443. Incorporating the Survivin Promoter in an Infectivity Enhanced CRAd-Analysis of Oncolysis in Five Different Cancer Cell Lines

Abstract

Replication-competent adenoviruses (Ad) are a promising new modality for the treatment of cancer. Selective replication of viral agents in tumor may lead to improved efficacy over non-replicating adenoviruses due to their inherent ability to multiply, lyse and spread to surrounding cells. We have previously shown that the E1B 55kDa-deleted adenovirus (YKL-1) exhibits tumor specific replication and cell lysis, but its cytolytic effects were reduced in comparison to the wild type adenovirus. To increase the oncolytic potency of YKL-1, we have reintroduced the adenovirus death protein (ADP) gene under the control of either the CMV or the MLP promoter at the E3 region of YKL-1, generating YKL-cADP and YKL-mADP adenovirus, respectively. ADP is an 11.6kDa protein encoded by the E3 transcription unit, and is required to efficiently kill adenovirus-infected cells. However to date, the mechanism by which ADP mediates cell death has not been clearly defined. In this study, we report that ADP-overexpressing Ad markedly enhanced cytolytic effect (up to 100-fold) against all tumor cell lines tested, but did not increase CPE in normal skin fibroblast, BJ. Moreover, plaque size formed by YKL-cADP was substantially larger than that of YKL-1, indicating enhancement in cell lysis. TUNEL assay and Annexin-V/PI double staining indicate that ADP-mediated cytotoxicity was largely driven by apoptosis. Finally, YKL-cADP adenovirus also showed superior antitumor effect than YKL-1 and YKL-mADP in C33A cervical and Hep3B hepatoma xenograft tumor models. Taken together, these lines of evidence demonstrate that the newly generated adenovirus expressing ADP under the CMV promoter induces efficient but tumor selective cell lysis, which is critical for adding therapeutic value to replicating adenovirus for cancer gene therapy.