Point Of Sickling Research Articles

Rheological Characteristics of Elderly Individuals with Sickle Cell Disease Compared with an Adult Sickle Cell Population

Introduction: Individuals with sickle cell disease (SCD) have a shorter life expectancy (median age 43 years in the US) compared with the general population. However, a subset of patients with SCD live ≥60 years. Prior case series examining conventional laboratory values found no significant differences between elderly and adult populations with SCD. Red blood cell (RBC) function, which is affected by RBC deformability and density, is abnormal in SCD, but the degree of impairment varies greatly among patients. Oxygen gradient ektacytometry measures RBC deformability as an elongation index (EI) under conditions of oxygenation (EI max) and deoxygenation (EI min). The oxygen partial pressure at which RBC deformability decreases is the point of sickling (PoS), and a higher PoS is associated with greater likelihood of a pain crisis. % dense RBCs (%DRBC), defined as RBCs containing >1.11 g/dL of hemoglobin (Hb), are associated with an increased likelihood of clinical complications. We hypothesized improved RBC function contributes to the longevity of patients with SCD and in this analysis we compared the RBC function of elderly patients with SCD vs an unselected adult cohort with SCD aged ≤45 years. Methods: Peripheral blood samples (transfused samples excluded) and clinical data were collected from consenting patients with SCD treated at Grady Memorial Hospital, Atlanta, GA, from August 2022 to June 2023. Patients were assigned to groups by age (elderly, ≥60 years; controls, 18-45 years) and genotype (HbSS or HbSC). Complete blood count, absolute reticulocyte count (ARC), and %DRBC were determined using an ADVIA hematology analyzer. EI max, EI min, and PoS were measured using a Laser Optical Rotational Red Cell Analyzer. Tests were performed within 24 h of blood collection. Hydroxyurea (HU) use and % fetal Hb were collected via chart review. Results: Thirteen elderly patients (HbSS, median age 63 years; 61.5% using HU) were compared with 78 control patients (HbSS, median age 28 years; 75.6% using HU). Apart from median ARC and absolute neutrophil count (ANC), which were significantly lower in the elderly group, all hematological parameters were comparable between HbSS groups (Table). The RBCs of the HbSS elderly group had improved deformability compared with the RBCs of the HbSS control group with a higher EI max (0.506 vs 0.410; P=0.001) and EI min (0.203 vs 0.125; P<0.001; Figure). PoS and %DRBC were significantly lower in the HbSS elderly group vs the HbSS control group (PoS: 20.48 mmHg vs 32.40 mmHg; %DRBC: 3.0% vs 7.4%; each P<0.001). Eleven elderly patients (HbSC, median age 64 years; 9.1% using HU) and 37 control patients (HbSC, median age 29 years; 18.9% using HU) were compared. Apart from median Hb, ANC, and mean corpuscular Hb concentration, which were significantly lower in the HbSC elderly group, all hematological parameters were comparable between HbSC groups (Table). Although there were no significant differences in EI max, EI min, or PoS between HbSC groups, %DRBC was significantly lower in the HbSC elderly group (8.5% vs 14.4%; P=0.003). Conclusions: This is the first study to compare rheology as well as hematologic parameters between elderly patients with SCD and control patients with SCD aged ≤45 years to determine if RBC function contributes to longevity. Significantly lower %DRBC in HbSS and HbSC elderly groups, vs respective control cohorts, may contribute to improved rheology as denser RBCs have higher concentrations of HbS and are more prone to sickle. In the elderly groups, %DRBC was numerically higher in the HbSC group vs the HbSS group, and significantly lower vs respective control groups, emphasizing the dehydrating effects of HbC and supporting phlebotomy to reduce %DRBC in HbSC disease. Only the HbSS elderly group demonstrated better RBC deformability regardless of oxygen conditions and a lower PoS, indicating less HbS polymerization at lower oxygen levels and suggesting increased relevance of deformability to pathophysiology and outcomes in HbSS vs HbSC disease. Even outside of SCD, older age is associated with more dehydrated, less deformable RBCs, and the reversal of this trend in HbSS groups is remarkable. Use of HU, a modifier of RBC function, was comparable between HbSS groups and cannot account for our findings. Overall, our results support the clinical relevance of RBC function testing and its potential role in assessing genotype-specific disease severity and expected longevity.

Hypertonicity and/or Acidosis Induce Marked Rheological Changes Under Hypoxic Conditions in Sickle Trait Red Blood Cells

Hemoglobin S (HbS) is an abnormal hemoglobin resulting from a single nucleotide substitution in the beta-globin gene. Individuals who are heterozygous for the HbS genotype are regarded as carriers of sickle cell anemia (SCA; HbSS) and are said to have sickle cell trait (SCT; HbAS). Recent data have established SCT as a risk factor for venous thromboembolism (VTE), chronic kidney disease (CKD), and end-stage renal disease (ESRD) (Naik RP, 2018). These complications have a relative risk of approximately 1.5 - 2.0 compared to race- and age-matched controls, but the mechanisms that account for these complications are not well understood. To study potential mechanisms for renal disease, we evaluated red blood cell (RBC) deformability from SCT donors under conditions designed to mimic the hypertonic, acidemic, and hypoxemic environment of the inner renal medulla. RBC deformability was measured using the Laser Optical Rotational Red Cell Analyzer (LORRCA; RR Mechatronics, The Netherlands). In SCA, LORRCA has been used to monitor RBC rigidity at baseline and under progressive hypoxia by calculating the Elongation Index (EI; the longitudinal dimension of the RBC along the axis of applied shear stress divided by transverse dimension of the cell along the same axis). The OxygenScan function measures EI during a cycle of continuously decreasing pO2 from about 150 mmHg (ambient air) to near 20 mm Hg, before reversing in a re-oxygenation phase. In SS RBCs, EI decreases from a stable baseline as RBCs start to sickle; this ‘point of sickling (PoS)‘ is defined as the pO2 at which there is a >5% decrease in EI. Similarly, the Osmoscan function can record the RBC EI at ambient oxygenation while osmolality of the blood progressively increases from approximately 100 to 600 mOsm. First, OxygenScan profiles were performed utilizing modified LORRCA buffers to evaluate four separate conditions: isotonic + neutral pH, isotonic + pH 6.9, 460 mOsm + neutral pH, and 460 mOsm + pH 6.9. Buffers were modified by the addition of sodium chloride, hydrochloric acid, or both. HbS percentage (HbS%) and RBC indices were recorded for each subject. HbS% and RBC indices were compared to PoS for all samples and conditions. EI from AS and AA samples were compared at physiologic serum osmolality (290 mOsm) and at 460 mOsm. AA samples demonstrated no measurable PoS under any condition. Some (11/21) AS samples demonstrated sickling under hypoxia alone, with mean PoS of 32.26 mmHg. Mean PoS for AS samples in isotonic buffer + pH 6.9 was 40.73 mmHg, but with sickling detected in all samples. Mean PoS in hypertonic buffer (460 mOsm) significantly increased to 60.68 mmHg, with notable inter-individual heterogeneity (Fig. 1). Mean PoS in hypertonic buffer + pH 6.9 was 79.04 mmHg, with less inter-sample heterogeneity. In exploratory experiments, we have observed similar changes in the Townes AS murine model of SCT (not shown). PoS was compared to HbS% in the human samples under all conditions. A positive correlation was observed between HbS% and PoS in isotonic buffer at pH 6.9 (r=0.566, p=0.012). PoS was also compared to RBC indices, with a trend towards a positive correlation with mean corpuscular hemoglobin concentration (MCHC) under hypertonic + neutral pH conditions ( p=0.11). Next, utilizing the Osmoscan function, no difference in EI at physiologic osmolality (290 mOsm) was observed between AA (n= 7) vs. AS (n= 8). However, at 460 mOsm, EI trended lower in AS RBCs ( p=0.12) (Fig. 2). Given the presence of severe tissue hypoxia (as low as 10 mmHg), hypertonicity (as high as 1,200 mOsm), and acidemia (as low as pH 5.5) in the inner renal medulla, these data provide an explanation for RBC-mediated occlusion of the vasa recta that is known to occur in the kidneys of subjects with SCT. We demonstrate that extracellular hypertonicity contributes significantly to SCT RBC sickling in the presence of hypoxemia, and that there is significant inter-individual susceptibility to sickling under these harsh biophysical conditions that is only partially explained by HbS%.

