157. A Mutant Type 2 Herpes Simplex Virus Deleted for the Protein Kinase Domain of the ICP10 Gene Is a Potent Oncolytic Virus
Authors: Xinping Fu, Lihua Tao, Xiaoliu Zhang
Journal: Molecular Therapy (2006)

Introduction: HSV-2 has several unique features that could be exploited to convert the virus to an oncolytic agent. The N-terminus of the HSV-2 ICP10 gene product contains a protein kinase (PK) domain, which can bind and phosphorylate the GTPase activating protein Ras-GAP, leading to activate the Ras/MEK/MAPK mitogenic pathway and thus facilitates efficient HSV-2 replication. Because Ras signaling system is aberrantly activated or over- expressed in many human tumors, and is inactivated in normal cells, we developed a Ras passway dependent oncolytic HSV-2 mutant (FusOn-H2) by deleting PK domain, which can selectively replicate in and lyse in the Ras activated or over-expressed tumor cells and be restricted in the Ras inactivated normal cells. Methods: We designed the mutant HSV-2 (FusOn-H2) by deleting the whole PK domain of ICP10 in HSV-2 (wt186) with replacement of a gene encoding the green fluorescent protein. The phenotype and replication capability of FusOn-H2 was in vitro characterized. Also its effectiveness of eradicating solid breast tumor was evaluated in a mouse model. Results: By Southern blot assay, we confirm that the modified ICP10 gene with PK domain deletion and GFP gene replacement in Fuson-H2 was correctly inserted into the viral genome, replacing the original wt ICP10 gene during the homologous recombination. Western blot assay also confirmed that the fusion gene consisting of EGFP and the remaining RR domain of ICP10 was correctly expressed, and produced an expected matching size of the chimeric protein of GFP and the RR domain. In vitro characterization study, compared to its parental wt186 virus or Baco-1, an oncolytic HSV- 1, we showed the highest the replication or killing abilities of FusOn- H2 in the human breast cancer cell line MDA-MB-435 or human ovarian cancer cell line SKOV-3, both having constitutively activated Ras signaling pathway due to elevated levels of Ras or PI3K expression. By using PD98059, a Ras signaling passway inhibitor, Fuson-H2 showed a significantly reduced replication and growth ability than wt186 did. We also compared the growth properties of FusOn-H2 and wt186 or Baco-1 in primary hepatocytes. By 24 and 48 h after infection, FusOn-H2 had totally disappeared from the hepatocytes, showing its safety in normal cells as hepatocytes; while the titers of both wt186 and Baco-1 had increased by 2 or 3 logs. To evaluate the antitumor effect of FusOn-H2 in vivo, we injected the virus (1x106 pfu) directly into orthotopically established human breast cancer xenografts. From 2-4 weeks after virus injection, FusOn-H2 produced significantly greater antitumor effects compared to Baco-1. All of the animals (8 of 8) became tumor free by week 2 after FusOn-H2 administration, while only 2 mice in the group injected with Baco-1 were tumor free. Conclusion: From the above results, we conclude that our first developed oncolytic virus-Fuson- H2 constructed from HSV-2 can be a safe and potent oncolytic agent useful for the treatment of malignant solid tumors such as breast cancer.