WO2012157239A1 - Gène chodl comme marqueur tumoral et cible thérapeutique pour le cancer - Google Patents

Gène chodl comme marqueur tumoral et cible thérapeutique pour le cancer Download PDF

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WO2012157239A1
WO2012157239A1 PCT/JP2012/003120 JP2012003120W WO2012157239A1 WO 2012157239 A1 WO2012157239 A1 WO 2012157239A1 JP 2012003120 W JP2012003120 W JP 2012003120W WO 2012157239 A1 WO2012157239 A1 WO 2012157239A1
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chodl
double
gene
stranded molecule
cancer
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Yataro Daigo
Yusuke Nakamura
Takuya Tsunoda
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Oncotherapy Science, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the field of biological science, more specifically to the field of cancer research, cancer diagnosis and cancer therapy.
  • the present invention relates to methods for assessing or predicting the prognosis of a subject with cancer and methods for treating and preventing cancer.
  • Priority The present application claims the benefit of U.S. Provisional Application No. 61/486,641, filed on May 16, 2011, the entire contents of which are hereby incorporated herein by reference.
  • Lung cancer is one of the most common cause of cancer death in the world, and non-small cell lung cancer (NSCLC) accounts for nearly 80% of those cases (Ahmedin J, et al., 2009. CA Cancer J Clin 2009; 59:225-49., Miki D, et al., Nat Genet 2010; 42:893-6.).
  • molecular-targeted agents including anti-EGFR or anti-VEGF monoclonal antibody, cetuximab or bevacizumab, and small molecule inhibitors of EGFR tyrosine kinase, such as gefitinib and erlotinib have been investigated in the clinical trials and/or were approved for clinical use. These agents are effective in the treatment of advanced NSCLC to a certain extent, but a proportion of patients who could receive a survival benefit is still limited (Dowell J, et al., Nat Rev Drug Discov 2005; 4:13-4., Pal SK, Pegram M., Anticancer Drugs 2005; 16:483-94.). Hence, new therapeutic strategies, such as the development of more selective and effective molecular-targeted agents against lung cancer are eagerly awaited.
  • Genome-wide cDNA microarray analysis of cancers enabled us to obtain their comprehensive gene expression profiles and to compare the gene expression levels with clinicopathological and biological information of cancers (NPL 1). This approach is also useful to identify unknown molecules involved in the carcinogenic pathway.
  • NPL 1 clinicopathological and biological information of cancers
  • CHODL a human ortholog of mouse chondrolectin (chodl) encodes a single-pass transmembrane protein with one carbohydrate recognition domain (CRD) of C-type lectins (NPL 37).
  • C-type lectin family is classified into 17 groups according to their domain architecture. They are divided into two major types; transmembrane C-type lectins and soluble ones. These lectins have a wide variety of functions at cytoplasm, cytoplasmic membrane, and extracellular space.
  • Mouse chodl was mainly expressed in embryonic tissues, and its expression level was tightly controlled during embryonic development.
  • Chodl transcripts were present at the stage of 7 days post-conception (dpc), and elevated up to the stage of 15 dpc, followed by decrease at the stage of 17 dpc (NPL 38). To date, the biological function of CHODL and its activation in human cancers were not reported.
  • NPL 1 Daigo Y, Nakamura Y., Gen Thorac Cardiovasc Surg 2008; 56:43-53.
  • NPL 2 Kikuchi T, et al., Oncogene 2003; 22:2192-205.
  • NPL 3 Kakiuchi S, et al., Mol Cancer Res 2003; 1:485-99.
  • NPL 4 Kakiuchi S, et al., Hum Mol Genet 2004; 13:3029-43.
  • NPL 5 Kikuchi T, et al., Int J Oncol 2006; 28:799-805.
  • NPL 6 Taniwaki M, et al., Int J Oncol 2006; 29:567 -75.
  • NPL 7 Suzuki C, et al., Cancer Res 2003; 63:7038-41.
  • NPL 8 Kato T, et al., Cancer Res 2005; 65:5638-46.
  • NPL 9 Furukawa C, et al., Cancer Res 2005; 65:7102-10.
  • NPL 10 Suzuki C, et al., Cancer Res 2005; 65:11314-25.
  • NPL 11 Ishikawa N, et al., Cancer Sci 2006; 97:737-45.
  • NPL 12 Takahashi K, et al., Cancer Res 2006; 66:9408-19.
  • NPL 13 Hayama S, et al., Cancer Res 2006; 66:10339-48.
  • NPL 14 Kato T, et al., Clin Cancer Res 2007; 13:434-42.
  • NPL 15 Suzuki C, et al., Mol Cancer Ther 2007; 6:542-51.
  • NPL 16 Hayama S, et al., Cancer Res 2007; 67:4113-22.
  • NPL 17 Taniwaki M, et al., Clin Cancer Res 2007; 13:6624-31.
  • NPL 18 Mano Y, et al., Cancer Sci 2007; 98:1902-13.
  • NPL 19 Kato T, et al., Cancer Res 2007; 67:8544-53.
  • NPL 20 Kato T, et al., Clin Cancer Res 2008; 14:2363-70.
  • NPL 21 Dunleavy EM, et al., Cell 2009; 137:485-97.
  • NPL 22 Hirata D, et al., Clin Cancer Res 2009; 15:256-66.
  • NPL 23 Sato N, et al., Clin Cancer Res. 2010; 16: 226-39.
  • NPL 24 Sato N, et al., Genes Chromosomes Cancer. 2010; 49: 353-67.
  • NPL 25 Nguyen MH, et al., Cancer Res. 2010; 70: 5337-47.
  • NPL 26 Ishikawa N, et al., Clin Cancer Res 2004; 10:8363-70.
  • NPL 27 Ishikawa N, et al., Cancer Res 2005; 65:9176-84.
  • NPL 28 Yamabuki T, et al., Cancer Res 2007; 67:2517-25.
  • NPL 29 Ishikawa N, et al., Cancer Res 2007; 67: 11601-11.
  • NPL 30 Takano A, et al., Cancer Res 2009; 69: 6694-703.
  • NPL 31 Sato N, et al., Cancer Res 2010; 70: 5326-36.
  • NPL 32 Suda T, et al., Cancer Sci 2007; 98:1803-8.
  • NPL 33 Mizukami Y, et al., Cancer Sci 2008; 99: 1448-54.
  • NPL 34 Harao M, et al., Int J Cancer 2008; 123: 2616-25.
  • NPL 35 Kono K, et al., Cancer Sci. 2009; 100: 1502-9.
  • NPL 36 Tomita Y, et al., Cancer Sci 2011 [Epub ahead of print]
  • NPL 37 Weng L, et al., Genomics. 2002 Jul; 80(1):62-7.
  • NPL 38 Weng, L., et al., Gene 2003; 308:21-9.
  • CHODL chondrolectin
  • the present invention relates to cancer-related gene CHODL, which is commonly up-regulated in tumors, and strategies for the development of molecular targeted drugs for cancer treatment using CHODL. Accordingly, it is an object of the present invention to provide methods of assessing or predicting a prognosis of a subject with cancer, using the expression level of the CHODL gene as an index.
  • the present invention provides the following [1] to [19]: [1] A method for assessing or predicting a prognosis of a subject with cancer, wherein the method comprises steps of: (a) detecting an expression level of the CHODL gene in a subject-derived biological sample; (b) comparing the expression level detected in step (a) to a control level; and (c) determining the prognosis of the subject based on the comparison of step (b); [2] The method of [1], wherein the control level is a good prognosis control level and an increase of the expression level compared to the control level indicates a poor prognosis; [3] The method of [1] or [2], wherein the expression level is determined by a method selected from a group consisting of: (a) detecting an mRNA of the CHODL gene; (b) detecting a CHODL protein;
  • Fig.1 demonstrates CHODL expression in lung cancers.
  • Part A depicts expression of CHODL in clinical lung cancers, examined by semi-quantitative RT-PCR.
  • Part B depicts expression of CHODL in lung-cancer cell lines, examined by semi-quantitative RT-PCR.
  • Part C depicts subcellular localization of endogenous CHODL protein in lung cancer cells.
  • CHODL was stained in the cytoplasm of cells with granular appearance in CHODL-positive A549 and NCI-H2170 cells, while CHODL was not stained in LC319 and BEAS-2B cells that did not express endogenous CHODL.
  • Part D depicts co-localization of endogenous CHODL with calreticulin as endoplasmic reticulum marker in A549 cells.
  • Fig. 2 demonstrates the expression of CHODL in normal tissues and lung tumors.
  • Part A depicts northern blot analysis of the CHODL transcript in 23 normal adult human tissues.
  • Part B depicts immunohistochemical evaluation of CHODL protein using anti-CHODL antibody in lung ADC, lung SCC, lung LCC and five normal tissues.
  • Part C depicts immunohistochemical staining of CHODL protein using anti-CHODL antibody in four representative paired lung tumors and adjacent normal lung tissues.
  • Part D depicts immunohistochemical evaluation of CHODL expression on tissue microarrays (X100). Examples are shown of strong, weak, absent, and normal lung tissue.
  • Fig. 3 demonstrates the growth effect of CHODL expression.
  • Part A-C depicts response of SBC-5 and NCI-H2170 cells to si-CHODL#A, si-CHODL#B or control siRNAs (si-LUC or si-SCR).
  • Part A depicts the level of CHODL mRNA expression detected by semi-quantitative RT-PCR in cells transfected with either control siRNAs or si-CHODLs.
  • Part B depicts the effect of siRNA against CHODL on cell viability, evaluated by MTT assay.
  • Part C depicts the effect of siRNA against CHODL on colony formation, evaluated by colony formation assays. All assays were performed thrice and in triplicate wells.
  • Part D-E depicts growth promoting effects of CHODL.
  • Part D depicts transient expression of CHODL in COS-7 and LC319 cells, detected by western blot analysis.
  • Part E depicts effect of CHODL on growth of COS-7 and LC319 cells. At each time point, cell viability was evaluated by MTT assay.
  • Fig. 4 demonstrates enhancement of cellular invasiveness by CHODL.
  • Part A depicts transient expression of CHODL in COS-7 and LC319 cells, detected by western blot analysis.
  • Part B and C depict assays demonstrating the invasive nature of COS-7 and LC319 cells in Matrigel matrix after transfection with expression plasmids for human CHODL.
  • Fig. 5 demonstrates the specificity of antibody to CHODL protein.
  • Part A depicts confirmation of the specificity of the CHODL signals in immunocytochemistry.
  • the cytoplasmic signals of CHODL of A549 cells were decreased by siRNA against CHODL.
  • Part B depicts antigen blocking assays to examine antibody specificity to CHODL. Assay was performed as follows; before immunohistochemical staining, 1 microgram anti-CHODL antibody was incubated with 5 microgram of CHODL antigen peptides (Catalog No. sc-54867P; Santa Cruz Biotechnology) that were used for immunization of goats to produce polyclonal anti-human CHODL antibodies for 60 min at 37 degrees C. The reaction product was centrifuged at 12,000 X g for 15 min at 4 degrees C to remove the immune complexes, and the supernatant was used as a neutralized antibody for further immunohistochemical analysis.
  • an isolated or purified antibody refers to antibodies that are substantially free of cellular material for example, carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • heterologous protein also referred to herein as a "contaminating protein”
  • the polypeptide is recombinantly produced, in some embodiments it is also substantially free of culture medium, which includes preparations of polypeptide with culture medium less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • the polypeptide is produced by chemical synthesis, in some embodiments it is substantially free of chemical precursors or other chemicals, which includes preparations of polypeptide with chemical precursors or other chemicals involved in the synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation.
  • That a particular protein preparation contains an isolated or purified polypeptide can be shown, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining or the like of the gel.
  • proteins including antibodies of the present invention are isolated or purified.
  • biological sample refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • body fluids including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen.
  • Biological sample further refers to a homogenate, lysate, extract, cell culture or tissue culture prepared from a whole organism or a subset of its cells, tissues or component parts, or a fraction or portion thereof.
  • biological sample refers to a medium, for example, a nutrient broth or gel in which an organism has been propagated, which contains cellular components, for example, proteins or polynucleotides.
  • a biological sample may be a tissue sample, such as tumor biopsy specimen or collection of lung cells or, alternatively, a serum sample.
  • polypeptide polypeptide
  • peptide protein
  • protein polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, for example, an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly functions to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine).
  • amino acid analog refers to substances that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium).
  • modified R group or modified backbones e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium.
  • amino acid mimetic refers to chemical substances that have different structures but similar functions to general amino acids. Amino acids can be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • nucleic acid and nucleic acid molecule
  • gene refers to the amino acids referred to by their commonly accepted single-letter codes. Similar to the amino acids, they encompass both naturally-occurring and non-naturally occurring nucleic acid polymers.
  • the gene, polynucleotide, oligonucleotide, nucleic acid, or nucleic acid molecule can be composed of DNA, RNA or a combination thereof.
  • CHODL gene encompasses polynucleotides that encode human CHODL gene or any of the functional equivalents of the human CHODL gene.
