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 40 days – 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 perform 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 “actionable” analytes (e.g., markers of inflammation post-surgery).

In this project, we sought to develop eRBC biosensors than detect highly toxic agents, with the long-term goal of enabling one to detect exposure to these agents prior to the onset of physical symptoms. As a first step towards this goal, we designed and evaluated a novel biosensor strategy that is suitable to 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. Ultimately, eRBC fluorescence may be monitored non-invasively using established technologies for fluorescent imaging of the retina.  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. In this study, we comparatively evaluated a range of biosensor architectures that implement the proposed mechanism, identified design biosensor features that successfully conferred significant ligand-induced generation of fluorescent output, and investigated strategies for improving biosensor performance (e.g., 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.