Azul, Melissa

Abstract Background: Sickle cell disease (SCD) is a group of genetic disorders in which sickle hemoglobin polymerizes under deoxygenation, altering red blood cell (RBC) morphology and behavior. The properties of sickle RBCs contribute to increased viscosity of blood and occurrence of vaso-occlusions, a major aspect of SCD pathophysiology. Voxelotor is a novel FDA-approved treatment for SCD which modulates hemoglobin O 2 affinity, and while its known mechanism inhibits sickle polymerization, the impact on other aspects of SCD pathophysiology remain unknown. Thus, despite the new treatment option, highly variable clinical manifestation continues to be a hallmark of sickle cell and there is consequently a need to optimize the use of current therapies based on patient-specific factors. In this work, we leverage datasets generated from a unique microfluidic assay that measures blood flow behavior under varying oxygen tension in conjunction with novel statistical approaches to model and assess sources of variability in sickle blood flow response to voxelotor. Methods: RBCs from patients with SCD (n=28) were treated with voxelotor at 500 uM concentration. Treated samples and untreated controls were perfused through a microfluidic platform that dynamically modulates oxygen tension and measures flow velocity (Wood et al, 2012; Valdez et al, 2019). The area between curves (ABC) of the normalized velocity across the range of oxygen tension between treated and control conditions was calculated to quantify the effect of voxelotor for each sample (figure 1). A paired t-test was used to assess the difference in response between treated and untreated samples. Where available, clinical data including the hemoglobin fractions and complete blood count (CBC) were collected for each sample as predictor variables, and partial least squares regression (PLSR) modeling was used to assess the correlation of predictors and responses. Results: Voxelotor increased the velocity ABC from untreated to treated conditions (p<.0001). We observed that there were differences in response for velocity ABC between sickle cell genotypes (figure 2). Thus, generating separate PLSR models for distinct SCD genotypes revealed differences in sets of clinical factors that explained the most variance in response to voxelotor treatment. A 2-component model was constructed for the HbSC samples (n=6) that best explained variance in the data and had good predictive abilities (R 2X=.69, R 2Y=.97, Q 2=.74). Within this subset, clustering of variables related to hemolysis and inflammation were observed (figure 3). An equivalent model constructed for the HbSS samples (n=15) characterized the predictor variables but lacked predictive power of the response (R 2X=.74, R 2Y=.25, Q 2=.-0.21). Response to voxelotor for this model was most strongly correlated with HbA. Due to low sample size (n=2 samples with full set of predictors), predictive modeling was not performed for HbSβ 0 samples, however, these samples responded the least to voxelotor treatment. Conclusions: Our analysis quantified patient-specific differences in the blood flow response to voxelotor, showing a wide variability in response despite treatment by the same drug concentration. Genotype-specific multivariable models that take into account easily measurable clinical variables such as the CBC have the potential to explain the variability in patient response to voxelotor treatment. In HbSC samples, the WBC, platelet, and reticulocyte counts were highly correlated and strong predictors of response to voxelotor, which may point to markers of hemolysis and inflammation being useful in determining patients that can be optimally treated with this drug. In HbSS, response to voxelotor was mainly inversely correlated with HbA levels, which is a surrogate marker for blood transfusions, indicating that the effect of voxelotor is lessened for patients who are receiving transfusions. However, the low R2Y of this model highlights the clinical variability in this SCD genotype and consequent need for additional biomarkers of disease severity. In conclusion, our hybrid experimental-computational approach is able to identify clinical factors that highly impact the response of patient blood samples to treatment with voxelotor for HbSC patients, and highlights the need for precision therapy recommendations in SCD. Figure 1 Figure 1. Disclosures Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company. Kemp: Parthenon Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Introduction Sickle cell disease affects a large population both nationally and globally. The disease is characterized by the presence of sickle hemoglobin, HbS, which polymerizes the red blood cell into a stiff, sickle shape upon deoxygenation. This polymerization causes several complications, most notably, vaso-occlusion. Voxelotor (Oxbryta, Global Blood Therapeutics) is a newly FDA approved therapeutic for the treatment of sickle cell disease that, when bound to HbS, maintains the oxy-Hb state and inhibits polymerization. Previous studies have demonstrated voxelotor's ability to improve the deformability of the sickle red blood cell (sRBC) via micropippeting and reduce viscosity under hypoxia through using a viscosmeter(Dufu et al, 2018), however its effect under dynamic flow conditions has yet to be explored. Microfluidic devices have served as useful tools to study sickle cell disease, allowing investigation under physiologic conditions of the rheological properties of the sRBC. In this experimental study we aim to examine voxelotor's effect on rheological properties of blood using a microfluidic platform that allows for direct observation of sickled blood flow in a physiologic relevant system. Materials and Methods Whole blood was drawn from 6 patients with sickle cell disease (HbSS or HbSC) as a part of routine blood work under an IRB approved protocol. The cohort included both pediatric and adult patients both on and off hydroxyurea. A stock solution of voxelotor in DMSO (dimethylsulfoxide) was mixed and stored in -20C until use. Red blood cells (RBCs) were isolated using centrifugation and fixed to 25% hematocrit with saline. Voxelotor was added to the blood samples for a final concentration of 500 uM. Voxelotor treated samples were then incubated at 37C for one hour. An untreated, non-incubated aliquot from each patient sample was also obtained to serve a control. From two patient samples, a DMSO vehicle control was also incubated at 37C for one hour to serve as an additional control. Using an electronic pressure regulator, blood from each treatment was then driven through a microfluidic device at a constant pressure and was exposed to hypoxic conditions while RBC velocity data was collected. The microfluidic device design and fabrication in this experiment is described in previously published studies(Wood et al, 2012; Valdez et al, 2019). Briefly, a 3-layer microfluidic device constructed of polydimethylsiloxane (PDMS) consists of a blood, hydration, and gas layer. Saline is perfused through the hydration layer to prevent blood evaporation throughout the experiment. Oxygen gas is pushed through the gas layer, exposing flowing blood to a specific oxygen tension achieved using a mixing setup supplied by air and nitrogen tanks. A fiber optic sensor records oxygen tension within the gas layer throughout the experiment. Deoxygenation-oxygenation cycles were conducted using oxygen saturations from 0 to 21% (0 to 160mmHg pO2). With each deoxygenation cycle after 0%, oxygen saturations were up titrated in a stepwise fashion until oxygen-independent flow was observed. RBC velocity was evaluated by tracking cell movement in the microchannel using high frame-rate imaging and computation video processing. Results and Conclusion A reduction in velocity occurs when sickle RBCs are exposed to deoxygenated conditions as seen in one sample example tracing in figure 1. However, the addition of voxelotor at 500 uM improved the blood flow response to deoxygenation, as RBCs treated with voxelotor had a reduction in velocity change compared to vehicle control and untreated samples when exposed to hypoxic conditions as low as 0 mmgHg oxygen (figure 2). Additionally, voxelotor treated samples began to experience oxygen-independent velocity at lower oxygen tensions compared to the controls. By inhibiting polymerization, voxelotor improves sensitivity of sickle RBC blood flow response in hypoxic conditions. While polymerization is one aspect of sickle cell disease, we would like to explore further effects of voxelotor on other aspects of the understood pathophysiology of the disease such as effects on adhesion in future experiments. Disclosures No relevant conflicts of interest to declare.