Sickle Cell Disease Red Blood Cells Research Articles

Functional and multi-omics signatures of mitapivat efficacy upon activation of pyruvate kinase in red blood cells from patients with sickle cell disease.

Mitapivat, a pyruvate kinase (PK) activator, shows great potential as a sickle cell disease (SCD)- modifying therapy. Safety and efficacy of mitapivat as a long-term maintenance therapy is currently being evaluated in two open-label studies. Here we apply a comprehensive multi-omics approach to investigate the impact of activating PK on red blood cells (RBCs) from 15 SCD patients. HbSS patients were enrolled in one of the open label, extended studies (NCT04610866). Leuko-depleted RBCs obtained from fresh whole blood at baseline (visit 1, V1), prior to drug initiation and longitudinal time points over the course of the study were processed for multiomics through a stepwise extraction of metabolites, lipids and proteins. Mitapivat therapy had significant effects on the metabolome, lipidome and proteome of SCD RBCs. Mitapivat decreased 2,3-diphosphoglycerate (DPG) levels, increased adenosine triphosphate (ATP) levels, and improved hematologic and sickling parameters in patients with SCD. Agreement between omics measurements and clinical measurements confirmed the specificity of mitapivat on targeting late glycolysis, with glycolytic metabolites ranking as the top correlates to parameters of hemoglobin S (HbS) oxygen affinity (p50) and sickling kinetics (t50) during treatment. Mitapivat markedly reduced levels of proteins of mitochondrial origin within 2 weeks of initiation of drug treatment, with minimal changes in the reticulocyte counts. The first six months of treatment also witnessed transient elevation of lysophosphatidylcholines and oxylipins with depletion in free fatty acids, suggestive of an effect on membrane lipid remodeling. Multi-omics analysis of RBCs identified benefits for glycolysis, as well as activation of the Lands cycle.

Fetal Hemoglobin Decrease during Voxelotor Treatment

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene leading to the formation of hemoglobin S (HbS). The polymerization of deoxygenated HbS is the first event in the molecular pathogenesis of SCD resulting in sickling of red blood cells (RBC), and hemolytic anemia which has been shown to be associated with organ damage and early death. Hemolytic anemia is defined by an accelerated rate of destruction RBC resulting in a lowered Hb level and a reduced gas transport capacity. Because Hb S in its oxy (R) conformation do not polymerize, drugs modifying hemoglobin (Hb) to increase the proportion of oxy-HbS have been propose to inhibit HbS polymerization. Voxelotor is such a molecule, which allosterically modifies Hb-oxygen affinity improving anemia and reducing hemolysis. However, the impact of Voxelotor on HbF levels is unknown. We describe here variations of %HbF and Mean corpuscular HbF (MCHbF) in a cohort of homozygous SCD patients treated with Voxelotor in our Sickle Cell Referral center. We conducted a prospective register study to analyze clinical and biological evolution of SCD patients treated with Voxelotor at 1500mg taken orally once daily. From December 2021 through December 2022, 25 SCD adult patients were followed during a median of 7 months [1-12]. Among them, HbF values were analyzable for 15 patients (SS gentype n=14; women in 73%; median age of 50 years [24-61]) who completed 6 months of Voxelotor with no transfusion during the same period; and with a stable dose of hydroxyurea (HU), for at least 3 months before inclusion and during the study in case of patients treated by HU (n=10). The dose of Voxelotor was stable for 13/15 patients and was decreased in 2 patients due to skin allergy (n=1) and moderate digestive intolerance. One patient died after 6 months of treatment in a context of end-stage renal failure and severe pulmonary hypertension not related to Voxelotor treatment. Mean corpuscular HbF (MCHbF) was calculated (MCHbF (pg) = MCH x %HbF). HbF percentage was measured by high performance liquid chromatography (Biorad, Variant II, beta-thal dual program). All participants provided written informed consent (CPP Erythropedie). From baseline to 6 months of follow up mean Hb (g/dL) concentration increased from 7.4 [5.4-8.5] to 8 [5-11.9] (p=0,0003; Figure A); absolute reticulocyte count (ARC ; Giga/L) decreased from 158 to 145 (p=0,005), %HbF decreased by 63% in all patients from 7,1% to 3,3% (p=0,008; Figure B) and MCHbF (pg) decreased from 3,36 to 1,03 (p=0,001; Figure D). A decrease in % HbF was observed from the first 15 days of treatment in both groups of patients treated or not with HU (Figure C) ; no changes in mean corpuscular volume (p=0.15) was observed in patients treated with HU during follow up. The HbF decrease in SCD patients treated with Voxelotor could be explained by different mechanisms: - Reduction of stress erythropoiesis which is due to an increase in erythroid output in response to anemic stress associated with a reduced expression of BCL11A, a transcriptional repressor that inhibits adult-stage HbF expression. This hypothesis is supported by the reduction in reticulocytes in patients treated with Voxelotor - The loss of an HbF-related survival advantage for SCD RBCs, while the normal RBC lifespan is approximatively 120 days, in SCD RBCs it is only a sixth of it. HbF is a major modulator of SCD and prolonge SCD RBCs survival. Its protective effect relies on its quantity and distribution among the RBC population, which is often heterogeneous in SCD patients. Protection against polymerisation of Voxelotor is probably more homogeneous among RBCs. As a result, RBCs containing low levels of HbF, which would normally disappear, have a prolonged lifespan. Similarly, it was presumed that sickling of erythroblasts could lead to ineffective terminal erythroid differentiation with an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. However we found no significant difference for index of ineffective erythropoiesis (soluble transferrin receptor / absolute reticulocyte count) before and after 6 months of treatment. The question raised is therefore that of the risk of a rebound effect upon discontinuation of Voxelotor, which has a short half-life, during the time it takes for HbF to increase. To better study this hypothesis, distribution of HbF/RBC will be studied in the HEMOPROVE trial (NCT05199766) currently in progress.

