Pathogenesis Of Sickle Cell Disease Research Articles

P-059: TRIAL IN PROGRESS: THE RANDOMIZED, DOUBLE-BLIND, MULTICENTER, PLACEBO-CONTROLLED PHASE 3 RESOLVE TRIAL INVESTIGATING THE EFFICACY OF VOXELOTOR WITH STANDARD OF CARE IN THE RESOLUTION OF LEG ULCERS IN PATIENTS WITH SICKLE CELL DISEASE

Purpose: Sickle cell disease (SCD) is a heritable blood disorder caused by a point mutation in the β-globin gene. Polymerization of deoxygenated mutated hemoglobin is the major molecular process in SCD pathogenesis that leads to red blood cell sickling and hemolysis. In addition to the major complications of SCD—hemolytic anemia, inflammation, and vascular occlusion—patients often experience secondary complications such as stroke, organ damage, shortened lifespan, and painful and debilitating skin ulcers. The prevalence of leg ulcers among people with SCD is likely underestimated, given the lack of registries and large prospective studies examining this complication. Current estimates of leg ulcers vary widely by geographic region: 43% in Brazil, 30% in Jamaica, 27% in Nigeria, 19% in Ghana, 13% in Sierra Leone, 8% in Saudi Arabia, and 1%-5% in the US. Among Americans with SCD, an estimated 14%-18% may develop leg ulcers. Voxelotor, a sickle hemoglobin polymerization inhibitor, is approved in the US for the treatment of SCD in adults and pediatric patients aged ≥4 years and in the EU for the treatment of hemolytic anemia due to SCD in adults and pediatric patients aged ≥12 years as a monotherapy or in combination with hydroxycarbamide. In a post hoc analysis of voxelotor-treated patients from the phase 3 HOPE study (NCT03036813), leg ulcers resolved within 24 weeks in 10 out of 14 patients, and leg ulcers improved or resolved in 13 out of 14 patients by week 72. Materials and methods: RESOLVE is an ongoing phase 3, randomized, double-blind, placebo-controlled, multicenter trial investigating the efficacy of voxelotor with standard of care (SOC) in the resolution of leg ulcers in patients with SCD. Target enrollment is 80 patients from Nigeria, Kenya, and Brazil with a confirmed diagnosis of SCD (HbSS or HbSβ0 genotype), aged ≥12 years, and with ≥1 cutaneous ulcer on the lower extremity (leg, ankle, or dorsum of foot) that meets the following criteria: ≥2 weeks’ and <6 months’ duration at screening and >2 cm2 in area before randomization. After a 2-week run-in period, participants are randomized 1:1 to receive once-daily oral voxelotor 1500 mg or placebo in addition to SOC for 12 weeks. After the randomized treatment period, all participants receive open-label voxelotor 1500 mg plus SOC for 12 weeks (Figure). Results: The primary objective of the study is to assess the efficacy of voxelotor plus SOC compared with placebo plus SOC on leg ulcer healing, measured by the proportion of patients with resolution of target ulcer(s) in each treatment group by week 12. Key secondary endpoints include days to resolution of target ulcer(s) up to week 12, change from baseline in total surface area of target ulcer(s) at week 12, and incidence of new ulcers by week 12. Conclusion: Results from this study will further guide clinicians and patients regarding the clinical use of voxelotor for the treatment of leg ulcers in patients with SCD. Participants will have the option to enroll in a separate open-label extension study after the 24-week treatment period.Figure. RESOLVE Study Design, Population, and Timeline J. DOSS declares a conflict of interest: Stock shareholder: Global Blood Therapeutics, Roche

