Engineering and Delivery of Programmable Protein Circuits as Potential Therapeutic Devices

Citation

Chong, Lucy Shin (2021) Engineering and Delivery of Programmable Protein Circuits as Potential Therapeutic Devices. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/jdhe-by95. https://resolver.caltech.edu/CaltechTHESIS:06072021-211522720

Abstract

Cell-specific targeting of therapeutics is a fundamental challenge in biomedicine. The use of engineered proteins that interact with one another as designed, synthetic circuits represents a promising solution to this challenge. These circuits can be constructed to directly sense endogenous cell signals, act on these signals to classify cellular state, and produce a specific response such as conditional triggering of cell death or targeted expression of a reporter. Synthetic protein circuits can also be delivered in mRNA vectors transiently to avoid permanent gene modification.

We recently showed viral proteases can be engineered to regulate one another in a composable manner, permitting the construction of diverse protein-level circuits (Circuits of Hacked Orthogonal Modular Proteases). CHOMP could perform a wide range of computations including Boolean logic, analogue signal processing, and dynamic signal processing. Using this system we were also able to directly sense key cellular pathways and conditionally respond to trigger apoptosis in cancer-like cells. Further expansion of synthetic protein circuits to include nonlinear signal processing enables new system-level behaviors.

Protein-based circuits are compatible with innovative delivery methods including mRNA encapsulated in lipid-nanoparticle formulations and engineered viruses. As a proof of principle, we were able to develop a controllable, transient RNA-virus delivery system that allowed for targeted delivery to defined cell populations. This paradigm requires control over multiple aspects of the viral delivery system, including (1) production and release of viral particles, (2) target cell entry based on cell-surface proteins, (3) replication within the cell depending on intracellular proteins, and (4) drug-dependent elimination of the virus. Here, we integrate each of these distinct levels of control can into a single system based on the well-characterized negative stranded RNA virus. This RNA-virus platform will enable synthetic protein circuit delivery.

Combining viral engineering and protein circuit construction, the work described here suggests a roadmap towards “smarter” circuit-based therapies that can integrate multiple cues to maximize therapeutic specificity and establishes a role for post-translational circuits as future therapeutic devices.

Thesis Files