The Effect of Voxelotor on Erythrocyte Deformability, Neutrophil Phenotype and Endothelial Activation

Introduction Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis, caused by polymerization of abnormal sickle hemoglobin. The ongoing intravascular hemolysis leads to release of cell-free heme and iron, resulting in widespread oxidative damage, inflammation, neutrophil activation, endothelial activation and hypercoagulability, all contributing to vaso-occlusive events and end organ damage. Voxelotor, a small molecule reversibly binding to hemoglobin S (HbS) increases its oxygen affinity, thereby decreasing HbS polymerization and sickling, resulting in decreased hemolysis. The decrease in erythrocyte sickling might lead to improved erythrocyte deformability and decreased adhesion, while reductions in cell-free heme and labile plasma iron levels might result in decreased neutrophil activation, adhesion and aging and decreased endothelial activation. The aim of this study is to assess the effects of voxelotor on erythrocyte deformability, neutrophil activation, adhesion and aging and endothelial activation. Methods SCD patients (HbSS and HbSβ 0-thalassemia), with hemoglobin (Hb) ≤ 10.5 g/dL were treated with voxelotor 1500 mg in a single arm interventional study. Blood was collected at baseline and after 3 months of treatment. Erythrocyte deformability was assessed by ektacytometry (Lorrca, RR mechatronics), measuring elongation index (EI) and point of sickling (PoS). EI max and EI min indicate the highest and lowest erythrocyte deformability upon oxygenation and deoxygenation respectively. PoS is the oxygen tension at which 5% reduction of the EI max is observed. Antigen expression of neutrophil markers for activation, adhesion and aging were assessed with flow cytometry. Reactive oxygen species (ROS) production of neutrophils was measured by Amplex Red Assay (Molecular probes), measuring hydrogen peroxide (H 2O 2) production under unstimulated and stimulated (zymosan, streptozotocin (STZ), factor-n-formyl-MET-LEU-Phe (fMLP), Phorbol myristate acetate (PMA)) and platelet-activating factor-fMLP (PAF-fMLP)). Endothelial activation was assessed by measuring von Willebrand Factor antigen (vWF-Ag) using enzyme-linked immunosorbent assay (ELISA) (Glostrup). Prothrombin fragment 1+2 (F 1+2), thrombin-antithrombin complexes (TAT) and D-dimer were measured using ELISA. Data are presented as median with interquartile range (IQR), unless stated otherwise. A P value of <0.05 was considered significant. Results 15 SCD patients (11 HbSS, 4 HbSβ 0-thalassemia), 5 female, median age 35 years (IQR 27-46)) completed 3-month follow-up. Hb levels increased significantly after 3 months of treatment (from 7.4 (6.9 - 8.1) g/dL to 9.7 (7.9 - 11.0) g/dL, p=0.002), corresponding to significant decreases in LDH and bilirubin levels and reticulocyte counts as markers of hemolysis (p<0.05). PoS decreased significantly after 3 months (from 20.1 (17.5 - 26.1) mmHg to 16.1 (10.3 - 18.1) mmHg, p=0.006). Both EI max and EI min increased after 3 months of treatment (from 0.33 (0.24 - 0.42) to 0.38 (0.28 - 0.48), p=0.029 and from 0.13 (0.07 - 0.26) to 0.21 (0.16 - 0.41), p=0.007 respectively). Markers of neutrophil activation, adhesion and aging were generally lower after 3 months of treatment, though the differences were not statistically significant (Figure 1 A-C). Neutrophil ROS production in both unstimulated and stimulated condition remained unchanged (Figure 1D). After 3 months of treatment vWF-Ag significantly decreased (from 252 (187 - 284) % to 183 (153-205) %, p = 0.033). No changes were observed in markers of coagulation activation (F 1+2 and TAT, and D-dimer). Conclusion Voxelotor significantly improved erythrocyte deformability as suggested by decreased PoS and increased EI max and EI min, potentially leading to improved rheological properties. While markers of neutrophil activation seem to decrease, the differences are not statistically significant, possibly due to the relatively small sample size. A reduction in vWF-Ag was observed possibly reflecting diminished endothelial activation, due to a decrease in intravascular hemolysis.

Red Cell Rheology and Blood Viscosity in Pediatric Individuals Having Received Allogenic Hematopoietic Stem Cell Transplantation or Ex Vivo Autologous Gene Therapy for Sickle Cell Disease

Background: Allogenic hematopoietic stem cell transplantation (HCT) and ex vivo autologous gene therapies (GT) are potentially curative treatments for sickle cell disease (SCD). However, there is debate around how to define cure including what degree of donor chimerism or level of functional hemoglobin constitutes a cure. Information on red cell function post-HCT/GT and how it relates to donor chimerism and resolution of SCD complications is scarce. Red cell function tests are not currently part of follow-up care post-HCT but may provide helpful information on the degree of red cell correction and risk of SCD complications. To address this knowledge gap, we report rheology and whole blood viscosity data on HbSS patients post-HCT or GT. Methods: Blood samples were collected under an IRB-approved protocol at Baylor College of Medicine and Emory University School of Medicine. We analyzed elongation index maximum and minimum (EImax, EImin), point of sickling (PoS), hematocrit-viscosity ratio at shear rates of 45 and 225 s-1(HVR45 & HVR225), and dense red blood cell % (DRBC%). EImax, EImin, and PoS were measured using a Laser Optical Rotational Red Cell Analyzer (Lorrca, RR Mechatronics, The Netherlands). Chimerism was measured by short tandem repeat testing (STR) after cell density sorting and DRBC% was measured on ADVIA cell counter (Siemens, Germany). HCT patients were compared with controls matched to the donor's genotype, and patients post-GT were compared with HbAS controls (HCT: HbAA donor =8, HbAS donor=19, GT=2, HbAA controls=42, and HbAS controls=15). Stata 18.0 (College Station, Texas, USA) was used to perform statistical analyses. Median values and ranges were used to describe the data. Wilcoxon rank sum test was used to compare groups and a mixed model was for the longitudinal data. A p-value <0.05 was considered statistically significant. Results: There were 29 patients with a median age of 6.6 years (range: 2.0-16.3) at HCT/GT and a median follow-up of 2.4 years (range: 0.1-7). A total of forty-three samples were analyzed. Median donor myeloid chimerism was 94% (33-100). Overall, the HbS% ranged from 28.9 - 45.2% with HbAS donors and 0% with HbAA donors. Two HbAS HCT patients had myeloid chimerism ≤ 50% with a follow-up period of 3.3 and 1.9 years, respectively. The myeloid donor% and HbS% were 33% and 50%, and 45.2% and 41.6%, respectively. At the first post-HCT/GT time-point (median 1 year, range: 0.1-6.9), DRBC%, EImax, and EImin were lower in HbAA donor HCT patients compared to HbAA controls (0.593 vs 0.604, p=0.008; 0.581 vs 0.604, <0.001; and 0.75 vs 0.20, p=0.02, respectively). RBC function improved with a longer follow-up from HCT/GT (median 2.9 years, range 1.3-7) with EImax showing a trend of statistical significance (p=0.07). Patients with HbAS donors had significantly higher point of sickling (PoS) values compared to the patients with HbAA donors (p=0.02). There was a trend for a lower HVR225 in patients with HbAS donors compared to the patients with HbAA donors (p=0.08). Seven patients (24%) had one or more red cell function test values outside the genotype-matched control range despite having myeloid donor chimerism of > 25-30% - a value commonly used in clinical practice to define cure (Table & Figure). Conclusion: A substantial number of patients had abnormal red cell function tests following HCT/GT despite achieving donor engraftment. Donor myeloid chimerism ≥ 25% has been reported protective from SCD complications while a chimerism ≥50% normalized tests for hemolysis. Higher odds of SCD-related complications have been reported with higher PoS and lower EImax/EImin. Thus, we propose that red cell function should be included in follow-up care of HCT/GT patients until values normalize and clinical relevance is further assessed.

Red Blood Cell Deformability and Morphological Dynamic Sickling As a Metric to Monitor Pharmacodynamic Response to Voxelotor

The recent introduction of new therapies, such as hemoglobin (Hb) oxygen affinity modifiers, fetal Hb (HbF) inducers, and gene therapy (replacement of HbS with non-sickling Hb), that affect the Hb polymerization rate for sickle cell disease (SCD), highlight the need for new metrics to assess changes in sickling characteristics as therapeutic response. Primary outcomes in clinical trials often focus on indirect clinical improvement i.e. decrease in vaso-occlusive events (VOC), Hb increase, fatigue and pain score. There is currently a lack of direct biomarkers to assess the pharmacodynamic impact of these therapies. Two methods have been developed to assess downstream Hb polymerization, LoRRca (oxygenscan), a commercial oxygen gradient ektacytometry that measures deformability as a function of oxygen tension (pO 2), and dynamic sickling assay (DSA), an enzymatic oxygen scavenging system that directly assesses morphological sickling. In the present study, we investigate the relationship between red blood cell (RBC) deformability and kinetics of sickling morphology after a treatment with different doses of voxelotor (GBT440, Global Blood Therapeutics), the only FDA approved Hb oxygen modifier. Blood samples from eight SCD patients were collected following the IRB #041718MP2E & IRB #Pro00066220 protocols. RBCs were incubated with voxelotor at five different doses based on sample hematocrit (Hct): 25% (0.32 ± 0.06 mM), 50% (0.65 ± 0.11 mM), 75% (0.97 ± 0.17 mM), 100% (1.29 ± 0.23 mM) and 125% (1.62 ± 0.28 mM), or with an equal volume of DMSO-containing buffer (vehicle). Oxygenscan is performed (LoRRca, RR Mechatronics, The Netherlands) as per manufacturer's instructions. The following parameters are calculated: i) elongation index (EImin), measured at minimum RBC deformability, ii) point of sickling (PoS), the pO 2 at which RBC deformability decreases below 5% of EImax during deoxygenation and iii) ΔEI, the difference between EImin and EImax. DSA uses an enzymatic reaction that allows for tight control of the rate and depth of hypoxia, resulting in RBC morphological changes observed through time-lapse photography. Sickling profiles were constructed to represent: i) morphological points of sickling at 50% of induced fraction (mPoS i@50%), ii) rate of sickling at 50% of induced fraction (rate i@50%) and iii) area under the curve at 15 minutes (AUC 15). Paired t-test analysis of the LoRRca elongation curves show a significant dose response in Elmin (Vehicle: 0.16 ± 0.16; 25%: 0.20 ± 0.16, p=0.0033; 50%: 0.23 ± 0.15, p=0.0001; 75%: 0.27 ± 0.15, p=0.0004; 100%: 0.30 ± 0.14, p=0.0009; 125%: 0.35 ± 0.13, p=0.0015), ΔEl (Vehicle: 0.26 ± 0.11; 25%: 0.22 ± 0.11, p=0.0016; 50%: 0.18 ± 0.11, p=0.0003; 75%: 0.15 ± 0.08, p=0.0002; 100%: 0.11 ± 0.06, p=0.0004; 125%: 0.09 ± 0.07, p=0.0011) and PoS (Figure 1; Vehicle: 35 ± 11 mmHg; 25%: 32 ± 6, p=0.0092; 50%: 28 ± 7, p=0.0038; 75%: 25 ± 6, p<0.0001; 100%: 17 ± 5, p=0.0007; 125%: 14 ± 4, p=0.0015). These findings indicate improvements in RBC deformability under hypoxic conditions when the drug is present. Furthermore, paired t-test of DSA data showed a significant dose response with AUC 15 (Vehicle: 958 ± 104; 25%: 909 ± 105, p=0.0061; 50%: 845 ± 146, p=0.0004; 75%: 768 ± 194, p=0.0014; 100%: 672 ± 179, p<0.0001; 125%: 623 ± 192, p=0.0002), rate i@50% (Vehicle: 0.87 ± 0.26; 25%: 0.77 ± 0.30, p=0.1236; 50%: 0.61 ± 0.29, p=0.0003; 75%: 0.33 ± 0.13, p<0.0001; 100%: 0.28 ± 0.10, p<0.0001; 125%: 0.25 ± 0.09, p=0.0004) and mPoS i@50% (Figure 2; Vehicle: 4.4 ± 0.7 minutes; 25%: 4.7 ± 0.9, p=0.0260; 50%: 5.0 ± 1.2, p=0.0407; 75%: 5.9 ± 1.7, p=0.0082; 100%: 7.0 ± 1.6, p=0.0002; 125%: 7.8 ± 1.8, p=0.0004). These results indicate a delay in morphological sickling due to drug treatment. Based on the results of our study there is a dose-dependent decrease in RBC deformability and in the kinetics of morphologic sickling based of DSA following voxelotor treatment. These two methods have demonstrated a comparable ability to assess downstream therapeutic effects on delaying/inhibiting HbS polymerization. The combination of these two unique properties, RBC deformability and morphology change, can possibly provide a comprehensive approach to describe the cellular level of phenotypes, which may predict SCD severity. With further development these complementary assays have the potential to become important tools for drug development and monitoring response to SCD therapies.

