539. Antitumor Activity of an hTERT-Specific, GFP Expressing Oncolytic Adenovirus

Abstract

Oncolytic viruses are designed to selectively replicate and lyse tumor cells by using mutations that restrict the lytic viral cycle to tumorigenic tissue or by using tumor-specific promoters to control essential early viral genes. However, various preclinical and clinical studies with oncolytics suggest that, though tumor specificity and replication are achieved, viral replication alone may not be sufficient to inhibit tumor growth. Also, several reports have indicated that oncolytics expressing additional therapeutic genes may also improve anti-tumor effects. Here we describe the construction and functional characterization of a green fluorescence protein (GFP)-expressing oncolytic adenovirus designated Ad/hTC-GFP-E1, whose transgene, GFP, and viral E1A gene are both under the control of a synthetic promoter (hTC) consisting of fusion sequences from the human telomorase reverse transcriptase (hTERT) promoter and minimal cytomegalovirus early promoter (CMV). hTC is used twice to express viral genes and transgenes separately, at high levels and in a tumor-specific manner, providing tumor restricted viral replication and additional effects mediated by the transgene of choice. Our virus' tumor specificity has been demonstrated by Western blot, fluorescence-activated cell sorting analysis, crystal violet staining, and cell viability assays. In addition, our virus has been shown to replicate and cause the eventual lysis of sensitive cell lines at multiplicities of infection (MOIs) as low as 10 viral particles per cell. In construction, no obvious cytotoxic effect was observed in normal human fibroblasts even at MOIs over 2000. The oncolytic vector, with the inclusion of an hTC-driven fluorescent reporter, GFP, can also be readily visualized in vitro and in vivo, in correlation with viral replication. Our results show that construction of an hTERT-specific, transgene-expressing oncolytic adenovirus is practical and useful for cancer therapy.