Roxyscan: A Novel Method to Assess Red Blood Cell Resilience to Oxidative Stress in Sickle Cell Disease

Background: Red blood cells (RBCs) are biconcave-shaped cells with viscoelastic membranes that are optimally adapted for oxygen delivery and gas exchange during their normal lifespan of 120 days. Reactive oxygen species (ROS) continuously challenge the viability of RBCs. Despite an arsenal of compounds to protect the RBC, including Glutathione (GSH), Vitamin E, superoxide dismutase (SOD) and Catalase, damage can easily be inflicted that will disrupt cellular deformability, structure, and membrane properties. The susceptibility to oxidative stress in individuals with Sickle Cell Disease (SCD) is believed to be linked to multiple factors. These include the release of heme and iron during hemolysis, inflammation processes, and a reduction in antioxidant defenses. Methods:Susceptibility of RBCs to oxidative stress was assessed with the RoxyScan. This novel application of the Lorrca MaxSis (RR Mechatronics) measures rheological behavior of RBCs in response to cumene hydroperoxide (CuHP, 100 µM) exposure during continuous shear (30 Pa). For this we used the Lorrca's peristaltic pump and suction needle, directing the blood and CuHP to the instrument's cup and mixing them for 30 seconds (s) before starting the run. Deformability of RBCs (EI, elongation index) was measured over time. PoX is defined as the time where a 10% decrease in EI is reached. EI start: EI t=0. EI final: EI t=1600s, ΔEI (EIstart-EIfinal). RoxyScan measurements were performed on blood samples from 16 adult SCD patients (HbSS, HbS/β0 or HbS/β+) and 19 healthy controls (HC). Outcome measurement PoX (calculated from curve fitted data) was correlated to laboratory parameters from complete blood count (Cell Dyn Sapphire, Abbott), RBC sickling tendency (Point of Sickling, PoS, as assessed by oxygen gradient ektacytometry on the Lorrca MaxSis), and levels of HbF/HbS (Tosoh G8). Results:RBCs from untreated HC show a stable EI over time (Fig 1A). Upon exposure to CuHP a similar decrease in EI is seen in 19 HC (Fig 1A), mean decrease 0.055, range (0.017 to 0.10). The mean and median calculated PoX in HC is 1723s and 1702s respectively. In contrast, SCD RBC showed an earlier and more pronounced decrease in EI in response to CuHP exposure (representative example, Fig 1A). Comparing the PoX between HC and SCD revealed significant differences: mean PoX HC: 1723s (SD 329s), SCD: 725s (SD 472s), ***p<0.0001, indicating that SCD RBCs are more susceptible to oxidative stress. Correlating PoX to routine laboratory parameters identified a correlation with Hb levels (***p=0.002), inverse correlation with reticulocyte count (***p=0.0014) and hemoglobin distribution width (HDW, ***p<0.001) (Fig 1B). Similarly strong inverse correlations were found for EI start and EI end (***p<0.001) (Fig 1B). PoX also showed an inverse correlation with red cell distribution width (RDW, ns p=0.07) and mean corpuscular volume (MCV, ns p=0.77). Interestingly, PoX showed a significant inverse correlation with PoS (***p=0.004), indicating that SCD RBCs which are more susceptible to oxidative stress tend to sickle at a higher oxygen tension. PoX showed no significant correlations with HbS/HbF levels (Fig 1B). Conclusion: In this study we demonstrate the applicability of the RoxyScan as a novel method to assess RBC resilience to oxidative stress. When applied to SCD, RBCs show an earlier and more pronounced response to oxidative stress than RBCs from HC. The inverse correlation of PoX, the major outcome parameter of this technique, with reticulocyte count could indicate a generally increased susceptibility to oxidative stress due to a higher level of hemolysis. The correlation with Hb levels affirms this. PoX showed other strong and significant correlations with routine laboratory parameters, such as HDW, implying that the greater variability in hemoglobin content is associated with higher susceptibility to oxidative stress. On a functional level, it is interesting to find strong correlations of PoX with PoS, showing a direct link between RBC sickling tendency and ability to withstand oxidative stress. On the other hand, HbS and HbF levels did not correlate significantly to PoX. Overall, our study highlights the potential use of the RoxyScan and its associated biomarkers in the analysis of the RBC's response to oxidative stress. Further research is in progress to investigate the clinical applicability of this new technique.

Functional and Multi-Omics Signatures of Mitapivat Efficacy upon Activation of Pyruvate Kinase in Red Blood Cells from Patients with Sickle Cell Disease

2,3-diphosphoglycerate (DPG) in red blood cells (RBCs) stabilizes deoxyhemoglobin to promote hemoglobin (Hb) oxygen off-loading and counteract hypoxia. In sickle cell disease (SCD), mutation of glutamate 6 to valine in the beta chain of Hb results in HbS that polymerizes, causing RBCs to sickle upon deoxygenation in the tissues. Elevated DPG promotes sickling by stabilizing HbS fibers. DPG is an intermediate substrate in the glycolytic pathway in which pyruvate kinase (PK) is a rate-limiting enzyme. In a previous Phase 1 study (NCT04000165, Xu et al, 2022), we established proof-of-concept for activating PK as a viable anti-sickling therapeutic approach; mitapivat, an investigational oral allosteric activator of PK, decreased DPG and increased adenosine triphosphate (ATP) levels, improved hematologic and sickling parameters in patients (pts) with SCD (HbSS). Here, we investigated the changes in the metabolome, lipidome and proteome of RBCs from patients with SCD currently enrolled in an open label, Phase 1/2 study (NCT04610866) that evaluates long-term safety and tolerability of mitapivat in pts with SCD. Fifteen pts with HbSS (aged 25 - 57 years, 10 males) were evaluated. All pts started mitapivat at 50 mg twice daily (BID), escalating after 4 wks to 100 mg BID; dose adjustments were performed for safety and tolerability, per PI discretion. At the time of data cutoff (March 23, 2023), all 15 pts completed the core period of 24 wks, 14 pts completed 48 wks ,10 pts completed 72 wks and 6 pts completed 92 wks. Leuko-depleted RBCs (LDRBCs) obtained from fresh whole blood in EDTA at baseline (V1, prior to drug initiation) and various longitudinal time points over the course of the study were flash-frozen in liquid nitrogen and kept frozen at -80°C until analysis. Clinical measurements of DPG, ATP, red cell PK (PKR) protein and activity levels, oxygen affinity (p50) and sickling kinetics (t50) were accompanied by multi-omics (metabolomics, lipidomics, proteomics) characterization of the LDRBCs. In total, 150 (6x12, 4x10, 4x8 and 1x6) timepoint samples were analyzed (Fig 1A). Multi-omics analyses were performed through a stepwise extraction of metabolites, lipids and proteins on 50 µl of packed LDRBC pellets. Extracts were separated via ultra-high-pressure liquid chromatography coupled to mass spectrometry, prior to peak picking and identification of ddMS2 data as previously described (Thomas et al 2020). Statistical analyses were conducted using MetaboAnalyst v 5.0 and Rstudio upon normalization of relative quantitative data to visit 0 levels for each variable. Network analysis and pathway analysis were performed in OmicsNet v 2.0. Mitapivat therapy had significant effects on the metabolome, lipidome and proteome of SCD RBCs. Overall, the temporal trends of omics responses to mitapivat treatment across visits was evident, especially when focusing on the top 50 metabolites/lipids or proteins by time series ANOVA. Specifically, results suggested a positive correlation between mass spectrometry-based quantitation of RBC mitapivat levels and DPG consumption, ATP accumulation, PKR activity (measured via enzymatic assays), and PKR protein levels, with internal consistency between omics data and antibody-based or enzymatic assays (Fig. 1A). The treatment also promoted activation of the Lands cycle, transient elevation of lysophosphatidylcholines and oxylipins, and depletion in free fatty acids within the first 6 months, suggestive of an effect on membrane lipid remodeling. SCD RBCs abnormally retain mitochondria; here, we showed a consistent decrease in the levels of residual mitochondrial proteins in RBCs within 4 wks of mitapivat treatment (Fig. 1B). In keeping with the predicted activation of PKR upon mitapivat treatment, we observed decreases in DPG and increases in ATP, with elevation in glycolytic fluxes, as inferred from longitudinal changes in glycolytic intermediates at the hexose and triose phosphate steps, as well as lactate. Unsupervised analyses of omics data confirmed the specificity of mitapivat on targeting late glycolysis, with glycolytic metabolites ranking as the top correlates to parameters of oxygen affinity (p50) and sickling kinetics (t50) during mitapivat treatment. Multi-omics analysis of RBCs from HbSS patients treated with mitapivat identified benefits for glycolysis, DPG consumption and ATP generation, as well as activation of the Lands cycle.

