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A new strategy to package "problematic" transgenes in adenovirus was developed that was based on modifications of the tetracycline-inducible system. This strategy used two components: the adenoviral genome containing the transgene under control of a hybrid TRE promoter/SV40 enhancer and a trans-encoded tTS suppressor Using luciferase reporters, expression of tTS in 293A cells reduced transcription from the promoter/enhancer 25-fold. Procaspase 8 adenovirus was then tested, since it is known to package poorly with standard adenoviral systems. Expression of tTS in 293A cells increased the titer of procaspase 8 adenovirus by 22-fold in initial viral packaging (using transiently transfected tTS) and 9-fold in subsequent viral reamplification (using 293A cells stably expressing tTS). The Tac antigen gene (i.e., CD25), which packages in adenovirus without difficulty, was also tested as a control. In contrast to that observed with procaspase 8, tTS expression did not alter the titer obtained when packaging the CD25 gene, thus excluding nonspecific effects of tTS expression on adenoviral titer Since tTS was provided in trans and did not package in the resulting adenoviruses, strong transcription of the transgenes occurred in transducted cells without the need of additional reagents.

Improving the titer of recombinant adenovirus by suppressing problematic transgene transcription during packaging