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Myeloproliferative neoplasms (MPN) are mainly sporadic but inherited variants have been associated with higher risk development. Here, we identified an EPOR variant (EPOR

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.

Sickle cell disease (SCD) is caused by a mutation in the β-globin gene leading to polymerization of the sickle hemoglobin (HbS) and deformation of red blood cells. Autologous transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically modified using lentiviral vectors (LVs) to express an anti-sickling β-globin leads to some clinical benefit in SCD patients, but it requires high-level transgene expression (i.e., high vector copy number [VCN]) to counteract HbS polymerization. Here, we developed therapeutic approaches combining LV-based gene addition and CRISPR-Cas9 strategies aimed to either knock down the sickle β-globin and increase the incorporation of an anti-sickling globin (AS3) in hemoglobin tetramers, or to induce the expression of anti-sickling fetal γ-globins. HSPCs from SCD patients were transduced with LVs expressing AS3 and a guide RNA either targeting the endogenous β-globin gene or regions involved in fetal hemoglobin silencing. Transfection of transduced cells with Cas9 protein resulted in high editing efficiency, elevated levels of anti-sickling hemoglobins, and rescue of the SCD phenotype at a significantly lower VCN compared to the conventional LV-based approach. This versatile platform can improve the efficacy of current gene addition approaches by combining different therapeutic strategies, thus reducing the vector amount required to achieve a therapeutic VCN and the associated genotoxicity risk.

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International audience; Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by clonal expansion of abnormal hematopoietic stem cells leading to hyperproliferation of one or more myeloid lineages. The main complications in MPNs are high risk of thrombosis and progression to myelofibrosis and leukemia. MPN patients with high risk scores are treated by hydroxyurea (HU), interferon-α, or ruxolitinib, a tyrosine kinase inhibitor. Polycythemia vera (PV) is an MPN characterized by overproduction of red blood cells (RBCs). ABCG2 is a member of the ATP-binding cassette superfamily transporters known to play a crucial role in multidrug resistance development. Proteome analysis showed higher ABCG2 levels in PV RBCs compared to RBCs from healthy controls and an additional increase of these levels in PV patients treated with HU, suggesting that ABCG2 might play a role in multidrug resistance in MPNs. In this work, we explored the role of ABCG2 in the transport of ruxolitinib and HU using human cell lines, RBCs, and in vitro differentiated erythroid progenitors. Using stopped-flow analysis, we showed that HU is not a substrate for ABCG2. Using transfected K562 cells expressing three different levels of recombinant ABCG2, MPN RBCs, and cultured erythroblasts, we showed that ABCG2 potentiates ruxolitinib-induced cytotoxicity that was blocked by the ABCG2-specific inhibitor KO143 suggesting ruxolitinib intracellular import by ABCG2. In silico modeling analysis identified possible ruxolitinib-binding site locations within the cavities of ABCG2. Our study opens new perspectives in ruxolitinib efficacy research targeting cell types depending on ABCG2 expression and polymorphisms among patients.

Purpose of review Sickle cell disease (SCD) is a hemolytic anemia caused by a point mutation in the β globin gene leading to the expression of an abnormal hemoglobin (HbS) that polymerizes under hypoxic conditions driving red cell sickling. Circulating red cells have been extensively characterized in SCD, as their destruction and removal from peripheral blood are the major contributors to anemia. However, few reports showed cellular abnormalities during erythropoiesis in SCD, suggesting that anemia could also be influenced by defects of central origin. Recent findings El Hoss et al. demonstrated ineffective erythropoiesis (IE) in SCD and deciphered the molecular mechanism underlying cell death during the hemoglobin synthesis phase of terminal differentiation. They showed that HbS polymerization induces apoptosis of differentiating erythroblasts and that fetal hemoglobin rescues these cells through its antipolymerization function. Summary IE is the major cause of anemia in β-thalassemia patients, and it is generally surmised that it contributes little to anemia of SCD. Recent reports demonstrate the occurrence of IE in SCD patients and show important alterations in the hematopoietic and erythroid niches, both in SCD patients and in the humanized Townes SCD mouse model. This implies that therapeutic strategies initially designed to improve red cell survival in the circulation of SCD patients would also positively impact erythropoiesis and bone marrow cellularity.