A Pilot Study for the Kidney Effects of Voxelotor in Sickle Cell Anemia

The kidneys are among the most commonly affected organ systems in sickle cell anemia (SCA) and an estimated 15% of deaths in patients with SCA are attributed to kidney failure (PMID 7993409). Angiotensin converting enzyme-inhibitors and angiotensin receptor blockers have been adopted to treat SCA-related chronic kidney disease (CKD) from the non-SCA literature, although demonstrated efficacy in SCA CKD has been marginal in a limited number of small, short-term studies (PMID 28589652). Therapies that directly target the pathophysiology of SCA-related kidney disease may stabilize or improve kidney function. Both the rate of hemolysis and the degree of anemia are associated with the development and progression of CKD in SCA (PMID 24329963, 21523807). Voxelotor inhibits hemoglobin polymerization in SCA red blood cells in the deoxygenated state, leading to a reduction in the rate of hemolysis and an increase in hemoglobin concentration. We conducted a pilot study to determine whether voxelotor can improve kidney function in adults with SCA (NCT 04335721). SCA (HbSS or HbSβ 0-thalassemia) patients ≥ 18 years old and with urine dipstick-defined hemoglobinuria (+ for blood & ≤ 2 red blood cells/high power field), CKD stage 1 or 2 (urine albumin-to-creatinine ratio [ACR] ≥ 30 mg/g & estimated glomerular filtration rate [eGFR] ≥ 60 mL/min/1.73m 2), and hemoglobin concentrations of 5.5 - 10.5 g/dL were eligible. The eGFR was calculated using the CKD-EPI 2009 creatinine-based equation without the race coefficient and the 2012 cystatin C-based equation. Patients were randomized to voxelotor 1,500mg/day for 48 weeks or standard of care, as defined by their primary SCA provider. First or second void urines were collected for urine ACR measurements. The primary endpoint was the change in urine ACR from the mean values at baseline and screening versus the mean values at 47 and 48 week visits. One patient randomized to standard of care was started on voxelotor at week 24 by his provider to improve his hemoglobin concentration. This patient is included in the voxelotor treatment group in combined analysis; the change in laboratory measures in this patient is based upon changes from the mean of values at week 12 and 24 to week 47 and 48 visits. Median and range values are provided. Between 3/2021 and 6/2022, 8 SCA patients with hemoglobinuria and urine ACR > 30mg/g crt observed during routine clinic visits were screened and 6 met the inclusion criteria. The median age of the enrolled cohort was 36 (27 - 63) years, 4 were male, 4 were on stable doses of hydroxyurea (median dose 1000mg/day; 500 - 1500mg/day) and 2 were on stable doses of lisinopril (5, 20mg/day) for at least 90 days before screening. Two other patients had been on lisinopril in the past but developed hyperkalemia prior to enrollment. The median values of kidney function measures at screening and baseline study visits were as follows: urine ACR, 251 (32 - 3132) and 392 (80 - 3101) mg/g crt; creatinine-based eGFR, 109 (93 - 136) and 115 (88 - 137) mL/min/1.73m 2; and cystatin C-based eGFR, 82 (61 - 141) and 85 (61 - 128) mL/min/1.73m 2, respectively. SCA patients who received voxelotor (n = 4) had a median increase in hemoglobin concentration of +0.9 g/dL (-0.4 - +2.6 g/dL) and reductions in indirect bilirubin concentration of -2.2 mg/dL (0 - -4 mg/dL) and reticulocyte % of -2.7% (-1.2 - -4.9%). Urine ACR (-59%, -21% - -100%) improved in SCA patients treated with voxelotor but worsened in those who received standard of care without voxelotor (n = 2) (+86.3%, 64 - 108%) (P=0.009) (Figure 1A). 24-hour urine protein concentrations also improved in those that received voxelotor (-45%, -25% - -100%) but increased in those who did not receive voxelotor (+122%, 113 - 131%) (P = 0.002) (Figure 1B). Changes in creatinine- and cystatin-C based eGFR from baseline were not significantly different between patients who received voxelotor and those who did not. This pilot study demonstrates that SCA patients with hemoglobinuria and early stages of CKD treated with voxelotor may have significant improvements in urine ACR and proteinuria after 24 - 48 weeks of therapy. These preliminary results support the concept that improving hemolytic anemia could be a targeted therapeutic approach to reduce biomarkers of kidney dysfunction, and they warrant larger, prospective clinical studies.

