Modeling oxygen-dependent hemoglobin polymerization in sickle cell disease at the single cell level

Sickle cell disease (SCD) was identified as the first molecular disease more than half a century ago. Polymerization of sickle hemoglobin (HbS) in red blood cells (RBCs) is established as the critical pathologic event. There is an urgent unmet need for mechanistic models of sickle hemoglobin polymerization that can guide experimental drug development and treatment personalization for each patient. It is well known that the equilibrium and kinetics of HbS polymerization are sensitive to several intracellular factors including HbS concentration ([HbS]), concentrations of other hemoglobin (Hb) isoforms, 2,3-bisphosphoglycerate (2,3-BPG) concentration, other oxygen affinity modulators, and more. Despite several decades of study and development of many sophisticated single-cell measurement methods, we still lack a mechanistic model that can explain heterogeneity in polymerization across cells in the same patient and across patients with different clinical outcomes. Without an accurate single-cell polymerization model, there is no effective way to prioritize SCD drug candidates in development, or to compare efficacy of new or existing treatments, or to identify the best treatment or combination for particular patients. To answer these questions, we currently have to resort to massive randomized clinical trials (RCTs) with subjective clinical endpoints that take a decade or more to plan and perform, require hundreds or thousands of patients, and cost tens of millions of dollars or more. The end results is that despite development of new SCD treatments over the past decades, including antibiotic prophylaxis and hydroxyurea (HU) as standards of care, SCD is still associated with a >30-year reduction in quality-adjusted life expectancy in the US. Aim 1. Develop a mathematical model for the oxygen-dependent equilibrium solubility of sickle hemoglobin in single red blood cells: Eaton developed a population average model 50 years ago that has generated significant insight into Hb polymerization. Modern single-cell methods have recently shown that there is significant heterogeneity at the single-RBC level in Hb-oxygen affinity, and Eaton’s ground-breaking 50-year-old model can now be updated. Aim 2. Develop a single- cell model for the kinetics of Hb polymer formation as a function of oxygen tension and the covariates mentioned above: Decades of work has established great heterogeneity in the kinetics of polymer formation in static in vitro systems. We will develop a single-cell model for the kinetics of Hb polymer formation as function of oxygen tension for Hb mixtures of HbS with either HbF or HbA. Aim 3. Develop a model of single-cell Hb-oxygen affinity and the covariates that determine its heterogeneity in circulating cell populations: New measurement methods have shown over the past 10 years that there is significant unexplained cellular heterogeneity Hb-oxygen affinity. We will develop a Hb-oxygen saturation model that accounts for Hb variants and includes the effects of variations in Hb concentration and 2-3-BPG concentrations. Project outcomes: The models developed during this study will dramatically advance our understanding of the biophysical mechanisms for SCD pathophysiology at the single-cell level, explain the unexpected intra-patient heterogeneity in cellular distributions of oxygen saturations, and ultimately provide guidance for the development of biomarkers that will accelerate treatment development and enable personalization of existing SCD treatments. Project Number: 1R01HL178560-01 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: John Higgins (+1 co-PI) | Institution: MASSACHUSETTS GENERAL HOSPITAL, BOSTON, MA | Award Amount: $719,128 | Activity Code: R01 | Study Section: Modeling and Analysis of Biological Systems Study Section[MABS] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01HL17856001