Analyses of melanoma-targeted oncolytic adenoviruses with tyrosinase enhancer/promoter-driven E1A, E4, or both in submerged cells and organotypic cultures.

We have generated novel conditionally replicative adenoviruses (CRAds) targeted to melanoma cells. In these adenoviruses, the E4 region (AdDelta24TyrE4) or both E1 and E4 regions (Ad2xTyr) were controlled by a synthetic tyrosinase enhancer/promoter (Tyr2E/P) specific for melanocytes. The properties of these CRAds were compared with wild-type adenovirus (Adwt) and our previous CRAd with a targeted E1A CRII mutation (AdTyrDelta24) in submerged cultures of melanoma cells and nonmelanoma control cells. We showed that AdDelta24TyrE4 had a cell type selectivity similar to AdTyrDelta24 but had a distinct block in viral reproduction in nonmelanoma cells and that Ad2xTyr had an augmented selectivity for melanoma cells. These viruses were additionally tested in organotypic cultures of melanoma cell lines, primary human keratinocytes (PHKs), or mixed cell populations. Unexpectedly, the CRAds exhibited somewhat different cell type selectivity profiles in these cultures relative to those observed in submerged cultures, demonstrating the importance of multiple assay systems. Specifically, AdTyrDelta24 and Ad2xTyr were selective for melanoma cells, whereas AdDelta24TyrE4 exhibited no selectivity, similar to Adwt. AdTyrDelta24 and Ad2xTyr were strongly attenuated in their ability to lyse PHKs in organotypic cultures. Furthermore, Ad2xTyr had a superior melanoma selectivity in organotypic cultures of cocultivated melanoma cells and PHKs. The enhanced selectivity for melanoma cells exhibited by Ad2xTyr provides a window of opportunity for therapeutic application. These studies also demonstrate that organotypic cultures derived from mixtures of tumor and normal cells represent a promising new model for analysis of CRAd specificity and toxicity.