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Sickle cell disease (SCD) is characterized by sickle hemoglobin (HbS) which polymerizes under deoxygenated conditions to form a stiff, sickled erythrocyte. The dehydration of sickle erythrocytes increases intracellular HbS concentration and the propensity of erythrocyte sickling. Prevention of this mechanism may provide a target for potential SCD therapy investigation. Ionophores such as monensin can increase erythrocyte sodium permeability by facilitating its transmembrane transport, leading to osmotic swelling of the erythrocyte and decreased hemoglobin concentration. In this study, we treated 13 blood samples from patients with SCD with 10 nM of monensin ex vivo. We measured changes in cell volume and hemoglobin concentration in response to monensin treatment, and we perfused treated blood samples through a microfluidic device that permits quantification of blood flow under controlled hypoxia. Monensin treatment led to increases in cell volume and reductions in hemoglobin concentration in most blood samples, though the degree of response varied across samples. Monensin-treated samples also demonstrated reduced blood flow impairment under hypoxic conditions relative to untreated controls. Moreover, there was a significant correlation between the improvement in blood flow and the decrease in hemoglobin concentration. Thus, our results demonstrate that a reduction in intracellular HbS concentration by osmotic swelling improves blood flow under hypoxic conditions. Although the toxicity of monensin will likely prevent it from being a viable clinical treatment, these results suggest that osmotic swelling should be investigated further as a potential mechanism for SCD therapy.

Sickle cell disease (SCD) is caused by a variant hemoglobin molecule that polymerizes inside red blood cells (RBCs) in reduced oxygen tension. Treatment development has been slow for this typically severe disease, but there is current optimism for curative gene transfer strategies to induce expression of fetal hemoglobin or other nonsickling hemoglobin isoforms. All SCD morbidity and mortality arise directly or indirectly from polymer formation in individual RBCs. Identifying patients at highest risk of complications and treatment candidates with the greatest curative potential therefore requires determining the amount of polymer in individual RBCs under controlled oxygen. Here, we report a semiquantitative measurement of hemoglobin polymer in single RBCs as a function of oxygen. The method takes advantage of the reduced oxygen affinity of hemoglobin polymer to infer polymer content for thousands of RBCs from their overall oxygen saturation. The method enables approaches for SCD treatment development and precision medicine., Competing Interests: The authors declare no competing interest.

The complex, systemic pathology of sickle cell disease is driven by multiple mechanisms including red blood cells (RBCs) stiffened by polymerized fibers of deoxygenated sickle hemoglobin. A critical step toward understanding the pathologic role of polymer-containing RBCs is quantifying the biophysical changes in these cells in physiologically relevant oxygen environments. We have developed a microfluidic platform capable of simultaneously measuring single RBC deformability and oxygen saturation under controlled oxygen and shear stress. We found that RBCs with detectable amounts of polymer have decreased oxygen affinity and decreased deformability. Surprisingly, the deformability of the polymer-containing cells is oxygen-independent, while the fraction of these cells increases as oxygen decreases. We also find that some fraction of these cells is present at most physiologic oxygen tensions, suggesting a role for these cells in the systemic pathologies. Additionally, the ability to measure these pathological cells should provide clearer targets for evaluating therapies. [ABSTRACT FROM AUTHOR]

We previously reported initial clinical results of post-transcriptional gene silencing of BCL11A expression (NCT 03282656) reversing the fetal to adult hemoglobin switch. A goal of this approach is to increase fetal hemoglobin (HbF) expression while coordinately reducing sickle hemoglobin (HbS) expression. The resulting combinatorial effect should prove effective in inhibiting HbS polymerization at lower physiologic oxygen values thereby mitigating disease complications. Here we report results of exploratory single-cell analysis of patients in which BCL11A is targeted molecularly and compare results with cells of patients treated with hydroxyurea (HU), the current standard of care. We use single-cell assays to assess HbF, HbS, oxygen saturation, and hemoglobin polymer content in RBCs for nine gene therapy trial subjects (BCLshmiR, median HbF% = 27.9) and compare them to 10 HU-treated subjects demonstrating high and comparable levels of HbF (HU High Responders, median HbF% = 27.0). All BCL11A patients achieved the primary endpoint for NCT 03282656, which was defined by an absolute neutrophil count greater than or equal to 0.5 × 109 cells/L for three consecutive days, achieved within 7 weeks following infusion. Flow cytometric assessment of single-RBC HbF and HbS shows fewer RBCs with high HbS% that would be most susceptible to sickling in BCLshmiR vs. HU High Responders: median 42% of RBCs with HbS%>70% in BCLshmiR vs. 61% in HU High Responders (p = 0.004). BCLshmiR subjects also demonstrate more RBCs resistant to HbS polymerization at lower physiologic oxygen tension: median 32% vs. 25% in HU High Responders (p = 0.006). Gene therapy-induced BCL11A down-regulation reverses the fetal-to-adult hemoglobin switch and induces RBCs with higher HbF%, lower HbS%, and greater resistance to deoxygenation-induced polymerization in clinical trial subjects compared with a cohort of highly responsive hydroxyurea-treated subjects. The authors report in full the primary endpoint data of a pilot clinical trial (NCT 03282656) that used post-transcriptional gene silencing of BCL11A expression to reverse the fetal to adult hemoglobin switch in sickle cell disease. They develop new single-cell flow cytometry and microfluidic techniques to predict the efficacy of HbF induction and show that red blood cells from these patients exhibit greater resistance to deoxygenation-induced polymerization than red blood cells from hydroxyurea-responsive patients. [ABSTRACT FROM AUTHOR]