| Co-Investigator(Kenkyū-buntansha) |
YOSHIKAWA Hideki Osaka University School of Medicine, Department of Orthopaedic Surgery, Professor, 医学系研究科, 教授 (60191558)
TOGUCHIDA Junya Kyoto University, Institute for Frontier Medical Science, Department of Tissue Regeneration, Professor, 再生医科学研究所, 教授 (40273502)
YAMAMURA Hisako Osaka Medical Center for Cancer & Cardiovascular Diseases, Department of Molecular Medicine, Associate director, 主任研究員 (50342994)
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| Budget Amount *help |
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2004: ¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 2003: ¥7,500,000 (Direct Cost: ¥7,500,000)
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| Research Abstract |
We have previously described a thymidine kinase (TK)-defective type I herpes simplex virus (HSV-1) mutant d12. CALP in which smooth muscle-specific calponin promoter drives expression of the RS1 gene encoding an essential trans-activating factor (ICP4) for viral genes (Yamamura et al. Cancer Res. 61 ; 3969-3977, 2001). In order to improve efficacy and safety for pre-clinical and clinical testing, we have developed a new conditionally replicating oncolytic HSV-1 (d12.CALPΔRR) in which smooth muscle-specific calponin transcriptional regulatory sequence drives the RS1 gene and the enhanced green fluorescent protein (EGFP) cDNA via bicistronic expression using the internal ribosomal entry site (IRES2). The engineered HSV-1 is a derivative of ICP4-null mutant d120 carrying the intact TK gene and an insertional mutation in the U_L39 gene encoding a large subunit of ribonucleotide reductase (ICP6), an essential enzyme for viral replication. d12.CALPΔRR also contains the Escherichia coli lacZ
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gene under the control of the intrinsic U_L39 gene promoter to trace viral replication. We examined the cytopathic effects of d12.CALPΔRR at low multiplicity of infection (0.01〜0.1 plaque-forming unit/cell) on primary cultures from surgically removed human leiomyosarcoma cells with or without calponin expression. d12.CALPΔRR preferentially killed calponin- expressing tumor cells. For in vivo studies, 15 animals (BALB/c nude mice) harboring human uterine leiomyosarcoma (mean tumor volume 44 mm^3) were randomly divided and treated three times intraneoplastically with either 1 x 10^7 plaque-forming units (PFU) of d12.CALPΔRR/100 mm^3 of tumor volume or medium alone on days 21, 27 and 34 after xenograft transplantation. The viral treatment group showed significant inhibition of tumor growth by day 39 (tumor volume ; mean±S.E., 1281±153, n=8 vs. 342±32 mm^3, n=7). Treatment with 5 x 10^7 PFU of d12.CALPΔRR intravenously injected five times in every 4-5 days resulted in stable and significant inhibition of tumor growth by day 42 (tumor volume ; mean±S.E., 1708±199 vs. 521±111 mm^3 n=5). We have tested the safety of d12.CALPΔRR in mice. BALB/c nu/nu mice (n=23〜26) inoculated intravenously with 2 x 10^7 PFU of d12.CALPΔRR survived for over 2 months with no apparent abnormalities in blood chemical values [liver, kidney and metabolic (glucose and lipid) functions]. After single intravenous injection of 2 x 10^7 PFU, analysis of serum samples showed significant recovery of active viruses in the portal vein blood at 15 min (mean value of 4-6 mice ; 1.3 x 10^5 PFU/ml), which significantly decreased over the first hour (673 PFU/ml) and was absent from 24 h and beyond. Histochemical analysis at 24 h post d12.CALPΔRR intravenous injection demonstrated scant positive expression of LacZ and ICP4 in liver parenchyma and cells in lung and spleen with no single cell co-expressing both LacZ and ICP4, indicating the absence of viral replication. Indeed, extracts prepared from brain, lung, liver and spleen tissues harvested at 24 h post injection demonstrated absence of active viruses. Also, no LacZ and ICP4 staining were observed in all organs examined at 72 h post injection. There was no overall necrosis and inflammation at light microscopic level in these specimens. Conversely, strong LacZ and ICP4 expression was accumulated in all leiomyosarcoma xenografts that received 2 x 10^7 PFU via direct intratumoral injection or intravenous injection from tail vein. Furthermore, semi-quantitative PCR analysis revealed that 2 x 10^7 PFU intravenous injection did not result in persistence of the viral DNA (genes for LacZ and Glycoprotein E) for no more than 1 week in the trigeminal nerve ganglia. Finally, intraperitoneal administration of aciclovir (30 mg/kg/day) for 7 days significantly inhibits viral replication in the leiomyosarcoma xenografts as assessed by ICP4 protein expression (ICP4-positive cells ; control vs. aciclovir treatment, 300±30 vs. 53±27/mm^2, n=5, p<0.0005). We conclude that this novel anti-leiomyosarcoma agent d12.CALPΔRR at or above doses that were efficacious in mouse tumor studies can be delivered safely both intravenous and direct tumor injection. d12.CALPΔRR should be investigated further as a challenging therapy for metastasis of sarcoma tumors to remote organs via systemic vascular injection. Less
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