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WO2006013733A1 - PROCÉDÉ SERVANT À TRAITER LE SARCOME SYNOVIAL UTILISANT UN ARNsi POUR LE FZD10 - Google Patents

PROCÉDÉ SERVANT À TRAITER LE SARCOME SYNOVIAL UTILISANT UN ARNsi POUR LE FZD10 Download PDF

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WO2006013733A1
WO2006013733A1 PCT/JP2005/013444 JP2005013444W WO2006013733A1 WO 2006013733 A1 WO2006013733 A1 WO 2006013733A1 JP 2005013444 W JP2005013444 W JP 2005013444W WO 2006013733 A1 WO2006013733 A1 WO 2006013733A1
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fzdlo
double
seq
stranded rna
rna molecule
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PCT/JP2005/013444
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English (en)
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Yusuke Nakamura
Toyomasa Katagiri
Chikako Fukukawa
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The University Of Tokyo
Oncotherapy Science, Inc.
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Priority to EP05766513A priority Critical patent/EP1789549A1/fr
Priority to JP2007507592A priority patent/JP2008509076A/ja
Publication of WO2006013733A1 publication Critical patent/WO2006013733A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the invention relates to a method for inhibiting or reducing an expression of FZDlO gene. Also, the present invention relates to a method for treating and/or preventing FZDIO-associated disease in a subject, particularly synovial sarcoma, colorectal cancer, gastric cancer, chronic myeloid leukemia, and acute myeloid leukemia. Furthermore, the present invention relates to a pharmaceutical composition comprising a double-stranded RNA molecule for FZDlO or an expression vector capable of expressing a double-stranded RNA molecule for FZDlO.
  • Frizzled homologue 10 is a member of Frizzled family, which is a receptor of Wnt signaling. We previously reported that FZDlO was significantly overexpressed in synovial sarcoma (SS) (Nagayama S, et al. (2002). Cancer Res, 62:5859-5866; WO2004/020668).
  • the present invention provides a method for inhibiting or reducing an expression of FZDlO gene in a cell or tissue in vitro, in vivo or ex vivo, comprising introducing into the cell or tissue a double-stranded RNA molecule for FZDlO or an expression vector capable of expressing a double-stranded RNA molecule for FZDlO, wherein the double-stranded RNA molecule for FZDlO comprises a nucleotide sequence targeted to the 10 to 100 continuous nucleotides, preferably 10 to 50 continuous nucleotides, more preferably 10 to 30 continuous nucleotides of SEQ ID NO: 1.
  • the double-stranded RNA molecule for FZDlO may comprise a short nucleotide sequence as a short-interfering RNA (siRNA).
  • siRNA short-interfering RNA
  • the double-stranded RNA molecule comprises a nucleotide sequence targeted to nucleotides nos. 1481 to 1499 (SEQ ID No. S) or nucleotides nos. 1595 to 1613 (SEQ ID No.6) of SEQ ID No I .
  • the double-stranded RNA molecule for FZDlO can be expressed by the expression vector having the sequence of SEQ ID Nos. 7, 8, 9, or 10.
  • the present invention provides a method for preventing or treating synovial sarcoma in a subject, comprising administering therapeutically effective amount of a double-stranded RNA for FZDlO or an expression vector capable of expressing a double-stranded RNA for FZDlO to the subject.
  • the present inventors found that the FZDlO protein can bind to importin- ⁇ . Therefore, a compound that inhibits an interaction or binding between FZDlO protein and importin- ⁇ may affect in development of synovial sarcoma.
  • the present invention provides a method for screening for a compound that inhibits an interaction and/or binding between FZDlO protein and importin- ⁇ protein, comprising: (a) contacting FZD 10 protein or a partial peptide thereof and importin- ⁇ protein or a partial peptide thereof in the presence of a test sample; and
  • the present invention provides a method for screening for a compound that can be used for treating or preventing synovial sarcoma, comprising:
  • Figs. IA to 1C show growth-inhibitory effects of small-interfering RNAs (siRNAs) designed to reduce expression of FZDlO in SS cell line, SYO-I.
  • siRNAs small-interfering RNAs
  • A Semi-quantitative RT-PCR showing suppression of endogenous expression of FZDlO in SYO-I cell. ⁇ 2MG was used as an internal control. Expression of FZD9 is not affected by these siRNAs.
