7-alkenylpurines. In another embodiment, if the double-stranded molecule is a double-stranded molecule with a 3 ′ overhang, the 3 ′ terminal nucleotide overhanging nucleotide can be replaced with deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15 (2): 188-200). For further details, published literature such as US20060234970 is available. The present invention is not limited to these examples, and any known chemical modification can be utilized for the double-stranded molecule of the present invention as long as the resulting molecule retains the ability to inhibit target gene expression. can do.

  Furthermore, the double-stranded molecules of the invention can include both DNA and RNA (eg, dsD / R-NA or shD / R-NA). In particular, hybrid polynucleotides of DNA and RNA strands or DNA-RNA chimeric polynucleotides exhibit increased stability. A mixture of DNA and RNA, that is, a hybrid double-stranded molecule comprising a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera containing both DNA and RNA in one or both of the single strand (polynucleotide) A type double-stranded molecule or the like may be formed to improve the stability of the double-stranded molecule.

  A hybrid of a DNA strand and an RNA strand is such that the sense strand is DNA and the antisense strand is RNA, or vice versa, as long as it can inhibit expression of the gene when introduced into a cell expressing the target gene. One of the structures. Preferably, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA. In addition, a chimeric double-stranded molecule has a structure in which both the sense strand and the antisense strand are composed of DNA and RNA as long as it has an activity of inhibiting the expression of a target gene when introduced into a cell that expresses the gene, or sense One of the strand and the antisense strand has a structure consisting of DNA and RNA. In order to improve the stability of the double-stranded molecule, the molecule preferably contains as much DNA as possible, but in order to induce inhibition of target gene expression, induce sufficient inhibition of expression. It is necessary that the molecule is RNA within the range.

As a preferred example of the chimeric double-stranded molecule, the upstream partial region of the double-stranded molecule (ie, the region adjacent to the target sequence or its complementary sequence in the sense strand or antisense strand) is RNA. Preferably, the upstream partial region indicates the 5 ′ side (5 ′ end) of the sense strand and the 3 ′ side (3 ′ end) of the antisense strand. Alternatively, the region adjacent to the 5 ′ end of the sense strand and / or the 3 ′ end of the antisense strand is referred to as the upstream partial region. That is, in a preferred embodiment, both the region adjacent to the 3 ′ end of the antisense strand, or the region adjacent to the 5 ′ end of the sense strand and the region adjacent to the 3 ′ end of the antisense strand are composed of RNA. For example, the chimeric or hybrid double-stranded molecule of the present invention includes the 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 and sense strand: 5 ′-(RNA)-[DNA] -3 ′
3 '-(--- RNA ---)-5': antisense strand.

  Preferably, the upstream partial region is a domain consisting of 9 to 13 nucleotides counted from the end of the target sequence or its complementary sequence in the sense strand or antisense strand of the double-stranded molecule. In addition, preferred examples of such chimeric double-stranded molecules include at least the upstream half region of the polynucleotide (the 5 ′ region in the sense strand and the 3 ′ region in the antisense strand) is RNA and the other half. Is a DNA having a length of 19 to 21 nucleotide chains. In such a chimeric double-stranded molecule, when the entire antisense strand is RNA, the effect of inhibiting the expression of the target gene is considerably increased (US20050004064).

  In the context of the present invention, double-stranded molecules may form hairpins, such as short hairpin RNA (shRNA) and short hairpins composed of DNA and RNA (shD / R-NA). shRNA or shD / R-NA is a sequence of RNA or a mixture of RNA and DNA that creates a tight hairpin turn that can be used to stop gene expression via RNA interference. The shRNA or shD / R-NA includes a sense target sequence and an antisense target sequence on a single strand, and these sequences are separated by a loop sequence. In general, the hairpin structure is cleaved by cellular machinery to become dsRNA or dsD / R-NA, which subsequently binds to the RNA-induced silencing complex RISC. This complex binds to and cleaves mRNA that matches the target sequence of dsRNA or dsD / R-NA.

In order to form a hairpin loop structure, a loop sequence consisting of any nucleotide sequence can be placed between the sense and antisense sequences. Therefore, the present invention relates to the general formula 5 ′-[A]-[B]-[A ′]-3 ′.
[A] is a sense strand containing a nucleotide sequence corresponding to the target sequence, [B] is an intervening single strand, and [A ′] is an antisense strand containing a complementary sequence to the target sequence. Certain double-stranded molecules are also provided. The target sequence can be selected from among the nucleotide sequences of SEQ ID NO: 7 and 8, for example for ERCC6L.

The present invention is not limited to these examples, and the target sequence in [A] is a modified sequence derived from these examples as long as the double-stranded molecule retains the ability to suppress the expression of the target ERCC6L gene. obtain. Region [A] hybridizes with region [A ′] to form a loop composed of region [B]. The intervening single-stranded portion [B], ie the loop sequence, can preferably be 3 to 23 nucleotides in length. For example, the loop sequence can be selected from the following sequences (http://www.ambion.com/techlib/tb/tb_506.html). In addition, a loop sequence of 23 nucleotides also provides an 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.

Examples of preferred double-stranded molecules of the present invention having a hairpin loop structure are shown below. In the following structure, the loop sequence can be selected from among AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGGA; however, the invention is not limited to these:
CUGCACAACCAACUCUUG- [B] -CAAAGAGUGUGGUUGCAG
(Target sequence SEQ ID NO: 7).
AUGUUAAUCAAUAUACUGGC- [B] -GCCAGUUAAUUGAUUAACAU
(Target sequence SEQ ID NO: 8).

  Furthermore, in order to increase the inhibitory activity of the double-stranded molecule, several nucleotides can be added as 3 ′ overhangs to the 3 ′ end of the sense and / or antisense strand of the target sequence. Preferred examples of nucleotides comprising a 3 ′ overhang include, but are not limited to “t” and “u”. The number of nucleotides to be added is at least 2, generally 2 to 10, preferably 2 to 5. The added nucleotide forms a single strand at the 3 ′ end of the antisense strand of the double stranded molecule. If the double-stranded molecule consists of a single polynucleotide and forms a hairpin loop structure, a 3 ′ overhang sequence can be added to the 3 ′ end of the single polynucleotide.

  The method for preparing the double-stranded molecule is not particularly limited, but it is preferable to use any chemical synthesis method known in the art. According to the chemical synthesis method, single-stranded sense and antisense polynucleotides are synthesized separately, and then annealed together by an appropriate method to obtain a double-stranded molecule. Examples of annealing include synthesized single stranded polynucleotides, preferably in a molar ratio of at least about 3: 7, more preferably in a molar ratio of about 4: 6, and most preferably substantially equimolar amounts (ie Mixing in a molar ratio of about 5: 5). Next, the mixture is heated to a temperature at which the double-stranded molecules dissociate, and then gradually cooled. The annealed double-stranded polynucleotide can be purified by commonly used methods known in the art. Examples of purification methods include methods that utilize agarose gel electrophoresis, or methods in which the remaining single-stranded polynucleotide is optionally removed, for example, by degradation with an appropriate enzyme.

The regulatory sequences adjacent to the ERCC6L sequence may be the same or different so that their expression can be regulated independently or in a temporal or spatial manner. Double-stranded molecules can be transcribed intracellularly by cloning the ERCC6L gene template into a vector containing, for example, an RNA polyIII transcription unit derived from nuclear small RNA (snRNA) U6 or a human H1 RNA promoter. .
Alternatively, the coding sequence of the double-stranded molecule may be replaced with a regulatory sequence that supports expression of the double-stranded molecule in appropriate cells (eg, RNA polyIII transcription unit from human nuclear RNA (snRNA) U6 or human The double-stranded molecule so that its expression can be regulated independently or in a temporal or spatial manner by cloning into a vector containing the H1 RNA promoter) adjacent to the coding sequence. May be transcribed inside the cell. The regulatory sequences adjacent to the coding sequence of the double-stranded molecule may be the same or different. Details regarding vectors capable of producing double-stranded molecules are described below.

