Design of a self-tunable mRNA circuit to control cellular protein levels

Project Summary Imbalances in protein levels disrupt the cellular homeostasis, leading to impaired cell function and the onset of diseases. Maintaining precise control over protein concentrations in cells, both the endogenous and therapeutic exogeneous proteins, is critical for therapeutic homeostasis. While the proteolysis-targeting chimera (PROTAC) technology is a newly emerged approach that removes disease-associated proteins by leveraging cellular programmed degradation machinery, the challenge lies in achieving long-term, precise control of the target protein within the therapeutic concentrations. The proposal aims to address this challenge by developing an mRNA- based gene circuit capable of sustained, accurate regulation of a target protein in cells. Utilizing advanced in silico peptide docking and innovative cell-based screening approaches, the circuit is designed to implement the PROTAC strategy for optimal protein concentration control. This proof- of-concept study aims to develop a modular, self-tuning mRNA gene circuit adaptable across diverse biological systems. By integrating experimental validation with a mathematical kinetic model for regulating target protein concentrations, this approach will facilitate the design of personalized therapeutic regimens and provide predictive insights into treatment outcomes. Aim 1 of the proposal focuses on the design and validation of one of the two modules that directs programmable degradation of the mRNAs expressing PROTAC proteins. Aim 2 will combine the two modules, measure the efficiency of the gene circuit in degrading and maintaining the cellular level of the target protein, and validate the mathematic kinetic model. The platform developed in this study holds significant potential for advancing precision medicine in the future.