A novel targeted gene therapy to incurable sarcomas

为了构建在钙钙蛋白阳性细胞和增殖细胞中选择性复制的HSV载体，将含有4F2增强子/-260钙钙蛋白启动子/ICP4/IRES-EGFP的DNA片段通过同源重组插入到ICP4缺陷的HSV突变体d120的RR （ICP6）位点（U_L36）和d12 calp中。构建ΔRR病毒载体。d12.CALP。ΔRR病毒载体在ICP6启动子控制下表达β-半乳糖苷酶，在钙钙蛋白启动子控制下可表达ICP4蛋白和EGFP蛋白。用表达钙钙蛋白的人平滑肌肉瘤细胞株SK-LMS-1和不表达钙钙蛋白的人骨肉瘤细胞株OST对d12.CALP病毒复制的细胞选择性进行了评价。ΔRR病毒载体。d12.CALP。ΔRR病毒载体在钙钙蛋白阳性的SK-LMS-1细胞中复制，但d12.CALP滴度。与SK-LMS-1细胞相比，感染后72小时，ΔRR病毒载体在钙钙蛋白阴性OST细胞中下降约为1/100000。当d12.CALP。ΔRR病毒载体应用于人类恶性肿瘤的治疗，其最重要的特性是具有完整的TK基因，对抗疱疹病毒药物更昔洛韦具有敏感性。d12.CALP的复制。ΔRR病毒载体在更昔洛韦存在下被抑制，用于SK-LMS-1细胞和引入ICP4 cDNA的Vero E5细胞。在SK-LMS-1细胞中，40 ng/ml的更昔洛韦完全抑制了细胞复制。B-3（体内治疗及组织学分析）d12.CALP的体内抗肿瘤作用。研究了从MFH-AI细胞分离的抗皮下移植肿瘤异种移植物（MFH-AI- lm） ΔRR病毒载体。单次静脉注射d12.CALP的治疗效果。ΔRR针对MFH-AI-LM细胞系肾下移植肿瘤的病毒载体在图7中按时间顺序表达。第0天，d12.CALP。将1×10^7 pfu/小鼠的ΔRR病毒载体感染到尾静脉。静脉注射治疗后第29天各组肿瘤体积（平均值±S.E,n=6） （d12.CALP）。ΔRR给予病毒载体)和未处理组（给予PBS）分别为500±136 mm^3和183±33 mm^3。与未治疗组相比，治疗组有明显的抗肿瘤作用。d12.CALP的治疗效果。ΔRR体内静脉注射抗人肺转移瘤病毒载体。d12.CALP。将1×10^7 pfu/mouse的ΔRR病毒载体注射到肺转移瘤模型小鼠的尾静脉中，其中使用从人恶性纤维组织细胞瘤MFH-AI细胞中分离的对肺具有高转移活性的MFH-AI- lm细胞，并于第13天在肺内转移肿瘤和当天切除的正常组织即脑、心、肝进行X-Gal染色。通过静脉注射d12.CALP。ΔRR病毒载体，X-Gal染色表明d12.CALP复制。ΔRR病毒载体在肺转移灶和组织学肿瘤坏死中观察。然而，X-Gal染色表明d12.CALP感染和复制。ΔRR正常组织如脑、心、肝未见病毒载体。随后，对人肺转移瘤的治疗效果，其中MFH-AI-LM细胞的给药数量设定为1X10^6或5X10^5， d12.CALP。观察MFH-AI-LM细胞给药后第17天、第27天、第34天静脉注射1×10^7 pfu/小鼠ΔRR病毒载体3次。通过尾静脉注射1X10^6或5X10^5 MFH-AI-LM肿瘤细胞构建肺转移瘤模型，观察d12给药组对肺转移瘤的抑制作用。CALPΔRR矢量是显而易见的。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