A vector comprising the double-stranded molecule of the present invention:
As noted above, the present invention contemplates vectors containing one or more of the double-stranded molecules described herein, and cells containing such vectors.
The following vectors [1] to [10] are of particular interest for the present invention:
[1] A vector encoding a double-stranded molecule that inhibits in vivo expression and cell proliferation of ERCC6L when introduced into a cell, wherein the molecules hybridize to each other to form a double-stranded molecule And a vector comprising an antisense strand complementary thereto;
[2] The vector according to [1], which encodes a double-stranded molecule that acts on mRNA corresponding to the target sequence of SEQ ID NO: 7 or 8;
[3] The vector according to [1], wherein the sense strand comprises a sequence corresponding to the target sequence of SEQ ID NO: 7 or 8;
[4] The vector according to [3], which encodes a double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes with an antisense strand in a target sequence, and has a length of less than about 100 nucleotide pairs. A vector that forms a double-stranded molecule.
[5] The vector according to [4], which encodes a double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes with an antisense strand in the target sequence, and has a length of less than about 75 nucleotide pairs. A vector that forms a double-stranded molecule.
[6] The vector according to [5], which encodes a double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes with an antisense strand in a target sequence, and has a length of less than about 50 nucleotide pairs. A vector that forms a double-stranded molecule.
[7] The vector according to [6], which encodes a double-stranded molecule, wherein a sense strand of the double-stranded molecule hybridizes with an antisense strand in a target sequence, and has a length of less than about 25 nucleotide pairs. A vector that forms a double-stranded molecule.
[8] The vector according to [7], which encodes a double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes with an antisense strand in a target sequence, and has a length of about 19 to about 25 nucleotide pairs. A vector that forms a double-stranded molecule having a length.
[9] The vector according to [1], wherein the double-stranded molecule is composed of a single polynucleotide having both a sense strand and an antisense strand linked by an intervening single strand;
[10] General formula 5 ′-[A]-[B]-[A ′]-3 ′
Wherein [A] is a sense strand comprising a sequence corresponding to the target sequence of SEQ ID NO: 7 or 8, and [B] is composed of 3 to 23 nucleotides The vector according to [9], wherein the intervening single strand and [A ′] is an antisense strand comprising a sequence complementary to the target sequence.

  The vector of the present invention preferably encodes the double-stranded molecule of the present invention in an expressible form. As used herein, the phrase “in an expressible form” indicates that a vector expresses the molecule when introduced into a cell. In a preferred embodiment, the vector contains the regulatory elements necessary for the expression of a double-stranded molecule. Such vectors of the present invention can be used to produce the double-stranded molecules of the present invention, or can be used directly as active ingredients for treating cancer.

  The vectors of the invention can be used, for example, to clone an ERCC6L sequence into an expression vector such that regulatory sequences are operably linked to the ERCC6L sequence in a manner that allows expression of both strands (by transcription of the DNA molecule). (Lee NS et al., Nat Biotechnol 2002 May, 20 (5): 500-5). For example, an RNA molecule that is antisense to mRNA is transcribed by a first promoter (eg, a promoter sequence adjacent to the 3 ′ end of the cloned DNA), and an RNA molecule that is the sense strand for the mRNA is second Transcribed by a promoter (eg, a promoter sequence adjacent to the 5 ′ end of the cloned DNA). The sense and antisense strands hybridize in vivo to produce a double stranded molecular construct for silencing the gene. Alternatively, the sense and antisense strands are each expressed using two vector constructs that encode the sense and antisense strands of the double-stranded molecule, respectively, and then a double-stranded molecular construct is formed. In addition, the cloned sequence may encode a single transcript of a construct having secondary structure (eg, a hairpin), ie, a vector containing both the sense and complementary antisense sequences of the target gene.

  The vectors of the present invention may also include those for achieving stable insertion into the genome of the target cell (see, eg, Thomas KR & Capecchi MR, Cell 1987 for a description of homologous recombination cassette vectors). , 51: 503-12). For example, Wolff et al., Science 1990, 247: 1465-8, US Pat. Nos. 5,580,859, 5,589,466, 5,804,566, 5,739, No. 118, No. 5,736,524, No. 5,679,647, and WO 98/04720. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivacaine, polymer, peptide mediated) delivery, cationic lipid complexes, and particle mediated (“gene gun”) or pressure mediated Sex delivery is included (see, eg, US Pat. No. 5,922,687).

  The vectors of the present invention include, for example, viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts such as cowpox virus or fowlpox virus (see, eg, US Pat. No. 4,722,848). This approach involves the use of cowpox virus, for example as a vector to express nucleotide sequences encoding double-stranded molecules. When introduced into a cell that expresses the target gene, the recombinant cowpox virus expresses the molecule, thereby inhibiting the growth of the cell. Another example of a vector that can be used includes Bacillus Calmette Guerin (BCG). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60. A wide range of other vectors are useful for therapeutic administration and production of double-stranded molecules. Examples include adenovirus vectors and adeno-associated virus vectors, retrovirus vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. For example, 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. Please refer.

Methods of inhibiting or reducing cancer cell growth or treating cancer using the double-stranded molecules of the invention:
In the present invention, dsRNA against ERCC6L was tested for the ability to inhibit cell proliferation. DsRNA against ERCC6L (FIGS. 2B, C) effectively knocked down expression of the gene in several cancer cell lines, consistent with inhibition of cell proliferation.
Thus, the present invention provides a method of inhibiting cancer cell growth by inducing ERCC6L gene dysfunction through inhibition of ERCC6L expression. ERCC6L gene expression can be inhibited by any of the aforementioned double-stranded molecules of the invention that specifically target the ERCC6L gene.

  Such ability of the present double-stranded molecules and vectors to inhibit cell growth of cancerous cells indicates that they can be used in methods of treating cancers such as lung cancer. Thus, the present invention does not cause adverse effects by administering a double-stranded molecule against the ERCC6L gene or a vector expressing the molecule, because the ERCC6L gene is minimally detected in normal organs (FIG. 1C). A method of treating a patient having cancer is provided.

