Autoregulated, bidirectional and multicistronic gas-inducible mammalian as well as lentiviral expression vectors

Abstract

We present a novel set of autoregulated, bidirectional and multicistronic mammalian as well as lentiviral expression vectors which enable transgene expression fine-tuning by gaseous acetaldehyde. The acetaldehyde-inducible regulation (AIR) technology capitalizes on Aspergillus nidulans components evolved to convert ethanol into metabolic energy. AIR is based on functional interaction of the fungal transactivator AlcR and AlcR-specific chimeric promoters (P AIR) which drive desired transgene expression in mammalian cells only in the presence of gaseous acetaldehyde. We have engineered AIR technology into a variety of different mammalian and lentiviral expression vector systems including (i) a most compact autoregulated expression format harboring alcR and the transgene in a single P AIR-driven transcription unit, (ii) a bidirectional P AIR derivative supporting expression of two transgenes with strict 1:1 transcription stoichiometry and (iii) a multicistronic expression arrangement providing simultaneous translation of three independent transgenes from a single P AIR-controlled transcript. All expression vectors have been validated in Chinese hamster ovary (CHO-K1), baby hamster kidney (BHK-21) and human HeLa cells for gas-inducible (co-)expression of the reporter transgenes such as Bacillus stearothermophilus-derived secreted α-amylase (SAMY), human vascular endothelial growth factor 121 (VEGF 121), human placental-secreted alkaline phosphatase (SEAP) and Escherichia coli-derived chloramphenicol acetyl-transferase (CAT). The panoply of mammalian/lentiviral vectors presented here provides a robust and versatile expression platform for the first gas-inducible transgene control system which we expect to foster future advances in gene therapy, tissue engineering as well as biopharmaceutical manufacturing.