  • the CHODL gene or its functional equivalent can be obtained from nature as naturally occurring proteins via conventional cloning methods or through chemical synthesis based on the selected nucleotide sequence. Methods for cloning genes using cDNA libraries and such are well known in the art.
  • cancer refers to cancers over-expressing the CHODL gene, such as lung cancer, including non small-cell lung cancer (NSCLC) small-cell lung cancer (SCLC).
  • NSCLC includes lung squamous cell carcinoma (SCC), adenocarcinoma (ADC) and large cell carcinoma (LCC).
  • the CHODL (chondrolectin) gene encodes a type I membrane protein with a carbohydrate recognition domain characteristic of C-type lectins in its extracellular portion.
  • the protein encoded by the CHODL gene localizes predominantly to the perinuclear region.
  • CHODL protein the protein encoded by the CHODL gene is referred to as "CHODL protein", and sometimes as "CHODL” or "CHODL polypeptide”.
  • nucleotide sequences of the CHODL gene and the amino acid sequence of the CHODL protein are known to those skilled in the art, and obtained, for example, from gene databases on the web site such as GenBank TM .
  • Exemplified nucleotide sequences of the human CHODL gene are shown in SEQ ID NO: 11 (GenBank accession No. NM_001204174.1), SEQ ID NO: 13 (GenBank accession No. NM_001204175.1), SEQ ID NO: 15 (GenBank accession No. NM_001204176.1), SEQ ID NO: 17 (GenBank accession No. NM_001204177.1), SEQ ID NO: 19 (GenBank accession No.
  • CHODL Functional equivalents of CHODL include those wherein one or more amino acids, e.g., 1-5 amino acids, e.g., up to 5% of amino acids, are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the CHODL protein.
  • amino acids e.g., 1-5 amino acids, e.g., up to 5% of amino acids
  • modifications of one or more amino acid in a protein do not influence the function of the protein (Mark DF, et al., Proc Natl Acad Sci U S A. 1984 Sep;81(18):5662-6; Zoller MJ & Smith M. Nucleic Acids Res. 1982 Oct 25;10(20):6487-500; Wang A, et al., Science.
  • amino acid side chains examples include hydrophobic amino acids (alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, valine), hydrophilic amino acids (arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, serine, threonine), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (glycine, alanine, valine, leucine, isoleucine, praline); a hydroxyl group containing side-chain (serine, threonine, tyrosine); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (aspartic acid, asparagine, glutamic acid, glutamine); a base containing side-chain (arginine, lysine
  • conservative substitution tables providing functionally similar amino acids are well known in the art.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: (1) Alanine (A), Glycine (G); (2) Aspartic acid (D), Glutamic acid (E); (3) 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, e.g., Thomas E. Creighton, Proteins Publisher: New York: W.H. Freeman, c1984).
  • Such conservatively modified polypeptides are included in the CHODL protein.
  • the present invention is not restricted thereto and the CHODL protein includes non-conservative modifications so long as they retain any one of the biological activity of the CHODL protein.
  • the number of amino acids to be mutated in such a modified protein is generally 10 amino acids of less, for example, 6 amino acids of less, for example, 3 amino acids or less.
  • Fusion proteins include fusions of the CHODL protein and other peptides or proteins, which also can be used in the present invention. Fusion proteins can be made by techniques well known to a person skilled in the art, for example, by linking the DNA encoding the CHODL gene with a DNA encoding other peptides or proteins, so that the frames match, inserting the fusion DNA into an expression vector and expressing it in a host. There is no restriction as to the peptides or proteins fused to the CHODL protein so long as the resulting fusion protein retains any one of the objective biological activity of the CHODL protein.
  • FLAG Hopp TP, et al., Biotechnology 6: 1204-10 (1988)
  • 6xHis containing six His (histidine) residues 10xHis
  • Influenza agglutinin HA
  • human c-myc fragment VSP-GP fragment
  • p18HIV fragment T7-tag
  • HSV-tag HSV-tag
  • E-tag E-tag
  • proteins that can be fused to a protein of the invention include GST (glutathione-S-transferase), Influenza agglutinin (HA), immunoglobulin constant region, beta-galactosidase, MBP (maltose-binding protein), and such.
  • GST glutthione-S-transferase
  • Influenza agglutinin HA
  • immunoglobulin constant region beta-galactosidase
  • beta-galactosidase beta-galactosidase
  • MBP maltose-binding protein
  • Methods known in the art to isolate functional equivalent proteins include, for example, hybridization techniques (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Lab. Press, 2001).
  • One skilled in the art can readily isolate a DNA having high homology (i.e., sequence identity) with a whole or part of the human CHODL DNA sequences (e.g., SEQ ID NO: 11, 13, 15, 17, 19 or 21) encoding the human CHODL protein, and isolate functional equivalent proteins to the human CHODL protein from the isolated DNA.
  • the CHODL protein used for the present invention include those that are encoded by DNA that hybridize under stringent conditions with a whole or part of the DNA sequence encoding the human CHODL protein and are functional equivalent to the human CHODL protein.
  • These proteins include mammal homologues corresponding to the proteins derived from human or mouse (for example, proteins encoded by monkey, rat, rabbit or bovine genes).
  • isolating a cDNA highly homologous to the DNA encoding the human CHODL gene lung cancer tissues or cell lines, or tissues from testis can be used.
  • hybridization refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will differ under different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • stringent conditions are selected to be about 5-10 degrees Centigrade lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • Stringent conditions can also be achieved with the addition of destabilizing agents for example, formamide.
  • a positive signal is at least two times of background, for example, 10 times of background hybridization.
  • hybridization can be performed by conducting prehybridization at 68 degrees C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 degrees C for 1 hour or longer.
  • the following washing step can be conducted, for example, in a low stringent condition.
  • a low stringent condition is, for example, 42 degrees C, 2x SSC, 0.1% SDS, for example, 50 degrees C, 2x SSC, 0.1% SDS.
  • high stringent condition is used.
  • a high stringent condition is, for example, washing 3 times in 2x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37 degrees C for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50 degrees C for 20 min.
  • temperature and salt concentration can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.
  • a gene amplification method for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a DNA encoding a protein functional equivalent to the human CHODL gene, using a primer synthesized based on the sequence information of the DNA encoding the human CHODL, examples of primer sequences are pointed out in Semi-quantitative RT-PCR in [EXAMPLE].
  • PCR polymerase chain reaction
  • Proteins that are functional equivalents to the human CHODL protein encoded by the DNA isolated through the above hybridization techniques or gene amplification techniques normally have a high homology (also referred to as sequence identity) to the amino acid sequence of the human CHODL protein.
  • “High homology” typically refers to the degree of identity between two optimally aligned sequences (either polypeptide or polynucleotide sequences).
  • high homology or sequence identity refers to homology of 40% or higher, for example, 60% or higher, for example, 80% or higher, for example, 85%, 90%, 95%, 98%, 99%, or higher.
  • the degree of homology or identity between two polypeptide or polynucleotide sequences can be determined by following the algorithm (Wilbur WJ & Lipman DJ. Proc Natl Acad Sci U S A. 1983 Feb; 80 (3):726-30).
  • BLAST and BLAST 2.0 algorithms are described (Altschul SF, et al., J Mol Biol. 1990 Oct 5; 215 (3):403-10; Nucleic Acids Res. 1997 Sep 1;25(17):3389-402).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the worldwide web at ncbi.nlm.nih.gov/).
  • the algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff S & Henikoff JG. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10915-9).
  • a protein useful in the context of the present invention can have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has any one of the biological activity of the CHODL protein, it is useful in the present invention.
  • a partial peptide has an amino acid sequence specific to the CHODL protein and consists of less than about 400 amino acids, usually less than about 200 and often less than about 100 amino acids, and at least about 7 amino acids, for example, about 8 amino acids or more, for example, about 9 amino acids or more.
  • the CHODL protein or functional equivalent thereof can be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on the selected amino acid sequence.
  • conventional peptide synthesis methods that can be adopted for the synthesis include: (1) Peptide Synthesis, Interscience, New York, 1966; (2) The Proteins, Vol. 2, Academic Press, New York, 1976; (3) Peptide Synthesis (in Japanese), Maruzen Co., 1975; (4) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985; (5) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991; (6) WO99/67288; and (7) Barany G. & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.
  • the CHODL protein can be obtained adopting any known genetic engineering methods for producing polypeptides (e.g., Morrison DA., et al., J Bacteriol. 1977 Oct;132(1):349-51; Clark-Curtiss JE & Curtiss R 3rd. Methods Enzymol. 1983;101:347-62).
  • a suitable vector comprising a polynucleotide encoding the objective protein in an expressible form (e.g., downstream of a regulatory sequence comprising a promoter) is prepared, transformed into a suitable host cell, and then the host cell is cultured to produce the protein.
  • the CHODL gene is expressed in host (e.g., animal) cells and such by inserting the gene into a vector for expressing foreign genes, for example, pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8.
  • a promoter can be used for the expression. Any commonly used promoters can be employed including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic engineering, vol. 3. Academic Press, London, 1982, 83-141), the EF- alpha promoter (Kim DW, et al. Gene. 1990 Jul 16;91(2):217-23), the CAG promoter (Niwa H, et al., Gene. 1991 Dec 15;108(2):193-9), the RSV LTR promoter (Cullen BR. Methods Enzymol. 1987;152:684-704), the SR alpha promoter (Takebe Y, et al., Mol Cell Biol.
  • the introduction of the vector into host cells to express the CHODL gene can be performed according to any methods, for example, the electroporation method (Chu G, et al., Nucleic Acids Res. 1987 Feb 11;15(3):1311-26), the calcium phosphate method (Chen C & Okayama H. Mol Cell Biol. 1987 Aug;7(8):2745-52), the DEAE dextran method (Lopata MA, et al., Nucleic Acids Res. 1984 Jul 25;12(14):5707-17; Sussman DJ & Milman G. Mol Cell Biol. 1984 Aug;4(8):1641-3), the Lipofectin method (Derijard B, et al., Cell.
  • electroporation method Chou G, et al., Nucleic Acids Res. 1987 Feb 11;15(3):1311-26
  • the calcium phosphate method Choen C & Okayama H. Mol Cell Biol. 1987 Aug;7(8):2745-52
  • the proteins can also be produced in vitro by using an in vitro translation system.
  • the present invention is based, in part, on the discovery that the (over)expression of CHODL gene is significantly associated with poorer prognosis of subjects with cancer, e.g., lung cancers, especially NSCLC.
  • the present invention provides a method for assessing or predicting the prognosis of a subject with cancer, by determining the expression level of the CHODL gene in a subject-derived biological sample; and comparing the expression level to a control level.
  • prognosis refers to a forecast as to the probable outcome of the disease as well as the prospect of recovery from the disease as indicated by the nature and symptoms of the case. Accordingly, a less favorable, negative or poor prognosis is defined by a lower post-treatment survival term or survival rate. Conversely, a positive, favorable, or good prognosis is defined by an elevated post-treatment survival term or survival rate.
  • assessing the prognosis refer to the ability of predicting, forecasting or correlating a given detection or measurement with a future outcome of cancer of the patient (e.g., malignancy, likelihood of curing cancer, estimated time of survival, and the like).
  • a determination of the expression level of the CHODL gene over time enables a predicting of an outcome for the patient (e.g., increase or decrease in malignancy, increase or decrease in grade of a cancer, likelihood of curing cancer, survival, and the like).
  • the phrase "assessing (or predicting) the prognosis” is intended to encompass predictions and likelihood analysis of cancer, progression, particularly cancer recurrence, metastatic spread and disease relapse.
  • the present method for assessing or predicting the prognosis is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria for example, disease staging, and disease monitoring and surveillance for metastasis or recurrence of neoplastic disease.
  • the present invention provides the following methods [1] to [7]: [1] A method for assessing or predicting a prognosis of a subject with cancer, wherein the method comprises steps of: (a) determining an expression level of the CHODL gene in a subject-derived biological sample; (b) comparing the expression level determined in step (a) to a control level; and (c) predicting the prognosis of the subject based on the comparison of (b); [2] The method of [1], wherein the control level is a good prognosis control level and an increase of the expression level compared to the control level indicates poor prognosis; [3] The method of [1], wherein the control level is a poor prognosis control level and a similar expression level to the control level indicates poor prognosis; [4] The method of [2], wherein the increase is at least 10% greater than said control level; [5] The method of any one of [1] to [4], wherein the expression level is determined by a method selected from a method selected
  • cancer is lung cancer. More preferably, lung cancer is NSCLC.
  • the subject for the method of the present invention can be a mammal and includes human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • the subject-derived biological sample used for the method of the present invention can be any sample derived from the subject with cancer so long as transcription product or translation product of the CHODL gene can be detected in the sample.
  • a subject-derived biological sample may be a bodily tissue sample or a bodily fluid sample. Examples of bodily fluid samples include sputum, blood, serum, plasma, pleural effusion, and so on.
  • a subject-derived biological sample is a tissue sample containing a cancerous area.
  • a lung cancer tissue sample is a preferable sample.
  • a subject-derived biological sample can be cells purified or obtained from a tissue.