PI-10: DIETARY IRON RESTRICTION PROTECTS AGAINST VASO-OCCLUSION AND ORGAN DAMAGE IN SICKLE CELL DISEASE

Purpose: Sickle cell disease (SCD) is the most commonly inherited monogenic hemoglobinopathy and results from a mutation in the β-globin gene (HbS), leading to red blood cell (RBC) sickling, vaso-occlusive episodes (VOE), and organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron-overload, and another subpopulation of SCD patients manifest iron deficiency. SCD mice exhibit increased iron absorption via upregulation of the intestinal transcription factor hypoxia inducible factor-2a(HIF-2a) leading to the development of iron overload. Disruption of intestinal HIF-2a or limiting iron intake by dietary iron restriction in SCD mice, significantly reduced systemic iron overload, lowered cellular sickle hemoglobin as reflected by reduced RBC mean corpuscular hemoglobin concentration (MCHC), and consequently improved hemolysis and ameliorated anemia. Understanding iron homeostasis is therefore critical to SCD pathogenesis and holds the promise for identifying novel approaches to treat SCD. Materials and methods: SCD mice were generated by transplanting bone marrow cells from Berkeley SCD (Tg[Hu-miniLCRα1GγAγδβS]Hba−/− Hbb−/−) mice into 8-week old, lethally-irradiated C57BL/6J mice. Immediately after transplantation, SCD mice were fed either a regular-iron diet (Ctrl) containing 185 parts per million (ppm) iron or an otherwise identical iron-restricted diet (IRD) containing 3 ppm iron. VOE and organ damage analysis were done 3-4 months after treatments. Results: IRD resulted in a reduced organ iron concentration (Fig-1A) in SCD compared to Ctrl group, confirming that dietary iron restriction ameliorates organ iron deposition in SCD mice. SCD mice treated with IRD exhibited reduced MCHC (Fig-1B) and serum indirect bilirubin(IDBILC) (Fig-1C) indicating a decrease in cellular sickle hemoglobin and hemolysis. Furthermore, IRD reduced the fraction of aged neutrophils by >50% in SCD mice (Fig-1D), which corresponded with improved VOE parameters. Specifically, intravital microscopy (IVM) demonstrated elevated mean centerline RBC velocity and blood flow rate and decreased leukocyte adhesion in IRD compared to Ctrl mice (Fig-1E-G). Importantly, IRD led to prolonged survival after TNF-a challenge in SCD mice (Fig-1H). IRD resulted in a dramatic reduction in lung leukocyte infiltration, indicating significantly reduced lung inflammation in SCD mice (Fig-2A). Compared to Ctrl liver samples, IRD resulted in significantly less liver necrosis, leukocyte infiltration and fibrosis (Fig-2B,C), decreased serum liver enzymes (ALT, AST; Fig-2D), and reduced levels of total and direct bilirubin (TBILC, DBILC; Fig-2D).We further hypothesized that, in addition to reducing iron concentration in organs, IRD ameliorates organ damage by preventing translocation of microbial compounds.IRD inhibits translocation of FITC-Dextran by 5-fold compared to Ctrl suggesting functionally improved gut permeability (Fig-2E). We detected significantly decreased TLR2 ligands in serum of IRD mice (Fig-2F) suggesting ligands from gram-positive bacteria (TLR2 agonists) may play a role in triggering SCD complications. Conclusion: Our study demonstrates for the first time that dietary iron restriction reduces VOE severity and alleviates chronic organ damage in SCD mice. Novel approaches that target iron availability/absorption in the gut, the gut microbiome and aged neutrophil crosstalk and gut barrier integrity, provide an important area of further investigation with the potential of improving hemolysis, VOE, and organ damage in SCD patients.(A)The bone-marrow/spleen/liver Non-heme iron content. (B)MCHC, (C)IDBILC,(D)aged neutrophils percentage in Ctrl/IRD SCD mice. IVM parameters (E) (Vrbc) (F) blood flow rate (J) the number of adherent leukocytes and (J) survival rate after TNF-a injection(A)Leukocyte infiltration in lung and (B)liver tissue. (C)Liver necrosis, leukocyte infiltration, and fibrosis. (D)Liver ALT and AST, TBILC, and DBILC from sera. (E)serum FITC-Dextran and (F) TLR2 ligands activity in Ctrl/IRD SCD mice The authors do not declare any conflict of interest

Metabolic Reprogramming in Sickle Cell Diseases: Pathophysiology and Drug Discovery Opportunities.

Sickle cell disease (SCD) is a genetic disorder that affects millions of individuals worldwide. Chronic anemia, hemolysis, and vasculopathy are associated with SCD, and their role has been well characterized. These symptoms stem from hemoglobin (Hb) polymerization, which is the primary event in the molecular pathogenesis of SCD and contributes to erythrocyte or red blood cell (RBC) sickling, stiffness, and vaso-occlusion. The disease is caused by a mutation at the sixth position of the β-globin gene, coding for sickle Hb (HbS) instead of normal adult Hb (HbA), which under hypoxic conditions polymerizes into rigid fibers to distort the shapes of the RBCs. Only a few therapies are available, with the universal effectiveness of recently approved therapies still being monitored. In this review, we first focus on how sickle RBCs have altered metabolism and then highlight how this understanding reveals potential targets involved in the pathogenesis of the disease, which can be leveraged to create novel therapeutics for SCD.

Genetic contribution and functional impairment of inflammasome in sickle cell disease.

Sickle cell disease (SCD), one of the most common single-gene disorders, is caused by mutations in the hemoglobin ß-chain gene. Clinical presentation is heterogeneous, and inflammation is a common condition. Thereby, we hypothesized that inflammasome and related cytokine IL-1ß could represent significant SCD pathogenesis contributors. 161 SCD (SS/Sβ) patients were enrolled for the study. Seven single nucleotide polymorphisms (SNPs) in 5 inflammasome genes (NLRP1, NLRP3, NLRC4, CARD8, IL1B) were selected based on minor allele frequency. Total peripheral blood mononuclear cells (PBMC) and monocytes were isolated from 10 out of 161 SCD patients (HbSS) and 10 healthy donors (control group, Ctrl) for inflammasome analysis. SCD patients presented a functional impairment of inflammasome, with monocytes and peripheral blood mononuclear cells (PBMC) exhibiting a different NLRP3 inflammasome activation rate. Gain-of-function variants in NLRP1 and IL1B genes resulted associated with a mild SCD clinical presentation. Our results can contribute to the understanding of SCD inflammation. SCD patients showed possible exhaustion of monocytes due to chronic inflammation, moreover others cells in PBMC can contribute to the NLRP3 inflammasome activation. NLRP1 gain-of-function was associated with mild clinical presentation, suggesting that other inflammasome receptors can be involved in SCD. This is the first study reporting a significant contribution of inflammasome SNPs in SCD.

