Developing conditional protein rescue for synthetic proten-based logic circuits towards the detection and treatment of cancer
Date
2020
Authors
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Publisher
University of Delaware
Abstract
Cancer is a complicated set of diseases marked by drastic changes in the cellular proteome. To treat cancer, clinicians still strongly rely on chemotherapy, which kills mitotic cells indiscriminately, leaving formidable side effects in its wake. Recently, there has been a drive for “smart therapies” that can distinguish between healthy and cancerous cells. To head this call, several researches have sought to leverage proteomic differences to deploy a therapeutic protein, but most have focused on individual differences in expression levels of cell surface markers only. Probing intracellular proteins or multiple proteins simultaneously still remains challenging. This dissertation attempts to alleviate these barriers by engineering an autonomous protein switch triggered by intracellular proteomic information. To this end, we have developed a modular platform that enables the control of protein half-life based on the presence of a target protein. Important stages in the development of this technology and their broader implications are investigated herein. ☐ First, foundational work was completed to determine that a prodrug converting enzyme, a commonly proposed option for a protein-based cancer therapy that enzymatically converts an innocuous prodrug into a cytotoxic drug, can be regulated via protein degradation. Using Traceless Shielding, a method for inducing the rescue of a protein of interest (POI) via a small molecule, we explored the kinetics of initial protein degradation and rescue. Next, we showed that cell viability was unaffected by the presence of the prodrug in the absence of the rescuing molecule. Thus, protein degradation is a viable strategy for managing prodrug converting enzymes. ☐ With this important cornerstone laid, we sought a clever, facile method for translating cellular protein inputs into a POI rescue output. This lead to the development of conditional protein rescue, in which a degron is genetically fused to a POI followed by a short, sensing domain. In the absence of the sensing domain’s target, the degron is exposed and the POI is fated for degradation. However, in the presence of the target, the degron is concealed and the protein is rescued. The first versions of this platform used SpyTag as the sensing domain, which resulted in a dramatic increase in POI concentration upon the co-expression of SpyCatcher, which spontaneously forms an isopeptide bond with SpyTag. We expanded upon this by using nanobodies—small, monomeric, and specific antigen-sensing domains—as the sensing domain. Nanobodies varied POI concentration based on the presence of co-expressed fluorescent proteins. Nicely, CPR is adept at distinguishing between cancer cells and non-cancer cells in vitro. ☐ Finally, we further improve the capabilities of CPR by constructing Boolean AND gate architecture into the framework. Fluorescent protein targets are initially employed to block N- and C-terminal degrons. We show that both targets are necessary to raise the concentration of the POI. We prove the therapeutic feasibility of this design by detecting a synthetic cancer-model system: HeLa cells constitutively expressing GFP from both HeLa cells and HEK293T cells. This promising results shows that our design is an initial step at addressing some of the challenges impeding next-generation therapies. ☐ This technology shows that the power of engineered protein degradation to yield rapid, definite changes in the concentration of a POI can be effectively harnessed to detect multiple, intracellular cancer protein targets. Paired with developments in targeted delivery, it is now possible to offer a two-pronged cancer sensing device requiring both the overexpression of a cell surface receptor and multiple intracellular proteins to activate. This should result in low background in off-target cells and an overall reduction of side effects. Furthermore, multi-input CPR can be expanded to any system of interest, and it may be practical in the study of other biological systems as well.
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Keywords
Prodrug converting enzymes, Protein circuits, Protein engineering, Synthetic biology