Pyruvate Kinase Thermostability Is Associated with Red Blood Cell Adhesion, Deformability and Oxygen Affinity in Patients with Sickle Cell Disease

Introduction: Sickle cell disease(SCD) is caused by a mutation in the beta globin gene that causes formation of abnormal hemoglobin S (HbS). Red blood cells (RBCs) from patients with SCD are less deformable and take on the shape of sickle upon deoxygenation. This results in multiple downstream effects including RBC adhesion, microvascular occlusion and chronic hemolysis. RBCs rely solely on anaerobic glycolysis to produce adenosine triphosphate (ATP), which is needed to maintain the cell's structure and function. Pyruvate kinase (PK) is a key enzyme involved in the last step of glycolysis. We have previously demonstrated a decrease in PK activity and stability as a novel pathophysiological feature of SCD. Currently there are several clinical trials ongoing investigating the efficacy of PK activation by small molecules such as mitapivat (NL8517, NCT04000165, NCT05031780), AG-946 (NCT04536792) and etavopivat (NCT04624659, NCT04987489). In this study we aim to investigate how PK properties correlate to sickle RBC properties in order to explore if impaired PK function is associated with specific features of the complex SCD pathophysiology. Methods: Homozygous HbS (HbSS) and HbS/ β0 thalassemia patients were eligible to participate. Patients who received blood transfusion (<3 months) were excluded. Dense RBCs (MCHC >41 g/dL, Advia 120/2120 Siemens) and HbF and HbS levels (Tosoh G8) were measured. PK and hexokinase (HK) activity were measured on purified RBCs. PK thermostability (reflected by % residual PK activity) was measured on purified RBCs lysates after one hour of incubation (53°C). RBC adhesion to laminin (Biolamina) was measured using a microfluidic device (IBIDI µ-Slide I 0.4). RBC hydration (Ohyper), RBC deformability (EImax) and point of sickling (PoS) were assessed by osmotic and oxygen gradient ektacytometry using the Laser-Optical Rotational Red Cell Analyzer (Lorrca, RR Mechatronics). Oxygen affinity (P50) was measured with the Hemox-Analyzer (TCS). Statistical analysis was performed in Graphpad Prism (Pearson's correlation). Results: Fifty-seven patients with SCD (53 HbSS, 4 HbS/β 0 thalassemia) were included of which 17 were children (median age 14 years [range 6-17]) and 40 were adults (median age 36 years [range 18-58]). Baseline characteristics are depicted in Table 1. Figure 1 shows a heatmap with correlation coefficients of all laboratory parameters from Table 1 as measured in this cohort. When focusing on PK properties we found that patients with SCD and lower PK thermostability had significantly higher absolute reticulocyte counts (ARC, p<0.0001), lower hemoglobin (p=0.012) and HbF (p=0.020) levels and increased RBC adhesion to laminin (p=0.007). Furthermore, PK thermostability was significantly correlated with EImax (p=0.017) and inversely correlated with PoS (p=0.011) and P50 (p=0.003), indicating that patients with less stable RBC PK had less deformable RBCs which sickled at a higher oxygen tension. RBC adhesion to laminin was significantly correlated with both ARC (p=0.0003) and dense RBCs (p=0.019). To explore whether our findings were a reflection of reticulocytosis we fractionated whole blood samples of HbSS patients according to cell density. We found that more RBCs from the most dense fraction adhered to laminin than from the less dense, reticulocyte enriched, fraction, even though RBCs from this less dense fraction had reduced PK-thermostability. Additionally, the most dense RBCs had the lowest PK activity, were less deformable and had a higher PoS (data not shown). Altogether these findings indicate that the reduced PK thermostability we found in our SCD cohort is associated with an overall functionally impaired RBC population with increased RBC adhesion and ARC. We found no correlation of markers of RBC dehydration (Ohyper and % dense RBCs) with PK thermostability which suggests that RBCs with reduced PK thermostability are not more dehydrated. Discussion/conclusion: This study shows for the first time a significant correlation between PK thermostability in sickle RBCs and RBC functions, such as adhesion, deformability and oxygen affinity. Our results suggest that enhancing the activity and stability of PK, with PK activators, is an attractive target in SCD that might improve other pathophysiological targets outside RBC metabolism. Future studies are needed to explore how activation of PK will affect these altered sickle RBC properties in vivo.

Effects of L-Glutamine on Biomarkers of Response in Sickle Cell Disease: A Pharmacokinetics-Pharmacodynamics Analysis

OralL-glutamine (Endari®) has been approved by the US-FDA to reduce the acute complications of sickle cell disease (SCD) in patients at least 5 years of age. L-glutamine has several roles in the body including de novo synthesis of intracellular glutathione which acts as an antioxidant. However, the mechanisms of action by which L-glutamine could reduce complications in SCD require further investigation. Additionally, there are no known biomarkers to assess response to L-glutamine therapy. We conducted an open-label, dose-ascending trial (NCT04684381) of L-glutamine in pediatric (n=4) and adult (n=4) participants with SCD as well as in adult healthy volunteers (n=4) (Table 1). Over a three-week trial period with 4 study visits, serial blood samples were collected to define the pharmacokinetics (PK), pharmacodynamics (PD), and PK-PD interactions of L-glutamine using a broad panel of laboratory investigations including amino acid concentrations, blood counts, percentage of dense red blood cells (%DRBC), whole blood viscosity, osmotic and oxygen gradient ektacytometry, and reactive oxygen species (ROS) of reticulocytes (ROS retic) and mature red blood cells (ROS RBC). In PK-PD analysis of amino acids, we focused analysis on arginine (Arg), citrulline (Cit), and ornithine (Orn) based on the hypothesis that L-glutamine improves endothelial function by increasing arginine bioavailability and augmenting nitric oxide (NO) production. Peak arginine concentration was directly correlated with both the maximum L-glutamine concentration (C max p=0.001) and area-under-the curve (AUC) (p=0.047) indicating improvement in arginine bioavailability, but there was no significant linear correlation with either the Arg:Orn or Arg:(Orn+Cit) ratios. In PK-PD analysis of osmotic gradient ektacytometry, L-glutamine C max was directly correlated with the elongation maximum (EI max), noted in all participants at visit 4 (p=0.033). In SCD participants, L-glutamine C max was inversely correlated with O hyper (osmolality at EI max/2) at visits 3 (p=0.044) and 4 (p=0.004). In PK-PD analysis of oxygen gradient ektacytometry in SCD participants, L-glutamine C max was inversely correlated with the point of sickling (POS) at visit 3 (p=0.005) but not at visit 4 (p=0.054). Together, these findings suggest that although L-glutamine may decrease cellular hydration (decreased O hyper) it may increase RBC deformability (increased EI max) and delay the onset of sickling after hypoxia (decreased POS). In PK-PD analysis of viscometry, higher L-glutamine C max was associated with higher whole blood viscosity measurements at all shear rates (p<0.05). However, L-glutamine C max was also directly correlated with hematocrit-to-viscosity ratio (HVR) at the highest shear rates in the overall population at visit 4 (p<0.05). In addition, L-glutamine C max for SCD participants was associated with increased hemoglobin concentration, an effect that was observed at visit 4 (p=0.025), and increased %DRBC, an effect detected at visits 3 (p=0.008) and 4 (p=0.007). In PK-PD analysis of ROS, L-glutamine AUC was inversely correlated with ROS Retic (p=0.024), but not ROS RBC, in all participants at visit 3. When considered as total ROS Retic (calculated as ROS retic × absolute reticulocyte count), there was a similar inverse correlation with glutamine AUC in SCD participants, although not statistically significant. In contrast to all other biomarkers, L-glutamine C max was not correlated with any ROS measurement. This PK-PD analysis of L-glutamine reveals biological effects that alter RBC characteristics and could modify SCD-related complications, with a complex interplay summarized in Figure 1. Prospective studies can validate these biomarkers and be used to monitor the effects of L-glutamine therapy in SCD patients.