A critical evaluation of crizanlizumab for the treatment of sickle cell disease

ABSTRACT Introduction P-selectin is a key adhesion molecule in the pathogenesis of sickle cell disease, including acute painful event(s). Many of the mediators activated in prototypical pain crisis are also involved in other complications seen in this population. Crizanlizumab is a monoclonal antibody approved in the US in 2019 for patients of all genotypes of sickle cell disease. By blocking P-selectin, it effectively prevents acute painful event(s) and has a manageable safety profile. Areas covered In this review, we provide an overview of the (i) biology of P-selectin in sickle cell disease, (ii) various agents inhibiting P-selectin, (iii) pharmacology of crizanlizumab, (iv) preclinical and clinical data on crizanlizumab, and (v) its potential for other indications, ongoing studies, regulatory status, and cost issues. Further, we describe its position among other approved agents in sickle cell disease and project future directions as well. Expert opinion Crizanlizumab holds great promise in modulating the natural history of sickle cell disease and may have pleotropic effects. Studies are ongoing to define its role in preventing other sickle cell-related complications, non-sickle cell inflammatory states, and thrombotic disorders.

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

Association of Thrombospondin-1 Gene Polymorphism with Elevated Tricuspid Regurgitant Velocity in Sickle Cell Anemia

Background: Pulmonary hypertension (PH) is associated with premature mortality in adults with sickle cell anemia (SCA). Decreased nitric oxide bioavailability has been implicated in the pathogenesis of SCA associated PH. Thrombospondin-1 (TSP1) protein encoded by THBS1 gene inhibits nitric oxide/cGMP signaling and has been hypothesized to promote PH in SCA (Novelli et al. Am J Physiol Lung Cell Mol Physiol 2019). Recently in a cross-sectional targeted gene analysis in adults with SCA, THBS1 gene single nucleotide polymorphisms (SNPs) rs1478605-G and rs1478604-T were found to be associated with PH, as estimated by its surrogate echocardiogram marker tricuspid regurgitant velocity (TRV). However, neither of these SNPs were validated in an external cohort (Jacob et al. Am J Hematol. 2017).Objective: To test the hypothesis that THBS1 gene SNPs were associated with TRV, we first validated the association of previously reported THBS1 gene SNPs (rs1478605-G and rs1478604-T) with TRV as quantitative and binary variables (elevated TRV: < or ≥ 2.5m/sec) in a longitudinal, pediatric, SCA patient cohort. We then assessed the associations of 8 other common THBS1 SNPs with false discovery rate controlled at 0.1. Finally, we sought to generate a polygenic score (PGS) incorporating this region genomic data and determine its association with TRV.Methods: Our cohort was comprised of children aged 5-18 years with HbSS or HbS/β 0thalassemia enrolled in either Long-Term Effects of Erythrocyte Lysis Trial (ELYSIS, NCT00842621) (Yates et al. Pediatr Blood and Cancer. 2019) and Sickle Cell Clinical and Intervention Program (SCCRIP, NCT02098863). All participants underwent prospective measurement of TRV by 2D-echocardiogram at baseline steady state (≥ 4 weeks from transfusion, hospitalization) and then repeated two years later (Rai et al. Blood Adv. 2021). Generalized linear mixed models were used to assess the association between quantitative or elevated TRV and genetic markers, after adjusting for age at baseline, sex, time point, NT-pro-BNP, creatinine and 5 principal components (PC). Three SNPs, rs1478605-G, rs753599-A and rs1051442-T statistically significant at levels given above for either quantitative or elevated TRV and whose pairwise linkage disequilibrium R 2 ≤ 0.1 were selected to build a PGS. The PGS was defined as the summed number of TRV-increasing unfavorable alleles carried by each individual across all selected SNPs. Two-stage iterative resampling (TSIR) approach was used to internally discover and validate the associations of PGS with TRV at an overall type I error rate of 0.05 with randomly sampled half of samples as the discovery cohort and the remaining as the validation cohort (Kang et al. J Hum Genet. 2015).Results: Our cohort included 138 children (276 data points) with SCA with mean age of 9.9 years (SD 3.25 years). We validated the association of quantitative TRV with previously reported two SNPs rs1478605 (estimate = -0.11, standard error [SE] = 0.04, p = 0.003) and rs1478604 (estimate = -0.09, SE = 0.04, p = 0.014). We further identified new associations of elevated TRV with THBS1 SNPs rs10551442 odds ratio (OR) 0.26 (95% CI 0.09, 0.77; p = 0.017), rs17633107 OR 0.26 (95% CI 0.09, 0.77; p=0.017), rs3743125 OR 4.4 (95% CI 1.2, 1.7; p = 0.03), and rs753599 OR 5.3 (95% CI 1.4, 2.1; p=0.018). Two of these SNPs rs10551442 (estimate = -0.13, SE = 0.05, p = 0.013) and rs17633107 (estimate = -0.13, SE = 0.05, p = 0.013) were also associated with quantitative TRV. One unfavorable allele increase in PGS was associated with a 0.1 m/sec increase in TRV (Figure 1A) and a 2.1 increase in log-odds of having an elevated TRV (Figure 1B). The associations of PGS with quantitative and elevated TRV were discovered and validated in 36 and 21 out of 100 repetitions by the TSIR analysis, respectively.Conclusion: We validated two previously published variants in the THBS1 gene rs1478605 and rs1478604 in an external and independent pediatric cohort with quantitative TRV. We also identified associations of common SNPs rs10551442, rs17633107, rs3743125, and rs753599 with TRV ≥ 2.5m/sec and of rs10551442 and rs17633107 with quantitative TRV in a pediatric population and created a PGS incorporating these data. The PGS is significantly associated with TRV, which was internally validated using TSIR approach. Our genomic findings further support the role of TSP1 in the pathophysiology of the pulmonary vasculopathy seen in SCA. [Display omitted] DisclosuresAtaga: Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Consultancy; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees. Hankins: Global Blood Therapeutics: Consultancy; Vindico Medical Education: Consultancy; Bluebird Bio: Consultancy; UpToDate: Consultancy. Estepp: Global Blood Therapeutics: Consultancy, Research Funding.