International audience; Bioimpedance measurements using microfluidic systems play an increasingly important role in cell analysis. In this paper, a microfluidic device mimicking the vasculature, in particular blood microcapillaries, and equipped with electrodes for bioimpedance sensing is described. Red blood cells, from patients suffering from sickle cell disease, were analyzed and discriminated in this device according to the physical parameters. The statistical position of different subpopulations on the Gaussian distribution of the whole sickle cell population was analyzed. A discrimination between sickle cell subpopulations, related to different density states, was achieved.

Sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT) are the most prevalent monogenic disorders worldwide. Trial HGB-205 ( NCT02151526 ) aimed at evaluating gene therapy by autologous CD34

While ineffective erythropoiesis has long been recognized as a key contributor to anemia in thalassemia, its role in anemia of sickle cell disease (SCD) has not been critically explored. Using in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. Modeling the bone marrow hypoxic environment, we found that hypoxia induces death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). Cell death was associated with cytoplasmic sequestration of heat shock protein 70 and was rescued by induction of HbF synthesis. Importantly, we document that in the bone marrow of SCD patients similar cell loss occurs during the final stages of terminal differentiation. Our study provides evidence for ineffective erythropoiesis in SCD and highlights an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. These findings imply that the beneficial effect on anemia of increased HbF levels is not only due to the increased life span of red cells but also a consequence of decreased ineffective erythropoiesis.

This paper describes the use of a microfluidic device comprising channels with dimensions mimicking those of the smallest capillaries found in the human microcirculation. The device structure, associated with a pair of microelectrodes, provides a tool to electrically measure the transit time of red blood cells through fine capillaries and thus generate an electrical signature for red blood cells in the context of human erythroid genetic disorders, such as sickle cell disease or hereditary spherocytosis, in which red cell elasticity is altered. Red blood cells from healthy individuals, heated or not, and red blood cells from patients with sickle cell disease or hereditary spherocytosis where characterized at a single cell level using our device. Transit time and blockade amplitude recordings were correlated with microscopic observations, and analyzed. The link between the electrical signature and the mechanical properties of the red blood cells is discussed in the paper, with greater transit time and modified blockade amplitude for heated and pathological red blood cells as compared to those from healthy individuals. Our single cell-based methodology offers a new and complementary approach to characterize red cell mechanical properties in human disorders under flow conditions mimicking the microcirculation.

Currently, the majority of commercial recombinant therapeutic proteins are produced in nonhuman expression systems. Human cells are capable of producing recombinant proteins with post‐translational modifications that are more similar to native proteins which reduce immunogenicity. This article describes the potential of the human cell lines Sk‐Hep‐1, HKB‐11, and Huh‐7, cultured in serum‐free suspension conditions, to produce a complex recombinant glycoprotein, human erythropoietin (EPO). Recombinant cell lines were generated by lentiviral transduction and sorted by flow cytometry. All the recombinant cells presented high‐specific cell growth rates and a high level of rhEPO production. Maximum rhEPO concentrations achieved by Sk‐Hep‐1, HKB‐11, and Huh‐7 cells were 112.5 μg/mL, 112.7 μg/mL, and 571 μg/mL, respectively. The levels of rhEPO production by Huh‐7 cells were higher than the levels commonly reported in the literature for serum‐free suspension cultures. The rhEPO produced demonstrated biological activity measured through in vitro differentiation of CD34+ cells. In view of this, we demonstrate that Sk‐Hep‐1, HKB‐11, and Huh‐7 cell lines have good characteristics to be used as host cells for the production of complex recombinant glycoproteins, with special emphasis on Huh‐7 due to enhanced EPO production.

