Recent progress advancing sickle cell disease cures is remarkable: ? Two gene therapy approaches using viral vectors have case reports of significantly attenuated disease phenotype with admittedly short-term follow-up. ? Four different viral vector approaches and one CRISPR/Cas9-based approach are in clinical trials. ? Multiple academic and industry groups have announced planned trials. ? A hemoglobin oxygen affinity modulator is in phase III study. To move beyond this first stage of successful case reports to a second stage of widespread, sustained cures, the field will need to prioritize these approaches. Perfect realization of each approach will cure disease, but partial amelioration of symptoms may be more realistic and yet still be considered successful. A critical goal in this regard is to quantify results of each approach along the continuum to a fully curative therapy. Gene transfer will produce a chimeric population of RBCs and in this scenario a key question is: how much non-sickle hemoglobin is needed in what fraction of RBCs to cure individual patients? Fetal hemoglobin induction and hemoglobin oxygen affinity modulation will reduce polymer in some fraction of RBCs: what reduction in hemoglobin polymer is required in what fraction of RBCs to cure individual patients? To answer these questions, we need a way to quantify hemoglobin polymer in individual RBCs and to measure the effect of varying fractions of those polymer-containing RBCs on blood flow in vitro. This project builds on prior published work by us and others to develop both types of assays. This project will: I. Assess analytic performance of assays of single-RBC oxygen-dependent polymer distributions and of oxygen-dependent rheology of blood flow. II. Validate their use at least as a ?reasonably likely surrogate endpoint? while simultaneously working toward a goal of establishing these assays or some subset of their measurements as a ?validated surrogate endpoint? for sickle cell clinical trials.