The following methods [1] to [32] are of particular interest for the present invention:
[1] A method of inhibiting cancer cell proliferation or treating cancer, wherein the cancer cell or the cancer expresses at least one ERCC6L gene, and the method overexpresses the ERCC6L gene. Administering at least one isolated double-stranded molecule, or a vector encoding such a double-stranded molecule, that inhibits the expression and cell proliferation of the gene in the cell, wherein the double-stranded molecule comprises A method comprising a sense strand that hybridizes to each other to form a double-stranded molecule and an antisense strand complementary thereto;
[2] The method according to [1], wherein the double-stranded molecule acts on mRNA corresponding to the target sequence of SEQ ID NO: 7 or 8;
[3] The method according to [2], wherein the sense strand comprises a sequence corresponding to the target sequence of SEQ ID NO: 7 or 8;
[4] The method according to [1], wherein the cancer to be treated is lung cancer;
[5] The method according to [3], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of less than about 100 nucleotide pairs;
[6] The method according to [5], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of less than about 75 nucleotide pairs;
[7] The method of [6], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of less than about 50 nucleotide pairs;
[8] The method of [7], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of less than about 25 nucleotide pairs;
[9] The method according to [8], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of about 19 to about 25 nucleotide pairs;
[10] The method according to [1], wherein the double-stranded molecule is composed of a single polynucleotide comprising both a sense strand and an antisense strand linked by an intervening single strand;
[11] The double-stranded molecule is represented by the general formula 5 ′-[A]-[B]-[A ′]-3 ′.
[A] is a sense strand containing a sequence corresponding to the target sequence of SEQ ID NO: 7 or 8, and [B] is an intervening single strand composed of 3 to 23 nucleotides. And [A ′] is an antisense strand comprising a sequence complementary to the target sequence;
[12] The method according to [1], wherein the double-stranded molecule is RNA;
[13] The method according to [1], wherein the double-stranded molecule includes both DNA and RNA;
[14] The method according to [13], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide.
[15] The method according to [14], wherein the sense strand and the antisense strand polynucleotide are composed of DNA and RNA, respectively.
[16] The method according to [13], wherein the double-stranded molecule is a chimera of DNA and RNA;
[17] The region adjacent to the 3 ′ end of the antisense strand, or both the region adjacent to the 5 ′ end of the sense strand and the region adjacent to the 3 ′ end of the antisense strand are composed of RNA. [16] Described method;
[18] The method according to [17], wherein the adjacent region is composed of 9 to 13 nucleotides;
[19] The method of [1], wherein the double-stranded molecule comprises a 3 ′ overhang;
[20] The method according to [1], wherein the double-stranded molecule is contained in a composition containing, in addition to the molecule, a transfection enhancing agent and a pharmaceutically acceptable carrier;
[21] The method of [1], wherein the double-stranded molecule is encoded by a vector;
[22] The method according to [21], wherein the double-stranded molecule encoded by the vector acts on mRNA corresponding to the target sequence of SEQ ID NO: 7 or 8;
[23] The method according to [22], wherein the sense strand of the double-stranded molecule encoded by the vector comprises a sequence corresponding to a target sequence selected from among SEQ ID NOs: 7 and 8;
[24] The method according to [21], wherein the cancer to be treated is lung cancer;
[25] The method of [23], wherein the double-stranded molecule encoded by the vector has a length of less than about 100 nucleotide pairs;
[26] The method of [25], wherein the double-stranded molecule encoded by the vector has a length of less than about 75 nucleotide pairs;
[27] The method of [26], wherein the double-stranded molecule encoded by the vector has a length of less than about 50 nucleotide pairs;
[28] The method of [27], wherein the double-stranded molecule encoded by the vector has a length of less than about 25 nucleotide pairs;
[29] The method of [28], wherein the double-stranded molecule encoded by the vector has a length of about 19 to about 25 nucleotide pairs;
[30] The method according to [21], wherein the double-stranded molecule encoded by the vector is composed of a single polynucleotide comprising both a sense strand and an antisense strand linked by an intervening single strand;
[31] The double-stranded molecule encoded by the vector has the general formula 5 ′-[A]-[B]-[A ′]-3 ′
[A] is a sense strand containing a sequence corresponding to the target sequence of SEQ ID NO: 7 or 8, and [B] is an intervening single strand composed of 3 to 23 nucleotides. And [A ′] is an antisense strand comprising a sequence complementary to the target sequence; and [32] a double-stranded molecule encoded by the vector is attached to the molecule In addition, the method according to [21], which is contained in a composition comprising a transfection enhancing agent and a pharmaceutically acceptable carrier.

The method of the present invention will be described in more detail below.
Growth of cells expressing the ERCC6L gene can be inhibited by contacting the cells with a double-stranded molecule against the ERCC6L gene, a vector expressing the molecule, or a composition comprising 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 proliferation” indicates that the cells grow slower or have a reduced cell viability compared to cells not exposed to the molecule. Cell proliferation may be measured by methods known in the art, for example using the MTT cell proliferation assay.
As long as the cell expresses or overexpresses the target gene of the double-stranded molecule of the present invention, the proliferation of any kind of cell can be suppressed according to the method of the present invention. Exemplary cells include lung cancer.

  Accordingly, a patient suffering from or at risk of developing a disease associated with ERCC6L is treated as a double-stranded molecule of the present invention, at least one vector expressing the molecule, or a composition comprising the molecule Can be treated. For example, cancer patients can be treated according to the methods of the invention. The type of cancer can be identified by standard methods according to the particular type of tumor being diagnosed. More preferably, patients treated by the method of the invention are selected by detecting the expression of ERCC6L in patient-derived biopsy specimens by RT-PCR or immunoassay. Preferably, prior to treatment of the present invention, a biopsy specimen from the subject is confirmed for overexpression of the ERCC6L gene by methods known in the art, such as immunohistochemical analysis or RT-PCR.

  According to the method of the invention for inhibiting cell proliferation and thereby treating cancer, during administration of multiple types of double-stranded molecules (or a vector expressing it or a composition comprising it) Each may have a different structure, but may act on mRNA that matches the same target sequence of ERCC6L. Alternatively, multiple types of double-stranded molecules can act on mRNAs that match different target sequences of the same gene. Alternatively, for example, the method may utilize a double stranded molecule directed against one, two, or more target sequences of ERCC6L.

  In order to inhibit cell proliferation, the double-stranded molecule of the invention may be introduced directly into the cell in a form to achieve binding of the molecule to the corresponding mRNA transcript. Alternatively, as described above, DNA encoding a double-stranded molecule may be introduced into a cell as a vector. To introduce double-stranded molecules and vectors into cells, transfection enhancing agents such as FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wakopure) may be used. .

  A treatment is considered “effective” if it results in a clinical benefit such as decreased expression of the ERCC6L gene or decreased cancer size, prevalence, or metastatic potential in the subject. When the treatment is applied prophylactically, “effective” means that the formation of the cancer is delayed or inhibited, or the clinical symptoms of the cancer are prevented or alleviated. Efficacy is determined in conjunction with any known method for diagnosing or treating a particular tumor type.

  As long as the methods and compositions of the invention are useful in the context of “prevention and prophylaxis”, such terms are used interchangeably herein to reduce mortality or morbidity burden due to disease. Refers to any action to be made. Prevention and prevention may be performed at “primary, secondary, and tertiary prevention levels”. Primary prevention and prophylaxis avoid the development of disease, whereas secondary and tertiary levels prevention (prevention and prophylaxis) prevent disease progression and appearance of symptoms (prevention and In addition to prophylaxis), it encompasses activities aimed at reducing the adverse effects of previously established diseases by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at reducing the severity of certain disorders, eg, reducing tumor growth and metastasis.

  Treatment and / or prevention of cancer and / or prevention of its postoperative recurrence may be in the following stages, for example, surgical removal of cancer cells, inhibition of cancerous cell growth, tumor regression or regression, remission Including induction and suppression of cancer development, tumor regression, and reduction or inhibition of metastasis. Effective treatment and / or prevention of cancer reduces mortality, improves the prognosis of individuals with cancer, reduces the level of blood tumor markers, and provides detectable symptoms associated with cancer. ease. For example, symptom reduction or amelioration is a component of effective treatment and / or prevention includes 10%, 20%, 30%, or more of a reduced or stable disease.

  It will be appreciated that the double-stranded molecules of the present invention degrade ERCC6L mRNA in substoichiometric amounts. Without being bound by any theory, it is believed that the double-stranded molecule of the present invention causes degradation of the target mRNA in a catalytic manner. Therefore, compared to standard cancer treatment, the present invention requires that significantly fewer double-stranded molecules be delivered at or near the cancer site in order to exert a therapeutic effect.

The skilled artisan will consider the subject's weight, age, sex, type of disease, symptoms and other conditions; factors such as the route of administration; and whether the administration is local or systemic. The effective amount of the double-stranded molecule of the present invention to be administered can be readily determined. In general, an effective amount of a double-stranded molecule of the present invention is about 1 nanomolar (nM) to about 100 nM, preferably about 2 nM to about 50 nM, more preferably about 2.5 nM to about 10 nM at or near the cancer site. Is the intercellular concentration. It is contemplated that higher or lower amounts of double-stranded molecules can be administered. The exact dosage required for a particular situation can be readily and routinely determined by one skilled in the art.
The methods of the invention can be used to inhibit the growth or metastasis of cancers that express the ERCC6L gene; for example, lung cancer. Specifically, double-stranded molecules comprising the target sequence of the ERCC6L gene (eg, SEQ ID NOs: 7 and 8) are particularly preferred for the treatment of cancer.

  In order to treat cancer, the double-stranded molecule of the present invention can be administered to a subject in combination with a pharmaceutical composition different from the double-stranded molecule. Alternatively, the double-stranded molecule of the present invention can be administered to a subject in combination with another treatment designed to treat cancer. For example, the double-stranded molecules of the present invention can be used to treat currently used therapeutics for cancer or prevention of cancer metastasis (eg, radiation therapy, surgery, and cisplatin, carboplatin, cyclophosphamide, 5 -In combination with chemotherapeutic agents such as fluorouracil, adriamycin, daunorubicin, or tamoxifen).