5589320 FOLLOW-UP RESULTS OF A PHASE 2 STUDY ASSESSING THE SAFETY AND EFFICACY OF MITAPIVAT TREATMENT, AN ORAL PYRUVATE KINASE ACTIVATOR, FOR UP TO 60 WEEKS IN SUBJECTS WITH SICKLE CELL DISEASE

Background: Sickle cell disease (SCD) is a devastating red blood cell (RBC) disorder. The root cause of SCD is polymerization of sickle hemoglobin (HbS) upon deoxygenation, resulting in poorly deformable sickled RBCs with shortened lifespan. 2,3-diphosphyglycerate (2,3-DPG), a glycolytic intermediate in RBCs, promotes deoxygenation by lowering hemoglobin (Hb)-oxygen affinity. Mitapivat (AG-348) is an oral allosteric activator of pyruvate kinase (PK), a key enzyme in RBC glycolysis generating adenosine triphosphate (ATP) and reducing 2,3-DPG levels. Aims: To report follow-up data of the safety and efficacy of mitapivat treatment in subjects with SCD (ESTIMATE study; www.trialregister.nl NL8517; EudraCT 2019-003438-18). Methods: The ESTIMATE study is a phase 2, investigator initiated, open-label study. Subjects ≥16 years with SCD (HbSS, HbS/β0 or HbS/β+), 1-10 vaso-occlusive crises (VOCs) in the prior year and/or prior SCD-related complications, a Hb level >6.1 g/dL and ≤11.1 g/dL, and no chronic transfusions are eligible and treated with mitapivat after obtaining informed content. In the 8-week dose finding period, initial dosing is 20 mg twice daily, escalated to max. 100 mg twice daily, and followed by an up to 52-week fixed dose extension period (FDEP). Primary endpoints are safety, based on adverse events (AEs), and efficacy of mitapivat treatment on RBC sickling (Point of Sickling (PoS), oxygen gradient ektacytometry). Key secondary endpoints are changes in Hb level, markers of hemolysis, ATP/2,3-DPG levels, and Hb-oxygen affinity (p50, Hemox Analyzer). Results: Until Jan. 2022, 9 subjects were treated with mitapivat. Baseline characteristics were: median age 22 years (range 16-59 years), 6/9 (67%) female, and 6/9 (67%) used hydroxyurea. 7/9 (78%) had HbSS, 1/9 (11%) had HbS/β0, and 1/9 (11%) had HbS/β+. One subject with a non-treatment related serious AE (SAE) of a urinary tract infection (grade 4), was lost to follow-up shortly after first dosing. No other SAEs or treatment-emergent AEs (TEAEs) grade ≥3 occurred. The remaining 8 subjects had a median treatment duration of 38 weeks (range 11-60 weeks). 6/8 (75%) took 100 mg mitapivat twice daily in the FDEP, and 2/8 (25%) took 50 mg mitapivat twice daily after a single dose reduction because of progressive transaminase increase. The most common TEAEs were (n>2 subjects): ALT or AST increase (both 5/9, 56%; all grade 1), and headache (4/9, 44%; grade 1-2). Three VOCs occurred: one related to excessive alcohol consumption with no need for hospitalization, and two in subjects with documented noncompliance the week before hospitalization. Mean annual VOC rate and SCD-related hospital admission days were, respectively, 1.5±1.3 and 5.9±7.1 days at baseline, and reduced to 0.5±0.7 and 1.6±3.1 days when weighting cases by follow-up duration (p=0.021 and p=0.134, respectively). Table 1 summarizes mean changes in parameters of primary and key secondary endpoints of scheduled visits in the FDEP compared to baseline. Hb level significantly increased, accompanied by a decrease in markers of hemolysis (absolute reticulocyte count, total bilirubin, lactate dehydrogenase). Mean PoS decreased, and p50, 2,3-DPG level and ATP/2,3-DPG ratio significantly improved. Conclusion: Treatment with mitapivat for up to 60 weeks in subjects with SCD showed no treatment related TEAEs grade ≥3. Improvements in anemia, markers of hemolysis, oxygen affinity, 2,3-DPG level and ATP/2,3-DPG ratio were seen.

GBT021601 Increases Hemoglobin and Improves Red Blood Cell Deformability without Rebound Hyperviscosity upon Drug Clearance in a Sickle Mouse Model

Background: Whole-blood viscosity and red cell deformability are markedly abnormal in sickle cell disease (SCD) due to sickling of red blood cells (RBCs). Increased whole-blood viscosity is a risk factor for vaso-occlusive crisis (VOC), avascular necrosis, and proliferative retinopathy. Blood viscosity is strongly impacted by hemoglobin (Hb) levels and RBC deformability. GBT021601 is a potent next-generation sickle hemoglobin (HbS) polymerization inhibitor, with anti-sickling properties, that increases the oxygen affinity of Hb. In preclinical studies, treatment with GBT021601 achieved higher levels of Hb than the US Food and Drug Administration-approved first-generation compound voxelotor, raising concern that this rise in Hb would substantially increase whole-blood viscosity and impose a VOC risk after treatment cessation. Objective: To investigate a potential rebound hyperviscosity effect from GBT021601 cessation in a murine model of SCD by determining the effect on whole-blood viscosity and on RBC deformability under a range of oxygen concentrations using oxygen gradient ektacytometry. Methods: Nine-week-old Townes HbSS mice were fed chows containing 0.2% GBT021601 for 5 weeks, then transitioned to control chow. Whole blood was collected in an EDTA tube. All tests were conducted within 4 hours of collection. Using an Element HT5 hematology analyzer (Heska), complete blood count was obtained from mice while on GBT021601 (day 0) and then on days 1, 3, and 5 after transition to control chow. Blood viscosity was measured with a cone/plate viscometer (AMETEK Brookfield) at 37 °C at shear rates of 45 s-1 and 225 s-1. Hematocrit to viscosity ratio (HVR) was calculated to determine RBC oxygen-carrying capacity. RBC deformability was measured by oxygen gradient ektacytometry (Lorrca, RR Mechatronics). Results: Treatment with GBT021601 significantly increased Hb from 8.0 to 12.9 g/dL (P<0.0001) without increasing HVR. Compared with previous studies with the voxelotor analog GBT1118, Hb dropped off more slowly with GBT021601 treatment; this allowed for the ideal assessment of whole-blood viscosity and HVR when the drug had cleared, and HbS was no longer modified, despite elevated Hb levels. HVR at both 45 s-1 and 225 s-1 showed no significant change with drug clearance. GBT021601 treatment also improved RBC deformability, as indicated by a higher elongation index (EI) under both normoxic (EImax, P<0.001) and hypoxic (EImin, P<0.0001) conditions. Point of sickling (PoS) was also reduced (P<0.0001), indicating that treated RBCs tolerated lower oxygen concentrations before sickling. When the drug was withdrawn, PoS and EImin returned to baseline levels, whereas EImax remained elevated until day 5 off treatment. Hb also remained elevated until day 5 off treatment, despite the return to baseline levels of the EImin, PoS, and p50 (partial oxygen pressure at which Hb is 50% saturated with oxygen), indicating drug clearance. Conclusions: In response to GBT021601, sickle cell mice showed a robust increase in Hb, comparable to that of a HbAS mouse, as well as a marked improvement in PoS and RBC deformability. When normalized to HVR, there was no significant difference in blood viscosity during treatment and after cessation of treatment. This finding suggests that oxygen-carrying capacity is not impaired and provides no indication for rebound hyperviscosity with drug clearance. Moreover, the observation that EImax remained elevated until 5 days after drug cessation suggests that some residual rheological improvement occurred in the RBC population that did not dissipate when the drug was cleared. In vitro addition of GBT021601 (and other related compounds) to blood rapidly increases the EImin and reduces the PoS of RBCs but does not alter the EImax. In contrast, an increase in the EImax of RBCs has been consistently observed with GBT021601 treatment in vivo, including in early clinical studies in patients with SCD. The precise mechanism by which this benefit is obtained requires further study. Funding: Global Blood Therapeutics.

An Endogenous Metabolic Modulator Composition Improves Hydration Status and Decreases Adhesion Properties of Red Blood Cells from Patients with Sickle Cell Disease

