A novel targeted gene therapy to incurable sarcomas

为了构建在钙调蛋白阳性细胞和增殖细胞中选择性复制的HSV载体，将含有4F 2增强子/-260钙调蛋白启动子/ICP 4/IRES-EGFP的DNA片段插入ICP 4缺陷型HSV突变体d120的RR（ICP 6）基因座（U_L36（J.Virol.56，558 - 570，1985），并构建d12. CALP.ΔRR病毒载体。d12.CALP.ΔRR病毒载体在ICP 6启动子控制下表达β-半乳糖苷酶，在钙调蛋白启动子控制下可表达ICP 4蛋白和EGFP蛋白。用表达calponin的人平滑肌肉瘤细胞系（SK-LMS-1）和不表达calponin的人骨肉瘤细胞系（OST）评价d12. CALP.ΔRR病毒载体复制的细胞选择性。d12.CALP.ΔRR病毒载体在钙调蛋白阳性的SK-LMS-1细胞中复制，但d12.CALP.ΔRR病毒载体的滴度在感染后72小时在钙调蛋白阴性的OST细胞中下降至约1/100000 ...更多信息 当d12. CALP.ΔRR病毒载体应用于人类恶性肿瘤的治疗时，最重要的性质是对更昔洛韦（一种抗疱疹病毒剂）的敏感性，因为它具有完整状态的TK基因。在更昔洛韦存在下，导入ICP 4 cDNA的SK-LMS-1细胞和Vero E5细胞中d12. CALP.ΔRR病毒载体的复制受到抑制。在SK-LMS-1细胞中，在存在40 ng/ml更昔洛韦的情况下复制被完全抑制。B-3（体内处理和组织学分析）检查了d12. CALP.ΔRR病毒载体对从MFH-AI细胞分离的皮下移植肿瘤异种移植物（MFH-AI-LM）的体内抗肿瘤作用。静脉注射d12.CALP.ΔRR病毒载体一次对MFH-AI-LM细胞系皮下移植肿瘤的治疗效果表示为图7中的时间变化。在第0天，将1×10^7 pfu/小鼠的d12.CALP.ΔRR病毒载体感染至尾静脉。肿瘤体积（平均值±S.E.，n=6）静脉注射后第29天（d12.CALP.ΔRR病毒载体给药组和未给药组（PBS给药组）分别为500±136 mm^3和183±33 mm^3。结果表明，经治疗组的抗肿瘤作用明显优于未治疗组，并观察了d12. CALP.ΔRR病毒载体静脉注射对人肺转移瘤的体内治疗作用。将1×10^7 pfu/小鼠的d12.CALP.ΔRR病毒载体注射到肺转移性肿瘤模型小鼠的尾静脉中，其中使用了从人恶性纤维组织细胞瘤MFH-AI细胞中分离的具有高肺转移活性的MFH-AI-LM细胞，并且在第13天肺和正常组织，即脑、心脏、对同一天切下的肝脏进行X-Gal染色。通过一次静脉注射d12.CALP.ΔRR病毒载体，观察到X-Gal染色，表明d12.CALP.ΔRR病毒载体在肺转移灶和组织学肿瘤坏死中复制。然而，未观察到表明d12. CALP.ΔRR病毒载体在正常组织如脑、心脏和肝脏中的感染和复制的X-Gal染色。随后，在第17天，静脉内注射1×10^7 pfu/小鼠的d12.CALP.ΔRR病毒载体总共3次，其中将待施用的MFH-AI-LM细胞的数目设定为1 × 10^6或5 × 10^5，在施用MFH-AI-LM细胞后第27天和第34天进行检查对于通过将1 × 10 - 6或5 × 10 - 5个MFH-AI-LM肿瘤细胞注射到尾静脉中构建的所有肺转移性肿瘤模型，给予d12.CALPΔRR载体的组的肺转移肿瘤抑制效果是明显的。少

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