WO2010095879A2 - Petit arn interferent a lier a des transcrits de genes associes a l'apoptose et compositions de traitement du cancer contenant ce petit arni - Google Patents

Petit arn interferent a lier a des transcrits de genes associes a l'apoptose et compositions de traitement du cancer contenant ce petit arni Download PDF

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WO2010095879A2
WO2010095879A2 PCT/KR2010/001031 KR2010001031W WO2010095879A2 WO 2010095879 A2 WO2010095879 A2 WO 2010095879A2 KR 2010001031 W KR2010001031 W KR 2010001031W WO 2010095879 A2 WO2010095879 A2 WO 2010095879A2
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sirna
cancer
seq
tab2
traf7
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PCT/KR2010/001031
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Korean (ko)
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WO2010095879A3 (fr
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한보람
김성열
박한오
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(주)바이오니아
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Publication of WO2010095879A2 publication Critical patent/WO2010095879A2/fr
Publication of WO2010095879A3 publication Critical patent/WO2010095879A3/fr

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    • 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
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • RNAi RNAi-binding protein
  • the present invention provides an siRNA wherein the mRNA is transcribed from a tumor necrosis factor receptor associated factor (TRAF7) gene or a TAB1 binding protein 2 (TAB2) gene.
  • TRF7 tumor necrosis factor receptor associated factor
  • TAB2 TAB1 binding protein 2
  • the present invention provides a method for inhibiting tumor cells, comprising treating a cell with a solution containing at least one siRNA selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 1-12.
  • siRNAs having the modified nucleotide sequence as follows were also identified. In addition, it was confirmed that it can still suppress the expression of TRAF7 mRNA.
  • the siRNA variants of SEQ ID NOS: 7-9 (sample names TRAF7-2449-M1, TRAF7-2449-M2, TRAF7-2449-M3, respectively) all replaced some bases in SEQ ID NO: 3 siRNA, as shown below.
  • the siRNA of the present invention can be used as a cancer-specific anticancer agent because it did not induce apoptosis in normal cells but not tumor cells.
  • the dosage of siRNA capable of complementarily binding to the mRNA encoding TRAF7 or TAB2 of the present invention is parenteral administration of 0.05 to 0.1 ⁇ g / kg body weight, depending on the patient's age, sex, symptoms, method of administration or prophylactic purpose. can do.
  • the level of dosage for a patient with specific symptoms can vary by the person skilled in the art depending on the weight, age, sex, health condition, diet, time of administration, method of administration, and the like of the patient.
  • a siRNA that inhibits the expression of TRAF7 or TAB2 of the present invention and a composition for treating cancer using the siRNA as an active ingredient can effectively inhibit tumor cell proliferation by inducing apoptosis of tumor cells and thus anticancer therapy for various uses. It can be widely used in.
  • 1 is a graph showing mRNA levels of TRAF7 expressed in cell lines 24 hours after transfection with three sequences of siRNA for TRAF7 of the present invention, wherein NC is transfected with negative control siRNA (negative control siRNA) Means control group.
  • Figure 2 is a graph showing the mRNA level of TRAF7 expressed over time after transfection with the SEQ ID NO: 2,3 siRNA of the present invention.
  • FIG. 3 is a graph showing the mRNA levels of TAB2 expressed in cell lines transfected with three sequences of siRNA for TAB2 of the present invention.
  • Figure 4 is a graph showing the mRNA level of TAB2 expressed over time after transfection with the SEQ ID NO: 4, 5 siRNA of the present invention.
  • FIG. 5 is a graph showing the mRNA level of TAB2 expressed after transfection with siRNA of SEQ ID NO: 4 and the prior art siRNA of the present invention.
  • TAB2-1301 siRNA of the present invention (SEQ ID NO: 4)
  • MCF7 human breast cancer cell lines
  • HeLa human cervical cancer cell lines