Blood Clots Are Mechanically Weak in Patients with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) causes a hypercoagulable state characterized by frequent thrombotic sequelae including pulmonary embolism (PE) (Connes et al. Blood. Rev. 2016). Although the coagulation cascade is well characterized in SCD, the engineering-based, biomechanical structural properties of sickle blood clots are poorly understood. Furthermore, the role of clot mechanics in both traditional thrombotic disease manifestations (in situ blood clot, PE) but also common sickle cell related sequelae (pain, stroke) is unknown. Since blood clot embolization is inherently a mechanical process, where clots embolize or “fracture” when hemodynamic forces exceed the internal “strength” of the clot (Tutwiler et al. Sci. Adv. 2020), we submit that investigating the mechanical properties of sickle cell blood clots will reveal insights into the disease-specific factors at play. We hypothesize that SCD clots are not only structurally abnormal, but that this abnormality informs an increased propensity for clot embolization and causes severe vascular complications. Methods: Under the guidance and approval of the IRB, we acquired venipuncture-derived human blood samples from 10 healthy control subjects (14.3±0.9 g/dL Hgb) and 10 patients with sickle cell disease (7 HbSS, 2 HbSC, 1 HbS beta; 10.5±2.3 g/dL Hgb). We coagulated blood into 3D-printed molds designed for uniaxial extension on our mechanical testing machine, see figure 1. To do so, we added calcium chloride to a concentration of 20mM to reverse the ACD anticoagulant and incubated samples for 60 minutes at 37°C and atmospheric oxygen conditions (Sugerman et al. Curr. Protoc. 2021). Samples were then mounted to our tensile testing machine (Instron, Norwood, MA, USA) and two tests were conducted: extension-to-failure in a simple pure shear geometry and in a mode-I fracture geometry. From this data, we calculated various mechanical metrics by quantifying the structural abnormalities and propensity for embolization of SCD blood clots. Results and Conclusion: We primarily compared the stiffness and fracture toughness of sickle cell and healthy blood clots. Stiffness measures how the blood clot deforms in response to force, while fracture toughness measures whether a blood clot is strong or weak (i.e its resistance to rupture). The measured stiffness of SCD clots is 7.27±1.68 kPa, which is significantly higher than the stiffness of healthy clots, 5.22±1.09 kPa (p < 0.001). Additionally, the blood from SCD patients makes clots with a fracture toughness of 2.91±0.85 J/m 2, which is significantly lower than the fracture toughness of healthy clots, 4.43±1.11 J/m 2 (p < 0.0001), see figure 2. The high stiffness of sickle cell blood clots suggests that they are more resistant to deformation than healthy clots. However, the low fracture toughness of sickle cell blood clots suggests that they are more likely to break or embolize when compared to healthy clots. In other words, sickle blood clots exhibit mechanical properties similar to ceramic which has high stiffness and low toughness as opposed to leather (low stiffness, high toughness) or diamond (high stiffness, high toughness). In conclusion, our biomechanical findings of higher stiffness and lower toughness in sickle blood clots are well aligned with established rheological data showing that SCD red blood cells are less deformable than healthy red blood cells (Lu et al. Exp. Biol. Med. 2020). That is, the low deformability of red blood cells in SCD is apparent even at the clot level, which may inform novel disease mechanisms. The sickle cell related determinants of these biomechanical abnormalities, their role in clinical outcomes (PE, pain and stroke) and their response to therapy is the object of future study.

Identifying Inflammatory Pathways That Promote RBC Alloimmunization in Patients with Sickle Cell Disease

Introduction: During red blood cell (RBC) transfusion, exposure to unmatched non-ABO antigens can lead to production of alloantibodies that promote potentially fatal hemolytic events. Patients with Sickle Cell Disease (SCD) have the highest incidence of RBC alloimmunization. Prior studies indicate that an altered immuno-regulatory and/or pro-inflammatory state may determine the risk of RBC alloimmunization. For example, one study reported that patients with SCD have inflammatory cytokine gene polymorphisms that could be associated with diverse disease presentations and alloimmunization. We previously reported that inflammatory type 1 interferons (IFNα/β) produced by innate immune cells are required for RBC alloimmunization in mice, while others have suggested that NFkB-induced cytokines play a role in murine transfusion models. However, the inflammatory factors that regulate RBC alloimmunization in patients with SCD are poorly understood. Alloimmunized patients with SCD also have elevated levels of anti-RBC autoantibodies, which facilitate erythrophagocytosis. Given the role of erythrophagocytosis by spleen macrophages in processing RBC antigens, we hypothesized that erythrophagocytosis of SCD RBCs may promote inflammatory pathways that regulate alloimmune responses. Methods: RBCs from control subjects (AA), and reticulocytes and mature RBCs from patients with SCD, were opsonized or not with anti-RBC antibodies (anti-RhD) and co-cultured for 3 hrs with human macrophages (hMac) differentiated from monocytes of healthy subjects. Phagocytosis of labeled RBCs was analyzed by flow cytometry. Bulk mRNA sequencing of co-cultured macrophages was performed to identify differential gene expression. Ingenuity Pathway Analysis (IPA) software was used to identify relevant pathways and upstream regulators of differentially expressed genes. To validate sequencing results, cytokine levels from co-culture supernatants were measured by multiplex cytokine assays. Results: Flow cytometric analysis revealed that opsonization was required for erythrophagocytosis in an FcR dependent manner. RNA-seq Differential Expression analysis of macrophages co-cultured with AA opsonized RBCs vs. non-opsonized RBCs identified 166 significantly upregulated and 24 downregulated genes (p<0.0001). IPA analysis revealed that the top 12 canonical pathways were related to inflammation, including TNF, IL-17, NF-kappa-B and NOD-like receptor signaling pathways. TNF signaling was the top predicted upstream regulator pathway with a fold enrichment score of 10.3 (16 genes) (p<0.0001). Co-cultures of mature RBCs from patients with SCD showed comparable results. When comparing macrophages from co-cultures of opsonized reticulocytes vs. non-opsonized reticulocytes from patients with SCD, 174 genes were upregulated and 31 were downregulated (p<0.0001). Pathway analysis also revealed unique signatures for opsonized reticulocytes from patients with SCD. The top 12 canonical pathways included TNF, NF-kappa-B and NOD-like receptor signaling pathways (Figure 1). The top predicted upstream regulators were TNF and the NF-kappa B signaling pathway with a fold enrichment score of 12.3 (22 genes) (p<0.0001) and 10.9 (18 genes), respectively (Figure 1). Finally, to validate the sequencing results, cytokines levels from supernatants of hMac co-cultures with the RBCs from the patients with SCD and control subjects were measured. Erythrophagocytosis of opsonized reticulocytes and AA RBCs, compared to non-opsonized co-cultures, led to production of NFκB-mediated cytokines, including TNF. Our results show that erythrophagocytosis of SCD RBCs promote NFκB-mediated TNF production. These results warrant further investigation of the role of NFκB-mediated inflammation in RBC alloimmunization in patients with SCD.

Red Blood Cell Pyruvate Kinase Properties in Sickle Cell Disease - of Mice and Men