Background Sickle cell disease (SCD) is a group of hemoglobinopathies caused by point mutations in the β-globin gene leading to abnormal red blood cells (RBCs). There are various forms of SCD; sickle cell anemia (SCA) is characterized by homozygosity for hemoglobin (Hb) S (HbSS) whereas HbSC disease results from compound heterozygosity for HbS and HbC. HbC does not lead to RBC sickling, but induces loss of intracellular K+ and H2O causing RBC dehydration. Similarly, in HbSS RBCs, through dehydration, an increase of the intracellular concentration of poorly soluble HbS enhances polymerization upon deoxygenation, causing RBCs to become rigid and to sickle. Until recently, the only approved therapy for SCD was hydroxyurea (HU). HU increases fetal Hb (HbF) levels and improves RBC hydration and deformability. For decades, amino acids have been explored as potential treatment to restore the disrupted pathophysiological pathways in SCD. RCitNacQCarLKHVS, an Endogenous Metabolic Modulator (EMM) composition of ten different amino acids and derivatives, was designed to target and support RBC metabolism. In this study, we investigated the effect of ex vivo treatment of this EMM on RBC properties and function in SCA and HbSC. Methods Blood of 12 different adult SCD patients (9 HbSS [of whom 6 were on chronic RBC exchange (RBCX)], 3 HbSC) and 2 healthy controls was collected. Whole blood was incubated at standardized hematocrit (20%, patient plasma) in the presence or absence of different RCitNacQCarLKHVS concentrations for 4h at 37°C. Routine hematological parameters were measured using the Cell-Dyn Sapphire (Abbott Diagnostics). Percentage of total dense RBCs (%DRBCs) was analyzed from 10/12 patients (4 HbSS on RBCX, 3 HbSS not on RBCX, 3 HbSC) using the Advia 120/2120 hematology analyzer (Siemens Healthcare Diagnostics). Point of sickling (PoS), RBC deformability (EImax), and hydration status (Ohyper) were assessed by oxygen gradient ektacytometry and osmotic gradient ektacytometry, respectively, in 12/12 patients using the Lorrca (RR Mechatronics). Blood viscosity was measured in 6/12 patients (3 HbSS not on RBCX and 3 HbSC) at 37°C using a cone-plate viscometer (Brookfield). The hematocrit-to-viscosity ratio (HVR) was calculated as follows: hematocrit/blood viscosity at 300 s-1. RBC adhesion to laminin (BioLamina) was assessed in 4/12 patients (3 HbSS not on RBCX, 1 HbSC) using a microfluidic device (IBIDI μ-Slide VI 0.4). Statistical analysis was performed in Graphpad Prism v9.3.0 using the Friedman test. Results Ex vivo treatment of blood of SCD patients with RCitNacQCarLKHVS showed a significant dose-dependent increase in Ohyper as measured by osmotic gradient ektacytometry (Figure 1), indicating an improvement of RBC hydration status of SCD RBCs. This was accompanied by a significant increase in mean corpuscular volume (MCV) and reduction in mean corpuscular Hb concentration (MCHC). RBCs of healthy controls showed a similar trend in Ohyper, MCV and MCHC. In addition, %DRBCs showed a significant dose-dependent decrease in both HbSS and HbSC RBCs as a result of RCitNacQCarLKHVS treatment. Oxygen gradient ektacytometry showed a significant increase in maximum deformability (EImax), but no significant change in point of sickling. The HVR did not significantly change during dose-response experiments. Median RBC adhesion to laminin decreased as a result of ex vivo treatment with RCitNacQCarLKHVS (Figure 2). Conclusion Ex vivo treatment with RCitNacQCarLKHVS demonstrated a significant dose-dependent increase in RBC hydration (Ohyper, MCV, MCHC and %DRBCs), deformability (EImax) and decreased adhesion properties in SCD RBCs. Dehydrated and dense RBCs with reduced deformability and increased adhesion are more prone to hemolysis, and associated with vaso-occlusive complications such as priapism, skin ulcers, and nephropathy. Results from this study therefore suggest benefit of EMM in modulating clinically-relevant rheological properties such as hydration state and adhesion of SCD RBCs in both SCA and HbSC. The fact that HVR did not change may represent an additional therapeutic benefit, especially for HbSC patients who have, in general, higher blood viscosity compared to HbSS patients. Moreover, it would be worthwhile to investigate if the concomitant use of select amino acid combinations with HU or other anti-sickling agents has an additional beneficial effect in SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Compound Nanoparticle 5HMF-Prodrug Formulations for Optimized Anti-Sickling Therapy

BACKROUND: Sickle cell disease (SCD) pathology stems from deoxygenation-induced hemoglobin (HbS) polymerization; allosteric Hb adducts that increase oxyHbS target this mechanistic trigger. 5-hydroxymethylfurfural (5HMF) binds to Hb αVal1, reducing O2 affinity & sickling and protecting hypoxic SS mice (survival, organ injury). However, 5HMF did not advance beyond PhI/II trials, due to pharmacokinetic/dynamic (PK/PD) limitations. A related molecule (Voxelotor) was approved based on surrogate endpoints (Hb level and hemolytic markers), but without improved vaso-occlusive morbidities. We reformulated 5HMF, a potentially superior agent, to address translation limitations arising from disfavorable PK/PD. 5HMF PK/PD: RBCs from SCD patients were incubated with 5HMF (20% Hct, 37C, HMF: 0-5mM, 0-3 hrs). 5HMF-induced Hb modification was quantified by HPLC (Waters e2695XE, Swift WCX-PEEK); O2 dissociation curves (ODC) were measured in intact RBCs (HEMOX); pO2's at the point of sickling (POS) and point of unsickling (POU) were determined during controlled Hb O2 unloading/loading, respectively (LORRCA Oxygenscan). We observed a dose dependent increase in 5HMF modified Hb, that was maximal (~75%) at 1 hr and 5mM [5HMF], and that resulted in: (1) 35% increase in HbO2 affinity (ODC p50 pH 7.4; 31.1 ± 3.1 Torr vs 20.3 ± 1.5 Torr; 0 vs 5mM 5HMF), (2) 30% POS reduction (56.9 ± 10.1 vs 39.8 ± 11.6 Torr; 0 vs 5mM 5HMF) and (3) 45% POR reduction (38.3 ± 9.1 vs 21.1 ± 5.7 Torr; 0 vs 5mM 5HMF) (p < 0.05 for all, RMANOVA). PRODRUG NPs: We designed two complementary 5HMF (phospholipid and graphitic) prodrugs (Lipid Prodrug; LP and carbon dot (CD)) that were evaluated individually and as an integrated self-assembled compound nanoparticle (CompNP). Phospholipid LP 'pro-HMF' synthesis: 5HMF was treated with NaH and THF (RT, N2), then mixed with 1-Lysophosphatidylcholine (16:0 lysoPC) and 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxy-succinimide (NHS) (anhydrous chloroform, RT, ON). Graphitic CD 'pro-HMF' synthesis: sucrose in water (0.2 μM, 18 MΩ cm) was heated (270C, 30m), followed by sonication (Q700, 20m: Amp, 1, on: 2s, off: 1s), filtering (0.2 μm) and dialysis (10 kD MWCO, 2d). Simple LNPs (comprised only of LP pro-5HMF) were prepared by combining 5HMF LP (5 mM) with L-α-Phosphatidylcholine (1:1) in chloroform/methanol, then drying (thin film) that was resuspended (PBS), vortexed and sonicated (amp 1, on 2s, off 1s, 10m) and dialyzed (2d, PBS). Compound Nanoparticle (CompNP) self-assembly: CDs in PBS were mixed with the above lipid film, followed by sonication and dialysis (as above) resulting 152±12nm (hydrodynamic size) particles with -34±6mV surface charge. All formulations were extensively characterized (physicochemical features and biodegradation mapping). NP PK/PD: We found that the prodrugs release 5HMF in RBCs through complimentary mechanisms (acutely from the LP shell by phospholipase A2 (PLA2) activity and slowly/sustained from the CD core by enzyme-triggered and peroxide-based degradation). Then, using human SS RBCs, we quantified time- and concentration-dependent in vitro PK/PD parameters (partially presented here): 3h data for the composite NP: Hb-5HMF modification (HPLC, 76.7±8.9% 5HMF modificatied HbS), HbO2 affinity (p50 15.4±3.8Torr) and antisickling potency (LORRCA deformability/elongation index rose by 0.28±3.8.07AU & point of sickling fell by 21.4±8.2Torr); imaging: ~75% reduced hypoxia-induced sickling (p<0.05, ANOVA, all). Notably, PK (5HMF modified SHb) for LNP & CompNP was sustained well beyond that for free 5HMF and was progressive beyond 24h (likely due to increased PLA2 and oxidizing milieu in SRBCs). PD (anti-sickling) for the CompNP formulation exhibited the greatest efficacy for (POS) (eg hypoxia tolerance) and POR (simulating O2 loading during pulmonary circulatory transit); as for our PK data, effect upon POS/POR progressed from 1h to 3h. NB: No produg/NP formulation resulted in hemolysis > 1-2%. SUMMARY: We developed first in class RBC-targeted, protected, biocompatible, self-assembled 5HMF prodrug nanoparticles for SCD therapy that are designed for eventual transdermal delivery. In vitro proof of concept data demonstrates: (1) improved potency, (2) targeted payload delivery and subsequent extended (>24h) prodrug release within RBCs, and (3) with prolonged pharmacodynamic (antisickling) effect.

The Pyruvate Kinase Activator Mitapivat Improves Red Blood Cell Deformability and Sickling Kinetics in Adult Patients with Sickle Cell Disease

