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molecular targeting therapy to skeletal muscule

Research Project

Project/Area Number 16591519
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Orthopaedic surgery
Research InstitutionLouis Pasteur Center for Medical Research

Principal Investigator

KISHIDA Tsunao  Louis Pasteur Center for Medical Research, Research Center of Medical, PhD., Researcher, 分子免疫研究部, 研究員 (00370205)

Co-Investigator(Kenkyū-buntansha) KISHI Atsuko  Louis Pasteur Center for Medical Research, Research Center of Medical, PhD., Researcher, 基礎研究部門, 研究員 (80260165)
KOKUBO Aoi  Louis Pasteur Center for Medical Research, Research Center of Medical, Researcher, 基礎研究部門, 研究員 (60342705)
Project Period (FY) 2004 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2004: ¥2,300,000 (Direct Cost: ¥2,300,000)
Keywordsatrophy of skeletal muscle / Cytokine / Molecular therapy / Myostatin / Regenerative Medicine
Research Abstract

Myostatin is secreted from muscle cells and blocks differentiation and proliferation of myoblasts, as a regulatory mechanism to maintain the number and volume of muscle fibers. We hypothesized that artificial inhibition of myostatin may induce regeneration and hypertrophy of skeletal muscle, providing novel therapeutic procedures to facilitate recovery of motility function in bedridden elderly whose muscles have been attenuated by long-term disuse. In this study we assessed feasibility of this molecular therapeutic strategy by administering short interfering RNA (siRNA) targeting myostatin into murine skeletal muscle. Synthetic siRNA duplex was labeled with Cy3 and mixed with RNAiFect (Qiagen) at various ratios. The resultant complex was transfected into cultured cells and fluorescent microscopic observation was performed to determine the optimal transfection condition as well as efficiency of delivery. As the results, more than 90% of the cells were successfully transfected with the s … More iRNA when 0.5 microgram of siRNA in combination with 3 microlitter of RNAiFect was added to ten thousand cells. Several siRNA duplexes were synthesized corresponding to various portions of the myostatin cDNA sequence (siMyostatin) and the siRNAs as well as a nonspecific siRNA as control were transfected into cultured cells under the optimal condition. Myostatin mRNA levels were estimated by Real-Time-PCR analysis to determine the most effective siRNA sequence for silencing of the target gene. The most remarkable silencing effect was obtained with siRNA that caused approximately 88% reduction in myostatin expression, while another siRNA, siMyostatin-2 was also comparably effective. Next, the siMyostatin-1 was transfected into C2C12 myoblast cell line and cell proliferation rate was estimated. Compared with untreated cells, or the cells given nonspecific siRNA, the siMyostatin-transfected cells showed significantly more vigorous proliferation. Based on these findings from the in vitro experiments, we examined transcriptional silencing effect in vivo of RNAi on myostatin expression. The siMyostatin-1 was labeled with Cy3 and transfected into the anterior tibial muscle of mice by means of electroporation. Fluorescence microscopic observation revealed that 60 to 90% of muscle cells contained the nucleic acid. Myostatin transcript was estimated by Real-Time PCR analysis, after transfection of siMyostatin-1 by the same protocol, and it was found that the message level of the protein was reduced to approximately 40% compared with that in the muscle electro-transfected with control siRNA. These results collectively indicate that specific knockdown of myostatin can be effectively achieved using the present methodologies, which may be applicable to RNAi-based molecular targeting therapy to promote muscular hypertrophy in bedridden elderly. Less

Report

(3 results)
  • 2005 Annual Research Report   Final Research Report Summary
  • 2004 Annual Research Report
  • Research Products

    (20 results)

All 2006 2005 Other

All Journal Article (19 results) Book (1 results)

  • [Journal Article] Absorption of Interferon alpha from patches in rats.2006

    • Author(s)
      Ito.Y et al.
    • Journal Title

      J Drug Target 13(6)

      Pages: 383-390

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Interleukin-21 Triggers both Cellular and Humoral Immune Response Leading to Therapeutic Antitumor Effects against Head and Neck Squamous Cell Carcinoma2006

    • Author(s)
      Nakano, H., et al.
    • Journal Title

      J.Gene Med. 8(1)

      Pages: 90-99

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Transthoracic direct current shock facilitates intramyocardial transfection of naked plasmid DNA infused via coronary vessels in canines.2006