In this method, the double-stranded molecule can be administered to the subject as a naked double-stranded molecule in combination with a delivery reagent or as a recombinant plasmid or viral vector that expresses the double-stranded molecule.
Suitable delivery reagents for administration in combination with the double-stranded molecules of the present invention include the Mirus Transit TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, or polycation (eg polylysine) or liposome. A preferred delivery reagent is a liposome.

  Liposomes can assist in the delivery of double-stranded molecules to specific tissues, such as lung tumor tissue, and can also increase the blood half-life of the double-stranded molecules. 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 sterols such as cholesterol. In general, the choice of lipid is guided by factors such as the desired liposome size and the half-life of the liposomes in the bloodstream. Regarding the preparation of liposomes, see, for example, Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467, and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028. And various methods, such as those described in US Pat. No. 5,019,369, the entire disclosure of which is hereby incorporated by reference.

Preferably, the liposome encapsulating the double-stranded molecule of the present invention comprises a ligand molecule capable of delivering the liposome to a cancer site. Preference is given to ligands that bind to receptors abundant in tumor or vesicular endothelial cells, such as monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens.
Particularly preferably, liposomes encapsulating the present double-stranded molecule are modified to avoid clearance by mononuclear macrophages and reticuloendothelial systems, for example by having opsonization-inhibiting moieties attached to the surface of the structure. . In one aspect, the liposomes of the invention can include both an opsonization inhibiting moiety and a ligand.

  The opsonization-inhibiting moiety used to prepare the liposomes of the present invention is typically a large hydrophilic polymer that binds to the liposome membrane. As used herein, an opsonization-inhibiting moiety is chemically or physically attached to a liposome membrane, for example, by intercalation of a lipid soluble anchor to the membrane itself, or to an active group of membrane lipids. It is “bound” to the liposome membrane by direct binding of. These opsonization-inhibiting hydrophilic polymers can be found in macrophage-monocyte systems (“MMS”) and, for example, as described in US Pat. No. 4,920,016, the entire disclosure of which is incorporated herein by reference. A protective surface layer is formed that significantly reduces liposome uptake by the reticulated system (“RES”). Thus, liposomes modified with opsonization-inhibiting moieties remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes referred to as “stealth” liposomes.

  Stealth liposomes are known to accumulate in tissues into which porous or “leaky” microvasculature flows. Therefore, these liposomes accumulate efficiently in target tissues characterized by such microvasculature defects, such as solid tumors. See Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53. Furthermore, the reduced uptake by RES reduces the toxicity of stealth liposomes by preventing significant accumulation in the liver and spleen. Thus, the liposomes of the present invention modified with opsonization-inhibiting moieties can deliver the double-stranded molecules of the present invention to tumor cells.

Opsonization-inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers having a molecular weight of about 500 Daltons to about 40000 Daltons, more preferably about 2000 Daltons to about 20000 Daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; for example, methoxy PEG or PPG, and stearic acid PEG or stearic acid PPG; synthetic polymers such as polyacrylamide or poly N-vinylpyrrolidone; linear polyamidoamine branched or dendrimer; polyacrylic acid; polyhydric alcohols, such as polyvinyl alcohol and polyvinyl xylitol carboxylic or amino groups are chemically bound, and, gangliosides, such as ganglioside GM _1. Also suitable are copolymers of PEG, methoxy PEG or methoxy PPG, or derivatives thereof. In addition, the opsonization-inhibiting polymer can also be a block copolymer of PEG and any of polyamino acids, polysaccharides, polyamidoamines, polyethyleneamines, or polynucleotides. Opsonization-inhibiting polymers are natural polysaccharides containing amino acids or carboxylic acids, such as galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharide or oligosaccharide (linear or branched) Or a carboxylated polysaccharide or a carboxylated oligosaccharide reacted with a derivative of a carboxylic acid from which a carboxylic acid group has been linked, for example.

Preferably, the opsonization inhibiting moiety is PEG, PPG, or a derivative thereof. Liposomes modified with PEG or PEG derivatives are sometimes referred to as “PEGylated liposomes”.
The opsonization inhibiting moiety can be bound to the liposome membrane by any one of a number of known techniques. For example, an N-hydroxysuccinimide ester of PEG can be coupled to a phosphatidyl-ethanolamine lipid soluble anchor followed by a membrane. Similarly, dextran polymers can be derivatized with stearylamine lipid soluble anchors by reductive amination at 60 ° C. using Na (CN) BH ₃ and a solvent mixture, eg, a 30:12 ratio of tetrahydrofuran and water.

  Vectors expressing the double-stranded molecules of the present invention have been described above. Such vectors that express at least one double-stranded molecule of the invention are also suitable, including directly or as a Mirus Transit LT1 lipophilic reagent, lipofectin, lipofectamine, cellfectin, or polycation (eg polylysine) or liposome. It can be administered in combination with a delivery reagent. Methods for delivering a recombinant viral vector expressing a double-stranded molecule of the invention to a cancerous area of a patient are within the skill of the art.

The double-stranded molecule of the present invention can be administered to a subject by any means suitable for delivering the double-stranded molecule to a cancer site. For example, the double-stranded molecule can be administered by gene gun, electroporation, or other suitable parenteral or enteral route of administration.
Suitable routes of enteral administration include oral delivery, rectal delivery, or intranasal delivery.

  Suitable parenteral routes of administration include intravesicular and intravenous (eg intravenous bolus injection, intravenous infusion, intraarterial bolus injection, intraarterial infusion and catheter instillation into the vasculature); peri-tissue and intra-tissue injection (Eg, peri-tumor tissue injection and intratumoral injection); subcutaneous injection or subcutaneous deposition including subcutaneous injection (eg, by osmotic pump); eg, catheter or other placement device (eg, porous, non-porous Direct application to or near the area of the cancer site with suppositories or implants containing gelatin-like materials; and inhalation. The injection or infusion of the double-stranded molecule or vector is preferably performed at or near the cancer site.

  The double-stranded molecule of the present invention can be administered in a single dose or multiple doses. When 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. It is preferred to inject the double-stranded molecule directly into the tissue at or near the cancer site. It is particularly preferred that the double-stranded molecule is injected multiple times into the tissue at or near the cancer site.

  One skilled in the art can also readily determine an appropriate dosing regimen for administering the double-stranded molecule of the invention to a given subject. For example, 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. Alternatively, the double-stranded molecule can be administered to the subject once or twice daily for a period of about 3 days to about 28 days, more preferably about 7 days to about 10 days. In a preferred dosing regimen, the double-stranded molecule is injected once a day for 7 days at or near the cancer site. Where multiple dosages are required for an administration regimen, it is understood that an effective amount of a double-stranded molecule administered to a subject can include the total amount of double-stranded molecules administered throughout the dosage regimen.

In the present invention, a cancer that overexpresses ERCC6L,
(A) the double-stranded molecule of the present invention,
It can be treated with at least one active ingredient selected from the group consisting of (b) the DNA encoding it, and (c) the vector encoding it.

Cancer includes, but is not limited to lung cancer. Therefore, prior to administration of the double-stranded molecule of the present invention as an active ingredient, whether or not the expression level of ERCC6L in the cancer cell or tissue being treated is increased compared to normal cells in the same organ. It is preferable to confirm. Accordingly, in one aspect, the invention provides a method for treating a cancer that (over) expresses ERCC6L, the method comprising:
i) measuring the expression level of ERCC6L in a cancer cell or tissue obtained from a subject having a cancer to be treated;
ii) comparing the expression level of ERCC6L to a normal control; and iii) for a subject having a cancer that overexpresses ERCC6L compared to a normal control,
(A) the double-stranded molecule of the present invention,
Administering (b) at least one component selected from the group consisting of (c) DNA encoding the same, and (c) a vector encoding the same.
Alternatively, the present invention is for use in administration to a subject having a cancer that overexpresses ERCC6L.
(A) the double-stranded molecule of the present invention,
Also provided is a pharmaceutical composition comprising at least one component selected from the group consisting of (b) DNA encoding it, and (c) a vector encoding it. In other words, the present invention relates to a subject to be treated (a) a double-stranded molecule of the present invention,
Further provided is a method for identification using (b) DNA encoding it, or (c) a vector encoding it, which measures the expression level of ERCC6L in a subject-derived cancer cell or tissue An increase in the level compared to a normal control level of the gene causes the subject to
(A) the double-stranded molecule of the present invention,
It is shown to have a cancer that can be treated with (b) the DNA encoding it, or (c) the vector encoding it.

