Bile acid-controlled transgene expression in mammalian cells and mice

Abstract

In recent years, using trigger-inducible mammalian gene switches to design sophisticated transcription-control networks has become standard practice in synthetic biology. These switches provide unprecedented precision, complexity and reliability when programming novel mammalian cell functions. Metabolite-responsive repressors of human-pathogenic bacteria are particularly attractive for use in these orthogonal synthetic mammalian gene switches because the trigger compound sensitivity often matches the human physiological range. We have designed both a bile acid-repressible (BEAROFF) as well as a bile-acid-inducible (BEARON) gene switch by capitalizing on components that have evolved to manage bile acid resistance in Campylobacter jejuni, the leading causative agent of human food-borne enteritis. We have shown that both of these switches enable bile acid-adjustable transgene expression in different mammalian cell lines as well as in mice. For the BEAROFF device, the C. jejuni repressor CmeR was fused to the VP16 transactivation domain to create a synthetic transactivator that activates minimal promoters containing tandem operator modules (Ocme) in a bile acid-repressible manner. Fusion of CmeR to a transsilencing domain resulted in an artificial transsilencer that binds and represses a constitutive Ocme-containing promoter until it is released by addition of bile acid (BEARON). A tailored multi-step tuning program for the inducible gene switch, which included the optimization of individual component performance, control of their relative abundances, the choice of the cell line and trigger compound, resulted in a BEARON device with significantly improved bile acid-responsive control characteristics. Synthetic metabolite-triggered gene switches that are able to interface with host metabolism may foster advances in future gene and cell-based therapies.