A novel binary adenovirus-based dual-regulated expression system for independent transcription control of two different transgenes.
BACKGROUND
Stringent multitransgene control is a prerequisite for future gene-therapy and tissue-engineering scenarios and requires constant improvements in design to achieve optimal conditional transcription profiles.


METHODS
We have pioneered a variety of recombinant adenoviruses which (i) enable streptogramin-responsive transgene transduction in a compact autoregulated one-virus format, (ii) manage independent streptogramin- and tetracycline-responsive control of two different transgenes from a single divergent expression unit, and (iii) control sense and antisense expression of the human cyclin-dependent kinase inhibitor p27(Kip1) to engineer conditional positive (enforced S-phase entry, p27(Kip1)-antisense expression) or negative (G1-phase-specific growth arrest, p27(Kip1)-sense expression) growth control in mammalian cell lines and human primary cells.


RESULTS
The transgene control performance of all adenoviral expression configurations has been rigorously optimized for tight, balanced and maximum expression levels and was validated for intracellular as well as for secreted product in a variety of biotechnologically relevant cell lines (Chinese hamster ovary cells [CHO-K1], baby hamster kidney cells [BHK-21]) as well as in human cell lines (human fibrosarcoma cells [HT-1080]) and primary cells (human aortic fibroblasts [HAFs]).


CONCLUSIONS
We believe that multiregulated multigene-controlled adenoviruses are important assets for successful therapeutic reprogramming of mammalian cells in clinically relevant scenarios.
Gonzalez-Nicolini
Valeria
V
Institute for Chemical and Bio-Engineering, Swiss Federal Institute of Technology, ETH Hoenggerberg, CH-8093 Zurich, Switzerland.
Fussenegger
Martin
M
D000256:Adenoviridae; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D025361:Streptogramins; D013752:Tetracycline; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000235:genetics
D025361:Streptogramins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013752:Tetracycline