Development of Strategies for Gene Repair Therapy for Inherited Hematopoietic Diseases
| Project/Area Number |
17590290
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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 |
Human genetics
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| Research Institution | Saitama Medical University |
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Principal Investigator |
MITANI Kohnoske Saitama Medical University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (10270901)
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| Co-Investigator(Kenkyū-buntansha) |
OHBAYASHI Fumi Saitama Medical University, Faculty of Medicine, Post-doctoral Fellow, 医学部, 研究員 (60406527)
岸本 充弘 埼玉医科大学, 医学部, 研究員 (70383289)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | adenoviral vector / adeno-associated virus vector / homologous recombination / gene repair / HPRT / hematopoietic / Fanconi anemia / gene therapy |
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| Research Abstract |
The aim of this study is to develop a strategy, which allows gene repair therapy via homologous recombination (H) using viral vectors, for treatment of inherited hematopoietic diseases.
1. Adenoviral vector
In order to analyze frequencies of HR in human primary fibroblasts using helper-dependent adenoviral vectors (HDAdVs), we targeted the HPRT locus as a model system. We obtained HPRT gene knock-outs at the frequencies of 10^<-5> to 10^<-6> per infected cells. However, when we tried to repair a mutant Fanconi anemia group A (FANCA) gene via HDAdV-mediated HR, we could not detect gene repair or even random chromosomal integration of the vector.
2. Adeno-associated virus (AAV) vector
HPRT knock-out and FANCA repair vectors, derived from AAV, were also constructed. The HPRT knock-out with various capsid serotypes were tested in a B cell line (BCL) from a normal male. Chromosomal integration of the vectors as well as gene knock-out was achieved at the frequencies of 1 x 10^<-5> -6 x 10^<-6> per cell and 1-2 x 10^<-6> per cell, respectively. Random chromosomal integration was also achieved with a BCL from the FANCA patient. However, we have not been able to obtain the FANCA gene repair using the AAV-FANCA.
3. Enhancement of HR and suppression of random integration
We tested strategies such as overexpression of UV damage endonuclease as well as the use of PARP1 inhibitor to enhance HR at the Hprt locus in mouse ES cells with electroporated plasmid DNA. We also treated the cells with a DNA-PKc inhibitor to suppress random integration of the plasmid. However, these strategies did not change the frequencies of HR or random chromosomal integration.
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Report
(3 results)
Research Products
(2 results)
