A novel targeted gene therapy to incurable sarcomas

一种针对无法治愈的肉瘤的新型靶向基因疗法

基本信息

批准号：
14571414
负责人：
YAMAMURA Hisako
金额：
$ 2.24万
依托单位：
Osaka Medical Center for Cancer and Cardiovascular Diseases
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

To construct an HSV vector that replicates selectively in calponin-positive cells and proliferative cells, a DNA fragment containing the 4F2 enhancer/-260 calponin promoter/ICP4/IRES-EGFP was inserted into the RR (ICP6) locus (U_L36) of the ICP4-deficient HSV mutant d120 (J.Virol.56,558-570,1985) by homologous recombination, and a d12.CALP.ΔRR viral vector was constructed. The d12.CALP.ΔRR viral vector expresses β-galactosidase under the control of an ICP6 promoter, and can express ICP4 protein and EGFP protein under the control of calponin promoter. The calponin-expressing human leiomyosarcoma cell line (SK-LMS-1) and calponin non-expressing human osteosarecma cell line (OST) were used to evaluate the cell selectivity of the viral replication of d12.CALP.ΔRR viral vector. The d12.CALP.ΔRR viral vector was replicated in calponin-positive SK-LMS-1 cells but the titers of d12.CALP.ΔRR viral vector decreased in calponin-negative OST cells 72 hours after infection to approximately 1/100000 … More compared to those of the SK-LMS-1 cells.When the d12.CALP.ΔRR viral vector is applied to therapies for human malignant tumors, the most important property is that sensitivity to ganciclovir, an anti-herpes viral agent, is indicated since it has TK genes in an intact state. The replication of d12.CALP.ΔRR viral vector was suppressed in the presence of ganciclovir, for SK-LMS-1 cells and Vero E5 cells introduced with ICP4 cDNA. In SK-LMS-1 cells, the replication was completely suppressed in the presence of 40 ng/ml ganciclovir.B-3 (In vivo treatment and histological analysis)The in vivo anti-tumor effect of the d12.CALP.ΔRR viral vector against subdermally transplanted tumor xenografts (MFH-AI-LM) that are isolated from MFH-AI cells was examined. The therapeutic effect by one intravenous injection of d12.CALP.ΔRR viral vector against subdennal transplanted tumors of MFH-AI-LM cell lines is expressed as a chronological change in Figure 7. On day 0, the d12.CALP.ΔRR viral vector of 1×10^7 pfu/mouse was infected into the tail vein. The tumor volume (means±S.E.,n=6) of the group on day 29 after being treated with intravenous injection (d12.CALP. ΔRR viral vector administered) and the non-treated group (PBS administered) were 500±136 mm^3 and 183±33 mm^3, respectively. The treated group showed significant anti tumor effect compared to the non-treated group.The therapeutic effect of d12.CALP.ΔRR viral vector against human lung metastatic tumor by intravenous injection in vivo was examined. The d12.CALP.ΔRR viral vector of 1×10^7 pfu/mouse was injected into the tail vein of a lung metastatic tumor model mouse wherein MFH-AI-LM cells with high metastatic activity to lung isolated from human malignant fibrous histiocytoma MFH-AI cells are used, and metastases tumor in the lung at day 13 and the normal tissues, that is, the brain, heart, liver excised at the same day were subjected to X-Gal staining. By conducting one intravenous administration of d12.CALP.ΔRR viral vector, X-Gal staining which indicates replication of d12.CALP.ΔRR viral vector in the lung metastatic focus and histological tumor necrosis was observed. However, X-Gal staining that indicate the infection and replication of the d12.CALP.ΔRR viral vector in normal tissues such as the brain, heart and liver was not observed. Subsequently, the therapeutic effect of human lung metastatic tumor wherein the number of MFH-AI-LM cells to be administered are set to 1X10^6 or 5X10^5, and the d12.CALP.ΔRR viral vector of 1×10^7 pfu/mouse was intravenously injected for a total of three tines on day 17,day 27 and day 34 after administration of MFH-AI-LM cells was examined For all the lung metastatic tumor models constructed by injecting 1X10^6 or 5X10^5 of MFH-AI-LM tumor cells into the tail vein, the lung metastatic tumor-suppressing effect of the groups administered with d12.CALPΔRR vector was apparent. Less

为了构建HSV，将在Calponin阳性细胞和增殖细胞中选择性地构建60钙蛋白启动子/ICP4/IRES-EGFP被插入ICP4-缺乏HSV突变体D120（J.Virol.56，J.Virol.56，J.Virol.56，，ICP6）基因座（U_L36）中558-570，1985）通过同源重组和D12.CALP.RR病毒载体的构造是LP.ΔRR载体载体在ICP6启动子的控制下表达β-半乳糖苷酶，并且可以表达ICP4和EGFP蛋白的控制。启动子-1）和钙蛋白酶的骨骨细胞系（OST）用于评估病毒载体的细胞选择性。与SK-LMS-1细胞相比，降低钙蛋白阴性的OST细胞72小时的感染约为1/100000…更多。当D12.RRRR对人类恶性肿瘤的治疗疗法时，最重要的特性是对Ganciclovir的敏感性，抗HERPES在完整状态下的TK基因在Ganciclovir的存在下，用于SK-LMS-1细胞，并在SK-LMS-1细胞中引入了vero E5细胞。分析D12的体内抗肿瘤效应。 D122.CALP。对MFH -AI -LM细胞系的亚登山肿瘤的静脉注射为图7中的时间变化。 @静脉注射后第29天的肿瘤体积（平均值±S.E.，n = 6）（d12.calp。（施用的PBS）为500±136 mm ^3和183±33 mm ^3，治疗组显示显示出与未处理的组相比，对静脉注射的静脉注射术的治疗量是1×10^7 pfu/小鼠，对人肺转移性肿瘤的治疗。使用尾静脉静脉静脉活性，从人类恶性纤维组织细胞瘤MFH-AAI细胞中分离出的肺，并在第13天和正常组织中的肺中的递载肿瘤，大脑，心脏，肝脏处切除至同一的肝脏，通过X-GAL进行X-GAL。进行D12的静脉内给药，X-GAL染色，X-GAL染色，D12.RR载体在肺部度量肿瘤坏死中。 d12.calp.rrp.rrr vial载体在正常组织（例如大脑，心脏和肝脏）中未观察到的f MFH-AI-LM细胞。 1×10^7 PFU/小鼠的ΔRR载体是静脉内注射MFH-AI-LM细胞F的组使用D12.CALPΔRR矢量较少