804. Expression of Green Fluorescent Protein Modifies the Antitumor Effect of Oncolytic HSV-1 Vectors

Abstract

Replication-competent, oncolytic herpes simplex virus type 1 (HSV-1) vectors are useful tools for cancer gene therapy. The virus genome is genetically engineered so that the vector selectively replicates in and destroys tumor cells while sparing the surrounding normal tissues. A marker gene is frequently used in order for replicating virus vectors to be easily detected. Green Fluorescent Protein (GFP) is now a popular marker, because it can be detected in living cells and also be used for selection. MGH-1 is a double-mutated HSV-1 with deletions in both copies of the γ34.5 gene and an insertion of the LacZ gene down-stream of the ICP6 promoter inactivating the ICP6 gene. Using this MGH-1 as a backbone and a bacterial artificial chromosome technology, we newly created multiple types of “armed” oncolytic HSV-1 vectors (vHSV series) in which the ICP6 and LacZ genes were replaced with the ICP6 promoter-driven GFP gene and a transgene cassette driven by the HSV IE4/5 promoter. Prior to testing “armed” oncolytic vectors, we compared the antitumor activity of vHSV-null, the control oncolytic vector expressing only the GFP marker, with that of MGH-1. The replication capability of vHSV-null in cultured Vero cells at a MOI of 0.01 was approximately 10 times lower than that of MGH-1. Despite decreased replication capability, vHSV-null showed slightly faster cytopathic effect than MGH-1 in Neuro2a murine neuroblastoma cells at MOI=0.1. A/J mice bearing bilateral subcutaneous Neuro2a tumors were used for in vivo evaluations. When vectors were inoculated directly into the left tumor at 2×106 plaque-forming units (days 0 and 3), vHSV-null was significantly more effective than MGH-1 for inhibiting the growth of inoculated tumors (p < 0.05). Contralateral non-inoculated tumors also tended to be smaller in vHSV-null-treated animals than MGH-1-treated animals although not statistically significant. Such difference in tumor growth inhibition was not observed in athymic mice, indicating that the greater antitumor activity of vHSV-null in vivo compared with MGH-1 is associated with T cell-mediated immune responses most likely due to expression of GFP. While this possible immunostimulatory effect by GFP may be beneficial to oncolytic virus therapy, a caution is needed to evaluate the antitumor activity of oncolytic HSV-1 vectors when the GFP gene is used as a marker for virus replication.