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Development of assay system to quantify insertional mutagendsis for development of safe retrovirus vector

开发定量插入诱变的测定系统，以开发安全的逆转录病毒载体

基本信息

批准号：
18590314
负责人：
HANAWA Hideki
金额：
$ 2.57万
依托单位：
Nippon Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

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 … More 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

X连锁严重联合免疫缺陷病（X-SCID）的基因治疗已在西方国家进行。虽然大多数患者在基因治疗后获得了临床改善，但据报道，T细胞白血病的发病率很高。白血病是整合的逆转录病毒载体增强子激活原癌基因（插入突变）的结果。提高载体和治疗程序的安全性被列为最优先事项。本项目建立了快速、灵敏的外显子捕获载体系统，用于批量检测逆转录病毒载体中插入基因的激活。将合成的外显子插入自失活（SIN）芽-逆转录病毒载体（MLV）或SIN人免疫缺陷病毒1型（HIV-1）载体中。在任一种情况下，将合成的外显子以反向方向插入内部启动子的上游。通过这种方式，外显子捕获载体从整合位点的下游“捕获”转录，并且捕获的转录物的量被检测到。 ...更多信息使用Taqmqn探针通过定量RTPCR定量。利用这种“外显子-陷阱载体系统”，定量分析了整合位点附近基因的插入激活，并比较了不同载体系统之间激活潜力的差异（MSCV vs. HIV 1）和内部启动子（延伸因子1 α启动子（EF 1）对γ逆转录病毒（MSCV）LTR启动子）进行比较。当使用MSCV-LTR内部启动子时，MLV载体对邻近基因的激活达到基线水平的38倍，而具有相同内部启动子的HEV-1载体仅激活1.8倍。人源启动子EF 1的利用将激活水平分别降低至2.4倍和1.5倍。为了证实体外结果，我们对X-SCID模型小鼠进行基因治疗，并比较了MSCV载体和HEV-1载体之间恶性疾病的发生率，MSCV载体和HEV-1载体都表达来自MSCV-LTR启动子的共同γ链。当使用MSCV载体治疗小鼠时，15只小鼠中有5只在治疗后33周至50周之间发生T细胞白血病/淋巴瘤。另一方面，HIV-1处理的小鼠（n=13）均未发生恶性肿瘤。这一结果表明，利用Efl启动子的HIV-1载体是潜在的安全的X-SCID基因治疗载体。少