The cancer treatment method of the present invention will be described in more detail below.
The subject to be treated by this method is preferably a mammal. Exemplary mammals include, but are not limited to, for example, humans, non-human primates, mice, rats, dogs, cats, horses and cows.

According to the present invention, the expression level of ERCC6L in a cancer cell or tissue collected from a subject is measured. Expression levels can be measured as transcription (nucleic acid) product levels using methods known in the art. For example, ERCC6L mRNA may be quantified by a hybridization method (eg, Northern hybridization) using a probe. Detection may be performed on a chip or array. For detection of the expression level of ERCC6L, the use of an array is preferred. One skilled in the art can prepare such probes using the sequence information of ERCC6L. For example, ERCC6L cDNA may be used as a probe. If necessary, the probe may be labeled with an appropriate label such as a dye, a fluorescent substance, and an isotope, and the expression level of the gene may be detected as the intensity of the hybridized label.
Furthermore, the transcript of ERCC6L (eg, SEQ ID NO: 9) may be quantified by a detection method (eg, RT-PCR) based on amplification using primers. Such primers may be prepared based on available sequence information about the gene.

  Specifically, the probe or primer used in the method of the present invention hybridizes with mRNA of ERCC6L under stringent conditions, moderately stringent conditions, or low stringent conditions. As used herein, the phrase “stringent (hybridization) conditions” refers to conditions under which a probe or primer hybridizes to its target sequence but not to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of stringent conditions is selected to be about 5 ° C. lower than the thermal melting temperature (Tm) of the specific sequence at a given ionic strength and pH. Tm is the temperature at which 50% of a probe complementary to a target sequence (under a given ionic strength, pH, and nucleic acid concentration) hybridizes with the target sequence in equilibrium. Since the target sequence is generally present in excess at Tm, 50% of the probes are occupied in equilibrium. Typically, stringent conditions include a salt concentration of less than about 1.0 M sodium ions at pH 7.0-8.3, typically about 0.01-1. The conditions are 0 M and the temperature is at least about 30 ° C. for short probes or primers (eg, 10-50 nucleotides) and at least about 60 ° C. for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

  Alternatively, the translation product may be detected for the diagnosis of the present invention. For example, the amount of protein (SEQ ID NO: 10) observed may be measured. A method for measuring the amount of a protein as a translation product includes an immunoassay method using an antibody that specifically recognizes the protein. The antibody may be monoclonal or polyclonal. Furthermore, as long as any fragment or modified antibody of the antibody retains the binding ability to the ERCC6L protein, the fragment or modification (eg, chimeric antibody, scFv, Fab, F (ab ′) 2, Fv, etc.) can be detected. Can be used. Methods for preparing these types of antibodies for detection of the proteins are well known in the art, and any method can be utilized in the present invention to prepare such antibodies and their equivalents. .

As another method for detecting the expression level of the ERCC6L gene based on its translation product, the staining intensity may be measured by immunohistochemical analysis using an antibody against the ERCC6L protein. That is, in this measurement, strong staining indicates an increase in protein abundance / level and at the same time a high expression level of the ERCC6L gene.
If the expression level of a target gene, eg, ERCC6L gene, in a cancer cell is increased by, for example, 10%, 25%, or 50% over a control level of the target gene (eg, a level in normal cells); or 1 It can be determined that it has increased if it has increased more than 1.times., 1.5.times., 2.0.times., 5.0.times., 10.0.times. Or more.

  By using the sample (s) previously collected and stored from the subject (s) with known disease state (cancerous or non-cancerous), the control level can be adjusted simultaneously with the cancer cells. You may decide. In addition, normal cells collected from non-cancerous areas of organs with cancer to be treated may be used as normal controls. Alternatively, a control level is determined by statistical methods based on results obtained by analyzing the expression level (s) of the ERCC6L gene previously measured in a sample of a subject with a known disease state. May be. Furthermore, the control level can be derived from a database of expression patterns from previously tested cells. Moreover, according to one aspect of the present invention, the expression level of the ERCC6L gene in a biological sample can be compared to a plurality of control levels determined from a plurality of reference samples. Preferably, a control level determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample is used. Furthermore, it is preferable to use a standard value of the expression level of the ERCC6L gene in a group whose disease state is known. The standard value can be obtained by any method known in the art. For example, the average value +/- 2S. D. Or average value +/- 3S. D. Can be used as standard values.

In the context of the present invention, a control level measured from a biological sample that has been found not to be cancerous is referred to as a “normal control level”. On the other hand, if the control level is measured from a cancerous biological sample, this is referred to as the “cancerous control level”.
A subject can be diagnosed with a cancer to be treated if the expression level of the ERCC6L gene is elevated compared to a normal control level or is similar / equivalent to a cancerous control level.

Composition comprising the double-stranded molecule of the present invention:
In addition to the above, the present invention also provides a pharmaceutical composition comprising the double-stranded molecule of the present invention or a vector encoding the molecule. The following compositions [1] to [32] are of particular interest for the present invention:
[1] inhibits the growth of cancer cells, comprising at least one isolated double-stranded molecule that inhibits ERCC6L gene expression and cell proliferation, or a vector encoding such a double-stranded molecule, or A composition for treating cancer, wherein the cancer and the cancer cell express the ERCC6L gene, and the molecule hybridizes to each other to form a double-stranded molecule and its complement A composition composed of a typical antisense strand.
[2] The composition according to [1], wherein the double-stranded molecule acts on mRNA corresponding to the target sequence of SEQ ID NO: 7 or 8;
[3] The composition according to [2], wherein the double-stranded molecule, the sense strand comprises a sequence corresponding to the target sequence of SEQ ID NO: 7 or 8;
[4] The composition according to [1], wherein the cancer to be treated is lung cancer;
[5] The composition of [3], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of less than about 100 nucleotide pairs;
[6] The composition of [5], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of less than about 75 nucleotide pairs;
[7] The composition of [6], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of less than about 50 nucleotide pairs;
[8] The composition of [7], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand in the target sequence to form a double-stranded molecule having a length of less than about 25 nucleotide pairs;
[9] The composition of [8], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand at the target sequence to form a double-stranded molecule having a length of about 19 to about 25 nucleotide pairs. ;
[10] The composition of [1], wherein the double-stranded molecule is composed of a single polynucleotide comprising a sense strand and an antisense strand linked by an intervening single strand;
[11] The double-stranded molecule is represented by the general formula 5 ′-[A]-[B]-[A ′]-3 ′.
Wherein [A] is a sense strand sequence containing a sequence corresponding to the target sequence of SEQ ID NO: 7 or 8, and [B] is an intervening single strand consisting of 3 to 23 nucleotides. And [A ′] is an antisense strand comprising a sequence complementary to the target sequence;
[12] The composition according to [1], wherein the double-stranded molecule is RNA;
[13] The composition according to [1], wherein the double-stranded molecule is DNA and / or RNA;
[14] The composition according to [13], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[15] The composition according to [14], wherein the sense strand and the antisense strand polynucleotide are composed of DNA and RNA, respectively.
[16] The composition according to [13], wherein the double-stranded molecule is a chimera of DNA and RNA;
[17] The region adjacent to the 3 ′ end of the antisense strand, or both the region adjacent to the 5 ′ end of the sense strand and the region adjacent to the 3 ′ end of the antisense strand are composed of RNA. [14] A composition as described;
[18] The composition according to [17], wherein the adjacent region is composed of 9 to 13 nucleotides;
[19] The composition of [1], wherein the double-stranded molecule comprises a 3 ′ overhang;
[20] The composition of [1], comprising a transfection enhancing agent and a pharmaceutically acceptable carrier.
[21] The composition of [1], wherein the double-stranded molecule is encoded by a vector and is included in the composition;
[22] The composition of [21], wherein the double-stranded molecule encoded by the vector acts on mRNA that matches the target sequence of SEQ ID NO: 7 or 8;
[23] The composition of [22], wherein the sense strand of the double-stranded molecule encoded by the vector comprises a sequence corresponding to the target sequence of SEQ ID NO: 7 or 8;
[24] The composition of [21], wherein the cancer to be treated is lung cancer;
[25] The composition of [23], wherein the double-stranded molecule encoded by the vector has a length of less than about 100 nucleotides;
[26] The composition of [25], wherein the double-stranded molecule encoded by the vector has a length of less than about 75 nucleotide pairs;
[27] The composition of [26], wherein the double-stranded molecule encoded by the vector has a length of less than about 50 nucleotide pairs;
[28] The composition of [27], wherein the double-stranded molecule encoded by the vector has a length of less than about 25 nucleotide pairs;
[29] The composition of [28], wherein the double-stranded molecule encoded by the vector has a length of about 19 to about 25 nucleotide pairs;
[30] The composition of [21], wherein the double-stranded molecule encoded by the vector is composed of a single polynucleotide comprising both a sense strand and an antisense strand linked by an intervening single strand;
[31] The double-stranded molecule is represented by the general formula 5 ′-[A]-[B]-[A ′]-3 ′.
[A] is a sense strand containing a sequence corresponding to the target sequence of SEQ ID NO: 7 or 8, and [B] is an intervening single strand composed of 3 to 23 nucleotides. And [A ′] is an antisense strand comprising a sequence complementary to [A], the composition according to [30]; and [32] a transfection enhancing agent and a pharmaceutically acceptable carrier. The composition of [21], comprising.