Background: Townes and Berkeley mouse models of sickle cell disease (SCD) are commonly used to study SCD pathophysiology and to develop novel therapies. Although the pathophysiology in these models is similar to the human disease, there are distinct differences. For example, Townes SS mice have higher ATP and lower 2,3-diphosphyglycerate (2,3-DPG) levels compared to Townes AA mice. This contrasts with SCD patients, who show lower ATP and higher 2,3-DPG levels, suggesting potential differences between human and murine red blood cell (RBC) metabolism. Pyruvate kinase (PK), a key ATP-generating enzyme in glycolysis, is a target for novel SCD therapies. While PK activators have been shown to increase hemoglobin in clinical trials, such treatments do not consistently lead to the same hematological change in mouse models, further indicating that PK properties may differ between SCD mouse models and human patients. A detailed comparative characterization of PK and glycolytic metabolites in murine SCD models and SCD patients is therefore important. Aim: To study PK properties in murine and human SCD RBCs. Methods: Wild-type (C57BL/6J), Townes (AA and SS) and Berkeley mice (non-sickling and sickling) were used. Human blood was obtained from healthy controls (HbAA) and untreated SCD patients (HbSS). Hematological parameters were measured using the Sysmex hematology analyzer. PK (Vmax, 5mM substrate) and hexokinase (HK) activity, and PK thermostability (53°C) were measured in purified RBCs. Levels of ATP, 2,3-DPG, as well as reduced glutathione (GSH) were measured by targeted LC-MS/MS and spectrophotometry, respectively. P50 (oxygen tension at which 50% of hemoglobin is saturated with oxygen) was measured with the Hemox analyzer (TCS). Statistical analysis was performed in Graphpad Prism 9.4.1. by Welch's t-test. Results: Samples from 17 wild-type mice, 21 Townes mice (10 AA, 11 SS), 15 Berkeley mice (7 non-sickling, 8 sickling) and 12 human subjects (6 HbAA, 6 HbSS) were included. Both Townes and Berkeley SCD mice showed increased PK activity (Table 1; p<0.0001). This may be attributed to their high reticulocyte counts (Townes SS vs. AA: 43% vs. 4%; Berkeley sickling vs. non-sickling: 30% vs. 6%), because when normalizing PK activity to that of HK, another RBC age-dependent enzyme, no genotype differences were found in these mouse models. This lack of PK/HK difference was in sharp contrast with our finding in human controls vs. SCD patients (Table 1). Notably, both Berkeley sickling and non-sickling mice had lower PK/HK ratio than their Townes counterparts (p<0.0001). Mouse PK thermostability was markedly reduced compared to that of human, and PK from the two sickling mice was less stable than their non-sickling controls (Fig. 1). Moreover, both sickling mice showed higher ATP/2,3-DPG ratio (Table 1; p<0.01). This was again in direct contrast with our observation using human blood, where SCD patients had lower ATP/2,3-DPG ratio than healthy controls (p<0.05). Even though there was no difference in ATP/2,3-DPG ratio between the two SCD models, Townes SS mice had significantly lower 2,3-DPG and higher ATP levels than Townes AA mice, whereas Berkeley sickling mice only had significantly higher ATP level compared to their controls. Furthermore, sickling mice had lower P50 value compared to non-sickling mice, reflecting a higher oxygen affinity. This also contrasted with human SCD patients, who had higher P50 value than healthy controls (Table 1). GSH levels were increased in both SCD patients and sickling mice, although the elevation was more pronounced in sickling mice (Table 1). Conclusions: This study reveals important distinctions between mouse models and humans: no differences in PK/HK ratio between sickling and non-sickling mice, and an overall lower PK thermostability in mice. Furthermore, this study shows significant changes in 2,3-DPG content (important for oxygen affinity), energy level (ATP), oxygen affinity (P50) and antioxidant level (GSH) when comparing sickling to non-sickling mice. Sickling mice also have higher ATP/2,3-DPG ratio, which contrasts with SCD patients. These differences should be considered when studying PK activators in SCD mouse models and extrapolating results from SCD mouse models to humans.

Erythrophagocytosis Mediated By Circulating Monocytes Is Induced By Distinct Mechanisms Depending on the Disease

Stress erythropoiesis (SE) occurs when the steady state erythropoiesis is no longer sufficient to sustain the number of healthy red blood cells (RBC) needed in the circulation. There is an extension of this activity to extramedullary sites, contributing to the increase in cell production in the erythroblastic island (EBI) niche, and altered or damaged RBCs are then cleared by macrophages. Some diseases have a state of chronic SE. Sickle cell anemia (SCA) affects multiple aspects of RBCs, leading to recurrent vaso-occlusive crisis, hemolytic anemia, and inflammation. Polycythemia Vera (PV), an acquired neoplastic disease with mutations in JAK2 kinase, is characterized by the overproduction of RBCs, increasing blood viscosity, and resulting in a higher risk of thrombotic events. We previously showed that circulating monocytes (MC) of both diseases have a phenotype similar to the EBIs macrophages, with higher expression of CD169, CD163, CD206 and VCAM-1, and also participate in the phagocytosis of RBCs, function typically carried out by macrophages. Therefore, the aim of this study was to determine whether the increased phagocytic activity found in these patients was due to the alterations on the MCs phenotype or if it was an influence of the RBCs, as well as investigate the consequences to the iron metabolism after phagocytosis. MCs from peripheral blood were isolated and incubated for 2 hours with previously stained RBCs, to allow the phagocytosis to happen. After this period, the remaining RBC were lysed and the antibodies anti-intracellular heme-oxigenase-1 (HO-1) and anti-membrane ferroportin (FPN) were used for flow cytometry analysis. The statistical analysis was done by paired t-student test (P<0.05). When exposed to SCA RBCs, MCs from SCA patients and healthy controls (HC) show higher phagocytic capability, than when exposed to HC RBCs (MC HC 22.9±8 vs 9.4±1.1, **P=0,0099; MC SCA 33.6±11.4 vs 16.4±11, **P=0,0054; respectively). The same happens to PV and HC MCs: when exposed to PV RBCs: they show higher phagocytosis compared to the presence of HC RBCs (MC HC 9.3±4.4 vs 2.8±4.7, **P=0.0065; MC PV 21.3±6.1 vs 11±6.8, *P=0.0146; respectively). Investigating the role of MCs in this activity, we compared SCA, PV and HC MCs in the presence of patients RBCs. SCA MCs internalized RBCs similarly to HC MCs (P=0.0699); on the other hand, PV MCs presented higher phagocytic capability when compared to HC MCs (**P=0.0022). In all groups, the expressions of both HO-1 and FPN were higher in those MCs that participated in the phagocytosis than in those that didn't contribute to this function (HO-1: MC SCA 91.9±4 vs 65.9±16.8, **P=0.0098, MC PV 92.1±5.5 vs 61.7±15.5, ***P=0.0007; FPN: MC SCA 81.8±13.8 vs 66.5±15.7, ***P=0.0003, MC PV 70.9±17 vs 53.2±16.5, **P=0.0013 respectively). This indicates an increase in the ability to degrade heme and export iron post-phagocytosis. The results suggest that RBCs from patients with SCA are the ones responsible for the increase in the phagocytic activity by MCs, indicating that RBCs clearance is not an innate function of SCA MCs, even though they are active participants in the circulation. For PV, both RBCs and MCs seem to contribute to the increase of this process in the circulation of patients. The higher expression of molecules related to the iron metabolism suggests a participation of circulating MCs to the iron disponibility, helping with the maintenance of stock for the erythrocytic production. Even though SCA and PV have different pathophysiology, they both present SE and the circulating MCs from both groups show an important participation in the clearance of RBCs, either by an intrinsic function of MCs or a direct influence of changes in the RBCs, suggesting the important contribution of MCs to avoid oxidative stress and help with iron disponibility for erythropoiesis during the course of SE.

