Potential and Optimization of Mammalian Splice Riboswitches for the Regulation of Exon Skipping-Dependent Gene Expression and Isoform Switching within the ALOX5 Gene

Synthetic riboswitches are attracting increasing interest for a diverse range of applications, including synthetic biology, functional genomics, and prospective therapeutic strategies. This study demonstrates that controlling alternative splicing with synthetic riboswitches represents a promising approach to effectively regulating transgene expression in mammalian cells. However, the function of synthetic riboswitches in the eukaryotic system in controlling gene expression is often limited to certain genes or cell types. So far, strategies to increase the dynamic range of regulation have been focused on adapting and modifying the riboswitch sequence itself without taking into account the context in which the riboswitch was inserted. In the present study, the tetracycline riboswitch was chosen to investigate the effects of the context and insertion site of a cassette exon within the gene to control the expression of an artificial arachidonate 5-lipoxygenase gene (ALOX5) in HEK293 cells. We demonstrate here that the use of riboswitch-controlled cassette exons for the control of gene expression via alternative splicing can be easily transferred to another gene through the process of contextual sequence adaptation. This was achieved through the introduction of gene-specific intronic and exonic sequences with different intron lengths and positions being tested. In contrast, the introduction of nonadapted constructs resulted in an unanticipated functionality outcome of the gene switch. Furthermore, we demonstrate that the combination of two cassette exons into a single gene resulted in a notable enhancement in the dynamic range. Finally, we generated a novel riboswitch-controlled splicing concept that enabled us to switch 5-LO wild-type to expression of an ALOX5 isoform that lacks exon 13 (5-LO Delta 13). Taken together, this study demonstrates that synthetic riboswitches that control alternative splicing are a powerful tool to regulate gene expression when applied in combination with gene-specific intronic and exonic sequences.