Development of a Toggle Switch Based on Post-Transcriptional Regulation That Resists Epigenetic Silencing

Synthetic biological circuits may enable sophisticated cell therapies that respond to patient biomarkers and inputs from clinicians. However, transcriptionally regulated genetic circuits are prone to epigenetic silencing, in which chromatin modifications often turn off transgene expression after a few weeks. This poses a significant challenge to the regulation of cell therapies that may persist in vivo for years. Constitutive transcription has been shown to reduce silencing, but tools to regulate gene expression post-transcriptionally are limited. To address this gap, we present the PERSIST platform for post-transcriptional regulation, in which RNA cleavage events activate or repress downstream gene expression. We take advantage of CRISPR proteins in the Cas6 and Cas13 families that cleave sequence-specific RNA hairpins, demonstrating a set of nine proteins that enact activation and repression, often with fold changes of two orders of magnitude. PERSIST genetic circuits show 28% less silencing than the transcriptionally regulated TetON system after 1 month and no more silencing than a constitutive control. We then used the PERSIST platform to implement a genetic toggle switch. Toggle switches could improve cell therapies by imparting them with memory, such that a transient input, such as a drug input from a clinician, can permanently change cell behavior.

We designed a system in which FDA-approved small molecule drugs could switch a cell between two behavioral states, and implemented this system by combining PERSIST logic processing with engineered small molecule-responsive fusion proteins. Preliminary results suggest that our circuit is able to switch between two stable states in response to clinically relevant small molecule drugs. These results will provide a platform to regulate the expression of transgenes in cell therapies over time, enabling optimized dosing schedules of therapeutic proteins in applications including encapsulated cell therapies, engineered immune cells, and gene therapies.