Suitable compositions of the present invention are described in further detail below.
The double-stranded molecules of the invention are preferably formulated as a pharmaceutical composition prior to administration to a subject according to techniques known in the art. The pharmaceutical composition of the present invention is characterized by being at least sterile and free of pyrogens. As used herein, “pharmaceutical composition” includes formulations for human and veterinary use. Methods for preparing the pharmaceutical compositions of the invention are within the skill of the art as described, for example, in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985). The entire disclosure of which is incorporated herein by reference.

  The pharmaceutical composition of the present invention comprises a double-stranded molecule of the present invention or a vector encoding the same (for example, 0.1 to 90% by weight) mixed with a pharmaceutically acceptable carrier medium, or the molecule Of a pharmaceutically acceptable salt. Preferred physiologically acceptable carrier media are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.

  According to the present invention, the composition may comprise multiple types of double-stranded molecules, each of which may be directed to the same or different target sequences of ERCC6L. For example, the composition can include a double-stranded molecule directed against ERCC6L. Alternatively, for example, the composition may comprise a double stranded molecule directed against one, two, or more target sequences ERCC6L.

Furthermore, the composition of the invention may comprise a vector encoding one or more double-stranded molecules. For example, a vector can encode one, two, or several types of double-stranded molecules of the invention. Alternatively, the composition of the present invention may comprise a plurality of types of vectors each encoding a different double-stranded molecule.
Furthermore, the double-stranded molecule of the present invention may be included as a liposome in the composition of the present invention. For details on liposomes, see the section entitled “Methods of treating cancer using double-stranded molecules”.

The pharmaceutical compositions of the present invention may also contain conventional pharmaceutical excipients and / or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmotic pressure adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers such as tromethamine hydrochloride, chelating agents such as DTPA or DTPA-bisamide, or calcium chelate complexes such as calcium DTPA, CaNaDTPA-bisamide. ) Or, optionally, addition of calcium or sodium salts (eg, calcium chloride, calcium ascorbate, calcium gluconate, or calcium lactate). The pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
The solid composition may be a conventional non-toxic solid carrier such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium carbonate and the like.

For example, a solid pharmaceutical composition for oral administration comprises one or more of any of the above carriers and excipients and 10-95%, preferably 25-75% of the double-stranded molecule of the invention. Can be included. A pharmaceutical composition for aerosol (inhalation) administration is one of 0.01-20 wt%, preferably 1-10 wt% of the double-stranded molecule of the invention encapsulated in liposomes as described above. Or it may contain multiple and propellants. If desired, a carrier such as lecithin may be included for intranasal delivery.
In addition to the above, the composition of the present invention may contain other pharmaceutically active ingredients as long as they do not inhibit the in vivo function of the double-stranded molecule of the present invention. For example, the composition can include chemotherapeutic agents conventionally used to treat cancer.

In another aspect, the present invention provides the use of a double stranded nucleic acid molecule of the present invention in the manufacture of a pharmaceutical composition for treating cancer characterized by the expression of the ERCC6L gene. For example, the present invention relates to the use of a double stranded nucleic acid molecule that inhibits expression of the ERCC6L gene in a cell to produce a pharmaceutical composition for treating a cancer that expresses the ERCC6L gene, wherein the molecule Includes a sense strand that hybridizes to each other to form a double-stranded nucleic acid molecule and an antisense strand complementary thereto, and targets the sequence of SEQ ID NO: 7 or 8.
The present invention further provides a double-stranded nucleic acid molecule of the present invention for use in treating a cancer that expresses the ERCC6L gene.

  Alternatively, the present invention further provides a method or process for producing a pharmaceutical composition for treating cancer characterized by expression of the ERCC6L gene, wherein the method or process is used as an active ingredient Formulating a pharmaceutically or physiologically acceptable carrier with a double stranded nucleic acid molecule that inhibits expression of the gene in cells that overexpress the ERCC6L gene, the molecules hybridizing to each other and It contains a sense strand that forms a double-stranded nucleic acid molecule and an antisense strand complementary thereto, and targets the sequence of SEQ ID NO: 7 or 8.

  In another aspect, the present invention provides a method or process for producing a pharmaceutical composition for treating cancer characterized by expression of the ERCC6L gene, wherein the method or process comprises an active ingredient And a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double stranded nucleic acid molecule that inhibits expression of the gene in cells that overexpress the ERCC6L gene, the molecule comprising: A sense strand that hybridizes to each other to form a double-stranded nucleic acid molecule and an antisense strand complementary thereto, and targets the sequence of SEQ ID NO: 7 or 8.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the following materials, methods, and examples only illustrate aspects of the invention and are in no way intended to limit the scope of the invention. Accordingly, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

  The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

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 adenocarcinoma A427, NCI-H1781, A549, and LC319; lung squamous cell carcinoma (SCC) NCI-H226, EBC-1, NCI-H520, and NCI-H2170; and SCLC DMS114, DMS273, SBC-3, SBC-5, H196, and H446. All cells were cultured in monolayers in appropriate media supplemented with 10% FCS and maintained at 37 ° C. in a humidified atmosphere containing 5% CO ₂ . Human peripheral airway epithelial cells (SAEC) were cultured in optimized media purchased from Cambrex Bio Science, Inc. Primary lung cancer tissue samples were obtained with informed consent as previously described (Kikuchi T, et al. Oncogene 2003; 22: 2192-205., Taniwaki M et al. Int J Oncol 2006; 29: 567-75.). This study and the use of all clinical materials were approved by individual institutional ethics committees.

Semi-quantitative reverse transcription-PCR
Total RNA was extracted from cultured cells using TRIzol reagent (Life Technologies, Inc.) according to the manufacturer's protocol. The extracted RNA was treated with DNase I (Nippon Gene) and reverse transcribed using oligo (dT) primer and SuperScript II. Semi-quantitative reverse transcription-PCR (RT-PCR) experiments were performed using the following ERCC6L specific synthetic primers or using β-actin (ACTB) specific primers as internal controls: ERCC6L, 5 ′ -AAAAATCCAGGAGGCCCTAA-3 '(SEQ ID NO: 1) and 5'-AGATCTTTCTTGCCCTTAAGCCTG-3' (SEQ ID NO: 2); 3 '(SEQ ID NO: 4). The PCR reaction was optimized for cycle number so that product intensity was within the logarithmic phase of amplification.

