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Development of rapid and high sensitivity detection methods for replication-competent viruses contaminated in viral vector products for gene therapy

Research Project

Project/Area Number 15590150
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Medical pharmacy
Research InstitutionNational Institute of Health Sciences

Principal Investigator

UCHIDA Eriko  National Institute of Health Sciences, Division of Cellular and Gene Therapy Products, Section Chief, 遺伝子細胞医薬部, 室長 (80176685)

Co-Investigator(Kenkyū-buntansha) YAMAGUCHI Taruhide  National Institute of Health Sciences, Division of Cellular and Gene Therapy Products, Head, 遺伝子細胞医薬部, 部長 (50111117)
NAGATA Ryuji  National Institute of Health Sciences, Division of Cellular and Gene Therapy Products, Senior Researcher, 遺伝子細胞医薬部, 主任研究官 (20370942)
ISHII Akiko (WATABE Akiko)  National Institute of Health Sciences, Division of Biological Chemistry and Biologicals, Section Chief, 生物薬品部, 室長 (50291117)
Project Period (FY) 2003 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
Keywordsgene therapy / retrovirus vector / adenovirus vector / replication-competent retrovirus / repication-competent adenovirus / infectivity-PCR / virus detection / polyethyleneimine / ポリエチレンイミン磁気ビーズ / ガラスビーズ法 / ガラスビーズ
Research Abstract

Contamination by replication-competent viruses is one of the most important issues for safety and quality of virus vector products for gene therapy clinical research. In order to detect replication-competent adenovirus(RCA) and replication-competent retrovirus(RCR) contaminated in vector products more sensitively and rapidly, we have developed a novel detection method, a hybrid method that combines the infectivity assay and real-time quantitative(RT-) PCR
Infectivity PCR was established for the detection of RCA. In this method, permissive cells were infected with RCA samples, and amplified RCA were quantified by real-time PCR. The glass-beads-based DNA extraction method was suitable for extracting DNA rapidly from RCA-infected cells. By infectivity PCR, 1 pfu of RCA spiked into 10^9 particles of adenovirus vectors could be detected within 3 days. In contrast, 1O^4 pfu of RCA could be detected by conventional cytopathic effect after 9 days of infection. These results showed that infectiv … More ity PCR was useful for the rapid and sensitive detection of RCA in adenovirus vector products.
Infectivity RT-PCR was established for the detection of RCR. In this method, permissive cells were infected with RCR samples, and amplified RCR were quantified by real-time RT-PCR. Polyethyleneimine(PEI)-conjugated magnetic beads were used for concentration of RCR in the culture supernatants. By infectivity RT-PCR, 1 infectious unit(iu) of RCR spiked into 10^6 cfu/ml of retroviral vectors could be detected within 3 days. The sensitivity for viral detection was increased more than 10-fold compared with the conventional S+L- assay. Therefore, infectivity RT-PCR was useful for the rapid and sensitive detection of RCR in retrovirus vector products In addition, RCR could be detected more rapidly when RCR RNA were directly extracted from RCR-infected cells. Moreover, the infection efficiency and the detection sensitivity could be improved when PEI-magnetic beads and magnetic field were used for the infection of RCR. Less

Report

(3 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • Research Products

    (9 results)

All 2004 2003 Other

All Journal Article (7 results) Publications (2 results)

  • [Journal Article] An improved method for detection of replication competent retrovirus in retrovirus vector products2004

    • Author(s)
      Uchida E et al.
    • Journal Title

      Biologicals 32

      Pages: 139-146

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] An improved method for detection of replication competent retrovirus in retrovirus vector products2004

    • Author(s)
      Uchida E. et al.
    • Journal Title

      Biologicals Vol.32

      Pages: 139-146

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] An improved method for detection of replication competent retrovirus in retrovirus vector products2004

    • Author(s)
      Uchida E, Sato K., Iwata A., Ishii-Watabe A., Mizuguchi H., Hikata M., Murata M., Yamaguchi T., Hayakawa T.
    • Journal Title

      Biologicals 32

      Pages: 139-146

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Detection of Replication-Competent Adenoviruses Spiked into Recombinant Adenovirus Vector Products by Infectivity-PCR2003

    • Author(s)
      Ishii-Watabe A et al.
    • Journal Title

      Molecular Therapy 8

      Pages: 1009-1016

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Detection of Replication-Competent Adenoviruses Spiked into Recombinant Adenovirus Vector Products by Infectivity-PCR2003

    • Author(s)
      Ishii-Watabe A et al.
    • Journal Title

      Molecular Therapy Vol.8

      Pages: 1009-1016

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Detection of Replication-Competent Adenoviruses Spiked into Recombinant Adenovirus Vector Products by Infectivity-PCR2003

    • Author(s)
      Ishii-Watabe A., Uchida E., Iwata A., Nagata R., Satoh K., Fan K., Murata M., Mizuguchi H., Kawasaki N., kawanishi T., Yamaguchi T., Hayakawa T.
    • Journal Title

      Molecular Therapy 8

      Pages: 1009-1016

    • Related Report
      2004 Annual Research Report
  • [Journal Article] 生物薬品のウイルス安全性を目的とした核酸増幅検査(NAT)のフィージビリティースタディー2003

    • Author(s)
      山口照英, 内田恵理子
    • Journal Title

      医薬品研究 34

      Pages: 763-769

    • Related Report
      2004 Annual Research Report
  • [Publications] Ishii-Watabe A. et al.: "Detection of Replication-Competent Adenoviruses Spiked into Recombinant Adenovirus Vector Products by Infectivity-PCR Combined with Glass Beads-Based DNA Extraction."Molecular Therapy. 8. 1009-1016 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 山口照英, 内田恵理子: "生物薬品のウイルス安全性を目的とした核酸増幅検査(NAT)のフィージビリティースタディー"医薬品研究. 34. 763-769 (2003)

    • Related Report
      2003 Annual Research Report

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Published: 2003-04-01   Modified: 2016-04-21  

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