  • FIG. 8 is a diagram illustrating lung cancer cell line (A549) and human articular synovial cell (synoviocyte), which are normal cells, were transfected with siRNA to compare whether siRNA SEQ IDs 3 and 4 induce apoptosis. It is a photograph observing whether death was induced.
  • FIG. 12 is a photograph showing the induction of apoptosis in a cell line transfected with three variant siRNAs to the SEQ ID NO: 3 siRNA of the present invention. * Indicates a substituted portion of the sequence.
  • SEQID 3 siRNA of the present invention affects the growth of tumors.
  • FIG. 16 compares tumor formation patterns of rats and controls treated with human oral cancer cell lines (KB) transfected with SEQID 3 siRNA after 14, 20, 30, and 43 days of cancer cell line administration. One picture.
  • Opti-MEM Opti-MEM medium
  • RPMI1640 culture medium or DMEM culture medium or 2.5 ml of EMEM was dispensed and then cultured at 37 ° C. and 5% (v / v) CO 2 for 24 and 72 hours, respectively.
  • the level of mRNA of TAB2 present in the cell lines transfected with three siRNA was 5.5% of the control group (SEQ ID NO: 4), 12.1% of the control group (SEQ ID NO: 5), 14.3% of the control group (SEQ ID NO: 6) I could confirm that.
  • the siRNA of SEQ ID NO: 4 showed 5.5% of the control group, and it was found that the expression of TAB2 was most effectively suppressed (FIG. 3).
  • mRNA amount was compared 72 hours after transfection. As a result, it was found that even after 72 hours, expression of TAB2 was effectively inhibited by siRNA (FIG. 4).
  • human uterine cancer cell line HeLa was transfected using the same method as in Example 2-2 to analyze the effect of apoptosis according to the concentration.
  • Modified TRAF7 2449-M1 (SEQ ID NO: 7), 2449-M2 (SEQ ID NO: 8), 2449-M3 (SEQ ID NO: 3) partially modified with the nucleotide sequence of siRNA (SEQ ID NO: 3), which showed excellent effects in Example 2-3. 9) were produced respectively.
  • the Ct values of the obtained TAB2 and GAPDH were measured, respectively, and the ⁇ Ct value, which is the difference between the two values, was obtained, and then the control group transfected with negative control siRNA (NC-siRNA, BIONEER, Korea)
  • the value of ⁇ Ct which is the difference between the values of ⁇ Ct and ⁇ Ct was determined. From this value, the value of 2 (- ⁇ Ct) X 100 was obtained, and the reduction rate of mRNA expression of TAB2 was compared.
  • Tumor cells were injected into nude mice to induce tumor formation and confirmed the tumor growth inhibition effect of TRAF7 siRNA in vivo.
  • Tumor cells transfected in Example 3-1 were injected subcutaneously with 3 ⁇ 10 6 cells per head in Balb / c nude mice (5 weeks old, CB, Korea), and then the size of the formed tumor was fixed. It was analyzed whether the siRNA of SEQ ID NO: 3 affects tumor formation inhibition.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
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  • Biotechnology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Biochemistry (AREA)
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  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

L'invention concerne un petit ARNi à lier à des transcrits de gènes associés à l'apoptose, ainsi que des compositions de traitement du cancer contenant ce petit ARNi. L'invention concerne plus particulièrement un petit ARNi qui se lie à l'ARNm de gènes du TRAF7 (facteur associé au récepteur du facteur de nécrose tumorale) ou de gènes de la TAB2 (protéine de liaison 2 à TAK1) pour inhiber l'expression de ces gènes, ce qui induit l'apoptose spécifique des cellules cancéreuses, ainsi que des compositions thérapeutiques contenant ce petit ARNi comme principe actif. Le petit ARNi et les compositions le contenant induisent l'apoptose spécifique des cellules cancéreuses et peuvent donc servir d'agents thérapeutiques anticancéreux efficaces qui n'endommagent pas les cellules saines.
PCT/KR2010/001031 2009-02-20 2010-02-19 Petit arn interferent a lier a des transcrits de genes associes a l'apoptose et compositions de traitement du cancer contenant ce petit arni WO2010095879A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020090014369A KR20100095206A (ko) 2009-02-20 2009-02-20 세포사멸 관련 유전자의 전사체에 결합하는 ｓｉＲＮＡ 및 이를 이용한 암 치료용 조성물
KR10-2009-0014369 2009-02-20

