(228cj) 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. A promising emerging frontier of this field is the development of engineered red blood cells (eRBCs) for therapeutic and diagnostic applications. RBCs have exceptionally long circulation times (around 120 days â?? far longer than synthetic vehicles), lack DNA (and thus are safe), and can be loaded with drugs, proteins, or other cargo. Technologies that enable one to engineer RBCs to act as biosensors, performing specific functions in vivo, could serve unmet diagnostic and therapeutic needs. In particular, new technologies are required for non-invasive, routine monitoring for pathogen exposure (e.g., in the context of first responders) and for other â??actionableâ? analytes (e.g., markers of inflammation post-surgery).

In this project, we are developing eRBC biosensors that generate a fluorescent output upon detection of the analyte of interest. Ultimately, eRBC biosensor fluorescent output may be monitored non-invasively using established technologies for fluorescent imaging of the retina. Using this simple imaging technology, a patient could perform regular self-analysis and enable real time, high frequency monitoring outside clinical settings, none of which is possible with existing technologies requiring specialized equipment, trained personnel, and/or sample collection. Thus, such biosensors that enable the detection of actionable analytes could benefit exposed personnel by accelerating the initiation of treatment (perhaps before obvious symptoms present) and reduction of further exposure risks when possible.

As a first step towards the goal of building eRBC biosensors, 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 novel cell-surface receptor protein in which ligand binding induces receptor dimerization, which then facilitates reconstitution of an intracellular split fluorescent protein. Importantly, our strategy involves modification of RBC-resident proteins, since retention of membrane proteins during RBC maturation is a tightly regulated and an incompletely understood process. We comparatively evaluated a range of biosensor architectures that implement the proposed mechanism, enabling us to identify biosensor designs and design features that successfully conferred significant ligand-induced generation of fluorescent output. We also evaluated and implemented strategies for improving biosensor performance, including minimization of background fluorescence and enhancing fold-induction upon exposure to ligand. This crucial proof-of-principle demonstration 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.