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

西方国家已经对X连锁Sevier联合免疫缺陷(X-SCID)进行了基因治疗。虽然大多数患者经过基因治疗后临床症状得到改善，但T细胞白血病的高发已有报道，白血病是整合逆转录病毒载体增强子(插入突变)激活原癌基因的结果。改进载体的安全性和治疗程序被列为当务之急。在本项目中，我们开发了快速、灵敏的“外显子陷阱载体系统”，用于批量检测逆转录病毒载体的插入基因激活。将合成外显子插入自失活(SIN)GEMA逆转录病毒载体(MLV)或SIN人类免疫缺陷病毒1型(HIV-1)载体，使合成外显子反向插入到内部启动子的上游。通过这种方式，外显子陷阱载体“陷阱”了整合位点下游的转录，并捕获了转录本的数量Wa…S用Taqmqn探针进行定量RT-PCR检测。利用该“外显子-陷阱载体系统”，对整合位点邻域基因的插入激活进行了定量，并比较了MSCV-LTR内启动子、MLV载体激活邻域基因最高可达基线水平的38倍，而具有相同内部启动子的HEV-1载体的激活潜能仅为原来的1.8倍。利用人源启动子EF1使激活水平分别降至2.4-FID和1.5倍。为了验证这一体外实验结果，我们对X-SCID模型小鼠进行了基因治疗，并比较了MSCV载体和HEV-1载体在治疗后33周和50周发生T细胞白血病/淋巴瘤的发生率。另一方面，HIV-1处理的小鼠(n=13)没有一只发生恶性反应。这一结果表明，利用EFL启动子的HIV-1载体是潜在安全的X-SCID基因治疗载体。较少

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