1020. Noninvasive Optical Monitoring of Conditionally Replivative Adenovirus Replication

Abstract

The employment of conditionally replicative adenoviruses (CRAds) offers a promising alternative for cancer treatment. To maintain safety in clinical trials, a noninvasive detection system of viral replication is required. Our previous studies revealed the effective and transcriptionally-restricted cytocidal effect of infectivity-enhanced cyclooxygenase-2 (Cox-2) promoter-driven CRAds. However, there are no currently available methods to noninvasively monitor dynamic adenovirus replication, spreading ability, and extent of persistence in vivo. In this study, we have developed a system which allows monitoring of CRAds replication via the detection of fluorescence or luminescence reporters expressed from the E3 region. We deleted or mutated most E3 genes except adenovirus death protein (ADP), and incorporated the enhanced green fluorescent protein (EGFP) or the firefly luciferase (Luc) into the E3 region under the control of the adenovirus major late promoter (MLP). Since MLP turns on only when the virus is replicating, we hypothesized that the transgene expression would closely represent the viral replication in this configuration. Finally, we combined E1 shuttle vector with E1 expression cassette under control of Cox-2 promoter with E3ADPLuc cassette and generated imaging capable Cox-2 CRAds with different fiber modifications in order to overcome coxsackie-adenovirus receptor deficiency of cancer cells. In addition, we considered the structure of the E3 region with and without ADP since a strong cytocidal effect of ADP may not be optimal for imaging. To assess the replication dependent expression of the reporter gene from the E3 region, a replication permissive human lung adenocarcinoma A549 cells and mouse hepatoma BNL-1NG-A.2 cells, in which human adenoviruses do not productively replicate, were applied in our experiments. In vitro, all replication-competent reporter viruses showed transgene expression augmentation over time and in a dose-dependent manner during infection of A549 cells. The detected signal closely correlated with the viral DNA quantity resulting from viral replication. On the other hand, no EGFP/Luc expression was observed in BNL-1NG-A.2 cells, thus indicating the dependence of fluorescence/luminescent signal on productive replication. In vivo optical studies performed with a custom made in vivo imaging system revealed that after intratumoral administration of adenoviruses with the E3 imaging cassette the EGFP expression could be detected two days post-injection. Cox-2 CRAds with the luciferase expression showed the signal on day one and maintained it over four weeks. Importantly, in vivo reporter detection strongly correlated with the underlying level of replication. These data validate the applicability of our E3 reporter system for noninvasive in vivo monitoring of CRAd replication.