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

To construct an HSV vector that replicates selectively in calponin-positive cells and proliferating 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.Viro.56,558-570,1985）通过同源重组和D12.CALP.ΔRR病毒载体。 D12.CALP.ΔRR病毒载体在ICP6启动子的控制下表达β-半乳糖苷酶，并且可以在CALPONIN启动子控制下表达ICP4蛋白和EGFP蛋白。使用表达钙蛋白的人平滑肌肉瘤细胞系（SK-LMS-1）和钙蛋白非表达的人骨肉核细胞系（OST）来评估D12.CALP.ΔRR病毒载体的病毒复制的细胞选择性。 D12.CALP.ΔRR病毒载体在钙蛋白阳性的SK-LMS-1细胞中复制，但D12.CALP.ΔRR病毒载体的滴度在感染后72小时降低了Calponin阴性细胞中72小时的calponin-1/100000的滴度，与Sk-LMS-1 Cell的速度相比，与Sk-lms-1 Cells的速度相比。恶性肿瘤是最重要的特性，即对Ganciclovir的敏感性（一种抗疱疹病毒剂），因为它具有完整状态的TK基因。 D12.CALP.ΔRR病毒载体的复制在存在Ganciclovir，SK-LMS-1细胞和ICP4 cDNA引入的VERO E5细胞的情况下被抑制。在SK-LMS-1细胞中，在存在40 ng/ml ganciclovir.b-3（体内治疗和组织学分析）的情况下，复制完全受到抑制。D12.CALP.ΔRR病毒载体的体内抗肿瘤效应抗体抗体移植肿瘤Xenosemics（MFH-ai-ai-ai ryed WAS MFINED）与MF的MFH-MFH-MF. 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.在接受静脉注射（D12.CALP。ΔRR病毒载体）治疗后第29天的肿瘤体积（平均值±S.E.，n = 6）和未经处理的组（PBS）分别为500±136 mm^3和183 mm^3和183 mm^33 mm^3。与未处理的组相比，治疗组显示出显着的抗肿瘤作用。D12.CALP.ΔRR病毒载体的治疗作用通过体内检查了静脉注射对人肺转移性肿瘤的治疗作用。 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,肝脏在同一天进行了极好的肝脏进行X-GAL染色。通过进行一次静脉内给药D122.CALP.ΔRR病毒载体，X-GAL染色表明在肺转移焦点和组织学肿瘤坏死中复制D12.CALP.ΔRR病毒载体。然而，未观察到X-GAL染色，表明未观察到正常组织（例如大脑，心脏和肝脏）中D12.CALP.ΔRR病毒载体的感染和复制。随后，人类肺转移性肿瘤的治疗作用，其中要施用的MFH-AI-LM细胞的数量设置为1x10^6或5x10^5，而D12.CALP.ΔRR病毒载体为1×10^7 Pfu/heme the Movene the Day 17，Day 17，Day 17，Day 17，Day 1的总计1×10^7 Pfu/小鼠的总体注射。检查了通过将MFH-AI-LM肿瘤细胞注射1x10^6或5x10^5构成的所有肺转移性肿瘤模型，并将肺转移性肿瘤抑制作用d12.calpΔRR载体施用。较少的