Editing the fetal γ-globin promoters in hematopoietic stem cells from sickle cell disease patients induces therapeutic γ-globin levels., Sickle cell disease (SCD) is caused by a single amino acid change in the adult hemoglobin (Hb) β chain that causes Hb polymerization and red blood cell (RBC) sickling. The co-inheritance of mutations causing fetal γ-globin production in adult life hereditary persistence of fetal Hb (HPFH) reduces the clinical severity of SCD. HPFH mutations in the HBG γ-globin promoters disrupt binding sites for the repressors BCL11A and LRF. We used CRISPR-Cas9 to mimic HPFH mutations in the HBG promoters by generating insertions and deletions, leading to disruption of known and putative repressor binding sites. Editing of the LRF-binding site in patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in γ-globin derepression and correction of the sickling phenotype. Xenotransplantation of HSPCs treated with gRNAs targeting the LRF-binding site showed a high editing efficiency in repopulating HSPCs. This study identifies the LRF-binding site as a potent target for genome-editing treatment of SCD.

International audience; Plasma MPs released during crisis in SS patients increase endothelial ICAM-1 levels and neutrophil adhesion. l HU treatment lowers PS exposure on MPs, which abolishes their proinflammatory properties. Microparticles (MPs) are submicron extracellular vesicles exposing phosphatidylserine (PS), detected at high concentration in the circulation of sickle cell anemia (SS) patients. Several groups studied the biological effects of MPs generated ex vivo. Here, we analyzed for the first time the impact of circulating MPs on endothelial cells (ECs) from 60 sickle cell disease (SCD) patients. MPs were collected from SCD patients and compared with MPs isolated from healthy individuals (AA). Other plasma MPs were purified from SS patients before and 2 years after the onset of hydroxyurea (HU) treatment or during a vasoocclusive crisis and at steady-state. Compared with AA MPs, SS MPs increased EC ICAM-1 messenger RNA and protein levels, as well as neutrophil adhesion. We showed that ICAM-1 overexpression was primarily caused by MPs derived from erythrocytes, rather than from platelets, and that it was abolished by MP PS capping using annexin V. MPs from SS patients treated with HU were less efficient to induce a proinflammatory phenotype in ECs compared with MPs collected before therapy. In contrast, MPs released during crisis increased ICAM-1 and neutrophil adhesion levels, in a PS-dependent manner, compared with MPs collected at steady-state. Furthermore, neutrophil adhesion was abolished by a blocking anti-ICAM-1 antibody. Our study provides evidence that MPs play a key role in SCD pathophysiology by triggering a proinflammatory phenotype of ECs. We also uncover a new mode of action for HU and identify potential therapeutics: annexin V and anti-ICAM-1 antibodies. (Blood. 2020;136(2):247-256

International audience; Vaso-occlusive crises are the hallmark of sickle cell disease (SCD). They are believed to occur in two steps, starting with adhesion of deformable low-dense red blood cells (RBCs), or other blood cells such as neutrophils, to the wall of post-capillary venules, followed by trapping of the denser RBCs or leukocytes in the areas of adhesion because of reduced effective lumen-diameter. In SCD, RBCs are heterogeneous in terms of density, shape, deformability and surface proteins, which accounts for the differences observed in their adhesion and resistance to shear stress. Sickle RBCs exhibit abnormal adhesion to laminin mediated by Lu/BCAM protein at their surface. This adhesion is triggered by Lu/BCAM phosphorylation in reticulocytes but such phosphorylation does not occur in mature dense RBCs despite firm adhesion to laminin. In this study, we investigated the adhesive properties of sickle RBC subpopulations and addressed the molecular mechanism responsible for the increased adhesion of dense RBCs to laminin in the absence of Lu/BCAM phosphorylation. We provide evidence for the implication of oxidative stress in post-translational modifications of Lu/BCAM that impact its distribution and cis-interaction with glycophorin C at the cell surface activating its adhesive function in sickle dense RBCs.

