Development of assay system to quantify insertional mutagendsis for development of safe retrovirus vector
| Project/Area Number |
18590314
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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 | Nippon Medical School |
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Principal Investigator |
HANAWA Hideki Nippon Medical School, Medical Department, Assistant (10256977)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥4,010,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Gene theranv / Sever combined immunodeficiency / Retrovirus vector / HIV-1 vector / Insertional mutaeenesis / Leukemia / HIV1ベクター / MLVベクター / SINベクター / インスレーター / エキソントラップ |
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| Research Abstract |
Gene therapy for the X-linked Sevier Combined ImmunoDeficiency (X-SCID) has been conducted in Western countries. Although most of the patient obtained clinical improvement after gene therapy, high incidence of T-cell leukemia has been reported The leukemia is the result of activation of proto-oncogene by integrated retrovirus vector enhancer (insertional mutagenesis). Improvements of the safety of the vector and therapy procedure are listed as top priority. In this project, we have developed quick and sensitive "exon trap vector system" for bulk detection of insertional gene activation by retrovirus vectors. Synthetic exon was inserted in self-inactivating (SIN) gemma-retrovirus vector (MLV) or SIN human immunodeficiency virus type 1 (HIV-1) vector In either ease the synthetic exon was inserted at upstream of internal promoter in reversed orientation. In this way, the exon-trap vector "traps" transcription from downstream of integration site, and the amount of the trapped transcript wa
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s quantified by quantitative RTPCR using Taqmqn Probe. Using this "exon-trap vector system", insertional activation of neighborhood gene of integration site was quantified and, and difference of the activation potential between the vector system (MSCV vs. HIV1) and internal promoter (elongation factor 1 alpha promoter (EF1) vs. gammaretrovirus (MSCV) LTR promoter) were compared When MSCV-LTR internal promoter was used, MLV vector activated neighborhood gene up-to 38-fold of base line level, while HEV-1 vector with same internal promoter activated only 1.8 fold. Utilization of human origin promoter EF1 reduced the activation level down to 2.4-fid and 1.5-fold respectively. To confirm the in vitro result, we performed gene therapy on X-SCID model mice and compared the incidence of malignant disease between MSCV vector and HEV-1 vector both of which express common-gamma chain from MSCV-LTR promoter When the mice were treated using MSCV vector 5 out of 15 mice developed T-cell Leukemia/Lymphoma between 33 week and 50 week after therapy. On the other hand, none of HIV-1 treated mice (n=13) developed malignancy. This result suggests that the HIV-1 vector utilizing Efl promoter is the potentially safe vector for gene therapy of X-SCID. Less
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Report
(3 results)
Research Products
(14 results)