Northern Blot Analysis Human multi-tissue blots (BD Biosciences Clontech) were hybridized with ERCC6L ³² P-labeled PCR product. ERCC6L cDNA probe was prepared by RT-PCR using the above primers. Prehybridization, hybridization, and washing were performed according to the supplier's recommendations. Blots were autoradiographed for 30 hours at room temperature using an intensifying BAS screen (Bio-Rad).

Immunofluorescence analysis Cells were plated on cover glass (Becton Dickinson Labware), fixed with 4% paraformaldehyde, and permeabilized for 3 minutes at room temperature with 0.1% Triton X-100 in PBS. . Nonspecific binding was blocked with CASBLOCK (ZYMED) for 10 minutes at room temperature. Cells were then incubated for 60 minutes at room temperature with the primary antibody of mouse monoclonal anti-myc antibody diluted in PBS containing 3% BSA. After washing with PBS, cells were stained with Alexa Fluor 488 conjugated secondary antibody (Molecular Probes) for 60 minutes at room temperature. After further washing with PBS, each specimen was encapsulated with Vectashield (Vector Laboratories, Inc.) containing 4 ′, 6′-diamidine-2′-phenylindole dihydrochloride (DAPI) and spectral confocal scanning system (TSC). Visualization with SP2 AOBS (Leica Microsystems).

RNA Interference Assay We previously established psiH1BX3.0, a vector-based RNA interference system designed to synthesize small interfering RNA (siRNA) in mammalian cells (Suzuki C et al Cancer Res 2003; 63: 7038-41.). Using 30 μL of LipofectAMINE 2000 (Invitrogen), 10 μg of siRNA expression vector was transfected into lung cancer cell line SBC-5. Transfected cells were cultured for 7 days in the presence of the appropriate concentration of geneticin (G418); colony counts were counted by Giemsa staining; after 7 days of treatment, cell viability was determined by 3- (4, bromide bromide). Evaluated by 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium assay. Briefly, Cell Count Kit-8 Solution (Dojindo) was added to each dish at a concentration of 1/10 volume and the plates were incubated at 37 ° C. for an additional 2 hours. The absorbance at 490 nm and 630 nm as a reference was then measured with a microplate reader 550 (Bio-Rad). In order to confirm suppression of ERCC6L mRNA expression, semi-quantitative RT-PCR experiments were performed according to standard protocols using the following ERCC6L-specific synthetic primers. The target sequence of the synthetic oligonucleotide for RNA interference was as follows: Control 1 (Luciferase / LUC: Photinus pyralis luciferase gene), 5′-CGTACCGGGATAACTTCGA-3 ′ (SEQ ID NO: 5) Control 2 (EGFP: gene, mutant of Aequorea victoria GFP), 5′-GAAGCAGCACGACTTCTCTC-3 ′ (SEQ ID NO: 6); siRNA-ERCC6L- # A, 5′-CTGCACAACACTACTTTTG-3 ′ (SEQ ID NO: 7); siRNA-ERCC6L- # B, 5′-ATGTTAATCAATAACTGGC-3 ′ (SEQ ID NO: 8).

Western blotting Cells were collected from a radioimmunoprecipitation assay buffer containing protease inhibitor cocktail set III (Calbiochem) [50 mmol / L Tris-HCl (pH 8.0), 150 mmol / L NaCl, 1% NP40, 0.5 % Deoxycholic acid-Na, 0.1% SDS]. Protein samples were separated by SDS-polyacrylamide gel and electroblotted onto Hybond-ECL nitrocellulose membrane (GE Healthcare Bio-Sciences). The blot was incubated with a mouse monoclonal anti-myc antibody. Antigen-antibody complexes were detected using a horseradish peroxidase-conjugated secondary antibody (GE Healthcare Bio-Sciences). Protein bands were visualized with a highly sensitive chemiluminescence western blotting detection reagent (GE Healthcare Bio-Sciences).

Cell Proliferation Assay The entire coding sequence of ERCC6L was cloned into the appropriate site of pcDNA3.1 myc-His plasmid vector (Invitrogen). COS-7 cells transfected with either myc-His tagged ERCC6L expressing plasmid or mock plasmid were cultured for 8 days in DMEM containing 10% FCS in the presence of appropriate concentrations of geneticin (G418). did. Cell viability was assessed by MTT assay; in brief, 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 ° C. for an additional 2 hours. The absorbance at 450 nm as a reference was then measured with a microplate reader 550 (BIO-RAD).

Example 2: ERCC6L Expression in Lung Tumors and Normal Tissues In order to explore new target molecules for developing therapeutic and / or diagnostic biomarkers for lung cancer, cDNA microarray analysis revealed that more than half of 101 lung cancer samples were 3 Genes with double or higher expression levels were first screened (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. Int J Oncol 2006; 28: 799-805 .; Taniwaki M, et al. Int J Oncol 2006; 29: 567-75.).
Of the 27,648 genes screened, overexpression of ERCC6L was identified in the majority of lung cancers examined, in 10 out of 15 additional lung cancer tissues and out of 14 lung cancer cell lines In 13 cases, semi-quantitative RT-PCR experiments confirmed its transactivation (FIGS. 1A and 1B). Northern blot analysis showed that ERCC6L is not expressed in normal tissues but strongly expressed in lung cancer cell lines (FIG. 1C). Immunofluorescence analysis was performed to examine the intracellular localization of exogenous ERCC6L in COS-7 cells. ERCC6L was weakly detected mainly in the cytoplasm and in the nucleus (FIG. 1D).

Example 3: Inhibition of lung cancer cell growth by siRNA against ERCC6L To assess whether ERCC6L is essential for lung cancer cell growth or survival, in addition to control plasmids (luciferase (LUC) and siRNA against EGFP), ERCC6L Plasmids were constructed to express siRNA against (si-ERCC6L- # A and si-ERCC6L- # B) and they were transfected into SBC-5 and A549 cells that strongly express ERCC6L. ERCC6L-mRNA levels in cells transfected with si-ERCC6L- # A or si-ERCC6L- # B were significantly reduced compared to cells transfected with either control siRNA (FIG. 2A). A significant decrease in the number of viable cells was observed (FIGS. 2B and 2C).

Example 4: Growth-promoting effect of ERCC6L In order to elucidate the potential role of ERCC6L in tumorigenesis, a plasmid (pcDNA3.1 / myc-His A vector) or mock vector designed to express ERCC6L was prepared. . In order to determine the effect of ERCC6L on the growth of mammalian cells, transient transfectants were made using COS-7 cells that hardly express endogenous ERCC6L. The cells transiently expressing exogenous ERCC6L showed significant growth promotion compared to cells transfected with the mock vector (FIGS. 3A and 3B).

Discussion Recent advances in the identification and characterization of new molecular targets for cancer treatment have created great interest in the development of new types of anticancer drugs (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. Int J Oncol 2006; 28: 799-805.; Taniwaki M, et al. Int J Oncol 2006; 29: 567-75. ).

To date, many targeted therapies have been investigated for lung cancer, but the range of tumor types that respond and the effectiveness of treatment remains very limited. Since molecular mechanism drugs have a clear mechanism of action, they are expected to have minimal adverse effects and high specificity for malignant cells. A promising strategy for this purpose is to combine the ability of genome-wide expression analysis to efficiently identify genes that are overexpressed in cancer cells and high-throughput screening for loss-of-function effects using RNAi techniques. (Suzuki C, et al. Cancer Res 2003; 63: 7038-41.; Ishikawa N, et al. Clin Cancer Res 2004; 10: 8363-70.; Kato T, et al. Cancer Res 2005; 65: 5638-46.; Furukawa C, et al. Cancer Res 2005; 65: 7102-10.; Ishikawa N, et al. Cancer Res 2005; 65: 9176-84.; Suzuki C, et al. Cancer Res 2005; 65: 11314-25.; Ishikawa N , et al. Cancer Sci 2006; 97: 737-45 .; Takahashi K, et al. Cancer Res 2006; 66: 9408-19.; Hayama S, et al. Cancer Res 2006; 66: 10339-48.).
Using this combined approach, it was shown that ERCC6L is frequently overexpressed in clinical lung cancer samples and cell lines, and that its gene product plays an essential role in the growth and progression of lung cancer cells.

