1. ¹Department of Cardiology & Pneumonology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany
2. ²Department of Dermatology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany

Correspondence: Henry Fechner, Fax: +49 (30) 8445 4582. E-mail: Henry.Fechner@medizin.fu-berlin.de

^*These authors have equally contributed to this work.

^†Current address: Center for Immunopathogenesis and Infectious Diseases, Mailman School of Public Health, Columbia University, New York, NY 10027, USA.

Received 14 June 2004; Accepted 26 October 2004.

Abstract

Restricted replication-competent adenoviruses (RRCAs) using tumor- and tissue-specific promoters (ttsP's) are new tools for cancer gene therapy. In this study we investigated viral and nonviral factors affecting "leakiness" of several ttsP's and their relevance for nonspecific ttsP-dependent RRCA (ttsP-RRCA) replication. The leakiness of the ttsP's in nontarget cells was per se highly variable and correlated with levels of nonspecific ttsP-RRCA replication. Transcriptional regulator elements fused to ttsP's showed variable effects: a hypoxic response element reduced leakiness of an -fetoprotein promoter. In contrast, a mouse tyrosinase enhancer increased leakiness of a tyrosinase promoter, although it was not affected by a human tyrosinase enhancer. Furthermore, leakiness of ttsP's was enhanced by 5'-terminal adenoviral E1A enhancers, and adenoviral E1A-13S was found to be a strong transactivator of ttsP's, leading to "autoactivation" of leaky ttsP-RRCAs. In a proof-of-principle study, ttsP-RRCA replication was shown to be inhibited by a tetracycline-controlled transcriptional silencer via direct ttsP silencing. This opens up the prospect of pharmacological regulation of ttsP-RRCAs. Together, these data indicate that leakiness of ttsP's induced by several factors is a major cause of nonspecific ttsP-RRCA replication. Consideration of these factors may help optimize ttsP-dependent RRCA vectors and may thereby improve their safety.