Background: A hallmark of sickle cell disease (SCD) is compromised red blood cell (RBC) deformability. Polymerization of hemoglobin S (HbS) underlies this "sickling" which results in altered biomechanical properties that impact rheology and hemodynamics (Barabino et al, 2010). There have been exciting clinical developments in anti-sickling therapies - including enhancement of red cell pyruvate kinase (PKR) modality. Importantly, deformability measurements of RBCs via ektacytometry are increasingly used to aid the assessment of their therapeutic efficacy. Here we employed the Laser assisted Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics), which permits the evaluation of RBC deformability as a function of shear stress, osmotic pressure, and oxygen tension, to evaluate the changes in RBC deformability in subjects undergoing treatment with mitapivat (AG-348), a PKR activator under clinical development for sickle cell disease. We analyzed changes in deformability measurements of RBCs in HbSS subjects after 4 weeks and 12 weeks of mitapivat therapy and compared them to t50 - a single parameter measure of sickling kinetics (Dunkelberger et al, 2018). Methods: We studied subjects currently enrolled under IRB approved clinical trial NCT04610866 that evaluates the long-term safety and tolerability of mitapivat in SCD patients. Fifteen subjects (aged 25 - 57 years; 5 females, 10 males; African/African-American) were evaluated, though not all endpoints were available for every patient across all assays. Fresh whole blood (collected in EDTA tubes) was obtained at baseline (prior to drug initiation), after 4 weeks and after 12 weeks of therapy. Cells were counted and diluted in polyvinylpyrrolidone (PVP) solution before undergoing same-day LORRCA assays. The Elongation Index (EI) is a measure of the cells' ability to undergo deformation at different physiologically relevant conditions. EI was assessed at increasing shear stresses of .3 Pascals (Pa), .95 Pa, 3 Pa, 9.49 Pa, and 30 Pa (elongation assay). Parameters derived under continuous osmotic changes (0-600mOsm/kg - osmoscan) include O min which informs on RBC fragility, O Hyper which informs on hydration state and corresponds to 50% of the maximum deformability in the hypertonic region, EI max (osmo) which reveals maximum deformability and informs on RBC cytoskeletal mechanics, and O Max which gives the osmolality at which EI Max is achieved. Point of Sickling (POS) gives the oxygen tension at which the cells start to sickle during nitrogen facilitated deoxygenation of the sample (oxyscan). These parameters of deformability are generated by LORRCA software. For all assays, we analyzed the % change from baseline after 4 weeks and 12 weeks of therapy (Wilcoxon signed rank test, R v1.4.1106) and compared these changes to those in t50 (Spearman correlation test, Prism v9) (Figure 1). Results: Mitapivat therapy resulted in increased elongation indices across all shear stress levels, with significance reached at shears of .3 Pa, .95 Pa, and 3 Pa (respective mean % changes of 78.5, 181.8, and 23.0 (4 weeks) and 101.2, 212.9, and 30.1 (12 weeks)) suggesting improved deformability of RBCs. Likewise, significant increases in O hyper (mean % changes of 2.8 and 3.4) and osmo EI Max (mean % changes 7.9 and 9.5) respectively, indicate improvements in RBC hydration and cytoskeletal dynamics in response to changing osmotic pressures. Furthermore, a significant decrease in the average point of sickling was noted at both 4 weeks and 12 weeks (-20.15% and -16.8%, respectively), suggesting RBCs were susceptible to sickling at partial pressures of oxygen lower than those observed before therapy. Conclusion: Altogether, these findings indicate that mitapivat therapy results in improved ability of RBCs to withstand deformational changes in response to shear stress, osmotic pressure, and decreasing oxygen tension, likely via bolstered membrane integrity. Interestingly, when compared to t50 values, significant correlation was observed at all levels of shear stress and at the maximal deformability (EI max) achieved during both the osmoscan and oxyscan assays. Acknowledgement: This study is part of a Cooperative Research and Development Agreement (CRADA) with AGIOS Pharmaceuticals, Inc., Cambridge, MA, United States Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pleiotropic Effects of Hydroxyurea on Red Blood Cell and Neutrophil Phenotype in Patients with Sickle Cell Disease; Data from the Notice Study

Introduction The pathophysiology of sickle cell disease (SCD) is complex and goes beyond red blood cell (RBC) sickling alone. Neutrophils have been proposed to play an important role in SCD pathophysiology, but their exact contribution remains to be elucidated. Factors such as chronic hemolysis and oxidative stress can influence neutrophil adhesion, activity and aging. We designed a prospective study (Notice) to investigate a broad set of neutrophil characteristics in SCD patients. To correlate treatment effects of hydroxyurea (HU) on neutrophils with those on RBCs in SCD, we also studied RBC sickling and adhesion. Here, we will focus on the effects of hydroxyurea on RBC deformability and neutrophil adhesion, activity and aging. Aim: To assess the effects of HU on RBC deformability and neutrophil phenotypes with respect to adhesion, activation and aging. Methods Blood (EDTA) was collected from SCD patients with HbSS or HbSβ0 in steady-state. A subgroup of patients was included before and during treatment with HU. For measurement of antigen expression by flow cytometry, total leukocyte fraction after lysis of red blood cells (without Percoll gradient) was used and neutrophils were gated based on their forward and side scatter. For in vitro neutrophil adhesion, neutrophils were isolated using a Percoll gradient and lysis buffer, as previously described. (Kuijpers et al. Blood 2007) Neutrophils were labeled with calcein and placed in an uncoated 96-well MaxiSorp plate. After washing, adherent cells were lysed and fluorescence was measured. (Kuijpers et al. J Allergy Clin Immunol 2017). Adhesion of RBCs was measured by flowing whole blood (20x106/ml)over laminin-coated Ibidi slides for 30 minutes with visualization by microscope. Percentage of mean RBC adhesion was normalized to a healthy control (HC). Data were analyzed using SPSS and GraphPad Prism and are presented as mean ±SE or ±SD as appropriate. Conventional statistics were used. Results A total of 49 patients (median age 25.0 (IQR 20.0 - 32.5) years, 39% female) were included. Twenty patients (41%) used a stable dose of HU at baseline. In 10 patients, blood was drawn before and after starting HU treatment. HU treatment improved hemoglobin F and markers of hemolysis and was associated with a decrease in the point of sickling (PoS) of RBCs (from median 24.8 mmHg [IQR 16.0-28.2] to 16.8 [15.0-20.8], P=0.13). Adhesion of RBC was more than 3-fold higher in SCD patients than in HC. At baseline, there was no difference in RBC adhesion between patients with and without HU (P=0.86). After starting HU treatment, RBC adhesion decreased significantly from a median of 318% (normalized to control) to 149% (P=0.016, Figure 1a-b). Adhesion of neutrophils was significantly higher in patients compared to HC (median 18.3% versus 12.6% P <0.0001). No difference was observed between patients with and without HU at baseline. Neutrophil adhesion did not decrease after starting HU treatment (Figure 1c-d). Neutrophil surface expression of CD11b/CD18 was measured as markers of adhesion. While CD11b expression was comparable between SCD patients and HC, CD18 expression was significantly higher in SCD patients (mean MFI 12193 vs 10586, P=0.016; Figure 2b. After treatment with HU both CD11b and CD18 expression decreased, though the differences did not reach statistical significance (P=0.22 and 0.56, respectively). Neutrophils of SCD patients displayed a more aged phenotype compared to neutrophils of HC with lower L-selectin (median MFI 3108 vs 4656, P=0.021) and higher CXCR4 (median MFI 1150 vs 886, P=0.003) expression. Activation marker CD64 was also higher in SCD patients compared to HC (median MFI 1841 vs 1065, P=0.0006). HU treatment seemed to modify all markers more towards a HC phenotype although no statistically significant changes were observed. Conclusion Adhesion of both RBCs and neutrophils is increased in SCD patients compared to HC. HU treatment was associated with significantly improved RBC rheology as shown by decreased PoS and decreased adhesion to laminin. Despite a trend to improvement in expression of adhesion markers (CD11b/CD18), in vitro adhesion of neutrophils did not decrease. These findings could be due to the small sample size of patients starting with HU or by a difference in vitro behaviour of changed composition of neutrophil populations after treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PI-05: PHARMACODYNAMIC EFFECTS OF AG-946, A HIGHLY POTENT NOVEL ACTIVATOR OF PYRUVATE KINASE, IN EX VIVO TREATMENT OF RED BLOOD CELLS FROM SICKLE CELL DISEASE PATIENTS

Purpose: Sickle cell disease (SCD) is a monogenetic red blood cell (RBC) disorder that is characterized by hemolytic anemia and vaso-occlusive crises. Among the many factors that contribute to disease pathophysiology is stiffening and sickling of RBCs, which is the direct result of the polymerization of abnormal hemoglobin S. Sickling is modulated by glycolytic intermediates such as 2,3-diphosphoglycerate (2,3-DPG) and ATP. Previously we showed that RBC pyruvate kinase (PKR), the key regulatory enzyme of glycolysis, is impaired in SCD and that ex-vivo treatment with mitapivat, an allosteric activator of PKR, increased enzymatic activity and thermostability, reduced 2,3-DPG levels, decreased p50, and subsequently reduced sickling (Rab et al, Blood 2021). Currently, mitapivat is being investigated in phase 1, phase 2, and phase 2/3 trials in patients with SCD (#NCT04000165, EudraCT#2019-003438-18, and NCT05031780). Recently, AG-946, a novel PK activator, has been developed. Here we investigate the pharmacodynamic effects of AG-946 in ex vivo treatment of RBC from SCD patients in comparison with mitapivat. Materials and methods: Buffy coat depleted whole blood obtained from five SCD patients was incubated for 20-24 hours in absence or presence of mitapivat (100mM) or AG-946 (1mM, 5mM, 50mM). After ex-vivo treatment, enzymatic activities of PKR and PKR-thermostability were measured. Glycolytic intermediates ATP and 2,3-DPG were measured using LC-MS/MS. Hemoglobin oxygen affinity (p50) was measured and RBC sickling was analyzed with oxygen gradient ektacytometry, a method that characterizes individual sickling behavior. Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO2 at which RBCs start to sickle during deoxygenation under shear stress. Results: PKR activity was increased compared to vehicle (DMSO) to a similar extent in the presence of mitapivat or AG-946 (Figure 1A). In addition, PKR thermostability was significantly increased compared to vehicle (mean 22%, SD 6%) in samples treated with mitapivat 100 mM (mean 78%, SD 11%), as well as AG-946 5mM (mean 66%, SD 23%), and AG-946 50mM (mean 95%, SD 17%, Figure 1B). After incubation with mitapivat or AG-946, 2,3-DPG decreased (Figure 1C), which was further illustrated by the improved ATP/2,3-DPG ratio (Figure 1D). Accordingly, p50 decreased significantly after incubation with mitapivat 100mM (mean 95%, SD 2%), as well as AG-946 1mM (mean 96%, SD 2%), AG-946 5mM (mean 94%, SD 2%), and AG-946 50mM (mean 95%, SD 3%, Figure 1E). The improved metabolic status and p50 was accompanied by a decreased PoS in RBCs treated with mitapivat or AG-946, indicating reduced RBC sickling tendency (Figure 1F). Conclusion: Ex vivo treatment of SCD RBCs with the novel PK activator AG-946 activates and stabilizes PKR, decreases 2,3-DPG levels, improves the ATP/2,3-DPG ratio, improves p50 and lowers the PoS. These beneficial effects are similar to ex-vivo treatment with mitapivat but, importantly, are obtained at much lower concentrations. Taken together, these results are the first in an ex-vivo model to demonstrate that the novel PK activator AG-946 has a similar favorable pharmacodynamic profile to mitapivat with enhanced PKR-stabilizing properties and, hence, might represent a potential novel therapeutic option in addition to mitapivat for the treatment of SCD.Pharmacodynamic effects of AG-946 in ex vivo treatment of red blood cells (RBC) from sickle cell disease (SCD) patients in comparison with mitapivat. Buffy coat depleted whole blood was incubated for 20-24 hrs in absence or presence of mitapivat (100?M, P. KOSINSKI declares a conflict of interest: Stock shareholder: employee and stock holder of Agios Pharmaceuticals, Inc

