Human papillomavirus E6E7-mediated adenovirus cell killing: Selectivity ofmutant adenovirus replication in organotypic cultures of human keratinocytes

Replication-competent adenoviruses are being investigated as potential anti cancer agents. Exclusive virus replication in cancer cells has been propose d as a safety trait to be considered in the design of oncolytic adenoviruse s. From this perspective, we have investigated several adenovirus mutants f or their potential to conditionally replicate and promote the killing of ce lls expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we h ave employed an organotypic model of human stratified squamous epithelium d erived from primary keratinocytes that have been engineered to express HPV- 18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B -de leted adenoviruses cause no deleterious effect regardless of the coexpressi on of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratino cytes. Finally, an adenovirus mutant deleted in the CRI and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressi ng cultures. We conclude that the organotypic keratinocyte culture represen ts a distinct model to evaluate adenovirus selectivity and that, based on t his model, further modifications of the adenovirus genome are required to r estrict adenovirus replication to tumor cells.