  • B MTT Assay of SYO-I cells transfected with psiU6BX3.0 vectors. A549 cell, in which undetectable FZDlO expression is observed, is an "off-target" control shows no growth-effect with these siRNAs.
  • FIG. 2A Colony-formation assay demonstrating a decrease in the numbers of colonies by knock-down of FZDlO expression in SYO-I cell.
  • A549 cell line is an "off-target" control.
  • Figs. 2A to 2C show that FZDlO can form homo-oligomer.
  • FIG. 2A to 2C show that FZDlO can form homo-oligomer.
  • FIG. 2A to 2C show that FZDlO can form homo-oligomer.
  • FIG. 2A to 2C show that FZDlO can form homo-oligomer.
  • FIG. 2A to 2C show that FZDlO can form homo-oligomer.
  • FIG. 2A to 2C show that FZDlO can form homo-oligomer.
  • Immunoprecipitates were analyzed by western blotting with ⁇ -HA and ⁇ -myc antibodies.
  • C Cytoplasmic region of FZDlO is not involved in oligomerization. Lysates from untransfected COS-7 cells or expressing HA-FLAG-FZD 10, HA-FLAG-FZDIO ⁇ C(l-578), HA-FLAG-FZD 10 AC(I -525) and FZDIO-myc/His were immunoprecipitated with ⁇ -HA F-7 antibody. Immunoprecipitates were analyzed by western blotting with ⁇ -HA F-7 and ⁇ -myc 9E10 antibodies. Figs.
  • FIG. 3A to 3C show TAP system purification of FZDlO and FZDIO-binding proteins.
  • A Schematic diagram of FZDlO-TAP and control TAP.
  • B TAP-purification of FZDlO identifies a new complex. Lysates from SNU-C5 cells expressing either control TAP or FZDlO-TAP were subjected to TAP system purification. Silver stained gel shows the proteins identified.
  • C The interaction between FZDlO and importin- ⁇ is also shown by co-immunoprecipitation.
  • Lysates from untransfected COS-7 cells or expressing either importin ⁇ -3XFLAG or FZDIO-myc/His, or both of them were immunoprecipitated with ⁇ -myc 9E10 antibody. Immunoprecipitates were analyzed by Western blotting with ⁇ -FLAG M2 and ⁇ -myc 9E10 antibodies.
  • Frizzled homologue 10 is a member of Frizzled family, which is a receptor of Wnt signaling.
  • RNA interference is known as a cellular event in which mRNA from an endogenous gene is degraded by introducing a double-stranded RNA (dsRNA) that has the sequence complementary to the sequence of the endogenous gene.
  • the present invention provides a method for inhibiting or reducing an expression of FZDlO gene in a cell or tissue in vitro, in vivo or ex vivo, comprising introducing into the cell or tissue a double-stranded RNA molecule for FZDlO or an expression vector capable of expressing a double-stranded RNA molecule for FZDlO, wherein the double-stranded RNA molecule for FZDlO comprises a nucleotide sequence targeted to the 10 to 100 continuous nucleotides, preferably 10 to 50 continuous nucleotides, more preferably 10 to 30 continuous nucleotides of SEQ ID NO: 1.
  • the double-stranded RNA molecule for FZDlO may comprise a short nucleotide sequence as a short-interfering RNA (siRNA).
  • the double-stranded RNA molecule comprises a nucleotide sequence targeted to nucleotides nos. 1481 to 1499 (SEQ ID No. 5) or nucleotides nos. 1595 to 1613 (SEQ ID No. 6) of SEQ DD No.l.
  • the double-stranded RNA molecule for FZDlO can be expressed by the expression vector having the sequence of SEQ ID Nos. 7, 8, 9, or 10 as indicated below.
  • the expression vector having the sequence of SEQ ID Nos. 7, 8, 9, or 10 may be useful in the method of the present invention.
  • the present invention provides a method for preventing or treating synovial sarcoma in a subject, comprising administering therapeutically effective amount of a double-stranded RNA for FZDlO or an expression vector capable of expressing a double-stranded RNA for FZDlO to the subject.
  • the method of the present invention can be performed ex vivo ⁇ e.g., by culturing the cell derived from a subject with the dsRNA or expression vector) or, alternatively, in vivo (e.g., by administering the dsRNA or expression vector to a subject).
  • the dsRN A can be delivered to a target location (such as a cancerous cell) by a variety of known administration methods.
  • the dsRNA can be delivered using an expression vector capable of expressing the dsRNA.
  • an expression vector that can be used in the present invention include, but are not limited to, adenovirus, herpes virus, vaccinia virus, and RNA viruses such as retrovirus.
  • a macromolecular complex, a nano-capsule, a microsphere, beads, oil-in-water type emulsion, micelle, mixed micelle, and liposome may be used as a delivery system.