Identification of Biomarkers That Are Associated with Clinical Complications of Hemoglobin SC Disease and Sickle Cell Anemia

Introduction: Sickle cell disease (SCD) is an umbrella term used to describe inherited anemias that originate from a mutation in HBB, the gene coding for β-globin, that causes the formation of the abnormal hemoglobin S (HbS). The two most common genotypes are HbSS, termed sickle cell anemia (SCA) and HbSC, termed hemoglobin SC disease. Due to the higher prevalence and more severe clinical symptoms in SCA, attention is focused on this latter group of patients. Patients with HbSC disease who report significant SCD related symptoms are treated largely the same as SCA patients, despite differences in disease pathophysiology.Objective: To evaluate rheological differences in red blood cells (RBC) and whole blood from individuals with SCA and HbSC, and examine for associations with clinical complications.Methods: We analyzed an adult (n=30) and pediatric cohort (n=226) of SCA and HbSC individuals. Blood samples were evaluated for percent dense red blood cells (% DRBC), whole blood viscosity, and several ektacytometry-derived parameters, such as the cell membrane stability test, osmotic gradient ektacytometry and Point of Sickling (PoS): an oxygen gradient ektacytometry-derived biomarker that depicts the oxygen tension at which sickling is initiated. Subsequently, we assessed in the pediatric cohort (189 SCA and 37 HbSC individuals) if PoS, dense RBCs or blood viscosity are associated with clinical complications.Results: In particular (micro)dense RBCs (Figure 1A and B), blood viscosity (Figure 1C) and point of sickling (PoS, Figure 1D-F), are notably different in HbSC disease compared to SCA. In SCA, PoS was associated with occurrence of acute chest syndrome (ACS, for every 10mmHg increase OR 1.84, p<0.0001, adjusted OR 1.67, p=0.002), but no such association was found in children with HbSC. In contrast, we found an association of blood viscosity and ACS (OR 9.3, p=0.012; adjusted OR 13.5, p=0.015) in HbSC children, while we found no such association in SCA. No significant association of micro dense RBCs was found in SCA or HbSC children.Conclusion: We found that associations between PoS and ACS found in SCA were not found in HbSC. Instead, in HbSC ACS was associated with higher whole blood viscosity. We therefore conclude that sickling is a key factor in the pathophysiology of SCA, whereas in HbSC disease blood viscosity might play a crucial role in development of certain complications like ACS. This study suggests that complications in SCA and HbSC disease are driven by different aspects of blood abnormalities, and may warrant a distinct treatment approach. [Display omitted] DisclosuresRab: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Schutgens: Shire/Takeda: Research Funding; Pfizer: Research Funding; OctaPharma: Research Funding; Novo Nordisk: Research Funding; CSL Behring: Research Funding; Bayer: Research Funding. Wijk: Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van Beers: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; RR Mechatronics: Research Funding; Pfizer: Research Funding. Sheehan: Beam Therapeutics: Research Funding; Novartis: Research Funding; Forma Therapeutics: Research Funding.

Pharmacodynamic Effects of AG-946, a Highly Potent Next-Generation Activator of Pyruvate Kinase, in Ex Vivo Treatment of Red Blood Cells from Sickle Cell Disease Patients

Background: Sickle cell disease (SCD) is a monogenetic red blood disorder that is characterized by hemolytic anemia and vaso-occlusive crises. Among the many factors that contribute to disease pathophysiology is stiffening and sickling of red blood cells (RBC), which is the direct result of the formation of abnormal hemoglobin S. Sickling is one of the core factors that cause vaso-occlusion and sickling is modulated by glycolytic intermediates such as 2,3-diphosphoglycerate (2,3-DPG) and ATP. Previously we showed that red blood cell pyruvate kinase (PKR), the key regulatory enzyme of glycolysis, is impaired in SCD and that ex vivo treatment with mitapivat, an allosteric activator of PKR, increased enzymatic activity and thermostability, reduced 2,3-DPG levels, decreased p50, and subsequently reduced sickling (Rab et al, Blood 2021). Currently, mitapivat is in phase 1 and phase 2 trials for SCD (#NCT04000165 and EudraCT#2019-003438).Aims: Recently, AG-946, a next-generation activator of PKR has been developed. Here we investigate the pharmacodynamic effects of AG-946 in ex vivo treatment of RBC from SCD patients in comparison with mitapivat.Methods: Buffy coat depleted whole blood obtained from five patients with SCD was incubated for 20-24 hrs in absence or presence of mitapivat (100 mM) or AG-946 (1 mM, 5 mM, 50 mM). After ex vivo treatment, enzymatic activities of PKR and PK-thermostability was measured. Glycolytic intermediates ATP and 2,3-DPG were measured using LC-MS/MS. Hemoglobin oxygen affinity (p50) was measured with the Hemox Analyzer. RBC sickling was analyzed with the oxygenscan, a newly developed method that characterizes individual sickling behavior by oxygen gradient ektacytometry. Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO 2 at which RBCs start to sickle during deoxygenation under shear stress.Results: PKR activity was increased compared to vehicle (DMSO) to a similar extent in presence of both mitapivat and AG-946 (Figure 1A). In addition, PKR thermostability was significantly increased compared to vehicle (mean 22%, SD 6%) in samples treated with mitapivat 100 mM (mean 78%, SD 11%), as well as AG-946 5 mM (mean 66%, SD 23%), and AG-946 50 mM (mean 95%, SD 17%, Figure 1B). The glycolytic intermediate 2,3-DPG decreased after incubation with both mitapivat and AG-946 (Figure 1C), which was further illustrated by the improved ATP/2,3-DPG ratio (Figure 1D). In line with these latter results p50 decreased significantly after incubation with mitapivat 100 mM (mean 95%, SD 2%), as well as AG-946 1 mM (mean 96%, SD 2%), AG-946 5 mM (mean 94%, SD 2%), and AG-946 50 mM (mean 95%, SD 3%, Figure 1E). The improved metabolic status and p50 was accompanied by a decreased PoS compared to vehicle in RBCs treated with mitapivat or AG-946, indicating reduced RBC sickling tendency in vitro (Figure 1F).Conclusion: Ex vivo treatment of SCD RBCs with the next-generation PKR activator AG-946 activates and stabilizes PK, decreases 2,3-DPG levels, improves the ATP/2,3-DPG ratio, improves p50 and lowers the PoS. These beneficial effects are similar to ex vivo treatment with mitapivat but, importantly, are obtained at much lower concentrations. Therefore, AG-946 may be a potent activator of PKR in SCD. Taken together, these results are the first in an ex vivo model to demonstrate that the next-generation PK activator AG-946 has a similar favorable pharmacodynamic profile to mitapivat with enhanced PK-stabilizing properties and, hence, represents a potential novel therapeutic option in addition to mitapivat for the treatment of SCD and other hemolytic anemias. [Display omitted] DisclosuresRab: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Van Dijk: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Kosinski: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Kung: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Van Beers: Pfizer: Research Funding; RR Mechatronics: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Dang: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Wijk: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Safety and Efficacy of Mitapivat (AG-348), an Oral Activator of Pyruvate Kinase R, in Subjects with Sickle Cell Disease: A Phase 2, Open-Label Study (ESTIMATE)