Magnetic Fractionation of Red Blood Cells Based on Oxygen Saturation in Sickle Cell Disease

Anemias are a broad classification of red blood cell (RBC) disorders which effectively result in inadequate oxygen transport in the body. Sickle Cell Disease (SCD) results from a point mutation in hemoglobin, and the hemoglobin-oxygen equilibrium becomes right shifted (i.e., SCD RBCs do not oxygen saturate to the same degree as healthy RBCs). RBC exchange transfusion is a common clinical practice to increase the oxygen carrying capacity of a SCD patient - a process by which the patient's own RBCs are exchanged with healthy donor blood. During this process, large amounts of donor RBCs are discarded along with the patients SCD RBCs. Here we present a new RBC separation modality based on hemoglobin-oxygen equilibrium.It is well known that RBCs become magnetized as a function of the oxygen saturation of its hemoglobin. Due to the difference in hemoglobin-oxygen equilibrium for healthy and SCD RBCs we demonstrate a magnetic method of fractionating RBCs based on oxygen saturation. In our study, we use the SCD transfusion waste product and sort this sample into healthy fractions and SCD fractions using a field flow permeant magnetic separator. Under variable oxygen tension of the sample, we preformed the magnetic sorting.By incorporation of magnetic separation into RBC exchange processes, more efficient selection, and removal of SCD RBCs can be accomplished. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeted Base Editing Strategies for Beta-Hemoglobinopathies

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult hemoglobin β-chain. In β-thalassemia, the reduced (β+) or absent (β0) production of β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) βS-chain causes Hb polymerization with consequent RBC sickling, vaso-occlusive crises, organ damage and reduced life expectancy. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors or CRISPR/Cas9 nuclease are not equally effective in all the patients and/or raise safety concerns. Base editing (BE) allows the introduction of point mutations (C>T by cytidine base editors, CBEs; A>G by adenine base editors, ABEs) without generating dangerous double strand breaks. In this study, we have developed BE strategies aiming either to correct the genetic defect or to target disease modifiers in patient cells, and provide a safe and effective treatment for β-hemoglobinopathies. First, we exploited the capacity of ABEs in combination with single guide RNAs (gRNAs) to correct prevalent β-thalassemic mutations (the CD39 and IVS2-1 β0 mutations and the IVS1-110 β+ mutation). RBCs derived from edited HSPCs exhibited high β-globin levels. The delayed erythroid differentiation typically observed in β-thalassemic cell cultures was corrected by our treatment. Measurement of Annexin+ cells by flow cytometry showed a substantially reduced apoptotic rate in edited β-thalassemic samples compared to untreated controls. Finally, off-target activity was minimal and by-stander edits (additional base conversions occurring close to the target base) did not impact the β-globin expression. In parallel, we used BE to target disease modifiers such as the fetal γ-globin genes. In fact, the co-inheritance of genetic mutations causing a sustained fetal β-like γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin (HbF), HPFH) reduces the clinical severity of β-hemoglobinopathies. HPFH mutations in the promoter of the two γ-globin genes, HBG1 and HBG2 eitherdisrupt the binding sites (BS) for transcriptional repressors (e.g., LRF) or generate BS for transcriptional activators (e.g., KLF1). BE allowed the introduction of HPFH mutations in the HBG promoters, which disrupt the LRF BS (via CBE) or generate the KLF1 BS (via ABE). This led to therapeutically relevant HbF levels in RBCs differentiated from edited β-thalassemia or SCD HSPCs, and rescued the pathological phenotypes (correcting ineffective erythropoiesis in β-thalassemia and the sickling phenotype in SCD RBCs). Finally, serial xenotransplantation experiments showed BE in long-term HSCs; however, the efficiency was reduced compared to input HSPCs. RNA-seq analysis showed no alteration for ABE-treated HSPCs, but activation of the p53 pathway and dysregulation of genes involved in HSC biology in CBE-treated samples. To increase the fitness of edited HSCs and editing efficiency, we optimized the RNA delivery of the BE machinery and used histone deacetylase, RNase and apoptosis inhibitors. Moreover, we developed a strategy to enrich edited cells. By way of example, two rounds of mRNA electroporation led to increased BE efficiency with minimal toxicity. Furthermore, highly processive base editors outperformed the original enzymes even upon only one round of electroporation. In conclusion, we developed efficient BE approaches to either correct highly prevalent mutations or induce HbF expression. Overall, our treatments led to correction of the pathological phenotypes and optimal BE efficiencies to finally enable the clinical development of base-edited HSPCs for β-hemoglobinopathies.

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

Profiling the E3 Ubiquitin Ligase Landscape in Normal and Sickle Red Blood Cells

Sickle cell disease (SCD) is the most common genetic disease affecting about 100,000 Americans and millions of people in developing countries. In sub-Saharan Africa, about 50% of children with SCD die before age five. The pathophysiology of SCD is associated with the formation of sickle hemoglobin (HbS) polymers during hypoxia which can be pharmacologically antagonized. Our goal is to exploit targeted protein degradation (TPD) as a novel modality for preventing or delaying sickling. As a first step, we utilized a label-free quantitative proteomic workflow to qualitatively and quantitatively analyze differential protein expression between red blood cells (RBC) from healthy and SCD patients. Since mature RBCs have a simple but active cellular proteome responsible for degrading proteins damaged by oxidative stress, which is significantly increased in SCD (D'Alessandro et al.,Blood Transfus, 2019), we focused on assessing the differential expression of proteins associated with the ubiquitin-proteasome pathway. We identified a total of 2090 proteins, including 41 proteins in the ubiquitin proteasome pathway as well as 27 and 35 E2 and E3 ligases, respectively. Identified proteins and their abundances correlated with previous literature findings in healthy samples (Bryk and Wiśniewski, J Proteome Res, 2017; Gautier et al., Blood Adv, 2018). We also found E3 ligases that demonstrated selective expression in RBC samples from SCD patients. These ligases have been implicated in various signaling activities including cell regulation pathway, metabolic control, heme synthesis, xenophagy and anti-tumor immunity. Based on this work, we will further explore the feasibility of developing innovative TPD based medicines for SCD and other RBC disorders.