  • the dsRNA or expression vector may be directly administered by intravenous injection (including continuous infusion), intramuscular injection, intraperitoneal injection, and subcutaneous injection, or via other route of administration.
  • the dsRNA or expression vector may be introduced into a cell or tissue obtained from a subject and then the cell may be administered to the subject (ex vivo method).
  • the introduction of the dsRNA or expression vector into the cell or tissue may be carried out by a conventional gene-introducing method such as, for example, a calcium phosphate method, a DEAE dextran method, electroporation, or lipofection.
  • the administration of the cell or tissue in which the dsRNA is expressed may also be carried out in the same manner as in the case of the direct administering of the dsRNA or expression vector.
  • the dosage of dsRNA or expression vector administered may vary depending on age, sex, symptoms, administration routes, administration frequency, and dosage form. However, a conventional method in the relevant art may be appropriately selected and used.
  • the present invention provides a composition comprising a double-stranded RNA molecule for FZDlO or an expression vector capable of expressing a double-stranded RNA molecule for FZDlO, and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier such as excipient, disintegrant, lubricant, surfactant, dispersing agent, buffering agent, preservative, solubilizer, antiseptic agent, stabilizing agent, and isotonizing agent.
  • a compound that inhibits an interaction or binding between FZDlO protein and importin- ⁇ may affect in development of synovial sarcoma.
  • the present invention provides a method for screening for a compound that inhibits an interaction and/or binding between FZDlO protein and importin- ⁇ protein, comprising:
  • FZDlO protein The amino acid sequence of FZDlO protein is shown in SEQ ID No.2 (GenBank Accession No.BAA84093).
  • the amino acid sequence of importin- ⁇ protein is shown in SEQ ID No.4 (GenBank Accession No.NM_002265; PDB ID No. IQGK-A).
  • a compound screened by the above method can be used for treating or preventing synovial sarcoma.
  • the present invention provides a method for screening for a compound that can be used for treating or preventing synovial sarcoma, comprising:
  • Test sample may include, but not limited to, proteins, peptides, non-peptide compound, synthetic compound, fermented product, and natural extract including cellular extract. Also, test sample may be a chemical library or peptide library. Detection of an interaction or a formation of binding between FZDlO and importin- ⁇ can be carried out according to the conventional methods known in the art.
  • the present invention provides a composition for treating or preventing synovial sarcoma, wherein the composition comprises a pharmaceutically effective amount of the compound obtained by any of screening method described above as an active ingredient, and a pharmaceutically acceptable carrier.
  • the invention encompasses pharmaceutical or therapeutic compositions containing one or more therapeutic compounds described herein.
  • Such therapeutic compound includes, but not limited to, a double-stranded RNA as described herein.
  • Pharmaceutical formulations may include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration, or for administration by inhalation or insufflation.
  • the formulations may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All such pharmacy methods include the steps of bringing into association the active compound with liquid carriers or finely divided solid carriers or both as needed and then, if necessary, shaping the product into the desired formulation.
  • compositions suitable for oral administration may conveniently be presented as discrete units, such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; or as a solution, a suspension or as an emulsion.
  • the active ingredient may also be presented as a bolus electuary or paste, and be in a pure form, i. e. , without a carrier.
  • Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrant or wetting agents.
  • a tablet may be made by compression or molding, optionally with one or more formulational ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be coated according to methods well known in the art. Oral fluid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.
  • the tablets may optionally be formulated so as to provide slow or controlled release of the active ingredient therein.
  • Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline water-for-injection, immediately prior to use. Alternatively, the formulations may be presented for continuous infusion.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter or polyethylene glycol.
  • Formulations for topical administration in the mouth include lozenges, comprising the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a base such as gelatin and glycerin or sucrose and acacia.
  • the compounds of the invention may be used as a liquid spray or dispersible powder or in the form of drops. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs.
  • the compounds are conveniently delivered from an insufflator, nebulizer, pressurized packs or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the compounds may take the form of a dry powder composition, for example, a powder mix of the compound and a suitable powder base such as lactose or starch.
  • a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form, in, for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflators.
  • compositions adapted to give sustained release of the active ingredient, may be employed.
  • the pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants or preservatives.
  • 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.
  • Preferred unit dosage formulations are those containing an effective dose, as recited below, or an appropriate fraction thereof, of the active ingredient.
  • the compositions may be administered orally or via injection at a dose of from about 0.1 to about 250 mg/kg per day.
  • the dose range for adult humans is generally from about 5 mg to about 17.5 g/day, preferably about 5 mg to about 10 g/day, and most preferably about 100 mg to about 3 g/day.
  • Tablets or other unit dosage forms of presentation provided in discrete units may conveniently contain an amount which is effective at such dosage or as a multiple of the same, for instance, units containing about 5 mg to about 500 mg, usually from about 100 mg to about 500 mg.
  • the pharmaceutical composition preferably is administered orally or by injection
  • the precise amount administered to a subject will be the responsibility of the attendant physician.
  • the dose employed will depend upon a number of factors, including the age and sex of the subject, the precise disorder being treated, and its severity. Also the route of administration may vary depending upon the condition and its severity.
  • Example 1 The present invention will be further illustrated by the following non-limiting examples: Example 1
  • siRNAs specific to FZDlO under the control of the U6 promoter (psiU6BX-FZD10).
  • siRNAs psiU6BX-FZD10B and C were constructed according to FZDlO ORF sequence.
  • snRNA U6 gene was reported to be transcribed by RNA polymerase III, which produces the short transcripts with uridines at the 3' end, we amplified the genomic fragment of snRNA U6 gene containing its promoter region by PCR using a set of primers,
  • the Bam ⁇ l, Xhol digested fragment containing the snRNA U6 gene was purified and cloned into nucleotide 1257 to 56 fragment of pcDNA3.1(+) plasmid, which was amplified by PCR with a set of primer, 5'-TGCGGATCCAGAGCAGATTGTACTGAGAGT-S' (SEQ ID NO.13) and 5'- CTCTATCTC GAGTGAGGCGGAAAGAACCA-S ' (SEQ ID No.14).
  • the ligated DNA was used as a template for PCR with primers, 5 ' -TTTAAGCTTGAAGACTATTTTTACATCAGGTTGTTT TTCT-3' (SEQ ID No.15) and 5 ' -TTTAAGCTTGAAGAC ACGGTGTTTCGTCCTTTCCAC A-3 ' (SEQ ID No.16) (underlines indicate HindHl site).
  • the product was digested with Hin ⁇ ill, which was subsequently self-ligated to produce psiU6BX3.0 vector plasmid.
  • PCR product was cloned into pcDNA3.1-myc/His vector (Invitrogen), and then the inserted FZDlO cDNA with myc/His-tag was further subcloned into pCAGGS/neo expression vector.
  • an internal-tagged HA-FLAG-FZDl OFL (residue 1-581; SEQ ID No.2), expression plasmid constructs were generated from the cloned full-length FZDlO cDNA by PCR amplification with following primer combinations:
  • FZD10-nt652 5'-AAGTCGACTACTGGAGCCGCGAGGACAAG-S' (SEQ ID NO.21)
  • FZD10 ⁇ C2 (residues 1-525) expression plasmid constructs were generated and C-terminal portion (residues 218-each end) of each construct was generated by the forward primer (FZD10-nt652) and the following reverse primers, respectively;
  • Plasmids expressing siRNAs specific to FZDlO were prepared by cloning the double- stranded oligonucleotides into psiU6BX3.0 vector (Table 1).
  • the target sequences for siRNA in each plasmid are shown in Table 2.