Imaging Blood-Brain Barrier Permeability Through MRI in Pediatric Sickle Cell Disease: A Feasibility Study.

Blood-brain barrier (BBB) disruption may lead to endothelium dysfunction and inflammation in sickle cell disease (SCD). However, abnormalities of BBB in SCD, especially in pediatric patients for whom contrast agent administration less than optimal, have not been fully characterized. To examine BBB permeability to water in a group of pediatric SCD participants using a non-invasive magnetic resonance imaging technique. We hypothesized that SCD participants will have increased BBB permeability. Prospective cross-sectional. Twenty-six pediatric participants (10 ± 1 years, 15F/11M) were enrolled, including 21 SCD participants and 5 sickle cell trait (SCT) participants, who were siblings of SCD patients. 3 T. Water extraction with phase-contrast arterial spin tagging with echo-planer imaging, phase-contrast and T1 -weighted magnetization-prepared rapid acquisition of gradient echo. Water extraction fraction (E), BBB permeability-surface area product (PS), cerebral blood flow, hematological measures (hemoglobin, hematocrit, hemoglobin S), neuropsychological scores (including domains of intellectual ability, attention and executive function, academic achievement and adaptive function, and a composite score). Regions of interest were drawn by Z.L. (6 years of experience). Wilcoxon rank sum test and chi-square test for group comparison of demographics. Multiple linear regression analysis of PS with diagnostic category (SCD or SCT), hematological measures, and neuropsychological scores. A two-tailed P value of 0.05 or less was considered statistically significant. Compared with SCT participants, SCD participants had a significantly higher BBB permeability to water (SCD: 207.0 ± 33.3 mL/100 g/minute, SCT: 171.2 ± 27.2 mL/100 g/minute). SCD participants with typically more severe phenotypes also had a significantly leakier BBB than those with typically milder phenotypes (severe: 217.3 ± 31.7 mL/100 g/minute, mild: 193.3 ± 31.8 mL/100 g/minute). Furthermore, more severe BBB disruption was associated with worse hematological symptoms, including lower hemoglobin concentrations (β=-8.84, 95% confidence interval [CI] [-14.69, -3.00]), lower hematocrits (β=-2.96, 95% CI [-4.84, -1.08]), and higher hemoglobin S fraction (β=0.77, 95% CI [0.014, 1.53]). These findings support a potential role for BBB dysfunction in SCD pathogenesis of ischemic injury. 2 TECHNICAL EFFICACY: Stage 2.

Endothelial dysfunction biomarkers in sickle cell disease: is there a role for ADMA and PAI-1?

Within the spectrum of sickle cell disease (SCD) are sickle cell anemia (SCA), presence of hemoglobin SS (HbSS), hemoglobin SC disease (HbSC), and sickle cell β-thalassemia (Sβ-thal). Asymmetric dimethylarginine (ADMA) competitively inhibits the binding of arginine to NOS, reducing NO production. In patients with HbSS, increased levels of ADMA have been reported, as well as changes in many hemostatic biomarkers, including the plasminogen activator inhibitor type 1 (PAI-1). We hypothesized that high levels of ADMA and PAI-1 may be associated with more severe SCD. Thus, ADMA and PAI-1 levels were determined in 78 individuals including 38 adult patients with SCD and 40 control subjects. Higher levels of ADMA were shown in HbSS and Sβ-thal patients compared to controls. Concerning PAI-1, all patients showed high levels of PAI-1 compared to controls. As a role of NO in the pathogenesis of SCD has already been established, we concluded that high levels of ADMA should compromise, at least in part, NO synthesis, resulting in endothelial dysfunction. Elevated plasma levels of PAI-1 in all patients may indicate not only endothelial dysfunction but also a hypofibrinolytic state favoring thrombotic complications. Finally, high levels of ADMA and PAI-1 may be associated with more severe SCD.

Expression of Netosis Regulators and Correlation with Inflammatory and Coagulation Biomarkers in Sickle Cell Anemia Patients

