Designing riboswitch-based gene control platforms for mammalian cells

Abstract

The capacity to precisely control the timing, duration, and levels of gene expression is critical for better understanding and manipulating complex biological systems. Therefore, synthetic gene expression control devices are valuable in basic and applied research areas, including gene therapy, cell therapy, tissue engineering, drug discovery, and functional genomics. In this regard, artificial riboswitches have emerged as modular tools with advantages such as small size and low risk of immunogenicity. Riboswitch-based gene expression control systems incor-porate allosterically addressable RNA structures that can bind small molecules, namely apta-mers, into post-transcriptional control mechanisms to regulate gene circuit behavior, namely the expression platforms. However, while there are naturally occurring examples in bacteria and de novo applications in the cell-free context, riboswitch-enabled gene expression control applications for eukaryotic systems have been limited. In the first part of the thesis, a novel expression platform for the tetracycline-responsive apta-mer was developed to regulate gene expression at the translational level. The developed plat-form is based on -1-programmed ribosomal frameshifting, a mechanism that is essential for the post-transcriptional regulation of gene expression in several organisms. By placing the apta-mer as a stimulatory structure on the -1 PRF platform, a switch has been constructed that enables robust induction of gene expression upon tetracycline addition. By varying the precise spacing and composition of the tetracycline aptamer and adapting -1 PRF modules from se-veral viruses, the characteristics and optimal frameshift layout for the tetracycline-inducible platform were characterized. Furthermore, dose-response analysis showed that the tetracyc-line-inducible -1-PRF platform has improved sensitivity at concentrations that fall within the clinically approved window. Next, a compact and modular tetracycline-inducible -1-PRF plat-form was developed by integrating accessory elements. Finally, the efficacy of the platform in various genetic contexts was confirmed by testing in different transcription units and cell lines and by controlling the production of therapeutically relevant genes. In the second part of the thesis, we aimed to expand the potential applications of mammalian exon-skipping riboswitches. Cis-regulatory elements on the mRNA are shown to be integral for refining outcomes of alternative splicing. By incorporating splicing-modulating G-quadruplexes into the splicing cassette, we have exploited these elements to fine-tune alternative splicing outcomes in exon-skipping riboswitches. Our work led to the improvement of the dynamic ran-ge in a number of different tetracycline-inducible exon-skipping riboswitches. Furthermore, we devised a method to convert the splicing-based ON switch to an OFF switch by integrating inhibitory competitor RNA structures into the splicing cassette, thereby enhancing the versatili-ty of the splicing-based switches. Lastly, we altered the splicing cassette to be readily incorpo-rated into the major late transcription unit of oncolytic adenoviruses. In our approach, an intron from the splicing cassette was first modified to be controlled by the essential splicing compo-nents of viral origin. As a next step, a series of tetracycline riboswitches were designed to mo-dulate gene expression by controlling the accessibility of these viral splicing elements. Collec-tively, the three design strategies discussed in this section have improved riboswitch perfor-mance and effectively exploited the modular "plug-and-play" features of riboswitches in mammalian systems. The systems presented in this thesis broaden the toolbox of genetic devices that can externally regulate gene expression with small molecules. The highly sensitive tetracycline-inducible -1-programmed ribosomal frameshifting platform developed in this study provides effective regu-lation at very low inducer concentrations and operates at the translational level. These features are particularly relevant for RNA-only therapeutic approaches and make these switches poten-tially compatible with therapeutic applications targeting diverse tissues. In addition, the opti-mization strategies described in this work could increase the practical value of artificial ri-boswitches in synthetic biology and enable new approaches to gene therapy.