β-hemoglobinopathies are caused by mutations affecting the adult hemoglobin production. In sickle cell disease (SCD), the β6 Glu→Val substitution leads to sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. In β-thalassemia, reduced β-globin production leads to precipitation of uncoupled α-chains causing ineffective erythropoiesis and the production of poorly hemoglobinized RBCs. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. The clinical severity of β-hemoglobinopathies is alleviated by the co-inheritance of mutations causing hereditary persistence of fetal Hb (HPFH). HPFH mutations clustering 200 nucleotides upstream of the TSS of the fetal γ-globin (HBG) genes either disrupt the binding site (BS) of the fetal Hb (HbF) repressor LRF or generate a de novo BS for the KLF1 activator. To reactivate γ-globin expression, nuclease-based approaches have been explored. However, nucleases generate double-strand breaks (DSBs), raising safety concerns for clinical applications. Base editing (BE) allows the introduction of point mutations without generating DSBs. In this study, we designed BE systems to introduce a variety of HPFH or HPFH-like mutations in the -200 region of the HBG promoters. First, we screened in erythroid cell lines known and novel BEs, and we selected combinations of BEs and guide RNAs that edit alternative bases of the -200 region. We then developed a clinically-relevant protocol based on RNA-transfection to deliver the BE system to HSPCs. The expression profile of genes activated by RNA stimuli revealed no immune response in HSPCs. A progenitor assay indicated no alteration in the growth and multilineage differentiation of edited HSPCs. We applied this protocol to SCD and β-thalassemia HSPCs, achieving editing efficiencies up to ~70% of the HBG promoters. In RBCs differentiated from edited SCD HSPCs, RT-qPCR, HPLC and flow cytometry showed a potent γ-globin reactivation with a high frequency of HbF + cells and a concomitant decrease in the HbS content/cell. Importantly, the pathological RBC sickling phenotype was corrected in the samples derived from edited HSPCs. Similarly, in β-thalassemia samples, RT-qPCR and HPLC analyses showed strong γ-globin induction and decrease of the α-globin precipitates. HbF expression rescued the delay in erythroid differentiation and ineffective erythropoiesis characterizing β-thalassemia, as demonstrated by the increased RBC enucleation rate and the reduced apoptosis and oxidative stress. We then compared BE strategies that either disrupt the LRF BS or create a de novo KLF1 BS in single colonies derived from erythroid progenitors. Generation of the KLF1 BS was associated with higher levels of HbF compared to the LRF BS disruption. These results suggest that eviction of the LRF repressor is sufficient to reactivate HBG genes, but recruitment of an activator is more effective to achieve high levels of gene expression. HbF expression induced by both LRF BS disruption and KLF1 BS generation was sufficient to rescue the SCD cell phenotype, but higher HbF levels - achieved only through KLF1 BS generation - were necessary to fully correct the β-thalassemia phenotype. In the majority of cases, we detected no DSB-induced insertions, deletions, or large genomic rearrangements in base-edited samples. Accordingly, DSB-induced DNA damage response (DDR) was absent in base-edited HSPCs, as measured by evaluating the expression of p21, a readout of p53-induced DDR. DNA off-target activity was assessed by GUIDE-seq and targeted sequencing of the potential off-target sites in edited HSPCs, while RNA off-target activity was evaluated by RNA-seq in HSPCs. Finally, BE-treated HSPCs were transplanted in immunodeficient mice to evaluate the engraftment and differentiation capability of edited HSCs. We detected good frequencies of human cells with up to ~60% of edited promoters in the peripheral blood of transplanted mice. In conclusion, we developed a clinically-relevant strategy to perform efficient BE in the HBG promoters that led to therapeutically-relevant HbF levels and rescued both the SCD and β-thalassemia phenotypes, thus providing sufficient proof of efficacy and safety to enable the clinical development of base-edited HSPCs for the therapy of β-hemoglobinopathies. Disclosures El Nemer: Hemanext: Consultancy.