Introduction: Sickle cell anemia (SCA) is a multi-systemic disease characterized by inflammation and chronic hemolysis. SCA is also associated with an increased risk of venous thromboembolism (VTE), with recent evidence suggesting that coagulation activation could also be involved in the pathogenesis of progressive organ failure in this condition. The generation of neutrophil extracellular traps, also termed NETosis, has been associated with the pathogenesis of VTE, and is also part of the immunothrombosis process. Furthermore, biomarkers of NETosis have been demonstrated in both animal models and SCA patients. NETosis is a complex process that involves the orchestrated participation of several proteins such as peptidyl arginine deaminase (PADI4), neutrophil elastase (ELANE) and myeloperoxidase (MPO). PADI4 mediates histone critulination, an essential step for NETosis, so that inhibition of PADI4 could be considered a potential therapeutic strategy for conditions in which NETosis play a relevant pathogenic role. Although attractive, investment in this strategy requires a more detailed knowledge of the role of PADI4 in pathogenesis of SCA. Objetives: in this study, we aimed to evaluate the expression of PADI4, ELANE, and MPO in steady state SCA patients, and to correlate these results with markers of inflammation and coagulation activation. Methods: mRNA was obtained from isolated neutrophils, and gene expression of PADI4, ELANE, and MPO were measured by qPCR in 54 steady state SCA patients and 40 healthy volunteers matched by age and geographic origin. Gene expressions levels were then correlated with inflammatory mediators and D-dimer. Results: no significant difference could be observed in the expression of PADI4 (0.55 vs 0.49; P = 0.42) and MPO (0.29 vs 0.39; P = 0.88) between healthy volunteers and patients respectively. In contrast, ELANE was up-regulated when patients compared with controls (1.46 vs 0.16; P <0.0001). PADI4 expression did not correlate with any biomarker of inflammation or coagulation (P>0.05). Interestingly, both ELANE and MPO were positively correlated with D-dimer (R=0.4; P=0.004 and R=0.32; P=0.004, respectively), and Von Willebrand factor (R=0.27; P=0.04 and R=0.30; P=0.04, respectively). ELANE was also correlated with TNF-α (R=0.30; P=0.03). Conclusion: gene expression of ELANE, but not PADI4 is up-regulated in steady state SCA patients, with both ELANE and MPO presenting significant associations with D-dimer and other biomarkers of inflammatory activation. Additional studies are ongoing to evaluate PADI4 expression and activity, both in steady state and during acute episodes in SCA. A more detailed evaluation of the role of PADI4 in the pathogenesis of SCA is warranted for the proper design of clinical trials based on PADI4 inhibition. DisclosuresNo relevant conflicts of interest to declare.

The Abnormal Presence of Mitochondria in Circulating Red Blood Cells Cause an Increased Oxygen Consumption Rate, ROS Generation and Hemolysis in Patients with Sickle Cell Disease

Sickle Cell Disease (SCD) is an inherited blood disorder that affects millions of people worldwide, and it is caused by a mutation of the β-globin gene which results in the polymerization of HbS when deoxygenated. Patients with sickle cell disease experience severe pain, multi organ damage, and shortened life span. Changes arising from sickle hemoglobin (HbS) inside the red blood cell (RBC) leads to an imbalance of the oxidative reactions which increases reactive oxygen species (ROS) generation. ROS causes hemolysis and damages blood vessels. Previously, our lab demonstrated that SCD patients have a significant fraction of mitochondria retaining-RBCs which were associated with excessive levels of ROS. In addition immature mitochondria containing reticulocytes, R1 reticulocytes, are increased in the peripheral blood of SCD patients compared to control subjects. Here we report that the percentage of mitochondria retaining-RBCs directly correlated with the absolute number of reticulocytes (n=18,r=0.69, P< 0.001). As would be expected we were able to link mitochondria-retention with hemolysis. In addition, when we investigated energy metabolism we made the novel finding that SCD RBCs have an increased oxygen consumption rate (OCR). Mitochondrial-RBC retention and markers of hemolysis (serum bilirubin, and LDH) were compared by using Pearson's correlation coefficient analysis. The total serum bilirubin was directly associated with the percentage of mitochondria-containing RBCs in SCD patients' blood samples (r=0.31, n=41, p<0.04). A subgroup analysis showed a strong association between bilirubin and mitochondria-retaining RBCs in SCD patients who were not taking Hydroxyurea (r=0.55, n=17, P<0.02). Lactate dehydrogenase (LDH) is a known biomarker for hemolysis-and associated disease complications and early mortality in patients with SCD. There was a less significant association of percentage of mitochondria- retaining RBCs in SCD with serum LDH (r=0.02, n=40, P<0.2) when all patients were analyzed, however, a strong association was seen with serum LDH and mitochondria retaining RBCs in SCD patients who were not taking Hydroxyurea (r=0.67, n=17, P<0.002). We further investigated the mitochondrial RBCs energy metabolism in SCD as compared to controls in both human and mice. RBCs obtained from Townes SCD or control mice were seeded in an Agilent Seahorse XF 24-well plate using the Seahorse XF Base Medium with supplements, and we monitored the OCR and extracellular acidification rate (ECAR), in real time by Seahorse XFe-extracellular flux analyzer. OCR(pmol/min) standardized for protein was significantly higher in sickle cell RBCs of both human and mice origin compared to control. [((SCD patients(Hb SS): 6.92± 1.23, n=2 and control subjects (Hb AA)2.55±0.17, n=2, p<0.006)). ((Townes SCD (HbSS): 169.3± 30.8, n=3 and Transgenic control (HbAA):5.0±0.8, n=2, p<0.001))]. OCR was reduced to normal levels with oligomycin which is a known blocker of mitochondrial respiration. ECAR, a measure of glycolysis, was significantly higher levels in SCD patient and mouse samples compared to control. These findings support our premise that mitochondria retaining RBCs play a role in intravascular hemolysis. In addition, they indicate that RBC from SCD patients have increased oxygen consumption. Mitochondrial retention in RBCs have two possible mechanism of damage to the RBC, increased ROS formation and increased deoxygenated HbS polymer formation due to mitochondrial oxygen consumption. These results support further investigation into the role of mitochondrial energy metabolism in the pathogenesis of sickle cell disease. DisclosuresNo relevant conflicts of interest to declare.