BackgroundSickle cell disease (SCD) is one of the most common and devastating inherited blood disorders characterized by a single nucleotide mutation in the beta-globin chain leading to the production of mutant hemoglobin S (HbS). HbS polymerizes upon deoxygenation causing red blood cells (RBC) to sickle which results in extremely painful episodes of vaso-occlusive crisis (VOC), severe hemolytic anemia, chronic multiorgan failure and a reduced life span. An important metabolic feature directly associated with RBC sickling is increased intracellular levels of the glycolytic intermediate 2,3-diphosphyglycerate (2,3-DPG) which promotes deoxygenation by lowering the oxygen affinity of hemoglobin (Hb). Pyruvate kinase R (PKR) is a key enzyme in RBC metabolism, generating adenosine triphosphate (ATP) to maintain energy homeostasis, membrane integrity and deformability, and modulates 2,3-DPG levels. Mitapivat (AG-348) is an oral, small molecule allosteric activator of PKR and shows promise as an anti-sickling agent in addition to its effect in PK deficiency and thalassemia. The safety and efficacy of mitapivat in subjects with SCD was evaluated in the dose finding period of this ongoing phase 2 study.MethodsThe ESTIMATE study is a phase 2, open-label study in which subjects ≥16 years with SCD (HbSS, HbS/β0, HbS/β+) with a baseline hemoglobin >6.1 g/dL and ≤11.1 g/dL, no chronic transfusion and adequate organ function were eligible. In the 8-week Dose Finding Period, initial dosing of mitapivat was 20 mg twice daily (BID). Subjects received a maximum of two sequential dose escalations of mitapivat (i.e. from 20 mg BID to 50 mg BID and 100 mg BID) depending on safety. The primary endpoints were safety, evaluated by frequency and severity of adverse events (AEs), and efficacy of mitapivat on RBC sickling. RBC sickling was evaluated by change in Point of Sickling (PoS), the pO2 at which sickling occurs as measured by oxygen gradient ektacytrometry on the Lorrca (RR Mechatronics). Secondary endpoints included changes in hematological parameters, levels of 2,3-DPG and ATP, Hb-oxygen affinity (p50) and surrogate markers of organ damage and mortality. Subjects who safely tolerated mitapivat and showed evidence of clinical improvement, were eligible to continue a 52-week follow-up period (Fixed Dose Extension Period).ResultsSix subjects have been enrolled as of September 2020 and completed the Dose Finding Period. All had homozygous HbSS except one patient who had HbS/β0-thalassemia. Baseline characteristics were: median age of 36 years (range 20-59 years), 4 (66.7%) were female and 5 (83.3%) were on stable-dose hydroxyurea. All subjects received dose escalation to a maximum dose of 100 mg BID. No serious adverse events (SAEs) occurred. Adverse events (AEs) were mild and often transient, with the most common treatment emergent AEs: transaminase increase (n=3 [50.0%], Grade 1), gastrointestinal disorders including dyspepsia, diarrhea and abdominal discomfort (n=3 [50.0%], Grade 1) and headache (n=2 [33.3%], Grade 1). One VOC occurred without hospital admission and did not require dose reduction or discontinuation. Table 1 summarizes the anti-sickling effect as well as the hematological and biochemical response to mitapivat treatment. Sickling occurred at lower pO2 levels in all 6 patients during the Dose Finding Period reflected by a significant decrease in treatment week 8 mean PoS compared to baseline. 5/6 subjects (83.3%) achieved a Hb increase of ≥1 g/dL during this period, which was accompanied by a decrease in hemolytic markers. Consistent with activation of PKR, 2,3-DPG levels decreased and ATP levels increased. Additional results including biomarker data will be presented.ConclusionMitapivat demonstrated an adequate safety profile during the 8-week Dose Finding Period in patients with SCD. The data show promising efficacy in terms of a decrease in the pO2 at which RBCs start to sickle, as well as increase in Hb from baseline and a concomitant decrease in markers reflecting hemolysis. The observed changes in 2,3-DPG and ATP levels are consistent with the proposed mechanism of the drug. The study is ongoing and further data including follow-up data, patient-reported outcomes, PKR activity and thermostability will be reported at a later stage. [Display omitted] Disclosuresvan Dijk: Agios Pharmaceuticals: Research Funding; Axcella Health: Research Funding. Rab: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Rijneveld: Servier: Research Funding; Amgen: Research Funding. Nur: Celgene: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Roche: Speakers Bureau. Schutgens: CSL Behring: Research Funding; Novo Nordisk: Research Funding; OctaPharma: Research Funding; Pfizer: Research Funding; Shire/Takeda: Research Funding; Bayer: Research Funding. Wijk: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van Beers: Pfizer: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; RR Mechatronics: Research Funding.

Etavopivat, an Allosteric Activator of Pyruvate Kinase-R, Improves Sickle RBC Functional Health and Survival and Reduces Systemic Markers of Inflammation and Hypercoagulability in Patients with Sickle Cell Disease: An Analysis of Exploratory Studies in a Phase 1 Study

Etavopivat, an Allosteric Activator of Pyruvate Kinase-R, Improves Sickle RBC Functional Health and Survival and Reduces Systemic Markers of Inflammation and Hypercoagulability in Patients with Sickle Cell Disease: An Analysis of Exploratory Studies in a Phase 1 Study

Rheological Impact of GBT1118 Cessation in a Sickle Mouse Model.

In sickle cell disease (SCD), higher whole blood viscosity is a risk factor for vaso-occlusive crisis, avascular necrosis, and proliferative retinopathy. Blood viscosity is strongly impacted by hemoglobin (Hb) levels and red blood cell (RBC) deformability. Voxelotor is a hemoglobin S (HbS) polymerization inhibitor with anti-sickling properties that increases the Hb affinity for oxygen, thereby reducing HbS polymerization. In clinical trials, voxelotor increased Hb by an average of 1g/dl, creating concern that this rise in Hb could increase viscosity, particularly when the drug was cleared. To investigate this potential rebound hyperviscosity effect, we treated SCD mice with GBT1118, a voxelotor analog, and stopped the treatment to determine the effect on blood viscosity and RBC deformability under a range of oxygen concentrations. GBT1118 treatment increased Hb, improved RBC deformability by increasing the elongation index under normoxic (EImax) and hypoxic conditions (EImin), and decreased the point of sickling (PoS) without increasing blood viscosity. The anti-sickling effects and improvement of RBC deformability balanced the effect of increased Hb such that there was no increase in blood viscosity. Forty-eight hours after ceasing GBT1118, Hb declined from the rise induced by treatment, viscosity did not increase, and EImin remained elevated compared to control animals. Hb and PoS were not different from control animals, suggesting a return to native oxygen affinity and clearance of the drug. RBC deformability did not return to baseline, suggesting some residual rheological improvement. These data suggest that concerns regarding viscosity rise above pre-treatment levels upon sudden cessation of voxelotor are not warranted.

Red Cell Rheology Biomarkers to Assess Cure in Gene-Based Therapies

Introduction Red cell rheology is abnormal in sickle cell disease (SCD); red blood cells (RBC) are rigid, dense, and the sickle hemoglobin (HbS) polymerizes with deoxygenation. There are several devices commercially available and under development to assess RBC rheology. One is an oxygen gradient ektacytometer (Lorrca with Oxygenscan, RR Mechatronics) which measures RBC deformability under oxygenated conditions (EImax) and deoxygenated conditions (EImin), and the oxygen concentration at which deformability begins to worsen, point of sickling (PoS). A commercially available hematology analyzer, the ADVIA (Siemens), measures hemoglobin (Hb) by flow and colorimetric methods, permitting automated calculation of the percent dense red blood cells (%DRBC). Allogeneic hematopoietic stem cell transplant (alloHSCT) can provide a cure for SCD, and viable gene-based therapy options are under investigation to serve the many patients without a matched related donor. However, the level of HbS correction or functional Hb induction necessary to achieve a cure is still unknown. Clinical endpoints such as prevention of pain events are important, but it is possible to be pain free for several years while still experiencing organ damage. As gene-based therapy clinical trials move forward, we must assess the level of functional improvement beyond Hb profile and conventional clinical labs. We propose that the goal of any gene-based SCD therapy should be to normalize blood rheology to the level of an individual with sickle cell trait (HbAS), and that EImax, EImin, PoS, and %DRBC may be used to distinguish between HbAS and HbSS/Sβ0 genotypes. Methods Subjects: Blood samples were collected from 257 unique patients (17 HbAS, and 240 HbSS/Sβ0) under IRB-approved protocols at Texas Children's Hospital and University Medical Center Utrecht. Patients were 56% male, ages 9 months to 22 years. Some HbSS/Sβ0 subjects were on transfusion and hydroxyurea (HU) (Table 1). Fetal hemoglobin (HbF) levels ranged from 0-41%. Oxygen gradient ektacytometry: Blood collected in EDTA and standardized to a fixed RBC count was suspended in 5 mL polyvinylpyrrolidone at room temperature. 1.5 mL of the sample solution was injected into test cup. Each sample was run in duplicate. ADVIA: 250µL of blood collected in EDTA at room temperature was aspirated to measure the %DRBC, defined as the percentage of RBCs with a Hb concentration &amp;gt;1.11 mg/mL. Analysis: Patient characteristics were summarized using median with 25th and 75th percentiles, and frequency with percentage. Characteristics and labs were compared by group with t-test, Wilcoxon rank sum test, or Fisher's exact test. Receiver operating characteristics (ROC) analyses were performed to identify HbAS versus HbSS/Sβ0 for each biomarker. All analyses were performed using Stata 15. Results The EImin, EImax, PoS, and %DRBC differed significantly between the HbAS and HbSS/Sβ0 groups, despite including transfused, very young, and HU-treated samples in the HbSS/Sβ0 cohort (p&amp;lt;0.001, p=0.002, p&amp;lt;0.001, and p&amp;lt;0.001, respectively; Table 1). ROC analysis showed that a threshold Elmin greater than or equal to 0.453 identified HbAS with 94.12% sensitivity and 95.43% specificity. EImax greater or equal to 0.569 identified HbAS with 94.12% sensitivity and 81.57% specificity. PoS less than 21.38 mmHg identified HbAS with 93.87% sensitivity and 93.33% specificity. %DRBC less than 1.6% identified HbAS with 88.13% sensitivity and 88.00% specificity (Table 2). Conclusion Conventional laboratory testing may not be able to establish if gene-based therapy has achieved a cure. We propose to define a cure as achievement of an HbAS level of RBC quality in a gene-based therapy edited RBC population. We show that rheological biomarkers EImin, EImax, PoS, and %DRBC differ significantly between individuals with HbAS and HbSS/Sβ0 of all spectrums of severity and treatment regimens. Not only are the rheological biomarker values significantly different, but there is little to no overlap in ranges of values obtained from the different genotypes, even in heavily transfused HbSS patients, with HbS as low as 31%. EImin, EImax, PoS, and %DRBC functionally identify genotype with high sensitivity and specificity; we propose that they be used not to diagnose SCD, but to determine if a HbSS or HbSS/Sβ0 individual who has undergone successful gene-based therapy has achieved the RBC functionality of a HbAS individual. Disclosures van Beers: Novartis: Research Funding; Pfizer: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding; RR mechatronics: Research Funding. Wijk:Agios Pharmaceuticals Inc.: Research Funding; RR mechatronics: Research Funding. Rab:RR Mechatronics: Research Funding. Sheehan:Global Blood Therapeutics: Research Funding; Novartis: Research Funding; Emmaus: Research Funding.