ATPS-98MONITORING ONCOLYTIC HSV-1 WITH NON-INVASIVE BIOLUMINESCENCE

Abstract

Oncolytic HSV-1 (hereinafter referred to as oHSV1) is one of the promising bioagents for cancer virotherapy. However, variability exists in oHSV1 mediated cytotoxicity and replication towards panels of patient-derived and mouse glioblastoma multiforme (GBM) cells. We hypothesized that cell-specific differences may exist in oHSV1 infection/replication kinetics accounting for the observed variability. To study this, we have engineered a novel oHSV1 that expresses different luciferase cDNAs for bioluminescence imaging (BLI) in vitro and in vivo. This new oHSV1 (NG34-duLuc) expresses both firefly luciferase (Fluc) and Renilla luciferase (Rluc) genes under the viral late gene and immediate early gene promoters, respectively, to monitor different phases of the viral replicative cycle. NG34-duLuc vector DNA was generated in E.coli with the fHsvQuik system (Gene Ther. 2006;13(8):705-14), based on the NG34 virus backbone (H Nakashima, EA Chiocca et al., in preparation). We confirmed that cells infected by isolated viral clones expressed both luciferases. We further validated the bioluminescence of each luciferase upon the infection in vitro using U251, U87 human glioma cells and CT2A mouse glioma cells. In vivo BLI after NG34-duLuc treatment was also performed using subcutaneous or intracranial U87 xenograft nude mice and CT2A syngeneic CL57/BL6 mice. Overall, our findings confirm that the duplicate bioluminescence imaging can monitor the viral late gene and immediate early gene promoters respectively by Fluc and Rluc.