Progress in Gene Therapy of Sickle Cell Disease Based on Hemoglobin F--Review

Sickle cell disease (SCD) is a single gene genetic disease, which seriously threatens the life span and quality of patients. On the basis of the pathogenesis of SCD and the alternative therapy based on fetal hemoglobin F (HbF), the research progress of transcription factors involved in the regulation of HbF gene expression, such as BCL11A, ZBTB7A, KLF-1, c-MYB and SOX6, as well as the application of CRISPR / Cas9, TALEN, zinc finger nuclease and other gene editing technologies in this field has been made, providing a solid theoretical basis for the exploration of new treatment schemes for β- like hemoglobin diseases, such as sickle cell disease and β- thalassemia.

Red blood cell mannoses as phagocytic ligands mediating both sickle cell anaemia and malaria resistance

In both sickle cell disease and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (Man5-9GlcNAc2), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. We find that extravascular hemolysis in sickle cell disease correlates with high mannose glycan levels on RBCs. Furthermore, Plasmodium falciparum-infected RBCs expose surface mannose N-glycans, which occur at significantly higher levels on infected RBCs from sickle cell trait subjects compared to those lacking hemoglobin S. The glycans are associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans as a response to cellular stress is a molecular mechanism common to both the pathogenesis of sickle cell disease and resistance to severe malaria in sickle cell trait.

Associations between TGF-β1 Levels and Markers of Hemolysis, Inflammation, and Tissue Remodeling in Pediatric Sickle Cell Patients.

Transforming growth factor beta (TGF-β) is a cytokine with important involvement in biological processes related to the pathogenesis of sickle cell disease (SCD), including endothelial and vascular dysfunction, inflammation, and hematopoietic homeostasis. This study is aimed at investigating associations between levels of TGF-β1 and classical laboratory biomarkers and inflammatory mediators, as well as the tissue inhibitor of metalloproteases-1 (TIMP-1) and matrix metalloproteinase-9 (MMP-9), in pediatric patients (n = 123) with SCD in steady state: 84 with sickle cell anemia (HbSS) and 39 with hemoglobin SC disease (HbSC). A healthy control (HC) group of 59 individuals was also included. Hematological and biochemical analyses were carried out using electronic methods. TGF-β1, TIMP-1, and MMP-9 plasma quantifications were performed by ELISA. TGF-β1 plasma levels were higher in HbSS individuals than in HbSC and HC. In individuals with HbSS, TGF-β1 levels were positively correlated with red blood cells, hemoglobin, hematocrit, platelets, and TIMP-1. In addition, HbSS individuals with TGF-β1 levels above the median (≥72.29 ng/mL) also presented increased monocyte counts and decreased albumin levels. In patients with HbSC, TGF-β1 levels were positively correlated with leukocytes, eosinophils, lymphocytes, monocytes, and platelets, as well as levels of TIMP-1, VLDL-C, triglycerides, heme, and AST. Additionally, HbSC individuals with TGF-β1 levels above the median (≥47.80 ng/mL) presented increased leukocyte and platelet counts, as well as increased levels of triglycerides, VLDL-C, MMP-9, and TIMP-1, and decreased HDL-C. Our findings suggest that TGF-β1 may play important roles in vascular remodeling, vasculopathy, angiogenesis, and inflammation in pediatric patients with SCD.

Neutrophil extracellular trap regulators in sickle cell disease: Modulation of gene expression of PADI4, neutrophil elastase, and myeloperoxidase during vaso‐occlusive crisis

BackgroundRecent evidence suggests that generation of neutrophil extracellular traps (NETosis), one of the components of immunothrombosis, is associated with the pathogenesis of both venous thromboembolism and sickle cell disease (SCD). NETosis is a complex process regulated by several proteins such as peptidyl arginine deaminase 4 (PADI4), neutrophil elastase (ELANE), and myeloperoxidase (MPO). Among these regulators, PADI4 is responsible of histone citrullination, an essential step for NETosis. Accordingly, its inhibition has been recently cited as a promising therapeutic strategy for diseases such as SCD. Although attractive, this strategy requires supportive evidence of its role in the pathogenesis of SCD. Patients and MethodsPatients from two independent cohorts were enrolled in this study. Samples were obtained at steady state (53 patients) or during acute episodes of vaso‐occlusive crisis (VOC; 28 patients) in patients from cohort 1. mRNA was extracted from granulocytes to analyze PADI4, ELANE, and MPO expression by qPCR. Furthermore, plasma activity of PADI4 was assessed from an independent cohort in 15 patients, within 24 hours from admission for VOC. Race‐matched healthy individuals from the same geographic regions were used as controls for each cohort. Results and ConclusionsHigher levels of gene expression of PADI4 and ELANE were observed during VOC. Furthermore, plasma activity of PADI4 was higher in acute VOC when compared to healthy individuals. These results demonstrate that NETosis regulators are modulated during acute VOC, and pave the way for studies of PADI4 inhibition as a therapeutic strategy for acute VOC in SCD.

