PROJECT SUMMARY Sickle Cell Disease (SCD) afflicts 100,000 Americans and millions of people worldwide, and is characterized by anemia, painful vaso-occlusive crises, ischemia, inflammation, significant morbidity, and early mortality. In SCD, deoxygenated sickle hemoglobin (deoxy-HbS) polymerizes, deforms red blood cells (RBCs), changes membrane properties, and triggers inflammation, thrombophilia, and vasculopathy. Traditional treatment approaches include routine blood transfusions and hydroxyurea therapy while both options exhibit inherent challenges and limitations. Recent efforts in developing new treatment approaches in SCD focused on targeted therapies, aiming to prevent hypoxia-mediated polymerization of sickle hemoglobin and the abnormal adhesive interactions between blood and endothelial cells. Accordingly, the Food and Drug Administration (FDA) has recently approved two new treatments for SCD: ADAKVEO (Crizanlizumab, Novartis) and Oxbryta (Voxelotor, Global Blood Therapeutics) to prevent abnormal RBC adhesion and sickling. In addition to those, many other drug candidates targeting a specific adhesion pathway are currently in the development stage. Because SCD pathophysiology is extremely complex and heterogenous, it is crucial to predict how/if a patient will benefit from a specific targeted therapy. An anti-adhesive therapy may not benefit a certain patient population or may need to be supplemented with additional therapeutic approaches (i.e., anti-sickling drugs). Further, patient-specific responses to such therapies may be impacted by or associated with clinical variables (e.g., lactate dehydrogenase level, reticulocyte count, hemoglobin levels, etc.). Being able to predict a patient's response to a certain targeted therapy, or combination of multiple, using biomimetic in vitro tools will substantially help the entire SCD patient population. Therefore, there is an urgent need for novel biomimetic in vitro assays that can serve as a drug screening platform, both in understanding the effect of emerging therapies on a single patient level and in screening drug candidates for potential mitigation of SCD pathophysiology. Our objective in this STTR Phase I/II proposal is to develop a standardized and validated endothelialized microfluidic cellular adhesion assay to predict in vitro patient-specific responses to a targeted therapy by probing blood cell adhesion to activated endothelial cells. In Phase I, we propose to standardize and validate the culture and activation of endothelial cells as well as blood cell adhesion within the Endothelium-on- a-chip under physiologically relevant flow conditions. Phase II aims and milestones focus on activities related to determining baseline cellular adhesion levels, establishing the testing methodologies for proof-of-concept, and clinically validating the Endothelium-on-a-chip. Our goal is to demonstrate the utility of the Endothelium-on- a-chip in predicting patient-specific responses to single and co...