Virus-Associated RNA I–Deleted Adenovirus, a Potential Oncolytic Agent Targeting EBV-Associated Tumors Yaohe Wang, 1 Shao-An Xue, 2 Gunnel Hallden, 1 Jennelle Francis, 1 Ming Yuan, 1 Beverly E. Griffin, 2,3 and Nick R. Lemoine 1 1 Cancer Research UK Molecular Oncology Unit, Institute of Cancer, Barts and the London School of Medicine and Dentistry, Queen Mary University of London; 2 Division of Medicine, Hammersmith Hospital, Imperial College London; and 3 Imperial College London at St. Mary’s, London, United Kingdom Abstract Given the growing number of tumor types recognizably associated with EBV infection, it is critically important that therapeutic strategies are developed to treat such tumors. Replication-selective oncolytic adenoviruses represent a promising new platform for anticancer therapy. Virus- associated I (VAI) RNAs of adenoviruses are required for efficient translation of viral mRNAs. When the VAI gene is deleted, adenovirus replication is impeded in most cells (including HEK 293 cells). EBV-encoded small RNA1 is uniformly expressed in most EBV-associated human tumors and can functionally substitute for the VAI RNAs of adenovirus. It enables replication to proceed through complementation of VAI-deletion mutants. We hypothesized that VAI-deleted adenovirus would selectively replicate in EBV-positive tumor cells due to the presence of EBV-encoded small RNA1 with no (or poor) replication in normal or EBV- negative tumor cells. In this report, we show that high levels of replication occurred in the VAI-deleted mutant in the EBV- positive tumor cells compared with low (or negligible) levels in EBV-negative and normal human primary cells. Corre- spondingly, high toxicity levels were observed in EBV-positive tumor cells but not in EBV-negative tumor or normal human primary cells. In vivo , VAI-deleted adenovirus showed superior antitumoral efficacy to wild-type adenovirus in EBV-positive tumor xenografts, with lower hepatotoxicity than wild-type adenovirus. Our data suggest that VAI-deleted adenovirus is a promising replication-selective oncolytic virus with targeting specificity for EBV-associated tumors. (Cancer Res 2005; 65(4): 1523-31) Introduction EBV, a ubiquitous human herpesvirus found latently expressed in 90% of the human population, is the causative agent of infectious mononucleosis and posttransplant immunoprolifera- tive disorders in immunocompromised patients. It is closely associated with the development of a variety of malignant diseases, including endemic Burkitt’s lymphoma, B-cell lympho- ma of immunocompromised patients, nasopharyngeal carcinoma, Hodgkin’s disease, T-cell lymphoma, natural killer cell leukemia/ lymphoma, smooth-muscle tumors, and gastric cancer (1, 2). More recent reports have linked EBV with conventional epithelial cancers of other sites, including breast (3–5), lung (6–9), prostate (7), liver (10), colon (7), and also with lymphoepithelioma-like carcinoma of the esophagus (11). Given the ever-growing number of tumor types associated with EBV infection, thera- peutic strategies to treat EBV-associated tumors may be considered a high priority in oncology. Replication-selective, oncolytic viruses provide a new platform to treat cancer. Promising clinical trial data with mutant adenoviruses have shown both their antitumor potency and safety (12, 13). Two main approaches are currently being used to engineer adenoviruses with tumor-selective replication. The first strategy limits the expression of the E1A gene to tumor tissues through the use of tumor- and/or tissue-specific promoters; consequently, E1A-induced S phase entry and activation of viral and cellular genes will only occur in tumor tissues. A second strategy optimizes tumor selectivity by deletion of viral genes that are critical for efficient replication in normal cells but expendable in tumor cells (14). The most commonly described oncolytic viruses explored over the past 10 years have been mutant adenoviruses where at least 25 different ones have been described for use in cancer treatments to date (15). Several studies have shown similarities in the function of adenovirus and EBV genes (16–20). Based on the similarities described, we believe that a window of opportunity exists in the design of replication-selective adenovirus for EBV-associated tumors. The adenovirus genome encodes two RNA polymerase III-directed, f160-nucleotide-long RNAs, the so-called virus- associated RNA I and RNA II. These accumulate to high levels during the late stages of viral infection (21–23). VAI RNA is obligatory for efficient translation of viral and cellular mRNAs (24, 25). It binds to and blocks the activation of cellular double- stranded RNA-dependent serine/threonine protein kinase (PKR), which phosphorylates protein translation initiation factor eIF-2a, enabling protein synthesis to proceed at normal levels (26–29). A mutant that fails to produce VAI RNA (dl 331) grows more poorly than its parent in human 293 cells (25). EBV also expresses two low-molecular-weight RNAs, designat- ed EBV-encoded RNAs (EBER) 1 and 2, transcribed by RNA polymerase III (30). Although there is no striking nucleotide sequence homology between virus-associated RNAs and EBERs, the RNAs are similar in size, genomic organization, and degree of secondary structure; they have similar functions (19, 20, 31). In particular, the two small RNAs encoded by EBV can efficiently complement the VAI RNA–mediated translational defect in adenovirus-infected cells, as shown by constructing an Ad5 substitution mutant in which the two virus-associated RNA genes have been deleted and replaced by an EBV DNA segment encoding the two EBERs (32, 33). Given the uniform expression Requests for reprints: Nick R. Lemoine, Cancer Research UK Molecular Oncology Unit, Institute of Cancer, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, John Vane Science Building, Charterhouse Square, London EC1 6BQ, United Kingdom. Phone: 44-20-7014-0420; E-mail: nick.lemoine@cancer.org.uk. I2005 American Association for Cancer Research. www.aacrjournals.org 1523 Cancer Res 2005; 65: (4). February 15, 2005 Research Article Research. on November 7, 2015. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from