  • Subject-derived biological samples can be obtained from a subject at various time points, including before, during, and/or after a treatment. For example, a lung cancer cell(s) obtained from a subject to be assessed is a preferable biological sample.
  • control level used for comparison can be, for example, the expression level of the CHODL gene detected before any kind of treatment in an individual or a population of individuals who showed good or positive prognosis of cancer, after the treatment, which herein will be referred to as "good prognosis control level".
  • control level can be the expression level of the CHODL gene detected before any kind of treatment in an individual or a population of individuals who showed poor or negative prognosis of cancer, after the treatment, which herein will be referred to as "poor prognosis control level".
  • the "control level” is a single expression pattern derived from a single reference population or from a plurality of expression patterns.
  • the control level can be determined based on the expression level of the CHODL gene detected before any kind of treatment in a patient of cancer, or a population of the patients whose disease state (good or poor prognosis) is known.
  • the cancer is lung cancer.
  • the standard value of the expression levels of the CHODL gene in a subject group with a known disease state is used.
  • the standard value can be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. can be used as standard value.
  • the control level can be determined at the same time with the test biological sample by using a sample(s) previously collected and stored before any kind of treatment from cancer subject(s) (control or control group) whose disease state (good prognosis or poor prognosis) are known.
  • the control level can be determined by a statistical method based on the results obtained by analyzing the expression level of the CHODL gene in samples previously collected and stored from a control group.
  • the control level can be a database of expression patterns from previously tested cells or patients.
  • the expression level of the CHODL gene in a subject-derived biological sample can be compared to multiple control levels, which control levels are determined from multiple reference samples.
  • a control level determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample is used.
  • a similarity in the expression level of the CHODL gene to the good prognosis control level indicates a more favorable prognosis of the subject and an increase in the expression level in comparison to the good prognosis control level indicates less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome.
  • a decrease in the expression level of the CHODL gene in comparison to the poor prognosis control level indicates a more favorable prognosis of the subject and a similarity in the expression level to the poor prognosis control level indicates less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome.
  • a lung cancer cell(s) obtained from a subject who showed good or poor prognosis of cancer after treatment is a preferable biological sample for good or poor prognosis control level, respectively.
  • cancer progression may be evaluated within 5 years.
  • a subject with less favorable, negative, or poor prognosis includes a subject who shows recurrence, metastatic spread or disease relapse of cancer within 5 years after the treatment.
  • the treatment of cancer may be surgical removal of cancerous tissues.
  • a cancerous tissue derived from such patient may be used as a control sample for poor prognosis in the present invention.
  • a subject with positive, favorable, or good prognosis includes a subject who does not show recurrence, metastatic spread and disease relapse of cancer within 5 years after the treatment.
  • the treatment of cancer may be surgical removal of cancerous tissues.
  • a cancerous tissue derived from such patient may be used as a control sample for good prognosis in the present invention.
  • an intermediate result may also be provided in addition to other test results for assessing the prognosis of a subject.
  • Such intermediate result may assist a doctor, nurse, or other practitioner to assess, determine, or estimate the prognosis of a subject. Additional information that may be considered, in combination with the intermediate result obtained by the present invention, to assess prognosis includes clinical symptoms and physical conditions of a subject.
  • the expression level of the CHODL gene is useful prognostic marker for assessing, predicting or determining the prognosis of a subject suffering from lung cancer (e.g. NSCLC). Therefore, the present invention also provides a method for detecting prognostic marker for assessing, predicting or determining the prognosis of a subject suffering from lung cancer including NSCLC, which comprises steps of: a) detecting or determining an expression level of a CHODL gene in a subject-derived biological sample, and b) correlating the expression level detected or determined in step a) with the prognosis of the subject.
  • an increased expression level to the control level is indicative of potential or suspicion of poor prognosis (poor survival).
  • the expression level of cancer marker gene including CHODL in a biological sample can be considered to be increased if it increases from the control level of the corresponding cancer marker gene (e.g., in a normal or non-cancerous cell) by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
  • An expression level of the CHODL gene in a subject- derived biological sample can be considered altered (i.e., increased or decreased) when the expression level differs from the control level by, for example, 10% or more, 25% or more, or 50% or more; or more than 1.1 fold or more, 1.5 fold or more, 2.0 fold or more, 5.0 fold or more, or 10.0 fold or more.
  • the difference in the expression level between the test biological sample and the control level can be normalized to a control, e.g., a housekeeping gene.
  • a control e.g., a housekeeping gene.
  • genes whose expression levels are known not to differ between the cancerous and non-cancerous cells including those coding for beta-actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1 can be used to normalize the expression levels of the CHODL gene.
  • the expression level of the CHODL gene can be determined by detecting the gene transcript in a subject-derived biological sample using techniques well known in the art.
  • the gene transcripts detected by the present method include both the transcription and translation products, for example, mRNA and protein.
  • the transcription product of the CHODL gene can be detected by hybridization, e.g., Northern blot hybridization analyses, that use a CHODL gene probe to the gene transcript.
  • the detection can be carried out on a chip or an array.
  • An array can be used for detecting the expression level of a plurality of genes including the CHODL gene.
  • amplification-based detection methods for example, reverse-transcription based polymerase chain reaction (RT-PCR) which use primers specific to the CHODL gene can be employed for the detection (see (b) Semi-quantitative RT-PCR in [EXAMPLE]).
  • the CHODL gene-specific probe or primers can be designed and prepared using conventional techniques by referring to the whole sequence of the CHODL gene.
  • the primers (SEQ ID NOs: 1,2,5 and 6) used in the Examples can be employed for the detection by RT-PCR, but the present invention is not restricted thereto.
  • a probe or primer used for the method of the present invention hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of the CHODL gene.
  • stringent (hybridization) conditions refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degrees Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH.
  • Tm thermal melting point
  • the Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degrees Centigrade for longer probes or primers. Stringent conditions can also be achieved with the addition of destabilizing agents, for example, formamide.
  • a probe or primer of the present invention is typically a substantially purified oligonucleotide.
  • the oligonucleotide typically includes a region of nucleotide sequence that hybridizes under stringent conditions to at least about 2000, 1000, 500, 400, 350, 300, 250, 200, 150, 100, 50, or 25 bases, consecutive sense strand nucleotide sequence of a nucleic acid including a CHODL sequence, or an anti sense strand nucleotide sequence of a nucleic acid including a CHODL sequence, or of a naturally occurring mutant of these sequences.
  • an oligonucleotide having 5-50 bases in length can be used as a primer for amplifying the genes, to be detected.
  • mRNA or cDNA of a CHODL gene can be detected with oligonucleotide probe or primer of a specific size, generally 15- 30 bases in length. In preferred embodiments, length of the oligonucleotide probe or primer can be selected from 15-25 bases.
  • Assay procedures, devices, or reagents for the detection of gene by using such oligonucleotide probe or primer are well known (e.g. oligonucleotide microarray or PCR). In these assays, probes or primers can also include tag or linker sequences. Further, probes or primers can be modified with detectable label or affinity ligand to be captured.
  • a polynucleotide having a few hundreds (e.g., about 100-200) bases to a few kilo (e.g., about 1000-2000) bases in length can also be used for a probe (e.g., northern blotting assay or cDNA microarray analysis).
  • the translation product can be detected for the method of the present invention.
  • the quantity of the CHODL protein can be determined.
  • a method for determining the quantity of the CHODL protein as the translation product includes immunoassay methods that use an antibody specifically recognizing the CHODL protein.
  • the antibody can be monoclonal or polyclonal.
  • any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody can be used for the detection, so long as the fragment retains the binding ability to the CHODL protein.
  • Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method can be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the intensity of staining can be observed via immunohistochemical analysis using an antibody against CHODL protein. Namely, the observation of strong staining indicates increased presence of the CHODL protein and at the same time high expression level of the CHODL gene.
  • the CHODL protein has a cell proliferation promoting activity. Therefore, the expression level of the CHODL gene can be determined using such cell proliferation promoting activity as an index. For example, cells which express the CHODL gene are prepared and cultured in the presence of a subject-derived biological sample, and then by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability, the cell proliferation promoting activity of the subject-derived biological sample can be determined. Moreover, in addition to the expression level of the CHODL gene, the expression level of other cancer prognostic marker genes can also be determined to improve the accuracy of the assessment or prediction. Examples of such cancer prognostic marker genes includes, but are not limited to, ANLN, CDCA1, DLX5, URLC8, URLC10, NPTX1, PKP3, KOC1, KNTC2, DKK1 and so on.
  • kits for Assessing or Predicting Cancer Prognosis The present invention also provides a kit for assessing or predicting the prognosis of a subject with cancer.
  • the present invention also provides a kit for determining a subject suffering from cancer that can be treated with the double-stranded molecule of the present invention or vector encoding thereof, which may also be useful in assessing and/or monitoring the efficacy of a cancer treatment.
  • kits of [1] to [4] [1] A kit for assessing or predicting a prognosis of a subject with cancer, wherein the kit comprises at least one regent selected from the group consisting of: (a) a reagent for detecting an mRNA of the CHODL gene; (b) a reagent for detecting a CHODL protein; and (c) a reagent for detecting a biological activity of a CHODL protein; [2] The kit of [1], wherein the reagent comprises an oligonucleotide that has a complementary sequence to a part of an mRNA of the CHODL gene and specifically binds to the mRNA; or an antibody against the protein encoded by the CHODL gene; [3] The kit of [1] or [2], wherein the kit further comprises either or both of a good prognosis control sample and a poor prognosis control sample; and [4] The kit of any one of [1] to [
  • kits of the present invention will be described more details bellow.
  • the kit of the present invention can applied to any cancer associated with over expression of the CHODL gene.
  • cancer is lung cancer. More preferably, lung cancer is NSCLC.
  • the kit of the present invention includes at least one reagent for determining the expression level of the CHODL gene in a subject-derived biological sample.
  • Such reagents can be selected from the group consisting of: (a) a reagent for detecting an mRNA of the CHODL gene; (b) a reagent for detecting a CHODL protein; and (c) a reagent for detecting a biological activity of a CHODL protein.
  • Suitable reagents for detecting an mRNA of the CHODL gene include nucleic acids that specifically bind to or identify the CHODL mRNA, for example, oligonucleotides which have a complementary sequence to a part of the CHODL mRNA. These kinds of oligonucleotides are exemplified by primers and probes that are specific to the CHODL mRNA. These oligonucleotides can be prepared based on methods well known in the art. If needed, such oligonucleotides can be immobilized on a solid matrix. Moreover, more than one reagent for detecting the CHODL mRNA can be included in the kit of the present invention.
  • the aforementioned oligonucleotides can be probes or primers against the CHODL mRNA.
  • Such probes or primers may have specific sizes.
  • the sizes of the probes and primers are preferably at least 10 nucleotides, at least 12 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides or at least 30 nucleotides.
  • the probes and primers may range in size from 5-50 nucleotides, 10-40 nucleotides, 15-30 nucleotides or 20-25 nucleotides.
  • the probe for Northern hybridization analysis can have any sizes so long as the probe specifically hybridizes the CHODL mRNA.
  • the size of the probe may be 50 nucleotides or more, 75 nucleotide or more, 100 nucleotide or more, 200 nucleotide or more, 300 nucleotide or more, 400 nucleotide or more, or 500 nucleotide or more.
  • cDNA of the CHODL gene may be used as a probe for the CHODL mRNA.
  • the reagent when the reagent is a probe against the CHODL mRNA, the reagent can be immobilized on a solid matrix, for example, a porous strip, to form at least one detection site.
  • the measurement or detection region of the porous strip can include a plurality of sites, each containing a nucleic acid (probe).
  • a test strip can also contain sites for negative and/or positive controls. Alternatively, control sites can be located on a strip separated from the test strip.
  • the different detection sites can contain different amounts of immobilized nucleic acids, i.e., a higher amount in the first detection site and lesser amounts in subsequent sites.
  • the number of sites displaying a detectable signal provides a quantitative indication of the amount of the CHODL mRNA present in the sample.
  • the detection sites can be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
  • suitable reagents for detecting the CHODL protein include antibodies against the CHODL protein.
  • antibody against the CHODL protein refers to an antibody that specifically binds to the CHODL protein.
  • the antibody can be monoclonal or polyclonal.
  • any fragments or modified antibodies (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody against CHODL protein can be used as such reagents, so long as such fragments or modified antibodies retain the binding ability to the CHODL protein.
  • Methods to prepare these kinds of antibodies are well known in the art, and any method can be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the antibody can be labeled with signal generating molecules via direct linkage or an indirect labeling technique.
  • Labels and methods for labeling antibodies and detecting the binding of antibodies to their targets are well known in the art and any labels and methods can be employed for the present invention.
  • more than one reagent for detecting the CHODL protein can be included in the kit.
  • the biological activity can be determined by, for example, measuring the cell proliferation promoting activity due to the expressed the CHODL protein in a subject-derived biological sample.
  • the cell is cultured in the presence of a subject-derived biological sample, and then by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability, the cell proliferation promoting activity of the subject-derived biological sample can be determined.
