Engineering Red Blood Cell-Based Biosensors for Physiological Monitoring

Cell-based therapies have a wide range of applications ranging from cancer immunotherapy to regenerative medicine. An emerging frontier of this field is the development of engineered red blood cells (eRBCs) for therapeutic and diagnostic applications. RBCs are an attractive platform because they have exceptionally long circulation times (120 days), lack DNA, and can be loaded with drugs, proteins, or other cargo. Recent technological advances have enabled the large-scale production of RBCs from precursor cells, which may potentially be harnessed to generate off-the shelf eRBC-based products to meet medical needs. The specific goal of this project is to generate eRBC-based technologies enabling non-invasive monitoring for pathogen exposure (e.g., in the context of first responders) and for other “actionable” physiological analytes (e.g., markers of acute inflammation). As a first step towards enabling RBCs to act as sensors, we designed and evaluated a novel biosensor strategy that is suitable for achieving biosensing in eRBCs, which lack DNA and thus require a readout other than gene expression. Towards this end, we engineered a cell-surface receptor protein in which ligand binding induces receptor dimerization, which then facilitates reconstitution of an intracellular split reporter protein. Importantly, our strategy involves modification of RBC-resident proteins, since retention of membrane proteins during RBC maturation is a tightly regulated and incompletely understood process. We comparatively evaluated a range of biosensor architectures, enabling us to identify biosensor designs and design features that successfully conferred significant ligand-induced generation of fluorescent output. We then carried forward the most promising architectures for testing in G1E cells—a murine erythroid cell line—to verify biosensor expression and functionality in a red blood cell model. This study establishes a foundation for developing eRBC biosensors that could ultimately address an unmet need for non-invasive monitoring of physiological signals for a range of diagnostic applications.