Serum Kallistatin Levels are Associated with the Pathogenesis of Sickle Cell Disease

Serum Kallistatin Levels are Associated with the Pathogenesis of Sickle Cell Disease

Prevalence of Sickle Cell

Background: Sickle cell disease (SCD) is a common and neglected inherited disorder in the Indian tribal and nontribal population. Prevalent in scheduled populations, these are socioeconomically disadvantaged communities. SCD pathogenesis is widely studied at national and international levels which are limited to pain episodes and vaso–occlusive crisis. Objectives: In the present study, we studied the prevalence of SCD in tribal and rural population from Palghar. Materials and Methods: Subjects from primary health centers of Palghar, Maharashtra, were included in this study. Informed written consent was obtained from the all subjects. The investigation was done by solubility test and high–performance liquid chromatography, along with complete blood count. Results: Population is divided into three groups: sickle cell homozygous (HbSS), sickle cell heterozygous (HbAS), and control (HbAA). In the sample size of 5000 subjects, 1% sample was found to be affected by SCD (HbSS) and 4.08% were sickle cell heterozygous (HbAS). Comparison among hemolytic events versus vaso–occlusive single events suggests that hemolytic events, pallor and yellow sclera, counted more than other single vaso–occlusive event. Detailed screening and awareness will be the key to early intervention to reduce morbidity and mortality due to SCD. Conclusions: As SCD is becoming an increasing health concern within India, identification and creating awareness is of paramount importance. In this pilot study, heterozygous and homozygous for the sickle cell gene were explained and it is clear that SCD is a major hemoglobinopathy among the tribal people of Palghar. Further in–depth study is necessary for a proper understanding of pathogenesis of SCD.

Identifying Potential Drug Targets for Sickle Cell Disease through Gene Expression and Pathway Analysis of GEO Data

Background: The pathogenesis of sickle cell disease (SCD) and its complications have been well characterized down to the molecular level. However, there remains a relative dearth of disease modifying therapies that reduce the frequency and number of vas-occlusive crises, hospitalizations, and deaths. Recent advancements in utilizing hydroxyurea and L-glutamine, which both impact unique disease pathways, should pave way for the identification of other molecular pathways as ideal drug targets. In this regard, our meta-analysis serves to identify key genes and associated pathways that are differentially expressed in SC patients. Methods: We employed our STARGEO platform to tag samples from the NCBI Gene Expression Omnibus and performed meta-analysis to compare SC and healthy control transcriptomes. For the meta-analysis, we tagged 285 peripheral blood samples from SC patients and 86 samples from healthy subjects as a control. We then analyzed the signature in Ingenuity Pathway Analysis to elucidate top disease functions from our analysis. Results: From our meta-analysis, we identified iron homeostasis signaling, NRF2-mediated oxidative stress response, cell senescence, and pyrimidine interconversion/biosynthesis as top canonical pathways that were upregulated in the peripheral blood samples from SC patients. Top upstream regulators included membrane associated protein and transporter ABCB6, non-coding RNY3, and erythroid maturation transcription factors GATA1, KLF1, and HIPK2 (with predicted activation). The most upregulated genes included inflammatory modulators RNF182 and IFI27, the latter of which has been shown to inhibit vascular endothelial growth and repair. Several membrane-associated protein coding genes such as GYPA, RAP1GAP, and PAQR9 were also upregulated in the SC samples. RAP1GAP is known to modulate neutrophil cell adhesion and homing while PAQR9 has roles in regulating protein quality control: a role also seen in similarly upregulated YOD1, a deubiquitinating enzyme involved in trafficking of misfolded proteins. Expectedly, also upregulated were HBBP1 and SOX6, which regulate globin genes and have been shown to silence γ-globin expression. Lastly, SLC6A19, the neutral amino acid transporter mutated in Hartnup disease, was also upregulated. Of the downregulated genes, WASF3, a member of the Wiskott-Aldrich syndrome protein family, has been linked to poor survival in many malignancies, including AML and CMML, but has not previously been linked to SCD pathogenesis. ENKUR was also downregulated and has been annotated as a tethering protein to cation channels as well as linked to pathways involving vascular leakage. SIGLEC10, which binds to vascular adhesion proteins, is a key suppressor of inflammatory responses to damage; it's downregulation along with ELAPOR1, a transmembrane protein involved in cellular response to stress, was also observed. Finally, based off the focus genes in our analysis we identified several networks with most being involved in amino acid metabolism, cellular assembly, function, and maintenance, hematological disease, and organismal injury. The top pathway is illustrated in Figure 1. Conclusions: Our study illustrates differentially expressed gene activity in SCD consistent with known pathophysiology such as immune response, endothelial damage and adherence, heme metabolism, and globin regulation. We also showed evidence of genes not previously studied in SCD, which may have novel roles such as those part of the ubiquitin-proteasome system like YOD1 and RNF182. Additionally, while some genes in our analysis like EKLF and GAT1 have been shown to enhance δ-globin expression, paving way for possible drug therapies for B-hemoglobinopathies, others like IFI27, PAQR9, RAP1GAP, ENKUR, SIGLEC10, WASF3, and SOX9 have yet to be studied as mediators of disease pathogenesis in SCD. A target to SOX9, a known suppressor of γ-globin, or ABCB6, a known modulator of erythroid cell shape and hydration, have particularly promising potential as disease modifying therapies. Finally, HIPK2, HBBP1, and SLC6A19 have previously been shown to have intriguing effects on hydroxyurea dosing and responsivity in SC patients and may also be candidate target molecules to enhance existing therapies. These data identify potential candidate pathways for mechanistic studies seeking to confirm a causative role in the pathogenesis of sickle cell disease. Disclosures No relevant conflicts of interest to declare.

