844. A Single Vector-Dependent, Doxycycline- Regulatable, Lung Cancer Specific Oncolytic Adenovirus

Tight regulation of oncolytic adenoviruses (oAdV) represents an important requirement for their safe application. We have developed a new doxycycline (Dox)-dependent oAdV with a bidirectional expression cassette, which drives the expression of the reverse tetracycline-controlled transactivator (rtTAs-M2) from a lung tumor-specific promoter and, in the opposite direction, the expression of the adenoviral E1A gene from a second generation tetO7 sequence linked to an isolated TATA box. In H441 lung cancer cells, this oAdV showed a strictly Dox-dependent E1A expression, adenoviral replication, cell killing activity and a 450-fold induction of progeny virus production. Furthermore, the virus could be shut off again by withdrawal of Dox and, in contrast to an oAdV expressing E1A directly from the SP-B promoter, did not replicate in non-target HeLa cells. In contrast, when the isolated TATA box in the E1A expression cassette was replaced by a CMV minimal promoter, the corresponding oAdV already expressed significant amounts of E1A in the absence of the inducer. These were sufficient to prevent any Dox-dependent regulation of adenoviral replication. These results underline the importance of non-leaky E1A expression for preventing undesirable oAdV replication and demonstrate, for the first time, Dox-dependent oAdV replication from a single adenoviral vector genome.