6-hydroxy-nicotine-inducible multilevel transgene control in mammalian cells

Abstract

The precise control of transgene expression is essential for biopharmaceutical manufacturing, gene therapy and tissue engineering. We have designed a novel conditional transcription technology, which enables reversible induction, repression and adjustment of desired transgene expression using the clinically inert 6-hydroxy-nicotine (6HNic). The 6-hydroxy-nicotine oxidase (6HNO) repressor (HdnoR), which manages nicotine metabolism in Arthrobacter nicotinovorans pAO1 by binding to a specific operator of the 6-hydroxy-nicotine oxidase (O(NIC)), was fused to the Krueppel-associated box protein of the human kox-1 gene (KRAB) to create a synthetic 6HNic-dependent transsilencer (NS) that controls chimeric mammalian promoters, which are assembled by cloning tandem O(NIC) operators 3' of a constitutive promoter. In the absence of 6HNic, NS binds to O(NIC) and silences the constitutive promoter, which otherwise drives high-level transgene expression when the NS-O(NIC) interaction stops in the presence of 6HNic. Generic NICE(ON) technology was compatible with a variety of constitutive viral and mammalian housekeeping promoters, each of which enabled specific induced, repressed, adjusted and reversible transgene expression profiles in Chinese hamster ovary (CHO-K1), baby hamster kidney (BHK-21) as well as in human fibrosarcoma (HT-1080) cells. NICE(ON) also proved successful in controlling multicistronic expression units for coordinated transcription of up to three transgenes and in the fine-tuning of transcription-translation networks, in which RNA polymerase II- and III-dependent promoters, engineered for 6HNic responsiveness, drove expression of siRNAs that triggered specific transgene knockdown. NICE(ON) represents a robust and versatile technology for the precise tuning of transgene expression in mammalian cells.