  • examples of reagent for detecting a biological activity of the CHODL protein include a medium for culturing cells.
  • the kit of the present invention can further comprise either or both of a poor prognosis control sample and a good prognosis control sample.
  • the poor prognosis control sample can be prepared by collecting cancerous tissues derived from the subject(s) who showed poor prognosis of cancer.
  • the good prognosis control sample can be prepared by collecting cancerous tissues derived from the subject(s) who showed good prognosis of cancer.
  • the kit of the present invention can include more than one of the aforementioned reagents. Furthermore, the kit of the present invention can include a solid matrix and reagent for binding a probe against the CHODL mRNA or antibody against the CHODL protein, a medium and container for culturing cells, good prognosis and/or poor control sample reagents, and a secondary antibody for detecting an antibody against the CHODL protein.
  • a kit of the present invention can further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts (e.g., written, tape, CD-ROM, etc.) with instructions for use. These reagents and such can be comprised in a container with a label. Suitable containers include bottles, vials, and test tubes. The containers can be formed from a variety of materials, for example, glass or plastic.
  • the kit of the present invention for assessing the prognosis of cancer may further include either of a good prognosis control sample or a poor prognosis control sample, or both.
  • a good prognosis control sample may be tissues or cells obtained from an individual or a population of individuals who showed good or positive prognosis of cancer, after the treatment.
  • a poor prognosis control sample may be tissues or cells obtained from an individual or a population of individuals who showed poor or negative prognosis of cancer, after the treatment.
  • a good or positive prognosis control sample may also be a clinical lung cancer tissue(s) obtained from a lung cancer patient(s) who showed good or positive prognosis of lung cancer, after treatment.
  • such lung cancer tissue may be an NSCLC tissue(s) obtained from a lung cancer patient(s).
  • NSCLC tissue may be a lung adenocarcinoma (ADC) tissue(s), a lung squamous cell carcinoma (SCC) tissue(s), and/or a large cell carcinoma tissue(s) (LCC).
  • ADC lung adenocarcinoma
  • SCC lung squamous cell carcinoma
  • LCC large cell carcinoma tissue
  • a good prognosis control sample may be prepared by determined a cut-off value and preparing a sample containing an amount of a CHODL mRNA or protein less than the cut-off value.
  • cut-off value refers to the value dividing between a good prognosis range and a poor prognosis range.
  • ROC receiver operating characteristic
  • the present kit may include a CHODL standard sample providing a cut-off value amount of a CHODL mRNA or polypeptide.
  • a poor or negative prognosis control sample may be a clinical lung cancer tissue(s) obtained from a lung cancer patient(s) who showed poor or negative prognosis of lung cancer, after the treatment.
  • lung cancer tissue may be an NSCLC tissue(s) obtained from a lung cancer patient(s).
  • NSCLC tissue may be a lung adenocarcinoma (ADC) tissue(s), a lung squamous cell carcinoma (SCC) tissue(s), and/or a large cell carcinoma tissue(s).
  • ADC lung adenocarcinoma
  • SCC lung squamous cell carcinoma
  • a poor prognosis control sample may be prepared by determined a cut-off value and preparing a sample containing an amount of a CHODL mRNA or protein more than the cut-off value.
  • double-stranded molecules refers to a nucleic acid molecule that inhibits the expression of a target gene including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g. double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)).
  • siRNA short interfering RNA
  • dsRNA double-stranded ribonucleic acid
  • shRNA small hairpin RNA
  • siD/R-NA short interfering DNA/RNA
  • siRNA refers to a double-stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed.
  • the siRNA includes a ribonucleotide corresponding to a sense nucleic acid sequence of a target gene (also referred to as “sense strand”), a ribonucleotide corresponding to an antisense nucleic acid sequence of a target gene (also referred to as "antisense strand”) or both.
  • the siRNA can be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, e.g., a hairpin.
  • the siRNA can either be a dsRNA or shRNA.
  • dsRNA refers to a construct of two RNA molecules comprising complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded RNA molecule.
  • the double-stranded RNA molecule may also refer to siRNA, or small interfering RNA molecule.
  • the sequence of two strands can comprise not only the "sense” or "antisense” RNAs selected from a protein coding sequence of a target gene sequence, but also RNA molecule having a nucleotide sequence selected from non-coding region of the target gene.
  • shRNA refers to an siRNA having a stem-loop structure, comprising a first and second regions complementary to one another, i.e., sense and antisense strands.
  • the degree of complementarity and orientation of the region is sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and can also be referred to as "intervening single-strand".
  • siD/R-NA refers to a double-stranded molecule which is composed of both RNA and DNA, and includes hybrids and chimeras of RNA and DNA and prevents translation of a target mRNA.
  • a hybrid indicates a molecule wherein an oligonucleotide composed of DNA and an oligonucleotide composed of RNA hybridize to each other to form the double-stranded molecule; whereas a chimera indicates that one or both of the strands composing the double stranded molecule can contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used.
  • the siD/R-NA includes a sense nucleic acid sequence of a target gene (also referred to as "sense strand"), an antisense nucleic acid sequence of a target gene (also referred to as “antisense strand”) or both.
  • the siD/R-NA can be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences from the target gene, e.g., a hairpin.
  • the siD/R-NA can either be a dsD/R-NA or shD/R-NA.
  • dsD/R-NA refers to a construct of two molecules comprising complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded polynucleotide molecule.
  • the nucleotide sequence of two strands can comprise not only the "sense” or "antisense” polynucleotides sequence selected from a protein coding sequence of target gene sequence, but also polynucleotide having a nucleotide sequence selected from non-coding region of the target gene.
  • One or both of the two molecules constructing the dsD/R-NA are composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules is composed of RNA and the other is composed of DNA (hybrid double-strand).
  • shD/R-NA refers to an siD/R-NA having a stem-loop structure, comprising a first and second regions complementary to one another, i.e., sense and antisense strands.
  • the degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the loop region of an shD/R-NA is a single-stranded region intervening between the sense and antisense strands and can also be referred to as "intervening single-strand".
  • Target sequence is a nucleotide sequence within an mRNA or cDNA sequence of a target gene, which will result in suppression of translation of the whole mRNA of the target gene if a double-stranded molecule targeting the sequence is introduced into a cell expressing the target gene.
  • a nucleotide sequence within an mRNA or cDNA sequence of a target gene can be determined to be a target sequence when a double-stranded molecule comprising a sequence corresponding to the target sequence inhibits expression of the target gene in a cell expressing the gene.
  • the double stranded polynucleotide by which suppresses the gene expression may consist of the target sequence and 3' overhang (e.g., uu).
  • the sense strand of the double-stranded cDNA i.e., a sequence that mRNA sequence is converted into DNA sequence
  • a double-stranded molecule is composed of a sense strand that has a sequence corresponding to a target sequence and an antisense strand that has a complementary sequence to the target sequence, and the antisense strand hybridizes with the sense strand at the complementary sequence to form a double-stranded molecule.
  • the phrase "corresponding to” means converting a target sequence according to the kind of nucleic acid that constitutes a sense strand of a double-stranded molecule.
  • a target sequence is shown in DNA sequence and a sense strand of a double-stranded molecule has an RNA region
  • base “t”s within the RNA region is replaced with base “u”s.
  • base "u"s within the DNA region is replaced with "t”s.
  • a sequence corresponding to a target sequence is "5'- AAAGUGGCAUGGAAGTATA-3'" (SEQ ID NO: 23) or "5'- GGUAUAAUUCCCAATCTAA-3'” (SEQ ID NO: 24).
  • a complementary sequence to a target sequence for an antisense strand of a double-stranded molecule can be defined according to the kind of nucleic acid that constitutes the antisense strand.
  • a target sequence is shown in the DNA sequence of SEQ ID NO: 9 or 10 and the antisense strand of the double-stranded molecule has the 5' side half region composed of DNA
  • "a complementary sequence to a target sequence” is "3'- UUUCACCGTACCTTCATAT -5'" (SEQ ID NO: 25) or "3'- CCAUAUUAAGGGTTAGATT-5'” (SEQ ID NO: 26).
  • the sequence corresponding to a target sequence of SEQ ID NO: 9 or 10 is the RNA sequence of SEQ ID NO: 9 or 10
  • the complementary sequence corresponding to a target sequence of SEQ ID NO: 9 or 10 is the RNA sequence of "3'- UUUCACCGUACCUUCAUAU-5'" (SEQ ID NO: 27)or "3'- CCAUAUUAAGGGUUAGAUU-5'" (SEQ ID NO: 28).
  • a double-stranded molecule may has one or two 3'overhangs having 2 to 5 nucleotides in length (e.g., uu) and/or a loop sequence that links a sense strand and an antisense strand to form hairpin structure, in addition to a sequence corresponding to a target sequence and complementary sequence thereto.
  • a double-stranded molecule against CHODL gene which molecule hybridizes to a CHODL mRNA, inhibits or reduces production of CHODL protein encoded by the gene by associating with the normally single-stranded mRNA transcript of the gene, thereby interfering with translation and thus, inhibiting expression of the CHODL protein.
  • the expression of CHODL gene in cancer cell lines was inhibited by each two double-stranded molecules (Fig. 3). Therefore, the present invention provides isolated double-stranded molecules having the property to inhibit or reduce the expression of CHODL gene in cancer cells when introduced into a cell.
  • the target sequences of double-stranded molecules may be designed by siRNA design algorithm mentioned below.
  • target sequences for CHODL include, for example, 5'-AAAGUGGCAUGGAAGUAUA -3' (SEQ ID NO: 9) or 5'- GGUAUAAUUCCCAAUCUAA-3' (SEQ ID NO: 10).
  • the present invention also provides a double-stranded molecule whose target sequence comprises or consisting of SEQ ID NO: 9 or 10.
  • the present invention provides the following double-stranded molecules [1] to [18]: [1] An isolated double-stranded molecule, which, when introduced into a cell, inhibits in vivo expression of a CHODL gene and cell proliferation, wherein the double-stranded molecule acts at mRNA which matches a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10; [2] An isolated double-stranded molecule, which, when introduced into a cell, inhibits in vivo expression of a CHODL gene and cell proliferation, wherein the double-stranded molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form a double strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9
  • [8] The double-stranded molecule of [7], which has a length of between about 19 and about 25 nucleotides.
  • [9] The double-stranded molecule of any one of [1] to [8], which consists of a single oligonucleotide comprising both the sense and antisense strands linked by an intervening single-strand.
  • [10] The double-stranded molecule of [9], which has a general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is a sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10 [B] is an intervening single-strand; and [A'] is an antisense strand comprising a nucleotide sequence corresponding to a sequence complementary to the target sequence selected in [A]. [11] The double-stranded molecule of any one of [1] to [10], which comprises RNA.
  • [12] The double-stranded molecule of any one of [1] to [11], which comprises both DNA and RNA.
  • the double-stranded molecule of [12] which is a hybrid of a DNA polynucleotide and an RNA polynucleotide.
  • the double-stranded molecule of [12] which is a chimera of DNA and RNA.
  • [16] The double-stranded molecule of [15], wherein a 5'-end region of the target sequence in the sense strand, and/or a 3'-end region of the complementary sequence of the target sequence in the antisense strand consists of RNA. [17] The double-stranded molecule of [16], wherein the RNA region consists of 9 to 13 nucleotides; and [18] The double-stranded molecule of any one of [1] to [2], which contains one or two 3' overhang(s).
  • the double-stranded molecule of the present invention will be described in more detail below.
  • Methods for designing double-stranded molecules having the ability to inhibit target gene expression in cells are known. (See, for example, US Pat No. 6,506,559, herein incorporated by reference in its entirety).
  • a computer program for designing siRNAs is available from the Ambion website (on the worldwide web at ambion.com/techlib/misc/siRNA_finder.html). The computer program selects target nucleotide sequences for double-stranded molecules based on the following protocol.
  • Target Sites 1. Beginning with the AUG start codon of the transcript, scan downstream for AA di-nucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al. recommend to avoid designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these can be richer in regulatory protein binding sites, and UTR-binding proteins and/or translation initiation complexes can interfere with binding of the siRNA endonuclease complex. 2. Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences.
  • BLAST which can be found on the NCBI server at: on the worldwide web at ncbi.nlm.nih.gov/BLAST/, is used (Altschul SF, et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-402). 3. Select qualifying target sequences for synthesis. Selecting several target sequences along the length of the gene to evaluate is typical.
  • Preferred target sequences for CHODL gene designed in "Examples” include 5'- AAAGUGGCAUGGAAGUAUA -3' (SEQ ID NO: 9) or 5'- GGUAUAAUUCCCAAUCUAA -3' (SEQ ID NO: 10).
  • the present invention provides the double-stranded molecules targeting any one of above-mentioned target sequences that were respectively examined for their ability to inhibit and reduce the growth of cancer cells expressing the target genes.
  • the present invention provides double-stranded molecules targeting a target sequence for CHODL gene selected from the group consisting of 5'- AAAGUGGCAUGGAAGUAUA-3' (SEQ ID NO: 9) and 5'-GGUAUAAUUCCCAAUCUAA-3' (SEQ ID NO: 10).