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

S267: PROPERDIN-BLOCKING ANTIBODIES ATTENUATE COMPLEMENT ALTERNATIVE PATHWAY ACTIVATION TRIGGERED BY CELL-FREE HEME IN SICKLE CELL DISEASE MODELS

Background: Complement activation may play an important role in sickle cell disease (SCD) pathophysiology. Heme has been identified as a trigger of complement activation in SCD patients and is present in excess during severe vaso-occlusive crises. Heme is also a DAMP (damage associated molecular pattern) and induces thrombo-inflammation through tissue factor expression. ALXN1820 is a bi-specific VHH nanobody that simultaneously binds human albumin and properdin, thereby effectively and selectively inhibiting alternative pathway (AP) activation and formation of C3 and C5 convertases. The safety of ALXN1820 has been assessed in non-human primates and is currently being tested in a phase 1 study in healthy participants. Aims: 1) To assess if complement activation and heme are functionally linked; 2) To investigate the therapeutic potential of ALXN1820 in in vitro SCD models and in in vivo SCD models using a mouse properdin-blocking monoclonal antibody (mAb), 14E1. Methods:In vitro, complement inactivated C3b (iC3b) and C5b-9 deposition on red blood cells (RBCs) from patients with SCD and complement iC3b and C5b-9 deposition on the endothelial cell line HMEC-1 were assessed by flow cytometry after exposure to heme +/- ALXN1820. In vivo, in Townes SCD mice, vaso-occlusion and hyper-hemolysis were induced by intravenous heme injection (50 µmol/kg) or hypoxia-reoxygenation (8% O2 for 3h then 21% O2 for 1h) in the presence and absence of 14E1 (40 mg/kg), a mouse-specific properdin inhibitor. Vaso-occlusion was measured by staining RBCs with immunofluorescence-labeled Ter-119 antibodies on lung and liver sections. C3b and C5b-9 deposition on SCD RBCs was assessed by flow cytometry. Markers of hemolysis (bilirubin, lactate dehydrogenase, free hemoglobin, free heme) were quantified in plasma using commercial assays. Results: In RBCs from patients with SCD, cell-free heme triggered marked iC3b and C5b-9 deposition (Table). Deposition was blocked in the presence of ALXN1820 by >95% for iC3b and by >85% for C5b-9. HMEC-1 cells exposed to heme showed marked deposition of iC3b and C5b-9. In the presence of ALXN1820, deposition was blocked by >70% for iC3b and >85% for C5b-9. Pretreatment of mice with 14E1 markedly ameliorated vaso-occlusion and reduced both C3b and C5b-9 deposition on RBCs, and hemolysis biomarkers. Image:Summary/Conclusion: These data strengthen the hypothesis that in SCD, cell-free heme is a potent trigger of complement AP activation, which can be blocked by targeting properdin. Investigating the efficacy and safety of anti-properdin ALXN1820 in patients with SCD is warranted as a novel approach to treatment of acute and chronic SCD complications.

Peroxiredoxin-2 recycling is slower in denser and pediatric sickle cell red cells.

Peroxiredoxin-2 (Prx-2) is a critical antioxidant protein in red blood cells (RBC). Prx-2 is oxidized to a disulfide covalently-bound dimer by H2 O2 , and then reduced back by the NADPH-dependent thioredoxin-thioredoxin reductase system. The reduction of oxidized Prx-2 is relatively slow in RBCs. Since Prx-2 is highly abundant, Prx-2s' peroxidase catalytic cycle is not considered to be limiting under normal conditions. However, whether Prx-2 recycling becomes limiting when RBCs are exposed to stress is not known. Using three different model systems characterized by increased oxidative damage to RBCs spanning the physiologic (endogenous RBCs of different ages), therapeutic (cold-stored RBCs in blood banks) and pathologic (RBCs from sickle cell disease (SCD) patients and humanized SCD mice) spectrum, basal levels of Prx-2 oxidation and Prx-2 recycling kinetics after addition of H2 O2 were determined. The reduction of oxidized Prx-2 was significantly slower in older versuin older versus younger RBCs, in RBCs stored for 4-5weeks compared to 1week, and in RBC from pediatric SCD patients compared to RBCs from control non-SCD patients. Similarly, the rate of Prx-2 recycling was slower in humanized SCD mice compared to WT mice. Treatment of RBC with carbon monoxide (CO) to limit heme-peroxidase activity had no effect on Prx-2 recycling kinetics. Treatment with glucose attenuated slowed Prx-2 recycling in older RBCs and SCD RBCs, but not stored RBCs. In conclusion, the reduction of oxidized Prx-2 can be further slowed in RBCs, which may limit the protection afforded by this antioxidant protein in settings associated with erythrocyte stress.

Dynamics of Individual Red Blood Cells Under Shear Flow: A Way to Discriminate Deformability Alterations.

In this work, we compared the dynamics of motion in a linear shear flow of individual red blood cells (RBCs) from healthy and pathological donors (Sickle Cell Disease (SCD) or Sickle Cell-β-thalassemia) and of low and high densities, in a suspending medium of higher viscosity. In these conditions, at lower shear rates, biconcave discocyte-shaped RBCs present an unsteady flip-flopping motion, where the cell axis of symmetry rotates in the shear plane, rocking to and fro between an orbital angle ±ϕ observed when the cell is on its edge. We show that the evolution of ϕ depends solely on RBC density for healthy RBCs, with denser RBCs displaying lower ϕ values than the lighter ones. Typically, at a shear stress of 0.08 Pa, ϕ has values of 82 and 72° for RBCs with average densities of 1.097 and 1.115, respectively. Surprisingly, we show that SCD RBCs display the same ϕ-evolution as healthy RBCs of same density, showing that the flip-flopping behavior is unaffected by the SCD pathology. When the shear stress is increased further (above 0.1 Pa), healthy RBCs start going through a transition to a fluid-like motion, called tank-treading, where the RBC has a quasi-constant orientation relatively to the flow and the membrane rotates around the center of mass of the cell. This transition occurs at higher shear stresses (above 0.2 Pa) for denser cells. This shift toward higher stresses is even more remarkable in the case of SCD RBCs, showing that the transition to the tank-treading regime is highly dependent on the SCD pathology. Indeed, at a shear stress of 0.2 Pa, for RBCs with a density of 1.097, 100% of healthy RBCs have transited to the tank-treading regime vs. less than 50% SCD RBCs. We correlate the observed differences in dynamics to the alterations of RBC mechanical properties with regard to density and SCD pathology reported in the literature. Our results suggest that it might be possible to develop simple non-invasive assays for diagnosis purpose based on the RBC motion in shear flow and relying on this millifluidic approach.