  • the complementary oligonucleotides were each phosphorylated by incubation with T4-polynucleotide kinase at 37 °C for 30 min, followed by boiling and cooling down to room temperature slowly to anneal the two oligonucleotides. Each product was ligated into psiU6BX3.0 to construct an FZDlO-siRNA expression vector.
  • Table 1 Sequences of specific double- stranded oligonucleotides inserted into siRNA expression vector psi-U6BX-FZD20-B
  • FZDlO constructs Two FZDlO constructs, HA-FLAG-FZD 10 (described below) and FZDIO-myc-His (prepared in Example 1; Fig. 2A) were co-transfected into COS-7 cell and were performed co-immunoprecipitation using ⁇ -HA antibody.
  • HA-FLAG-FZD 10 For the construction of HA-FLAG-FZD 10, a following set of oligonucleotides, 5 ' -ACGTGTCGACTACCCATACGACGTCCCAGACTACGCTATGGACTACAAGG ACGACGATGACAAGCTCGAGATGC-3' (SEQ ID No.37) (underline indicates Sail site) and 5'-GCATCTCGAGCTTGTCATCGTCGTCCTTGTAGTCCATAGCGTAGTC TGGGACGTCGTATGGGTAGTCGACACGT-S' (SEQ ID NO.38) (underline indicates Xfjol site) was annealed and digested with SaK and Xhol to generate the HA-FLAG tag.
  • 5 ' -ACGTGTCGACTACCCATACGACGTCCCAGACTACGCTATGGACTACAAGG ACGACGATGACAAGCTCGAGATGC-3' (underline indicates Sail site)
  • residues 1-217 and 218-sto ⁇ codon (or C-terminal deletion) fragments were individually PCR-amplified. PCR products were digested with Sail and Xtiol, and ligated sequentially into pCAGGS/neo expression vector. The HA-FLAG tag fragment digested with SaIL and Xhol was ligated between nt651 and nt652 in frame.
  • COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. FuGENE ⁇ (Roche) was used for transfection of COS-7 cells. The transfected cells were used 48-72h after transfection.
  • Transfected COS-7 cells were washed twice with cold PBS(-) and lysed in IPP buffer. The cell lysate was incubated with anti-HA F7 antibody (Santa Cruz) and protein G-sepharose (Sigma). Bound proteins were washed five times with IP buffer and eluted with SDS-sample buffer. Eluted proteins were analyzed by 10% SDS-PAGE. Proteins were electrophorestically transferred to nitrocellulose membranes (Amersham Biosciences). The membranes were incubated with anti-myc 9E10 monoclonal antibody, anti-FLAG M2 antibody or anti-HA F7 antibody followed by horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences). Bound secondary antibodies were visualized by ECLTM Western blotting detection reagents (Amersham Biosciences).
  • HA-FLAG-FZD 10 As shown in Fig. 2B, upper panel, immunoprecipitated HA-FLAG-FZD 10 seemed to appear as multiple bands that were once or twice the predicted molecular mass and the highest band was stacked at the top of the running gel for SDS-PAGE, suggesting that FZDlO are able to form oligomers. Furthermore, using a ⁇ -HA antibody to pull down HA-FLAG-FZD 10 resulted in co-immunoprecipitation of FZDlO-myc-His (Fig.
  • FZDlO contains two motifs, KTxxxW (Umbhauer M, et al. (2000). EMBO J. 19:4944-4954), which is located at two amino acids after the seventh transmembrane domain, and TCV motifs at the predicted C-terminal which are thought to be important for protein-protein interaction.
  • Fig. 2A To examine the involvement of these motifs in oligomerization, we designed two C-terminal deleted constructs (Fig. 2A) whether they can form oligomers. As shown in Fig. 2C, pull down with ⁇ -HA antibody resulted in co-immunoprecipitation of FZD10-myc-His, suggesting that C-terminus motifs of FZDlO might be not involved in oligomerization.
  • TAP Tandem affinity purification-system purification was performed according to previous report (Rigaut G, et al. (1999). Nat Biotechnol, 17:1030-1032). Briefly, pCAGGS/neo-FZDIO-TAP or pCAGGS/neo-TAP as control were transfected to SNU-C5 cells.
  • TAP expression vector For construct of TAP expression vector, cDNA for the TAP tag sequence, consisting of immunoglobulin G-binding domain and calmodulin-binding peptide separated with the cleavage site of Tabacco each virus protease (TEV) with SaH at 3' end was PCR-amplified according to standard protocols (Fig. 3A). First, TAP tag sequence mentioned above was cloned into pcDNA-3.1(+)-myc-His expression vector. Next, pcDNA-3.1(+)-myc-His-TAP was digested with Xhol and Sail and resulted myc-His-TAP fragment was inserted into pCAGGS/neo vector.
  • the FZDlO ORF cDNA was subcloned into pCAGGS-myc-His-TAP/neo expression vector.
  • Colon cancer cell line, SNU-C5 was maintained in RPMI1640 medium supplemented with 10% fetal bovine serum and antibiotics. FuGENE ⁇ (Roche) was used for transfection of SNU-C5 cells.
  • COS-7 cells (Fig.3C).