  • the double-stranded molecules of the present invention targeting the above-mentioned target sequence of CHODL gene include isolated polynucleotide(s) that comprises any one of the nucleic acid sequences of target sequences and/or complementary sequences to the target sequences.
  • Examples of a double-stranded molecule targeting CHODL gene include an oligonucleotide comprising the sequence corresponding to SEQ ID NO: 9 or 10, and complementary sequences thereto.
  • the present invention is not limited to these examples, and minor modifications in the aforementioned nucleic acid sequences are acceptable so long as the modified molecule retains the ability to suppress the expression of CHODL gene.
  • “minor modification" in a nucleic acid sequence indicates one, two or several substitution, deletion, addition or insertion of nucleic acids to the sequence.
  • a double-stranded molecule is composed of two polynucleotides, one polynucleotide has a sequence corresponding to a target sequence, i.e., sense strand, and another polypeptide has a complementary sequence to the target sequence, i.e., antisense strand.
  • the sense strand polynucleotide and the antisense strand polynucleotide hybridize to each other to form double-stranded molecule.
  • double-stranded molecules include dsRNA and dsD/R-NA .
  • a double-stranded molecule is composed of a polynucleotide that has both a sequence corresponding to a target sequence, i.e., sense strand, and a complementary sequence to the target sequence, i.e., antisense strand.
  • the sense strand and the antisense strand are linked by an intervening strand, and hybridize to each other to form a hairpin loop structure.
  • Examples of such double-stranded molecule include shRNA and shD/R-NA.
  • a double-stranded molecule of the present invention comprises a sense strand polynucleotide having a nucleotide sequence of the target sequence and anti-sense strand polynucleotide having a nucleotide sequence complementary to the target sequence, and both of polynucleotides hybridize to each other to form the double-stranded molecule.
  • a part of the polynucleotide of either or both of the strands may be RNA, and when the target sequence is defined with a DNA sequence, the nucleotide "t" within the target sequence and complementary sequence thereto is replaced with "u".
  • such a double-stranded molecule of the present invention comprises a stem-loop structure, composed of the sense and antisense strands.
  • the sense and antisense strands may be joined by a loop.
  • the present invention also provides the double-stranded molecule comprising a single polynucleotide containing both the sense strand and the antisense strand linked or flanked by an intervening single-strand.
  • double-stranded molecules targeting the CHODL gene may have a sequence selected from among SEQ ID NOs: 9 and 10 as a target sequence.
  • preferable examples of the double-stranded molecule of the present invention include polynucleotides that hybridize to each other at a sequence corresponding to SEQ ID NO: 9 or 10 and a complementary sequence thereto, and a polynucleotide that has a sequence corresponding to SEQ ID NO: 9 or 10 and a complementary sequence thereto.
  • a double-stranded molecule of the present invention can be tested for its ability using the methods utilized in the Examples (see, RNA interference assay in "EXAMPLES").
  • the double-stranded molecules comprising sense strands and antisense strands complementary thereto of various portions of mRNA of CHODL genes were tested in vitro for their ability to decrease production of CHODL gene product in cancers cell lines (e.g., using A549, LC319, NCI-H1373, PC14, NCI-H1781, LU61, NCI-H520, NCI-H1703, NCI-H1781, LU71, NCI-H520, NCI-H1703, NCI-H2170, SK-MES-1, LX1, DMS114, DMS273, SBC-3, SBC-5 etc.) according to standard methods.
  • CHODL gene product in cells contacted with the candidate double-stranded molecule compared to cells cultured in the absence of the candidate molecule can be detected by, e.g. RT-PCR using primers for CHODL gene mRNA mentioned (see,(b)) Semi-quantitative RT-PCR in "EXAMPLES"). Sequences which decrease the production of CHODL gene product in vitro cell-based assays can then be tested for there inhibitory effects on cell growth. Sequences which inhibit cell growth in vitro cell-based assay can then be tested for their in vivo ability using animals with cancer, e.g. nude mouse xenograft models, to confirm decreased production of CHODL gene product and decreased cancer cell growth.
  • the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a polynucleotide
  • binding means the physical or chemical interaction between two polynucleotides.
  • the polynucleotide comprises modified nucleotides and/or non-phosphodiester linkages, these polynucleotides can also bind each other as same manner.
  • complementary polynucleotide sequences hybridize under appropriate conditions to form stable duplexes containing few or no mismatches.
  • the sense strand and antisense strand of the isolated polynucleotide of the present invention can form double-stranded molecule or hairpin loop structure by the hybridization.
  • such duplexes contain no more than 1 mismatch for every 10 matches.
  • such duplexes contain no mismatches.
  • the polynucleotide of the invention is typically less than 500, 200, 100, 75, 50, or 25 nucleotides in length.
  • the isolated polynucleotides of the present invention are useful for forming double-stranded molecules against CHODL gene or preparing template DNAs encoding the double-stranded molecules.
  • the sense strand of the polynucleotide can be longer than 19 nucleotides, for example, longer than 21 nucleotides, for example, between about 19 and 25 nucleotides.
  • the present invention provides the double-stranded molecules comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence.
  • the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.
  • the double-stranded molecule serves as a guide for identifying homologous sequences in mRNA for the RISC complex, when the double-stranded molecule is introduced into cells.
  • the identified target RNA is cleaved and degraded by the nuclease activity of Dicer, through which the double-stranded molecule eventually decreases or inhibits production (expression) of the polypeptide encoded by the RNA.
  • a double-stranded molecule of the invention can be defined by its ability to generate a single-strand that specifically hybridizes to the mRNA of the CHODL gene under stringent conditions.
  • target sequence the portion of the mRNA that hybridizes with the single-strand generated from the double-stranded molecule is referred to as "target sequence", “target nucleic acid” or “target nucleotide”.
  • target sequence the portion of the mRNA that hybridizes with the single-strand generated from the double-stranded molecule
  • target nucleic acid or “target nucleotide”.
  • nucleotide sequence of the "target sequence” can be shown using not only the RNA sequence of the mRNA, but also the DNA sequence of cDNA synthesized from the mRNA.
  • the double-stranded molecules of the invention can contain one or more modified nucleotides and/or non-phosphodiester linkages.
  • Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule.
  • the skilled person will be aware of other types of chemical modification which can be incorporated into the present molecules (WO03/070744; WO2005/045037).
  • modifications can be used to provide improved resistance to degradation or improved uptake.
  • modifications include phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base” nucleotides, 5'-C- methyl nucleotides, and inverted deoxyabasic residue incorporation (US Pat Appl. No. 20060122137).
  • modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule.
  • Modifications include chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3' or 5' terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2 -fluoro modified ribonucleotides and 2'-deoxy ribonucleotides (WO2004/029212).
  • modifications can be used to increase or decrease affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (WO2005/044976).
  • an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine.
  • an unmodified purine can be substituted with a 7-deaza, 7-alkyl, or 7-alkenyl purine.
  • the double-stranded molecule when the double-stranded molecule is a double-stranded molecule with a 3' overhang, the 3'- terminal nucleotide overhanging nucleotides can be replaced by deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200).
  • deoxyribonucleotides Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200.
  • published documents for example, US Pat Appl. No.20060234970 are available.
  • the present invention is not limited to these examples and any known chemical modifications can be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene.
  • the double-stranded molecules of the invention can comprise both DNA and RNA, e.g., dsD/R-NA or shD/R-NA.
  • RNA e.g., dsD/R-NA or shD/R-NA.
  • a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability.
  • RNA i.e., a hybrid type double-stranded molecule made of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule comprising both DNA and RNA on any or both of the single strands (polynucleotides), or the like can be formed for enhancing stability of the double-stranded molecule.
  • the hybrid of a DNA strand and an RNA strand can be either where the sense strand is DNA and the antisense strand is RNA, or the opposite so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
  • the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA.
  • the chimera type double-stranded molecule may have the structure either where both of the sense and antisense strands are composed of DNA and RNA, or where any one of the sense and antisense strands is composed of DNA and RNA so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
  • the molecule contains as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule is required to be RNA within a range to induce sufficient inhibition of the expression.
  • an upstream partial region i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands
  • RNA is RNA
  • the upstream partial region means the 5' side (5'-end) of the sense strand and the 3' side (3'-end) of the antisense strand. That is, in some embodiments, a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand consists of RNA.
  • the chimera or hybrid type double-stranded molecule of the present invention comprise following combinations.
  • sense strand 5'-[---DNA---]-3' 3'-(RNA)-[DNA]-5' :antisense strand
  • sense strand 5'-(RNA)-[DNA]-3' 3'-(RNA)-[DNA]-5' :antisense strand
  • sense strand 5'-(RNA)-[DNA]-3' 3'-(---RNA---)-5' :antisense strand
  • the upstream partial region can be a domain of about 9 to 13 nucleotides counted from the terminus of the target sequence or complementary sequence thereto within the sense or antisense strands of the double-stranded molecules.
  • examples of such chimera type double-stranded molecules include those having a strand length of 19 to 21 nucleotides in which at least the upstream half region (5' side region for the sense strand and 3' side region for the antisense strand) of the polynucleotide is RNA and the other half is DNA.
  • the effect to inhibit expression of the target gene is much higher when the entire antisense strand is RNA (US Pat Appl. No. 20050004064).
  • the double-stranded molecule can form a hairpin, for example, a short hairpin RNA (shRNA) and short hairpin made of DNA and RNA (shD/R-NA).
  • shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • the shRNA or shD/R-NA comprises the sense target sequence and the antisense target sequence on a single strand wherein the sequences are separated by a loop sequence.
  • the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the target sequence of the dsRNA or dsD/R-NA.
  • RISC RNA-induced silencing complex
  • a loop sequence composed of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form the hairpin loop structure.
  • the present invention also provides a double-stranded molecule having the general formula 5'-[A]-[B]-[A']-3' or 5'-[A]'-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence, [B] is an intervening single-strand and [A'] is the antisense strand containing a complementary sequence to the target sequence.
  • the target sequence can be selected from the group consisting of, for example, SEQ ID NO: 9 and SEQ ID NO: 10.
  • the present invention is not limited to these examples, and the target sequence in [A] can be modified sequences from these examples so long as the double-stranded molecule retains the ability to suppress the expression of the targeted CHODL gene and result in inhibits or reduces the cell expressing these genes.
  • the region [A] hybridizes to [A'] to form a loop comprising the region [B].
  • the intervening single-stranded portion [B], i.e., the loop sequence can be 3 to 23 nucleotides in length.
  • the loop sequence for example, can be selected from group consisting of following sequences (on the worldwide web at ambion.com/techlib/tb/tb_506.html).
  • loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26): CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26; UUCG: Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003 Feb 18, 100(4): 1639-44, Epub 2003 Feb 10; and UUCAAGAGA: Dykxhoorn DM et al., Nat Rev Mol Cell Biol 2003 Jun, 4(6): 457-67.
  • the loop sequence can be selected from group consisting of AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA; however, the present invention is not limited thereto: GUUAGUACCUUGUACCAAA-[B]-UUUGGUACAAGGUACUAAC (for target sequence of SEQ ID NO: 9); CUUCAUCCGUGAGAUCAGA-[B]-UCUGAUCUCACGGAUCAAG (for target sequence of SEQ ID NO: 10); Furthermore, in order to enhance the inhibition activity of the double-stranded molecules, several nucleotides can be added to 3'end of the sense strand and/or the antisense strand, as 3' overhangs.
  • the number of nucleotides' to be added is at least 2, generally 2 to 10, for example, 2 to 5.
  • the added nucleotides form single strand at the 3'end of sense strand and/or the antisense strand of the double-stranded molecule.
  • the nucleotides for 3' overhang are preferably "u" or "t", but are not limited to.
  • a 3' overhang is added to the 3' end of the antisense strand.
  • the method of preparing the double-stranded molecule can use any chemical synthetic method known in the art. According to the chemical synthesis method, sense and antisense single-stranded polynucleotides are separately synthesized and then annealed together via an appropriate method to obtain a double-stranded molecule. Alternatively, a double stranded molecule or siRNA molecule of the present invention may also be synthesized with in vitro translation. In this embodiment, DNA encoding a nucleotide sequence that comprises the target sequence and antisense thereof is transcribed into the double stranded molecule in vitro.
  • the synthesized single-stranded polynucleotides are mixed in a molar ratio of at least about 3:7, for example, about 4:6, for example, substantially equimolar amount (i.e., a molar ratio of about 5:5).
  • the mixture is heated to a temperature at which double-stranded molecules dissociate and then is gradually cooled down.
  • the annealed double-stranded polynucleotide can be purified by usually employed methods known in the art.
  • Example of purification methods include methods utilizing agarose gel electrophoresis or wherein remaining single-stranded polynucleotides are optionally removed by, e.g., degradation with appropriate enzyme.
  • the regulatory sequences flanking target sequences can be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner.
  • the double-stranded molecules can be transcribed intracellularly by cloning CHODL gene templates into a vector containing, e.g., a RNA pol III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.
