Valeria Gonzalez-Nicolini1, Martin Fussenegger. 1. Institute for Chemical and Bio-Engineering, Swiss Federal Institute of Technology, ETH Hoenggerberg, CH-8093 Zurich, Switzerland.
Abstract
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.
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 (humanfibrosarcoma 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.
Authors: Marija Vujadinovic; Kerstin Wunderlich; Benoit Callendret; Marina Koning; Mark Vermeulen; Barbara Sanders; Esmeralda van der Helm; Adile Gecgel; Dirk Spek; Karin de Boer; Masha Stalknecht; Jan Serroyen; Maria Grazia Pau; Hanneke Schuitemaker; Roland Zahn; Jerome Custers; Jort Vellinga Journal: Hum Gene Ther Date: 2017-11-30 Impact factor: 5.695