Development of platform technology to measure kinetics and equilibrium concentration of sickle hemoglobin polymerization in single RBCs for drug potency assessment and patient risk stratification - Sickle cell disease is caused by the pathologic polymerization of variant sickle hemoglobin (HbS) when deoxygenated. Polymerization of HbS inside red blood cells (RBCs) immediately compromises RBC morphology and blood flow properties, and it also triggers exacerbation of inflammation and cellular adhesion to endothelium, ultimately leading to ischemia, vaso-occlusion, stroke, tissue damage, and acute and chronic organ dysfunction. The only approved drugs are far from curative, with incompletely understood mechanisms, and the quality of life for most sickle cell patients remains poor with life expectancy in the US reduced by more than 30 years. The prospects for improvement in sickle cell treatments have increased significantly in recent years with scientific and technological developments making multiple promising treatment approaches more realistic including gene therapy, gene editing, fetal hemoglobin induction, and hemoglobin (Hb) affinity modulation. These new treatment approaches share the same basic mechanism of action: reduce the amount of Hb polymer in RBCs at physiologic oxygen tensions. Perfect realization of any one of these approaches will cure the disease, but partial realization is all we can expect, and it is essential for prioritizing and optimizing treatment development that we be able to measure the intracellular Hb polymer concentration, the kinetics of Hb polymerization, and the reduction in Hb polymer formation achieved by each regimen of each treatment. The process of intracellular Hb polymer formation has been studied extensively, and theory is well established with Eaton and Hofrichter's double-nucleation model. Both the kinetics of Hb polymer formation and its equilibrium levels are sensitive to several factors that complicate measurement including HbS concentration, concentration of other Hb isoforms, oxygen tension, pH, Hb concentration, temperature, intracellular flows, and more. We will develop a high-throughput platform to measure the equilibrium and kinetics of Hb polymerization in single RBCs while controlling and modulating these factors. We will calibrate the platform by comparing the characteristics of its measurements against existing standard clinical laboratory instruments and current theory. We will also use it to measure variation across a range of clinical phenotypes and sensitivity to variation in HbS concentration, Hb concentration, and more. This new platform technology will enable the development of in vitro markers to optimize treatment discovery, prioritize alternative strategies, ensure treatment product quality, assess patient response, and optimize individual patient management. Our study will enable development of quantitative in vitro endpoints for sickle cell disease derived from measurements of the kinetics and equilibrium of Hb polymerization in individual RBCs.
