Cell targeting with synthetic sense-and-respond protease circuits Abstract A fundamental challenge in biomedicine is developing therapeutics that can target specific cell populations. Synthetic protein circuits, based on engineered proteins that interact with one another and with endogenous cellular pathways, could provide a powerful platform to address this challenge. These circuits could directly sense key cellular pathways, process that information to classify the cellular state, and respond by conditionally triggering cell death or other beneficial responses. Synthetic protein circuits could also be encoded as mRNA and delivered transiently to avoid genome modification. We recently showed that viral proteases could be engineered to regulate one another in a modular way, allowing construction of diverse protein-level functions from a limited number of components. However, key challenges remain: Can engineered protease circuits be generalized to sense multiple cancer pathways with minimal perturbations to the cell? Can they implement a broader set of signal processing capabilities, including thresholding, integration, and dosage compensation to allow for versatile and precise function in diverse cell contexts? And, can they selectively target cancer cells when transiently delivered as mRNA? Here, we aim to develop this system into a broader platform for targeting cancer cells by creating new pathway sensing capabilities, designing flexible signal processing modules, and demonstrating the ability to sense and kill specific target cell types. We will focus on cellular models of hepatocellular carcinoma (HCC), a disease which remains challenging to treat but is relatively permissive for mRNA delivery. In Aim 1, we will design and validate protease sensors of major oncogenic pathways that play critical roles in HCC. These sensors conditionally activate viral proteases in response to the localization, clustering, activity, or abundance of target proteins. In Aim 2, we will create protease-based circuit modules that actively process these signals. We will engineer thresholding modules to suppress undesired responses to basal pathway activities in normal cells, and combinatorial logic modules to allow AND-like integration of signals from distinct sensors. In addition, we will design dosage compensation modules that make protein expression insensitive to circuit delivery, In Aim 3, we will design mRNA-delivered circuits that selectively kill HCC cell lines with minimal impact on normal hepatocytes. This research program will establish the end-to-end feasibility of mRNA-delivered protease circuits and provide the foundations for future programmable circuit-based therapeutics.

PROJECT NARRATIVE An ideal cancer therapeutic would be able to selectively target cancer cells based on the activities of key cellular pathways that support or drive cancer. Synthetic biology approaches now allow the engineering of protein circuits that can operate within the cell, sensing the activities of these pathways and using that information to selectively kill tumor cells. Here, we will develop the foundation for protein circuits as therapeutic devices, by engineering pathway sensors and signal integration modules, and validating protein circuits in models of hepatocellular carcinoma.