  • snRNA small nuclear RNA
  • the double-stranded molecules may be transcribed intracellularly by cloning its coding sequence into a vector containing a regulatory sequence that directs the expression of the double-stranded molecule in an adequate cell (e.g., a RNA poly III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter) adjacent to the coding sequence.
  • a regulatory sequence that directs the expression of the double-stranded molecule in an adequate cell e.g., a RNA poly III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter
  • the regulatory sequences flanking the coding sequences of double-stranded molecule may be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. Details of vectors which are capable of producing the double-stranded molecules will be described below.
  • Vector Also included in the invention is a vector containing one or more of the double-stranded molecules described herein, and a cell containing the vector.
  • a vector of the present invention encodes a double-stranded molecule of the present invention in an expressible form.
  • the phrase "in an expressible form” indicates that the vector, when introduced into a cell, will express the molecule.
  • the vector includes regulatory elements necessary for expression of the double-stranded molecule.
  • the expression vector encodes the double-stranded molecule of the present invention and is adapted for expression of the double-stranded molecule.
  • Such vectors of the present invention can be used for producing the present double-stranded molecules, or directly as an active ingredient for treating cancer.
  • the present invention provides vectors comprising each of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence corresponding to SEQ ID NOs: 9 or 10, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing the CHODL gene, inhibits expression of said gene.
  • the polynucleotide is an oligonucleotide of between about 19 and 25 nucleotides in length (e.g., contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 11, 13, 15, 17, 19 or 21). More preferably, the combination of polynucleotide comprises a single nucleotide transcript comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence.
  • the combination of polynucleotide has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is a nucleotide sequence comprising SEQ ID NO: 9 or 10; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotide; and [A'] is a nucleotide sequence complementary to the target sequence.
  • Vectors of the present invention can be produced, for example, by cloning a sequence comprising target sequence into an expression vector so that regulatory sequences are operatively-linked to the sequence in a manner to allow expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5).
  • RNA molecule that is the antisense to mRNA is transcribed by a first promoter (e.g., a promoter sequence flanking to the 3' end of the cloned DNA) and RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA).
  • a first promoter e.g., a promoter sequence flanking to the 3' end of the cloned DNA
  • RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA).
  • the sense and antisense strands hybridize in vivo to generate a double-stranded molecule constructs for silencing of the gene.
  • two vectors constructs respectively encoding the sense and antisense strands of the double-stranded molecule are utilized to respectively express the sense and antisense strands and then forming a double-stranded molecule construct.
  • the cloned sequence can encode a construct having a secondary structure (e.g., hairpin); namely, a single transcript of a vector contains both the sense and complementary antisense sequences of the target gene.
  • the vectors of the present invention can also be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; US Pat Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720.
  • DNA-based delivery technologies include "naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., US Pat No. 5,922,687).
  • the vectors of the present invention can be, for example, viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, for example, vaccinia or fowlpox (see, e.g., US Pat No. 4,722,848). This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the double-stranded molecule. Upon introduction into a cell expressing the target gene, the recombinant vaccinia virus expresses the molecule and thereby suppresses the proliferation of the cell.
  • Another example of useable vector includes Bacille Calmette Guerin (BCG).
  • BCG vectors are described in Stover et al., Nature 1991, 351: 456-60.
  • a wide variety of other vectors are useful for therapeutic administration and production of the double-stranded molecules; examples include adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14: 571-85.
  • the present invention provides methods for inhibiting cell growth, i.e., cancerous cell growth of a cell from a cancer resulting from overexpression of a CHODL gene, or that is mediated by a CHODL gene, by inhibiting the expression of the CHODL gene.
  • CHODL gene expression can be inhibited by any of the aforementioned double-stranded molecules of the present invention which specifically target the expression of CHODL gene or the vectors of the present invention that can express any of the double-stranded molecules of the present invention.
  • the present double-stranded molecules and vectors to inhibit cell growth of cancerous cells indicates that they can be used for methods for treating cancer, a cancer resulting from overexpression of a CHODL gene, or that is mediated by a CHODL gene.
  • the present invention provides methods to treat patients with a cancer resulting from overexpression of CHODL gene, or that is mediated by a CHODL gene by administering a double-stranded molecule, i.e., an inhibitory nucleic acid, against a CHODL gene or a vector expressing the molecule without adverse effect because those genes were hardly detected in normal organs.
  • a double-stranded molecule i.e., an inhibitory nucleic acid
  • the present invention provides the following methods [1] to [23]: [1] A method for inhibiting or reducing a growth of a cell (over)expressing a CHODL gene or a method for treating or preventing cancer (over)expressing CHODL gene, wherein the method comprising the step of administering to a subject at least one double-stranded molecule or vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell, inhibits or reduces in vivo expression of the CHODL gene.
  • the method of the present invention will be described in more detail below.
  • the growth of cells (over)expressing a CHODL gene is inhibited by contacting the cells with a double-stranded molecule against CHODL 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 growth" indicates that the cell proliferates at a lower rate or has decreased viability compared to a cell not exposed to the molecule.
  • Cell growth can be measured by methods known in the art, e.g., using the MTT cell proliferation assay.
  • the growth of any kind of cell can be suppressed according to the present method so long as the cell expresses or over-expresses the target gene of the double-stranded molecule of the present invention.
  • Exemplary cells include cancers cells.
  • patients suffering from or at risk of developing disease related to CHODL gene can be treated by administering at least one of the present double-stranded molecules, at least one vector expressing at least one of the molecules or at least one composition comprising at least one of the molecules.
  • patients of cancers can be treated according to the present methods.
  • the type of cancer can be identified by standard methods according to the particular type of tumor to be diagnosed.
  • patients treated by the methods of the present invention are selected by detecting the (over)expression of a CHODL gene in a biopsy from the patient by RT-PCR, hybridization or immunoassay.
  • the biopsy specimen from the subject is confirmed for CHODL gene over-expression by methods known in the art, for example, immunohistochemical analysis, hybridization or RT-PCR (see, Semi-quantitative RT-PCR, Western-blotting or Immunohistochemistry in "EXAMPLES”).
  • each of the molecules can direct to the different target sequence of same gene, or different target sequences of different gene.
  • the method can utilize different double-stranded molecules directing to CHODL gene transcript.
  • the method can utilize double-stranded molecules directed to one, two or more target sequences selected from same gene.
  • a double-stranded molecule of present invention can be directly introduced into the cells in a form to achieve binding of the molecule with corresponding mRNA transcripts.
  • a DNA encoding the double-stranded molecule can be introduced into cells by means of a vector.
  • transfection-enhancing agent for example, FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical
  • FuGENE FuGENE (Roche diagnostics)
  • Lipofectamine 2000 Invitrogen
  • Oligofectamine Oligofectamine
  • Nucleofector Nucleofector
  • a treatment is determined efficacious if it leads to clinical benefit for example, reduction in expression of a CHODL gene, or a decrease in size, prevalence, or metastatic potential of the cancer in the subject.
  • "efficacious” means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer. Efficaciousness is determined in association with any known method for diagnosing or treating the particular tumor type.
  • prevention and prophylaxis can occur “at primary, secondary and tertiary prevention levels.” While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g. reducing the proliferation and metastasis of tumors.
  • the treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence thereof include any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis.
  • Effectively treating and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer.
  • reduction or improvement of symptoms constitutes effectively treating and/or the prophylaxis includes 10%, 20%, 30% or more reduction, or stable disease.
  • the double-stranded molecule of the invention degrades the target mRNA (gene transcripts) in substoichiometric amounts. Without wishing to be bound by any theory, it is believed that the double-stranded molecule of the invention causes degradation of the target mRNA in a catalytic manner. Thus, compared to standard cancer therapies, significantly less a double-stranded molecule needs to be delivered at or near the site of cancer to exert therapeutic effect.
  • an effective amount of the double-stranded molecule of the invention to be administered to a given subject, by taking into account factors for example, body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is regional or systemic.
  • an effective amount of the double-stranded molecule of the invention comprises an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, for example, from about 2 nM to about 50 nM, for example, from about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of the double-stranded molecule can be administered.
  • the present methods can be used to inhibit the growth or metastasis of cancer; for example, a cancer resulting from overexpression of a CHODL gene or that is mediated by a CHODL gene, e.g., lung cancer.
  • a double-stranded molecule directed to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10 for CHODL find use for the treatment of cancers.
  • the double-stranded molecule of the invention can also be administered to a subject in combination with a pharmaceutical agent different from the double-stranded molecule.
  • the double-stranded molecule of the invention can be administered to a subject in combination with another therapeutic method designed to treat cancer.
  • the double-stranded molecule of the invention can be administered in combination with therapeutic methods currently employed for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents, for example, cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen).
  • chemotherapeutic agents for example, cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.
  • the double-stranded molecule can be administered to the subject either as a naked double-stranded molecule, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector which expresses the double-stranded molecule.
  • Suitable delivery reagents for administration in conjunction with the present a double-stranded molecule include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.
  • the delivery reagent is a liposome.
  • Liposomes can aid in the delivery of the double-stranded molecule to a particular tissue, for example, retinal or tumor tissue, and can also increase the blood half-life of the double-stranded molecule.
  • Liposomes suitable for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, for example, cholesterol. The selection of lipids is generally guided by consideration of factors for example, the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example as described in Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467; and US Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369, the entire disclosures of which are herein incorporated by reference.
  • the liposomes encapsulating the present double-stranded molecule comprises a ligand molecule that can deliver the liposome to the cancer site.
  • Ligands which bind to receptors prevalent in tumor or vascular endothelial cells for example, monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens, find use.
  • the liposomes encapsulating the present double-stranded molecule are modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example, by having opsonization-inhibition moieties bound to the surface of the structure.
  • a liposome of the invention can comprise both opsonization-inhibition moieties and a ligand.
  • Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane.
  • an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids.
  • These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); e.g., as described in US Pat. No.
  • Liposomes modified with opsonization-inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes.
  • Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature.
  • target tissue characterized by such microvasculature defects for example, solid tumors, will efficiently accumulate these liposomes; see Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53.
  • the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation in liver and spleen.
  • liposomes of the invention that are modified with opsonization-inhibition moieties can deliver the present double-stranded molecule to tumor cells.
  • Opsonization inhibiting moieties suitable for modifying liposomes can be water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, for example, from about 2,000 to about 20,000 daltons.
  • Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers for example, polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, for example, ganglioside GM 1 .
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • synthetic polymers for example, polyacrylamide or poly N-viny
  • Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable.
  • the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide.
  • the opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.
  • natural polysaccharides containing amino acids or carboxylic acids e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan
  • aminated polysaccharides or oligosaccharides linear or branched
  • the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof.
  • Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".
  • the opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques. For example, an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane.
  • a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH 3 and a solvent mixture for example, tetrahydrofuran and water in a 30:12 ratio at 60 degrees C.
  • Vectors expressing a double-stranded molecule of the invention are discussed above. Such vectors expressing at least one double-stranded molecule of the invention can also be administered directly or in conjunction with a suitable delivery reagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.
  • a suitable delivery reagent including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.
  • the double-stranded molecule of the invention can be administered to the subject by any means suitable for delivering the double-stranded molecule into cancer sites.
  • the double-stranded molecule can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.
  • Suitable enteral administration routes include oral, rectal, or intranasal delivery.
  • Suitable parenteral administration routes include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (for example, by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation.
  • injections or infusions of the double-stranded molecule or vector can be given at or near the site of cancer.
  • the double-stranded molecule of the invention can be administered in a single dose or in multiple doses.
  • the infusion can be a single sustained dose or can be delivered by multiple infusions.
  • Injection of the agent can be directly into the tissue or near the site of cancer. Multiple injections of the agent into the tissue at or near the site of cancer can be administered.
  • the double-stranded molecule can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site.
  • the double-stranded molecule can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, for example, from about seven to about ten days.
  • the double-stranded molecule is injected at or near the site of cancer once a day for seven days.
  • a dosage regimen comprises multiple administrations, it is understood that the effective amount of a double-stranded molecule administered to the subject can comprise the total amount of a double-stranded molecule administered over the entire dosage regimen.
  • a cancer overexpressing CHODL can be treated with at least one active ingredient selected from the group consisting of: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof.
  • the cancer includes, but is not limited to, lung cancer. Accordingly, prior to the administration of the double-stranded molecule of the present invention as active ingredient, it is preferable to confirm whether the expression level of CHODL in the cancer cells or tissues to be treated is enhanced as compared with normal cells of the same organ.
  • the present invention provides a method for treating a cancer (over)expressing CHODL, which method may include the steps of: i) determining the expression level of CHODL in cancer cells or tissue(s) obtained from a subject with the cancer to be treated; ii) comparing the expression level of CHODL with normal control; and iii) administrating at least one component selected from the group consisting of (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof, to a subject with a cancer overexpressing CHODL as compared with normal control.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one component selected from the group consisting of: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof, for use in administrating to a subject having a cancer overexpressing CHODL.
  • the present invention further provides a method for identifying a subject to be treated with: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, or (c) a vector encoding thereof, which method may include the step of determining an expression level of CHODL in subject-derived cancer cells or tissue(s), wherein an increase of the level compared to a normal control level of the gene indicates that the subject has cancer which may be treated with: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, or (c) a vector encoding thereof.