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WO2010095879A2 true WO2010095879A2 (fr) 2010-08-26
WO2010095879A3 WO2010095879A3 (fr) 2011-02-24

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114940992A (zh) * 2022-05-26 2022-08-26 华南农业大学 lncRNA TAB2-AS调控TAB2在猪卵巢颗粒细胞中的应用
CN114941010A (zh) * 2022-05-26 2022-08-26 华南农业大学 Tab2在猪卵巢颗粒细胞中的应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016516804A (ja) 2013-04-17 2016-06-09 ファイザー・インク 心血管疾患を治療するためのｎ−ピペリジン−３−イルベンズアミド誘導体

Citations (2)

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US20050095246A1 (en) * 2003-10-24 2005-05-05 Medtronic, Inc. Techniques to treat neurological disorders by attenuating the production of pro-inflammatory mediators
US20080248024A1 (en) * 2007-02-28 2008-10-09 Korea Research Institute Of Bioscience And Biotechnology Therapeutic agent for cancer, inflammation, and auto-immune disease containing inhibitor of zinc finger protein 91

Patent Citations (2)

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US20050095246A1 (en) * 2003-10-24 2005-05-05 Medtronic, Inc. Techniques to treat neurological disorders by attenuating the production of pro-inflammatory mediators
US20080248024A1 (en) * 2007-02-28 2008-10-09 Korea Research Institute Of Bioscience And Biotechnology Therapeutic agent for cancer, inflammation, and auto-immune disease containing inhibitor of zinc finger protein 91

Non-Patent Citations (5)

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Title
DATABASE GENBANK 10 February 2008 'Homo sapiens TNF receptor-associated factor 7 (TRAF7), mRNA' Database accession no. NM 032271 *
DATABASE GENBANK 28 September 2008 'Homo sapiens TGF-beta activated kinase 1/ MAP3K7 binding protein 2 (TAB2), mRNA' Database accession no. NM 015093 *
HIROKI YOSHIDA ET AL.: 'The Tumor Suppressor Cylindromatosis (CYLD) Acts as a Negative Regulator for Toll-like Receptor 2 Signaling via Negative Cross- talk with TRAF6 and TRAF7.' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 280, no. 49, 2005, pages 41111 - 41121 *
LIANG-GUO XU ET AL.: 'TRAF7 Potentiates MEKK3-induced AP 1 and CHOP Activation and Induces Apoptosis.' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 279, no. 17, 2004, pages 17278 - 17282 *
TEWIS BOUWMEESTER ET AL.: 'A physical and functional map of the human TNF - /NF-K B signal transduction pathway.' NATURE CELL BIOLOGY. vol. 6, no. 2, 2004, pages 97 - 105 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114940992A (zh) * 2022-05-26 2022-08-26 华南农业大学 lncRNA TAB2-AS调控TAB2在猪卵巢颗粒细胞中的应用
CN114941010A (zh) * 2022-05-26 2022-08-26 华南农业大学 Tab2在猪卵巢颗粒细胞中的应用
CN114941010B (zh) * 2022-05-26 2023-08-15 华南农业大学 Tab2在猪卵巢颗粒细胞中的应用
CN114940992B (zh) * 2022-05-26 2023-08-29 华南农业大学 lncRNA TAB2-AS调控TAB2在猪卵巢颗粒细胞中的应用

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WO2010095879A3 (fr) 2011-02-24

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