| Research Abstract |
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
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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
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