Differential Acetone Extraction of Total and Hemoprotein-Unbound Heme to Quantify Heme Binding Capacity of Plasma in Patients with Sickle Cell Disease: The Role of Heme Scavengers

Introduction: Heme, an iron-containing protoporphyrin, is an essential component of hemoglobin that binds oxygen for delivery to tissues. In sickle cell disease, intravascular hemolysis leads to the presence of cell-free hemoglobin and heme, which may contribute to oxidative damage and activation of inflammatory pathways. Hemoproteins such as haptoglobin and hemopexin provide pathways to remove hemoglobin and heme, respectively, from circulation. Due to its hydrophobic nature, heme also intercalates in cell membranes and binds to plasma components such as albumin and lipoproteins, though with varying affinity. Hemopexin has high affinity for heme and removes heme from other heme pools in blood to counter the highly toxic properties of heme unbound to hemoproteins. Due to chronic hemolysis, hemopexin is depleted in individuals with sickle cell disease. We hypothesize that the reduction in heme binding capacity leads to increased unbound heme in blood and contributes to the pathogenesis of sickle cell disease. To define the different heme binding pools in patients with sickle cell disease, we developed a method requiring small amounts of plasma which allows measurement of total and hemoprotein-unbound heme. With this method, we can quantify the binding capacity of plasma for heme and correlate that with measurement of heme scavenging proteins. Methods: Blood from healthy individuals and sickle cell patients was collected in EDTA as anticoagulant under IRB approval. Plasma was separated by centrifugation from whole blood, and either processed fresh or after freezing at -80°C. Plasma protein was precipitated with a 4-fold volume of acetone at neutral pH (NA) or acidic pH (AA). Under acidic condition, heme is released from all heme binding pools, including hemoglobin, and provides detection of the total heme present in plasma. Under neutral pH condition, only heme unbound to plasma proteins is extracted. Once extracted, samples were dried and resuspended in DMSO. Heme concentration was spectrophotometrically determined at 400nm using standard curves prepared from hemin added in AA or NA. To determine heme binding capacity, hemin was added to serial dilutions of plasma and extracted in NA and AA as above. The appearance of heme in NA relative to AA represents the point at which heme binding capacity of plasma was saturated. This was compared to measurement of hemopexin and haptoglobin using commercially available ELISA measurements. Hemopexin and albumin were added to samples to modulate heme binding capacity. Results: Heme concentration closely correlates with spectroscopic measurement of heme in DMSO confirming reliable quantification of total and unbound heme in acidic and neutral acetone extractions as low as 2.5µM. We next show that heme binding capacity can be determined. Heme added to plasma was effectively recovered in AA extracts and begins to appear in the NA extract when binding sites start to become saturated. We note that not all sites appear to be fully saturated before heme is detected in NA extract. Addition of hemopexin to plasma increased the binding capacity on an equimolar basis, indicating that hemopexin effectively binds heme present in plasma. In samples from patients with sickle cell disease, concentration of total and unbound heme varied widely, and did not necessarily correlate with degree of intravascular hemolysis, estimated based on the measurement of cell free hemoglobin. Both the capacity of plasma to bind heme and levels of hemopexin indicated that, in a number of patients, the amount of heme present was greater than the ability of hemopexin to bind cell free heme. Discussion: We present a novel method to quantitatively differentiate hemoprotein-bound and unbound heme in plasma, the latter of which is pathologically relevant in sickle cell disease. Our data show significant variation in the concentration of total and unbound heme in sickle cell patient samples, and that the binding capacity in sickle cell plasma only partially correlates to the degree of hemolysis measured based on cell free hemoglobin. Patients are currently enrolled in a clinical study to measure intra-patient differences in heme and heme-binding capacity during steady state and during acute sickle cell-related illness. Understanding the clinical implications of heme and heme scavengers may provide insights into diagnostic and therapeutic targets for patients with sickle cell disease. Disclosures Neumayr: Emmaus: Consultancy; Bayer: Consultancy; CTD Holdings: Consultancy; Pfizer: Consultancy; ApoPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Micelle: Other: Site principal investigator; GBT: Other: Site principal investigator; PCORI: Other: site principal investigator; Novartis: Other: co-investigator; Bluebird Bio: Other: co-investigator; Sangamo Therapeutics: Other; Silarus: Other; Celgene: Other; La Jolla Pharmaceuticals: Other; Forma: Other; Centers for Disease Control and Prevention: Other; Seattle Children's Research: Other; Imara: Other; National Heart, Lung, and Blood Institute: Other; Health Resources and Services Administration: Other. Vichinsky:Bluebird Bio: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding; GBT: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Kuypers:Forma Therapeutics, Inc.: Research Funding.

FT-4202, an Allosteric Activator of Pyruvate Kinase-R, Demonstrates Proof of Mechanism and Proof of Concept after a Single Dose and after Multiple Daily Doses in a Phase 1 Study of Patients with Sickle Cell Disease

FT-4202, an Allosteric Activator of Pyruvate Kinase-R, Demonstrates Proof of Mechanism and Proof of Concept after a Single Dose and after Multiple Daily Doses in a Phase 1 Study of Patients with Sickle Cell Disease