A safe oncolytic virus, suitable as a therapeutic agent, must target cancer cells for destruction but not propagate in normal tissues. We have recently reported the first use of organotypic "raft" cultures of primary human keratinocytes, which form fully stratified and differentiated epithelia, to test the properties of a conditional replication-competent adenovirus (CRAD) CB016 targeted to tissues expressing the human papillomavirus (HPV) oncogenes E6 and E7 (Balague et al., 2001). Mucosotropic HPVs cause a spectrum of hyper-proliferative ano-genital and oral lesions, including condylomata, papillomas, dysplasias, and carcinomas of cervical, penile, anal, and tonsillar epithelia with no effective treatment or vaccine. HPV E7 protein shares considerable functional homology with the adenovirus E1A proteins, in inactivating the host tumor suppressor protein pRB and related p107 and p130. This functional homology forms the conceptual basis for the development and investigation of Ad5 CB016. This virus is deleted of conserved regions CR1 and CR2 of E1A. It replicates and is cytolytic in raft cultures that express the HPV-18 oncogenes, but not in control raft cultures. However, in carefully conducted time course experiments, we further demonstrated that productive infection of CB016 was considerably delayed, but not eliminated, in normal raft cultures. Our long-term goal is to build upon these initial findings and design new safer CRAD having minimal cytopathic effects in normal raft cultures, but oncolytic in HPV oncogene-expressing raft cultures that simulate benign papillomas, dysplasias and cancers. Emphasis will be placed on the deletion of 19 kDa Ad E4-E6/7 protein, which complements E1A mutations by forming transcriptionally active complexes with the E2F/DP1 family of transcription factors, and on additional domains of the E1A protein, which also activate other early adenovirus promoters. We also intend to investigate the incorporation of Ad E1B deletions as an additional safety feature for normal tissues.