In the present invention, when lung cancer cells were treated with specific siRNA for knocking down ERCC6L expression, growth of cancer cells was suppressed. We have also shown further evidence supporting the significance of this pathway in carcinogenesis; for example, expression of ERCC6L significantly promoted cell proliferation in vitro. The results obtained suggested that ERCC6L is likely to be an important growth factor for lung cancer cells, but the molecular mechanism underlying the increase in ERCC6L expression levels in many cancer cells remains elucidated. Not. Since ERCC6L is classified as one of the cancer-specific antigens, selective inhibition of ERCC6L activity by molecular targeted drugs minimizes the risk of adverse events and has potent biological activity against cancer. It can be a promising therapeutic strategy that is expected to have.
In summary, ERCC6L activation has a specific functional role for cancer cell growth. The data strongly suggests the possibility of designing new anticancer agents to specifically target the carcinogenic activity of ERCC6L for the treatment of lung cancer patients.

INDUSTRIAL APPLICABILITY The data provided herein will provide a comprehensive understanding of cancer, facilitate the development of new diagnostic strategies, and provide clues for identifying molecular targets for therapeutic and prophylactic agents. give. Such information contributes to a deeper understanding of tumorigenesis and provides an indicator for developing new strategies for cancer diagnosis, treatment, and ultimately prevention.

  Specifically, the gene expression analysis of cancer described herein using a combination of laser capture dissection and genome-wide cDNA microarrays revealed that ERCC6L is a gene that is significantly elevated in cancer compared to normal organs. Was identified. It is therefore useful in the context of cancer prevention and treatment. For example, given its differential expression, ERCC6L can be advantageously used as a molecular diagnostic marker for identifying and detecting cancer, particularly lung cancer. Thus, the ERCC6L gene and the protein encoded thereby are useful in cancer diagnostic kits and assays.

The present invention further demonstrates that cell proliferation can be suppressed by double stranded nucleic acid molecules that specifically target the ERCC6L gene. Therefore, this double-stranded nucleic acid molecule is useful for the development of anticancer preparations. In addition, ERCC6L polypeptides are useful targets for the development of anticancer formulations. For example, a substance that blocks the expression of ERCC6L protein or prevents its activity may be therapeutically useful as an anticancer agent, particularly an anticancer agent for treating lung cancer.
All publications, databases, sequences, patents, and patent applications cited herein are hereby incorporated by reference.

  Although the invention has been described in detail and with reference to specific embodiments thereof, the foregoing description is illustrative and explanatory in nature and is intended to describe the invention and its preferred embodiments. It should be understood. Those skilled in the art will readily recognize through routine experimentation that various changes and modifications can be made therein without departing from the spirit and scope of the invention. Accordingly, the invention is intended to be defined not by the above description, but by the following claims and their equivalents.

Claims (27)

A method for detecting or diagnosing cancer or a predisposition to develop cancer in a subject, comprising measuring the expression level of the ERCC6L gene in a biological sample from the subject, as compared to a normal control level of the gene An increase in the level indicates that the subject has cancer or is at risk of developing cancer, wherein the expression level is
(A) detecting mRNA of the ERCC6L gene;
Measured by any one of the methods selected from the group consisting of: (b) detecting the protein encoded by the ERCC6L gene; and (c) detecting the biological activity of the protein encoded by the ERCC6L gene. The way it is.   The method of claim 1, wherein the increase is at least 10% higher than the normal control level.   The method of claim 1, wherein the subject-derived biological sample is a biopsy sample.   The method of claim 1, wherein the cancer is lung cancer. (A) a reagent for detecting mRNA of the ERCC6L gene;
(B) a reagent for detecting a protein encoded by the ERCC6L gene; and (c) a reagent selected from the group consisting of a reagent for detecting the biological activity of the protein encoded by the ERCC6L gene. Kit for diagnosing cancer.   The kit according to claim 5, wherein the reagent is a probe or primer set for a gene transcript of ERCC6L, or an antibody against a protein encoded by the ERCC6L gene. A method for screening candidate substances for treating and / or preventing cancer or inhibiting the growth of cancer cells,
(A) contacting a test substance with an ERCC6L polypeptide or fragment thereof;
(B) detecting a binding activity between the polypeptide or the fragment and the test substance; and (c) a test substance that binds to the polypeptide or the fragment for treating or preventing cancer. Selecting the candidate substance. A method for screening candidate substances for treating and / or preventing cancer or inhibiting the growth of cancer cells,
(A) contacting the test substance with a cell expressing the ERCC6L gene;
(B) detecting the expression level of the ERCC6L gene in the cells of step (a); and (c) detecting in step (b) compared to the expression level of the ERCC6L gene detected in the absence of the test substance Selecting a test substance that reduces the expressed expression level. A method for screening candidate substances for treating and / or preventing cancer or inhibiting the growth of cancer cells,
(A) contacting a test substance with an ERCC6L polypeptide or fragment thereof;
(B) detecting the biological activity of said polypeptide or fragment of step (a); and (c) comparing the biological activity detected in the absence of the test substance with step (b) And selecting a test substance that suppresses the biological activity of the polypeptide or fragment detected in (1).   10. The method of claim 9, wherein the biological activity is cell proliferation activity. A method for screening candidate substances for treating and / or preventing cancer or inhibiting the growth of cancer cells,
(A) contacting a test substance with a cell into which a vector containing a transcriptional regulatory region of the ERCC6L gene and a reporter gene expressed under the control of the transcriptional regulatory region is introduced;
(B) measuring the expression or activity of said reporter gene in step (a); and (c) detected in step (b) compared to the expression and / or activity level in the absence of the test substance Selecting a substance that reduces the level of expression and / or activity of the reporter gene.   The method according to any one of claims 7, 8, 9, and 11, wherein the cancer is lung cancer.   An isolated double stranded molecule comprising a sense strand and an antisense strand complementary thereto, wherein the strands hybridize to each other to form a double stranded molecule, the sense strand comprising SEQ ID NO: 7 and Isolation comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of 8, wherein said double-stranded molecule inhibits expression and cell proliferation of said gene when introduced into a cell expressing ERCC6L gene Double-stranded molecule.   14. The double-stranded molecule of claim 13, wherein the sense strand hybridizes with the antisense strand at the target sequence to form a double-stranded molecule having a length of 19-25 nucleotide pairs.   14. A double-stranded molecule according to claim 13, having one or two 3 'overhangs 3' of the sense and / or antisense strand.   14. A double-stranded molecule according to claim 13, which is a single polynucleotide comprising a sense strand and an antisense strand linked by a single-stranded nucleotide sequence. The polynucleotide has the general formula 5 ′-[A]-[B]-[A ′]-3 ′.
17. wherein [A] is the sense strand; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotides; and [A ′] is the antisense strand. Double-stranded molecule.   The double-stranded molecule according to claim 17, wherein [A] comprises a nucleotide sequence corresponding to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7 and 8.   A vector encoding the double-stranded molecule according to any one of claims 13 to 18.   A method for treating and / or preventing cancer in a subject comprising: a pharmaceutically effective amount of a double-stranded molecule against the ERCC6L gene or a vector encoding the same and a pharmaceutically acceptable carrier. Administering the ERCC6L gene and inhibiting cell growth when introduced into a cell that expresses the ERCC6L gene.   21. The method of claim 20, wherein the double-stranded molecule is that of claim 13.   21. The method of claim 20, wherein the vector is that of claim 19.   21. The method of claim 20, wherein the cancer to be treated is lung cancer.   A composition for treating and / or preventing cancer comprising a pharmaceutically effective amount of a double-stranded molecule against the ERCC6L gene or a vector encoding the same, and a pharmaceutically acceptable carrier. A composition comprising: wherein the double-stranded molecule inhibits ERCC6L gene expression and cell proliferation when introduced into a cell that expresses the ERCC6L gene.   25. The composition of claim 24, wherein the double-stranded molecule is that of claim 13.   25. The composition of claim 24, wherein the vector is that of claim 19.   25. The composition of claim 24, wherein the cancer to be treated is lung cancer.

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