Nicotinamide Improves Anemia and Decreases Reactive Oxygen Species in Sickle Cell Disease Mice

The polymerization of deoxygenated HbS molecules in the red blood cells (RBCs) of patients with sickle cell disease (SCD) causes RBC destruction resulting in chronic pain, debilitating acute pain crises, strokes, multi-organ damage and a reduced life span. Therapeutic options remain limited. Bone marrow transplantation can be curative but is not an option for the majority of patients. Gene therapy interventions that also offer the promise of a cure are under investigation but are not likely to be available to the vast majority of patients in the near future. Hydroxyurea, the first drug approved for treatment of SCD, increases levels of Fetal Hemoglobin (HbF) that inhibit polymerization of HbS molecules but is not effective in all patients while a more powerful HbF-inducing drug, the DNA methyltransferase inhibitor decitabine has yet to be approved. L-glutamine, another approved therapeutic option, increases NAD redox potential and decreases reactive oxygen species (ROS) in the sickle RBCs to reduce symptoms. In our laboratory we have observed that increased ROS is associated with the retention of mitochondria in the SCD RBCs and have hypothesized that the abnormal presence of mitochondria in these cells is a major source of ROS (Jagadeeswaran et al Exp Hematol 50:46-52, 2017). In this investigation we have tested the hypothesis that chronic oral supplementation with nicotinamide, a direct precursor of NAD synthesis, would improve NAD redox potential, decrease mitochondrial retention and ROS in SCD RBCs, and reduce anemia in the SCD mouse model. The effect of nicotinamide was tested in SCD mice whose drinking water was supplemented for three months with 1% nicotinamide. The percentage of RBCs retaining mitochondria and the levels of ROS were determined by flow cytometric assays using the mitochondrial-specific dye TMRM and the ROS probe CM-H2DCFDA, respectively. In SCD mice receiving nicotinamide the fraction of RBCs retaining mitochondria was reduced 22.1% (p<0.05) and the level of ROS in RBCs was reduced 41% (p<0.01) compared to control SCD mice. The reticulocyte percentage was reduced 28% in nicotinamide-treated SCD mice compared to control SCD mice (p<0.01). The total RBC count was 30% higher (p<0.05) in nicotinamide-treated mice (6.61±0.76 X 10 6/μl) compared to control SCD mice (5.08±0.70 X 10 6/μl). Similar differences in hematocrit and total hemoglobin were also observed but failed to reach statistical significance. Total NAD levels were not significantly different in SCD mice receiving nicotinamide compared to control SCD mice (p<0.05), but the NADH/NAD total ratio was increased 2 fold (p<0.05). These results show that oral administration of high doses of nicotinamide decreases mitochondrial retention and ROS in SCD RBCs and improves NAD redox potential and anemia in SCD mice. These effects strongly suggest that additional studies be performed to investigate nicotinamide as a therapeutic option in SCD. [Display omitted] DisclosuresSaunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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.

Pegylated Carboxyhemoglobin (SANGUINATE®) Restores Blood Flow and Tissue Oxygenation in a Novel Rat Model of Acute Vaso-Occlusive Crisis

INTRODUCTION: Sickle Cell Disease (SCD) hemoglobin S (HbS) promotes hemoglobin polymerization and red blood cell (RBC) sickling during hypoxia. PEGylated carboxyhemoglobin bovine (PEG-COHb; SANGUINATE®) has been previously shown to reverse sickling of SCD RBC in vitro by directed gas transfer to hypoxic RBC. A rat model was used to evaluate the capability of SANGUINATE® to restore cardiovascular rheology and tissue oxygenation (PISFO2) at sites distal to acute Vaso-Occlusions (VOC).OBJECTIVES: Evaluate the in vivo capability of PEG-COHb to improve rheology in rats placed in active VOC through transfusion of human SCD RBC while monitoring treatment effects on PISFO2 and systemic functions.METHODS: Human homozygous (HbSS) whole blood was obtained from consented SCD volunteers. The SCD RBC were isolated by centrifugation, triple-washed and resuspended in lactated Ringer's saline (LRS). Male Sprague Dawley rats were anesthetized, cannulated for fluid transfer, and surgically prepared for microscopic study of the spinotrapezius microcirculation. Isovolemic exchange transfusion of rat blood with suspended SC RBCs (27.5 ± 3.5% total blood volume) was conducted until PISFO2 at the distal portion of the spinotrapezius muscle was < 1 mmHg. VOC was visually confirmed as wide-spread precapillary arteriolar flow-arrest. Animals in VOC were subsequently treated 15 minutes later with PEG-COHb or LRS administered as a bolus of 8.0 ml/kg over 5 minutes. Animals were observed for up to 4 hours post-treatment and then euthanized. Measurements included systemic cardiovascular parameters, survival, blood chemistry, and PISFO2 via phosphorescence quenching microscopy and were obtained across treatment groups and the no treatment [Sham] control group.RESULTS: Exchange transfusion successfully produced a reduction in PISFO2 in the distal portion of the spinotrapezius muscle for all animals to <1 mmHg presumably through Vaso-Occlusions. VOC was visually apparent as flow arrest was initially observed in capillaries and most small venules and arterioles, and then progressed proximally into sites with larger feed vessels and higher intravascular pressures with subsequent declines in local PISFO2. Intervention with PEG-COHb and LRS both showed a visual improvement in microvascular flow, but yielded differential effects on PISFO2 in all sites (average return to 96 ± 6% and 64 ± 20% of baseline, respectively). PEG-COHb -treated animals survived the entire 4 h observation phase with a final global spinotrapezius PISFO2 level within 10% of baseline values. In contrast, global spinotrapezius PISFO2 declined to 0 mmHg for LRS treated animals by 3 h with rapid decline in mean arterial pressure and death occurring prior to 4 h. Serum lactate levels were monitored to provide an assessment of systemic ischemia from time 0 after exchange transfusion to the last collection point near the end of the experiment. Lactate increased for untreated Sham (34 ± 4.5 to 124 ± 43.0 mg/dl) and LRS groups (48 ± 15.0 to 110 ± 43.0 mg/dl), but remained stable for SANGUINATE®-treated animals (42 ± 1.5 to 40 ± 4.5 mg/dl). Total microcirculatory collapse occurred in Sham animals by 3h along with a null-reading of PISFO2, which was followed by a rapid decline in mean arterial pressure and death at 3.5 h similar to the LRS group.SUMMARY: This study was conducted in a novel rat model providing the ability to monitor real-time microcirculatory events and systemic parameters, which is difficult in murine models due to their smaller stature. PEG-COHb treatment promoted VOC resolution as evidenced through improved blood flow and concomitant effects on PISFO2. Importantly, the gas transfer properties of SANGUINATE® not only acutely resolved VOC, but provided the added benefit of restoring PISFO2 to near baseline values. In contrast, LRS animals showed transient rheology and minimal PISFO2 improvements, but levels declined over time and outcomes were similar to sham controls. Restoring blood flow and PISFO2 are key events to limiting VOC pain and complications and the treatment with PEG-COHb demonstrated the ability to achieve these critical functions suggesting it may provide improvements in treating acute VOC in human SCD individuals. DisclosuresSong:Prolong Pharmaceuticals: Consultancy, Research Funding. Nugent:Prolong Pharmaceuticals: Consultancy, Research Funding.