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  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé servant à inhiber ou à réduire une expression du gène FZD10. L'invention concerne également un procédé servant à traiter et/ou à prévenir chez un sujet une maladie associée au FZD10. L'invention concerne en outre une composition pharmaceutique comprenant une molécule d'ARN double brin pour le FZD10 ou un vecteur d'expression capable d'exprimer une molécule d'ARN double brin pour le FZD10.
PCT/JP2005/013444 2004-08-05 2005-07-14 PROCÉDÉ SERVANT À TRAITER LE SARCOME SYNOVIAL UTILISANT UN ARNsi POUR LE FZD10 WO2006013733A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP05766513A EP1789549A1 (fr) 2004-08-05 2005-07-14 Procédé servant à traiter le sarcome synovial utilisant un arnsi pour le fzd10
JP2007507592A JP2008509076A (ja) 2004-08-05 2005-07-14 ＦＺＤ１０に対するｓｉＲＮＡを用いた滑膜肉腫の治療方法

Applications Claiming Priority (2)

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US59883404P 2004-08-05 2004-08-05
US60/598,834 2004-08-05

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WO2006013733A1 true WO2006013733A1 (fr) 2006-02-09

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PCT/JP2005/013444 WO2006013733A1 (fr) 2004-08-05 2005-07-14 PROCÉDÉ SERVANT À TRAITER LE SARCOME SYNOVIAL UTILISANT UN ARNsi POUR LE FZD10

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EP (1) EP1789549A1 (fr)
JP (1) JP2008509076A (fr)
CN (1) CN1993466A (fr)
WO (1) WO2006013733A1 (fr)

Cited By (2)

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Publication number Priority date Publication date Assignee Title
EP2386577A2 (fr) 2006-06-21 2011-11-16 Oncotherapy Science, Inc. Anticorps monoclonaux de FZD10 ciblant des tumeurs et leurs utilisations
WO2018066585A1 (fr) 2016-10-06 2018-04-12 オンコセラピー・サイエンス株式会社 Anticorps monoclonal anti-fzd10, et application de celui-ci

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6155255B2 (ja) * 2012-04-17 2017-06-28 Hoya株式会社 Ｆｚｄ１０結合性ペプチド

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WO2004020668A2 (fr) * 2002-08-30 2004-03-11 Oncotherapy Science, Inc. Methode de traitement du synovialome

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WO2004020668A2 (fr) * 2002-08-30 2004-03-11 Oncotherapy Science, Inc. Methode de traitement du synovialome

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KIRIKOSHI HIROYUKI ET AL: "Expression profiles of 10 members of Frizzled gene family in human gastric cancer", INTERNATIONAL JOURNAL OF ONCOLOGY, vol. 19, no. 4, October 2001 (2001-10-01), pages 767 - 771, XP008055827, ISSN: 1019-6439 *
NAGAYAMA SATOSHI ET AL: "Identification of PDZK4, a novel human gene with PDZ domains, that is upregulated in synovial sarcomas", ONCOGENE, vol. 23, no. 32, 15 July 2004 (2004-07-15), pages 5551 - 5557, XP002354476, ISSN: 0950-9232 *
NAGAYAMA SATOSHI ET AL: "Therapeutic potential of antibodies against FZD 10, a cell-surface protein, for synovial sarcomas.", ONCOGENE, vol. 24, no. 41, 15 September 2005 (2005-09-15), pages 6201 - 6212, XP002354477, ISSN: 0950-9232 *
TERASAKI H ET AL: "Frizzled-10, up-regulated in primary colorectal cancer, is a positive regulator of the WNT - beta-catenin - TCF signaling pathway", INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, vol. 9, no. 2, February 2002 (2002-02-01), pages 107 - 112, XP002262585, ISSN: 1107-3756 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2386577A2 (fr) 2006-06-21 2011-11-16 Oncotherapy Science, Inc. Anticorps monoclonaux de FZD10 ciblant des tumeurs et leurs utilisations
US8221751B2 (en) 2006-06-21 2012-07-17 Oncotherapy Science, Inc. Tumor-targeting monoclonal antibodies to FZD10 and uses thereof
US9139655B2 (en) 2006-06-21 2015-09-22 Oncotherapy Science, Inc. Tumor-targeting monoclonal antibodies to FZD10 and uses thereof
WO2018066585A1 (fr) 2016-10-06 2018-04-12 オンコセラピー・サイエンス株式会社 Anticorps monoclonal anti-fzd10, et application de celui-ci
KR20190059318A (ko) 2016-10-06 2019-05-30 온코세라피 사이언스 가부시키가이샤 Fzd10에 대한 모노클로널 항체 및 이의 사용
US11079386B2 (en) 2016-10-06 2021-08-03 Oncotherapy Science, Inc. Monoclonal antibody against FZD10 and use thereof

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JP2008509076A (ja) 2008-03-27
EP1789549A1 (fr) 2007-05-30

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