  • a subject to be treated by the present method is preferably a mammal.
  • exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • the expression level of CHODL in cancer cell in cancer cells or tissues obtained from a subject is determined.
  • the expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art.
  • the mRNA of CHODL may be quantified using probes by hybridization methods (e.g., Northern hybridization). The detection may be carried out on a chip or an array.
  • an array is preferable for detecting the expression level of CHODL.
  • Those skilled in the art can prepare such probes utilizing the sequence information of CHODL.
  • the cDNA of CHODL may be used as the probes.
  • the probes may be labeled with a suitable label, such as dyes, fluorescent substances and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.
  • the transcription product of CHODL may be quantified using primers by amplification-based detection methods (e.g., RT-PCR). Such primers may be prepared based on the available sequence information of the gene.
  • a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of CHODL.
  • stringent (hybridization) conditions refers to conditions under which a probe or primer will hybridize to its target sequence, but not to other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences.
  • the temperature of a stringent condition is selected to be about 5 degree Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to their target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degree Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degree Centigrade for longer probes or primers.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • the translation product may be detected for the diagnosis of the present invention.
  • the quantity of CHODL protein SEQ ID NO: 16
  • Methods for determining the quantity of the protein as the translation product include immunoassay methods that use an antibody specifically recognizing the protein.
  • the antibody may be monoclonal or polyclonal.
  • any fragment or modification e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.
  • Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the intensity of staining may be measured via immunohistochemical analysis using an antibody against the CHODL protein. Namely, in this measurement, strong staining indicates increased presence/level of the protein and, at the same time, high expression level of CHODL gene.
  • the expression level of a target gene, i.e., the CHODL gene, in cancer cells can be determined to be increased if the level increases from the control level (e.g., the level in normal cells) of the target gene by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
  • the control level may be determined at the same time with the cancer cells by using a sample(s) previously collected and stored from a subject/subjects whose disease state(s) (cancerous or non-cancerous) is/are known.
  • normal cells obtained from non-cancerous regions of an organ that has the cancer to be treated may be used as normal control.
  • the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of CHODL gene in samples from subjects whose disease states are known.
  • the control level can be derived from a database of expression patterns from previously tested cells.
  • the expression level of CHODL gene in a biological sample may be compared to multiple control levels, which are determined from multiple reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample. Moreover, it is preferred to use the standard value of the expression levels of CHODL gene in a population with a known disease state. The standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as the standard value.
  • a control level determined from a biological sample that is known to be non-cancerous is referred to as a "normal control level”.
  • the control level is determined from a cancerous biological sample, it is referred to as a "cancerous control level”.
  • the expression level of CHODL gene is increased as compared to the normal control level, or is similar/equivalent to the cancerous control level, the subject may be diagnosed with cancer to be treated.
  • compositions comprising at least one of the present double-stranded molecules or the vectors coding for the molecules.
  • composition is used to refer to a product including that include the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • pharmaceutical when used in relation to the modifier "pharmaceutical” (as in “pharmaceutical composition”), are intended to encompass products including a product that includes the active ingredient(s), and any inert ingredient(s) that make up the carrier, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, in the context of the present invention, the term “pharmaceutical composition” refers to any product made by admixing a molecule or compound of the present invention and a pharmaceutically or physiologically acceptable carrier.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier”, as used herein, means a pharmaceutically or physiologically acceptable material, composition, substance or vehicle, including but not limited to, a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
  • active ingredient refers to a substance in composition that is biologically or physiologically active.
  • active ingredient refers to a substance that shows an objective pharmacological effect.
  • active ingredients in the agents or compositions may lead to at least one biological or physiologically action on cancer cells and/or tissues directly or indirectly.
  • such action may include reducing or inhibiting cancer cell growth, damaging or killing cancer cells and/or tissues, and so on.
  • the "active ingredient” may also be referred to as "bulk", “drug substance” or "technical product”.
  • compositions [1] to [24] [1] A composition for inhibiting or reducing a growth of cell expressing CHODL gene, or for treating or preventing a cancer expressing a CHODL gene, which comprises at least one double-stranded molecule against CHODL gene or vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell, inhibits or reduces in vivo expression of said gene.
  • composition of [1] wherein the double-stranded molecule acts at mRNA which matched a target sequence selected from the group SEQ ID NO: 9 and SEQ ID NO: 10.
  • composition of [1], wherein said double-stranded molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form a double strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10.
  • composition of any one of [1] to [3], wherein the cancer to be treated is lung cancer; [5] The composition of [4], wherein the lung cancer is small cell lung cancer or non-small cell lung cancer; [6] The composition of any one of [1] to [5], wherein the composition contains plural kinds of the double-stranded molecules; [7] The composition of [6], wherein the plural kinds of the double-stranded molecules target the same gene; [8] The composition of any one of [1] to [7], wherein the double-stranded molecule has a length of less than about 100 nucleotides; [9] The composition of [8], wherein the double-stranded molecule has a length of less than about 75 nucleotides; [10] The composition of [9], wherein the double-stranded molecule has a length of less than about 50 nucleotides; [11] The composition of [10], wherein the double-stranded molecule has a length of less than about 25 nucleotides
  • composition of [13], wherein said double-stranded molecule has a general formula 5'-[A]-[B]-[A']-3'or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10; [B] is the intervening single-strand; and [A'] is the antisense strand comprising an oligonucleotide corresponding to a sequence complementary to the target sequence selected in [A].
  • composition of any one of [1] to [14], wherein the double-stranded molecule comprises RNA; [16] The composition of any one of [1] to [14], wherein the double-stranded molecule comprises DNA and RNA; [17] The composition of [16], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide; [18] The composition of [17], wherein the sense and antisense strand polynucleotides are made of DNA and RNA, respectively; [19] The composition of [18], wherein the double-stranded molecule is a chimera of DNA and RNA; [20] The composition of [19], wherein at least a region flanking to the 5'-end of one or both of the sense and antisense polynucleotides consists of RNA.
  • [21] The composition of [20], wherein the flanking region consists of 9 to 13 nucleotides; [22] The composition of any one of [1] to [21], wherein the double-stranded molecule contains one or two 3' overhang(s); [23] The composition of any one of [1] to [22], wherein the double-stranded molecule is encoded by a vector; [24] The composition of any one of [1] to [23], which further comprising a transfection-enhancing agent, cell permeable agent or pharmaceutically acceptable carrier.
  • the double-stranded molecules of the invention can be formulated as pharmaceutical compositions prior to administering to a subject, according to techniques known in the art.
  • Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free.
  • pharmaceutical formulations include formulations for human and veterinary use.
  • suitable pharmaceutical formulations of the present invention include those suitable for oral, rectal, nasal, topical (including buccal, sub-lingual, and transdermal), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation.
  • Other formulations include implantable devices and adhesive patches that release a therapeutic agent.
  • the above-described formulations may be adapted to give sustained release of the active ingredient.
  • compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
  • the present pharmaceutical formulations comprise at least one of the double-stranded molecules or vectors encoding them of the present invention (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt of the molecule, mixed with a physiologically acceptable carrier medium.
  • physiologically acceptable carrier media include, for example, water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
  • the composition can contain plural kinds of the double-stranded molecules, each of the molecules can be directed to the same target sequence, or different target sequences of CHODL gene.
  • the composition can contain double-stranded molecules directed to CHODL gene.
  • the composition can contain double-stranded molecules directed to target sequences selected from CHODL gene.
  • the present composition can contain a vector coding for one or plural double-stranded molecules.
  • the vector can encode one, two or several kinds of the present double-stranded molecules.
  • the present composition can contain plural kinds of vectors, each of the vectors coding for a different double-stranded molecule.
  • the present double-stranded molecules can be contained as liposomes in the present composition. See under the item of "(iii) Methods Of Inhibiting Or Reducing A Growth Of Cancer Cells And Treating Or Preventing Cancer Using Double-Stranded Molecules r" for details of liposomes.
  • compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives.
  • Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (for example, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
  • a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10-95%, for example, 25-75%, of one or more double-stranded molecule of the invention.
  • a pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01-20% by weight, for example, 1-10% by weight, of one or more double-stranded molecule of the invention encapsulated in a liposome as described above, and propellant.
  • a carrier can also be included as desired; e.g., lecithin for intranasal delivery.
  • the present composition can contain other pharmaceutical active ingredients so long as they do not inhibit the in vivo function of the present double-stranded molecules.
  • the composition can contain chemotherapeutic agents conventionally used for treating cancers.
  • the pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants or preservatives.
  • the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question; for example, those suitable for oral administration may include flavoring agents.
  • the present invention also provides the use of the double-stranded nucleic acid molecules of the present invention or a vector(s) encoding the double-stranded nucleic acid molecule in manufacturing a pharmaceutical composition for treating a cancer (over)expressing the CHODL gene.
  • the present invention relates to the use of double-stranded nucleic acid molecule inhibiting the (over)expression of a CHODL gene in a cell, which over-expresses the gene, which molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets a sequence of SEQ ID NOs: 9 or 10, or a vector(s) encoding the double-stranded nucleic acid molecule for manufacturing a pharmaceutical composition for treating a cancer (over)expressing the CHODL gene.
  • the present invention further provides the double-stranded nucleic acid molecules of the present invention or a vector(s) encoding the double-stranded nucleic acid molecule for use in treating a cancer expressing the CHODL gene.
  • the present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a cancer (over)expressing the CHODL gene, wherein the method or process comprises step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the (over)expression of a CHODL gene in a cell, which over-expresses the gene, which molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets a sequence of SEQ ID NOs: 9 or 10 or a vector(s) encoding the double-stranded nucleic acid molecule as active ingredients.
  • the present invention also provides a method or process for manufacturing a pharmaceutical composition for treating a cancer (over)expressing the CHODL gene, wherein the method or process comprises step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double-stranded nucleic acid molecule inhibiting the expression of CHODL gene in a cell, which over-expresses the gene, which molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets a sequence of SEQ ID NOs: 9 or 10 or a vector(s) encoding the double-stranded nucleic acid molecule.
  • Example 1 General Methods Cell lines and clinical tissue samples.
  • the human lung-cancer cell lines used in this study included five adenocarcinomas (ADCs; A549, LC319, NCI-H1373, PC14, and NCI-H1781), five squamous cell carcinomas (SCCs; LU61, NCI-H520, NCI-H1703, NCI-H2170, SK-MES-1), one large cell carcinoma (LCC; LX1), and four small cell lung cancers (SCLCs; DMS114, DMS273, SBC-3, and SBC-5).
  • a human bronchial epithelial cell line (BEAS-2B) was used as a control (Table 1).
  • tumor samples were selected from patients who fulfilled all of the following criteria: (a) patients suffered from primary NSCLC with confirmed stage (only pT1 to pT3, pN0 to pN2, and pM0), (b) patients underwent curative surgery, but did not receive any preoperative treatment, (c) among them NSCLC patients with lymph node metastasis positive (pN1, pN2) tumors were treated with platinum-based adjuvant chemotherapies after surgery, whereas patients with pN0 did not receive adjuvant chemotherapies, and (d) patients whose clinical follow-up data were available.
  • This study and the use of all clinical materials mentioned were approved by individual institutional ethics committees.
  • Immunocytochemical analysis were done as previously described (Kato T, et al., Cancer Res 2005; 65:5638-46.), using 1 microgram/mL of a goat polyclonal anti-CHODL antibody (Catalog No. sc-54867; Santa Cruz Biotechnology) for detecting endogenous CHODL and 4 microgram/mL of a rabbit polyclonal anti-Calreticulin antibody (Catalog No. SPA-600; StressGen) for detecting endogenous carleticulin in the endoplasmic reticulum (ER) as a primary antibody.
  • a goat polyclonal anti-CHODL antibody Catalog No. sc-54867; Santa Cruz Biotechnology
  • a rabbit polyclonal anti-Calreticulin antibody Catalog No. SPA-600; StressGen
  • the cells were incubated with these primary antibodies for 1 h at room temperature, followed by incubation with Alexa 488-conjugated donkey anti-goat secondary antibodies (Molecular Probe) and an Alexa 594-conjugated donkey anti-rabbit secondary antibodies (Molecular Probe).
  • Each stained specimen was mounted with Vectashield (Vector Laboratories, Inc.) containing 4', 6-diamidino-2-phenylindole and visualized with Spectral Confocal Scanning Systems (TSC SP2 AOBS; Leica Microsystems).
  • the cDNA probe of CHODL was prepared by reverse transcriptase-PCR (RT-PCR) using primers CHODL-F (5'-GGTGCATAAACACTAATGCAGTC-3' (SEQ ID NO: 5)) and CHODL-R (5'-GTTAAAAGGAGCACAGGGACATA-3' (SEQ ID NO: 6)). Prehybridization, hybridization, and washing were performed according to the supplier's recommendations. The blots were autoradiographed with intensifying screens at -80 degrees C for 7 days.
