Synthetic mammalian RNA-based gene circuits for high-fold changes in expression

Synthetic and native switchable genetic expression systems are widely used in research and applications in biomedicine and biotechnology. However, they face challenges related to "leakiness," or the unintended baseline activity of a promoter in the absence of its specific activator, leading to issues from ineffective biological sensors to unwanted toxic effects.

To address this, we propose an auxiliary RNA-based system utilizing mutual inhibition to enhance existing genetic circuits without altering transcription factors, promoters, or output genes. Our system features a constitutively expressed small hairpin RNA (shRNA) that downregulates a target gene regulated by a responsive promoter. Additionally, we introduce a second promoter that produces a binding-site sponge, specifically designed to sequester the shRNA and alleviate its inhibitory effects on the target gene. This dual mechanism balances the shRNA’s silencing potential while allowing controlled gene expression in response to stimuli.

Mathematical modeling has been conducted to predict the system’s behavior, and experimental validation involves testing its effectiveness across various contexts, including the doxycycline-inducible Tet-On system and a native cancer-specific promoter, pH2A1. Preliminary results indicate that our auxiliary system successfully reduces unintended expression levels, enhancing gene activation specificity and reliability. This approach provides more precise control over gene expression in response to external stimuli, offering a promising direction for advancing targeted therapies and innovative biotechnological applications, with future research exploring additional binding-site sponges and applications in various cell lines.