755. Vaccinia Deleted Multiple Anti-Apoptosis Viral Genes Enhances Its Specificity as an Oncolytic Virus

Abstract

Top of pageAbstract We have previously studied vaccinia as an oncolytic virus and demonstrated enhanced tumor selectivity by deleting viral genes encoding thymidine kinase and vaccinia growth factor. Vaccinia virus encodes at least three anti-apoptosis genes, spi-1, spi-2 and F1L. Spi-2 is a homoloque of cowpox virus CrmA gene, whose product inhibits ICE and other caspase activity. Spi-1 is thought to inhibit the mitochondrial pathway of apoptosis by binding cathespin G. F1L is a newly characterized gene encoding a protein inhibiting apoptosis thorough stablization of the mitochondrial membrane. Cancer cells are well known to possess the ability to evade apoptosis. Therefore, the viral anti-apoptosis genes may be redundant for the virus to replicate in cancer cells, but essential for virus to replicate in normal cells. We have constructed mutant viruses deleted for either F1L alone (vF1L), for spi-1 and spi-2 (vSP), or for all three viral genes (vSPF). Infection of several tumor cell lines including murine colon adenocarcinoma (MC38), human colon adenocarcinoma (HT29) and human epithelial ovarian cancer cell (A2780) with vF1L, vSP, vSPF and a wild type vF13, yielded nearly equivalent viral recovery. The mechanism of cell death in infected cancer and normal cells was investigated by annexin V and PI staining utilizing flow cytometry. In the human colon carcinoma cells infected with WT virus, 11.5% were annexin V positive, while 30.4% were annexin V positive in vSPF-infected cells. However, both vF13L+ and vSPF induced more cell death and more apoptosis in human normal fibroblasts. The virus vF13L+ induced 34.5% and vSPF induced 58% of annexin V postive cells. These results suggest that normal cells are more sensitive to virus-induced apoptosis than cancer cells, and the cells-infected with vSPF are more prone to die via apoptosis. The biodistribution of these viruses were studied in nude mice bearing subcutaneous MC38 tumor. These mice were injected i.p. with either a mutant virus or WT at 107 plaque-forming units (pfu). The results demonstrated that vSPF reduced dramatically viral yields in brain and liver, as well as other normal tissues, while retaining similar yields in tumor. The deletion of F1L alone (vF1L) displayed significant reduction of virus in the liver. The triple deletion virus vSPF also displayed reduced pathogenicity. Naive nude mice receiving 108 pfu of vSPF i.p. lived more than 100 days without illness, whereas those receiving WT virus had median survivals of 7 days and died of viral pathogenicity. The efficiency for tumor regression is currently under study.