(725c) 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 are an attractive platform for diagnostics because they have exceptionally long circulation times (around 120 days – far longer than synthetic vehicles), lack DNA (and thus are safe), and can be loaded with drugs, proteins, or other cargo. Recent biotechnological 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, including both diagnostic and therapeutic applications.

Diagnostics based upon eRBCs comprise a novel, translatable modality for monitoring physiological state, which could save lives. Biosensors could benefit patients when non-invasive, frequent monitoring is needed and when detection before symptom onset could accelerate treatment initiation to reducing morbidity and mortality. Possible applications include: monitoring IL-6 to detect cytokine storms following CAR T Cell therapy; detecting graft vs. host disease following allogeneic stem cell transplant; and detecting circulating cancer cells following cancer treatment. In the envisioned application of this work, eRBC biosensors will be manufactured as off-the shelf products engineered to sense a specific ligand. Upon ligand detection, each eRBC will emit a near infrared fluorescent readout, which can be monitored non-invasively using established technologies for retinal imaging. Thus, a patient could perform regular self-analysis to enable real- time, high frequency monitoring outside clinical settings, which currently requires specialized equipment, trained personnel, and/or sample collection.

As a first step to enable 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 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 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. Further, we are generating modified primary red blood cells for in vivo evaluation of eRBC biosensor ligand-inducible output and eRBC persistence in a mouse model. Overall, this study establishes the feasibility of eRBC-based technologies enabling non-invasive monitoring for physiological signals and actionable analytes to address an unmet need for a range of diagnostic applications.