  • Tumor tissue microarrays were constructed using 295 formalin-fixed primary lung cancers, as published previously (Chin SF, et al., Mol Pathol 2003; 56:275-9.; Callagy G, et al., Diagn Mol Pathol 2003; 12:27-34.; Callagy G, et al., J Pathol 2005; 205:388-96.). The tissue area for sampling was selected based on visual alignment with the corresponding HE-stained section on a slide.
  • tissue cores Three, four, or five tissue cores (diameter 0.6 mm; height 3-4 mm) taken from a donor tumor block were placed into a recipient paraffin block using a tissue microarrayer (Beecher Instruments, Sun Prairie, WI). A core of normal tissue was punched from each case, and 5 micro-m sections of the resulting microarray block were used for immunohistochemical analysis.
  • CHODL protein was stained in the following manner. Briefly, 4 microgram/ml of a goat polyclonal anti-human CHODL antibody (Catalog No. sc-54867; Santa Cruz Biotechnology) was added after blocking of endogenous peroxidase and proteins. The sections were incubated with HRP-labeled anti-goat IgG as the secondary antibody. Substrate-chromogen was added and the specimens were counterstained with hematoxylin. On immunohistochemical analyses, it was confirmed that the antibody was specific for CHODL protein by antigen blocking assays using CHODL antigen peptides (Catalog No.
  • CHODL positivity semi-quantitatively without prior knowledge of clinicopathological data.
  • the intensity of CHODL staining was evaluated using the following criteria: strong positive (scored as 2+), brown staining in > 50% of tumor cells completely obscuring cytoplasm; weak positive (1+), any lesser degree of brown staining appreciable in tumor cell cytoplasm; and absent (scored as 0), no appreciable staining in tumor cells. Cases were accepted as strongly positive if two or more investigators independently defined them as such.
  • Statistical analysis were performed using the StatView statistical program (SaS) to compare patient characteristics with responses to therapy. Associations between clinicopathological variables and positivity for CHODL were compared by Fisher's exact tests. Tumor-specific survival and 95% confidence intervals (CIs) were evaluated with the Kaplan-Meier method, and differences between the two groups were evaluated with the log-rank test. Factors associated with the prognosis were evaluated using Cox's proportional-hazard regression model with a step-down procedure. Only those variables with statistically significant results in univariate analysis were included in a multivariate analysis. The criterion for removing a variable was the likelihood ratio statistic, which was based on the maximum partial likelihood estimate (default P value of 0.05 for removal from the model).
  • RNA interference assay To evaluate the biological functions of CHODL in lung cancer cells, small interfering RNA (siRNA) duplexes against the target genes were used.
  • the target sequences of the synthetic oligonucleotides for RNA interference were as follows: control 1 (si-LUC: siRNA against Photinus pyralis luciferase gene), 5'-CGUACGCGGAAUACUUCGA-3' (SEQ ID NO: 7); control 2 (si-SCR: siRNA against chloroplast Euglena gracilis gene coding for 5S and 16S (rRNAs), 5'-GCGCGCUUUGUAGGAUUCG-3' (SEQ ID NO: 8); si-CHODL-#A, 5'-AAAGUGGCAUGGAAGUAUA-3' (SEQ ID NO: 9); si-CHODL-#B, 5'-GGUAUAAUUCCCAAUCUAA-3'(SEQ ID NO: 10).
  • Lung cancer cell lines SBC-5 and NCI-H2170 were plated onto 10 cm dishes (1.0 X 10 6 per dish), and transfected with either of the siRNA oligonucleotides (100 nmol/L) using 24 micro-L of Lipofectamine 2000 (Invitrogen) according to the manufacturers' instructions. After 7 days of incubation, these cells were stained by Giemsa solution to assess colony formation, and cell viability was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.
  • MTT 5-diphenyltetrazolium bromide
  • COS-7 and LC319 cells which scarcely expressed endogenous CHODL were plated at densities of 1 X 10 6 cells/10 cm dish and transfected with plasmids designed to express myc-tagged CHODL or mock plasmids. Cells were selected in medium containing 0.4 mg/mL of geneticin (Invitrogen) for 7 days, and cell viability was assessed by MTT assay (cell counting kit-8 solution; Dojindo Laboratories).
  • Matrigel invasion assay COS-7 and LC319 cells transfected either with plasmids expressing myc-tagged CHODL or with mock plasmids were grown to near confluence in DMEM or RPMI respectively containing 10% FCS. The cells were harvested by trypsinization, washed in medium without addition of serum or protease inhibitor, and suspended in medium at a concentration of 2 X 10 5 /mL. Before preparing the cell suspension, the dried layer of Matrigel matrix (Becton Dickinson Labware) was rehydrated with medium for 2 h at room temperature.
  • Matrigel matrix Becton Dickinson Labware
  • Example 2 CHODL expression in lung cancers and normal tissues.
  • the present inventors To search for novel target molecules for the development of therapeutic agents and/or diagnostic biomarkers for NSCLC, the present inventors first screened genes that showed more than a 5-fold higher level of expression in cancer cells than in normal cells, in more than 50% of the lung cancers analyzed by cDNA microarray. Among 27,648 genes or ESTs screened, the CHODL transcript was identified as a transcript over-expressed commonly in lung cancers, and its transactivation was confirmed in 7 of 15 additional lung cancer tissues and 10 of 15 lung cancer cell lines by semi-quantitative RT-PCR experiments (Figs. 1A and 1B).
  • CHODL protein was localized in cytoplasm with granular appearance in A549 and NCI-H2170 cells, which expressed endogenous CHODL, but it was not detectable in CHODL-negative LC319 and BEAS-2B cells (Fig. 1C). Furthermore, it was confirmed that CHODL protein was mainly co-localized with calreticulin that was used as an ER marker (Fig. 1D), but not co-localized with Golgi and endosome markers (data not shown). It was confirmed that the positive signal by anti-CHODL antibody obtained in lung cancer A549 cells was markedly reduced by suppression of CHODL expression by siRNA against CHODL, which proved the specificity of antibody to CHODL protein (Fig. 5A).
  • CHODL protein was examined in five normal tissues (heart, liver, lung, kidney, and testis), as well as lung cancers using anti-CHODL antibody, and it was found to be hardly detectable in the former four tissues, whereas positive CHODL staining was detected at cytoplasm of testis and lung cancer tissues (Fig. 2B).
  • Example 3 Association of CHODL protein expression with poor clinical outcome of NSCLC patients.
  • the expression of CHODL protein was also examined by means of tissue microarrays containing lung cancer tissues from 295 NSCLC patients who underwent surgical resection. A pattern of CHODL expression on the tissue array ranging from absent (scored as 0) to weak/strong positive (scored as 1+ or 2+; Fig. 2D) was classified. As shown in Table 2, the number of NSCLC tissues scored as 2+, 1+, and 0 were 98 (33.2%), 131 (44.4%), and 66 (22.4%), respectively. Then, the association between CHODL status and clinicopathologic variables among the 295 patients was evaluated.
  • Example 4 Growth-promoting effect of CHODL.
  • siRNA oligonucleotides for CHODL the present inventors attempted to knock down the expression of endogenous CHODL in lung cancer cell lines SBC-5 and NCI-H2170, which showed high levels of endogenous CHODL expression.
  • Two CHODL-specific siRNAs (si-CHODL-#A and si-CHODL-#B) significantly suppressed expression of CHODL transcripts compared with control siRNAs (si-LUC and si-SCR) (Fig. 3A).
  • siRNAsi-LUC and si-SCR a control siRNAs
  • plasmids designed to express either CHODL (pcDNA3.1/myc-His vector) or mock vector were prepared, and transfected into COS-7 cells and LC319 cells which scarcely expressed endogenous CHODL.
  • Cells that transiently expressed exogenous CHODL revealed significant growth promotion compared with the mock-transfected cells (Figs. 3D and 3E).
  • Example 5 Enhanced cell invasion by CHODL. Since strong expression of CHODL in NSCLC tissues was associated with poor prognosis of patients, the present inventors next examined a possible role of CHODL in cellular invasion by Matrigel assays using COS-7 and LC319 cells. Transfection of CHODL cDNA into either of the cells significantly enhanced their invasive activity (Figs. 4A-4C). This result also suggests that CHODL could contribute to the more malignant phenotype of cells.
  • CHODL appears to belong to C-type lectin-containing protein family that is classified into 17 groups. They are involved in diverse processes including cell recognition and communication, cell-cell adhesion, and extracellular matrix-cell interactions (Zelensky, A.N. and Gready, J.E., FEBS Lett 2005; 272:6179-217.).
  • proteoglycans belong to soluble C-type lectin family member without transmembrane domain, play an important role in the structure and stability of the extracellular matrix (ECM) and the interaction between the ECM and the cell (Drickamer, K. and Taylor, M.E., Annu Rev Cell Biol 1993; 9:237-64.).
  • ECM extracellular matrix
  • Some lectins are also important in embryonic development and immune responses.
  • the selectines which belong to C-type lectin with a transmembrane domain, function at cell surface in leukocyte-leukocyte and leukocyte-endothelial interactions (Drickamer, K. and Taylor, M.E., Annu Rev Cell Biol 1993; 9:237-64.).
  • Some transmembrane C-type lectins such as calnexin play important roles as molecular chaperones like calreticulin in the ER. These proteins bind to misfolded proteins and prevent them from being exported from the ER to the Golgi apparatus (Ellgaard, L. and Helenius, A., Nat Rev 2003; 4:181-91.).
  • CHODL was partly localized in the ER of cancer cells, it might have a similar function in control system for some oncogenic proteins in the ER.
  • the present inventors demonstrated that CHODL was expressed only in testis among the 23 normal tissues examined and was highly expressed in surgically resected samples from NSCLC patients, indicating CHODL to be a typical cancer-testis antigen.
  • Clinicopathological evidences obtained through our tissue microarray experiments indicated that NSCLC patients with strong CHODL-positive tumors had shorter cancer-specific survival periods than those with weak/absent expression tumors.
  • Functional assays by knockdown of CHODL expression with siRNA or exogenous expression of CHODL revealed that CHODL was important for cellular growth and invasion.
  • CHODL functions as an oncogenic protein in lung carcinogenesis and tumor progression. Elucidation of the mechanism implied by these observations should reveal important new information about cancer cell proliferation and cancer progression.
  • the present inventors have shown that over-expressed CHODL is a contributor to a growth-promoting pathway and to aggressive features of lung cancers. CHODL is a convenient prognostic biomarker in the clinic.
  • the present invention provides a novel molecular diagnostic marker for identifying and detecting cancers as well as assessing the prognosis. Furthermore, as described herein, CHODL are involved in cancer cell survival. Therefore, the present invention also provides novel molecular targets for treating and preventing cancer. They may be useful for developing novel therapeutic drugs and preventative agents without adverse effects. The methods described herein are also useful for the identification of additional molecular targets for prevention, diagnosis, and treatment of cancers.
  • the data provided herein add to a comprehensive understanding of cancers, provide novel diagnostic strategies, and provide clues for identification of molecular targets for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of tumorigenesis, and provides indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of cancers.

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Abstract

La présente invention est basée sur la découverte que le gène CHODL est surexprimé dans le cancer et associé au pronostic du cancer, et impliqué dans la survie cellulaire. La présente invention concerne des procédés pour l'évaluation ou la prédiction du pronostic d'un sujet atteint de cancer, au moyen du gène CHODL comme marqueur de pronostic. La présente invention concerne également une molécule double brin dirigée contre le gène CHODL, un procédé ou une composition pour le traitement ou la prévention du cancer au moyen de telles molécules double brin ou des vecteurs codant pour une telle molécule double brin.
PCT/JP2012/003120 2011-05-16 2012-05-14 Gène chodl comme marqueur tumoral et cible thérapeutique pour le cancer WO2012157239A1 (fr)

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Citations (5)

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WO2004031413A2 (fr) * 2002-09-30 2004-04-15 Oncotherapy Science, Inc. Technique de diagnostic de cancers bronchopulmonaires « non a petites cellules »
JP2004187620A (ja) * 2002-12-13 2004-07-08 Sumitomo Pharmaceut Co Ltd 腎疾患の疾患マーカーおよびその利用
WO2006022722A1 (fr) * 2004-08-16 2006-03-02 Agensys, Inc. Acides nucléiques et protéines correspondantes appelés 58p1d12 utiles dans le traitement et la détection du cancer
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WO2004031413A2 (fr) * 2002-09-30 2004-04-15 Oncotherapy Science, Inc. Technique de diagnostic de cancers bronchopulmonaires « non a petites cellules »
JP2004187620A (ja) * 2002-12-13 2004-07-08 Sumitomo Pharmaceut Co Ltd 腎疾患の疾患マーカーおよびその利用
WO2006022722A1 (fr) * 2004-08-16 2006-03-02 Agensys, Inc. Acides nucléiques et protéines correspondantes appelés 58p1d12 utiles dans le traitement et la détection du cancer
WO2008104543A2 (fr) * 2007-02-26 2008-09-04 Inserm (Institut National De La Sante Et De La Recherche Medicale) Procédé de prévision de l'occurrence de métastases chez des patients souffrant de cancer du sein
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