    • Author(s)
      Iida, Y et al.
    • Journal Title

      Gene Therapy (In press)

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart2005

    • Author(s)
      Tsunoda, S. et al.
    • Journal Title

      Biochem. Biophys. Res. Commun. 336(1)

      Pages: 118-127

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Electrotransfer of small interfering RNA ameliorated arthritis in rats2005

    • Author(s)
      Inoue, A. et al.
    • Journal Title

      Biochem. Biophys. Res. Commun. 336(3)

      Pages: 903-908

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Interleukin-21 Triggers both Cellular and Humoral Immune Responses Leading to Therapeutic Antitumor Effects against Head and Neck Squamous Cell Carcinoma2005

    • Author(s)
      Nakano, H. et al.
    • Journal Title

      J. Genne Med. 8(1)

      Pages: 90-99

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Folding transition into a loosely collapsed state in plasmid DNA as revealed by single-molecule observation2005

    • Author(s)
      Sato, Y.T.et al.
    • Journal Title

      FEBS Lett. 279(14)

      Pages: 3095-3099

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Folding transition into a loosely collapsed state on plasmid DNA as revealed by single-molecuke observation2005

    • Author(s)
      Sato, Y.T.et al.
    • Journal Title

      FEBS Lett. 579(14)

      Pages: 3095-3099

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart2005

    • Author(s)
      Tsynoda, S., et al.
    • Journal Title

      Biochem.Biophys.Res.Common. 336(1)

      Pages: 118-127

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Electrotransfer of small interfering RNA ameliorated arthritis in rats2005

    • Author(s)
      Inoue, A., et al.
    • Journal Title

      Biochem.Biophys.Res.Common. 336(3)

      Pages: 903-908

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Functional Nucleotide Sequences Capable of Promoting Nonviral Genetic Transfer2005

    • Author(s)
      Mazda, O. et al.
    • Journal Title

      Non-viral Gene Therapy : Gene Desigh and Delivery. Springer-Verlag Tokyo

      Pages: 198-210

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart2005

    • Author(s)
      Tsunoda, S. et al.
    • Journal Title

      Biochem.Biophys.Res.Commun. 336(1)

      Pages: 118-127

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Electrotransfer of small interfering RNA ameliorated arthritis in rats2005

    • Author(s)
      Inoue, A. et al.
    • Journal Title

      Biochem.Biophys.Res.Commun. 336(1)

      Pages: 903-908

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Interleukin-21 Triggers both Cellular and Humoral Immune Responses Leading to Therapeutic Antitumor Effects against Head and Neck Squamous Cell Carcinoma2005

    • Author(s)
      Nakano, H. et al.
    • Journal Title

      J.Gene Med. 8(1)

      Pages: 90-99

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Absorption of interferon alpha from patches in rats.2005

    • Author(s)
      Iida, Y et al.
    • Journal Title

      J Drug Target 13(6)

      Pages: 383-390

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Folding transition into a loosely collapsed state in plasmid DNA as revealed by single-molecule observation2005

    • Author(s)
      Sato, Y.T.et al.
    • Journal Title

      FEBS Lett. 579(14)

      Pages: 3095-3099

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Transthoracic direct current shock facilitates intramyocardial reansfaction of naked plasmid DNA infused DNA infused via coronary vessels in canines.

    • Author(s)
      Iida, Y et al.
    • Journal Title

      Gene Therapy (In Press)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Transthoracic direct current(DC) shock facilities intramyocardial transfection of naked plasmid DNA infused via coronary vessels in canines

    • Author(s)
      Iida, Y. et al.
    • Journal Title

      Gene Therapy (in press)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Intracellular Interferon Triggers Jak/Stat Signaling Cascade and induces p53-dependent antiviral protection

    • Author(s)
      Shin-Ya, M., T.Kishida ほか
    • Journal Title

      Biochem.Biophys.Res.Commun. (in press)

    • Related Report
      2004 Annual Research Report
  • [Book] Proceedings of the 2005 International Symposium on Micro-NanoMechatronics and Human Science2005

    • Author(s)
      Kishida, T. et al.
    • Total Pages
      324
    • Publisher
      Institute for Electrical and Electronics Engineers
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Annual Research Report 2005 Final Research Report Summary

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Published: 2004-04-01   Modified: 2016-04-21  

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