| Project/Area Number |
15590150
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Medical pharmacy
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| Research Institution | National Institute of Health Sciences |
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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)
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| 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)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| 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)
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| Keywords | gene 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 |
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| 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
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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
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