Development of targeted gene therapy for incurable sarcoma using a novel replication-selective and oncolytic viral vector

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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 … More 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