Spleen dysfunction and susceptibility to pneumococcal infection is a well known feature in homozygous sickle cell disease (HbSS), whilst to date splenic function in hemoglobin SC disease (HbSC) has been poorly investigated. The aim of this study was to analyze spleen function in children with HbSC disease using a high-throughput validated method (1) and to examine if the current recommendations regarding pneumococcal risk are appropriate in this population. Spleen function was evaluated using a flow cytometry quantification of red blood cells (RBCs) with Howell-Jolly bodies (HJBs), in a cross-sectional study of patients at steady state during an outpatient visit in an expert center. Quantification of HJB-RBCs was performed in children with HbSC disease aged < 10 years and compared to children with HbSS disease or healthy children of the same age groups, or splenectomized children. Additional exploratory analysis was performed according to age (under or above the age of 5 years old) and treatment group (hydroxyurea). The median (Q1-Q3) HJB-RBCs count was 16 (11-28.25) /100.000 RBCs in 40 HbSC children (Figure 1). This result was not statistically different from the control group of 22 healthy children (p=0.96) nor in subgroups < or ≥ 5 years old, indicating that children with HbSC under 10 years have a preserved splenic function. Expectedly, the HJB-RBCs counts differed significantly from splenectomized children (419 (296-489)/100.000 RBCs, n=15, p By contrast, among the 53 HbSS children, the median HJB-RBCs count was 134 (29-216) /100.000 RBCs, differing significantly from HbSC children (p The result of this study suggests that spleen function in children under 10 years old with HbSC is not altered. The routine administration of prophylactic penicillin to young children with SC disease may therefore be questioned. Similarly, fever in children with HbSC under 3 years old may not require parenteral antibiotics as it is generally currently recommended by analogy to children with HbSS. Functional or anatomical asplenia in children with HbSC is delayed compared to those with HbSS at least after the first decade of life. Future large cohort studies using similar methodology will allow better evaluation of the pneumococcal risk in adolescents and adults with Hb SC disease. Bibliography (1) El Hoss S, Dussiot M, Renaud O, Brousse V, El Nemer W. A novel non-invasive method to measure splenic filtration function in humans. Haematologica. oct 2018;103(10):e436-9. Figure 1 Figure 1. Disclosures El Nemer: Hemanext: Consultancy.

In sickle cell disease (SCD), the β6 Glu→Val substitution leads to sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) represents a promising therapeutic option for patients lacking a compatible donor. We previously designed a lentiviral vector (βAS3 LV) expressing a potent anti-sickling βAS3 globin and demonstrated its safety and efficacy in SCD patient cells (Weber et al., 2018). In vitro and in vivo preclinical studies demonstrated the safety and efficacy of a gene therapy (GT) protocol based on the efficient transduction of plerixafor-mobilized SCD HSPCs by βAS3 LV. Based on these results a Phase 1/2 clinical trial (NCT03964792) started in January 2020. Three homozygous SCD patients (P1, P2 and P3) were recruited. Before GT, patients suffered from severe SCD symptoms that were not controlled despite of a strict adherence to the treatment. Plerixafor-mobilized HSPCs were transduced achieving a VCN of 0.9±0.2 in liquid culture, 1.6±0.3 in BFU-E and 0.8±0.2 in CFU-GM. The patients were hypertransfused to reach HbS levels Here, for the 3 patients we report the follow-up of 18 (P1), 10 (P2) and 6 (P3) months. No drug-related severe adverse events were observed. Importantly, no sign of clonal hematopoiesis was detected by NGS sequencing performed on the graft before and after transduction. Despite of the similar VCN in the drug product, gene marking in peripheral blood mononuclear cells was variable and ranged between 0.2 and 0.6, as measured at the last follow-up (0.4 in P1, 0.6 in P2 and 0.2 in P3). These results pinpoint the difficulty to estimate the gene marking, self-renewal and engraftment potential of HSCs in the grafts. P1 and P2 clinically benefit from the GT treatment despite they presented non-severe vaso-occlusive crises (VOCs) not requiring hospitalization (two for P1 at month 6 and 14 and one in P2 at month 8 post-GT). Despite the rapid resolution of these episodes, P1 started hydroxyurea at month 10 and stopped it at month 16. P2 initiated a phlebotomy program to treat liver iron overload and decrease viscosity. Intravascular hemolysis improved in P1 and P2, as shown by a decrease in plasma hemoglobin and heme, and an increase in hemopexin (HPX). Interestingly, in P1 a mild worsening occurred at the time of VOC. In P1 and P2, total bilirubin, a marker of extravascular hemolysis, decreased post-GT. Deformability, measured using osmoscan, improved in P1 and to a lesser extent in P2; however, both still exhibited some degree of dehydration compared to normal RBCs (Fig. 1). Adhesion to thrombospondin decreased after GT with an increase during VOC in P1 or prior the VOC in P2 (Fig. 1). In both patients, markers of inflammation (e.g., C reactive protein) and reticulocyte counts decreased upon GT. An in vitro sickling assays was performed for P1 and showed significant improvement, reaching a frequency of sickling cells similar to SCD heterozygous carriers (from 88% before GT to 45% 6 months post-GT). The GT treatment was not effective in P3 due to the modest intake and rapid decline of gene modified cells. In P1 and P2, who were no longer on transfusions, HbAS3 levels correlated with the VCN (2.1 g/dl for P1 and 3.3 g/dl for P2) and the therapeutic Hb accounted for 21.5%, and 28.7% of the total hemoglobins, respectively. For P3, who remained transfusion-dependent, this frequency was Overall, these clinical data indicate a variable efficacy of the DREPAGLOBE GT treatment, which likely depends on the extent of gene marking achieved in HSCs in vivo and on the engraftment capability of genetically modified HSCs in SCD patients' bone marrow. Single-cell RNA-seq analysis of HSPCs and evaluation of the bone marrow niche in SCD patients will aid to define critical parameters for achieving successful outcomes in GT clinical trials for SCD. Figure 1 Figure 1. Disclosures Joseph: bluebird bio: Consultancy. El Nemer: Hemanext: Consultancy. Bartolucci: INNOVHEM: Other: Co-founder; AGIOS: Consultancy; GBT: Consultancy; Bluebird: Consultancy, Research Funding; Jazz Pharma: Other: Lecture fees; Fabre Foundation: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Steering committee, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy; Hemanext: Consultancy; Addmedica: Consultancy, Other: Lecture fees, Research Funding; Emmaus: Consultancy. Cavazzana: Smart Immune: Other: co-founder.

Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a point mutation in the β globin gene resulting in a Glu-to-Val substitution at the 6th position of the β globin protein. The resulting abnormal hemoglobin (HbS) polymerizes under hypoxic conditions driving red blood cell (RBC) sickling (Pauling et al., 1949). While pathobiology of circulating RBCs has been extensively analyzed in SCD, erythropoiesis is surprisingly poorly documented. In β-thalassemia, ineffective erythropoiesis is characterized by high levels of apoptotic erythroblasts during the late stages of terminal differentiation, due to an accumulation of free β-globin chains (Arlet et al., 2016). Ineffective erythropoiesis is the major cause of anemia in β-thalassemia patients. In contrast, a marked decrease in life span of circulating red cells, a feature of sickle red cells, is considered to be the major determinant of chronic anemia in SCD. It is generally surmised that ineffective erythropoiesis contributes little to anemia. The bone marrow environment has been well documented to be hypoxic (0.1 to 6% O2) (Mantel et al., 2015). As hypoxia induces HbS polymerization, we hypothesized that cell death may occur in vivo because of HbS polymer formation in the late stages of differentiation characterized by high intracellular hemoglobin concentration. In the present study, using both in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. We explored the mechanistic basis of the ineffective erythropoiesis in SCD using biochemical, cellular and imaging techniques. In vitro erythroid differentiation using CD34+ cells isolated from SCD patients and from healthy donors was performed. A 2-phase erythroid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e. normoxia and partial hypoxia (5% O2). We found that hypoxia induces cell death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). This inference was supported by flow cytometry data showing higher percentages of dead cells within the non-F-cell population as compared to the F-cell population for SCD cells. Moreover, SCD dead cells showed higher levels of chaperon protein HSP70 in the cytoplasm than live cells, while no difference was detected between both subpopulations for control cells, suggesting that cell death of SCD erythroblasts was probably due to HSP70 cytoplasmic sequestration. This was supported by western-blot experiments showing less HSP70 in the nucleus of SCD erythroblasts under hypoxia, associated with decreased levels of GATA-1. At the molecular level, HSP70 was co-immunoprecipitated with HbS under hypoxia indicating that both proteins were in the same complex and suggesting interaction between HSP70 and HbS polymers in the cyotplasm. Importantly, we confirm these results in vivo by showing that in bone marrow of SCD patients (n = 5) cell loss occurs during terminal erythroid differentiation, with a significant drop in the cell count between the polychromatic and the orthochromatic stages (Figure 1). In order to specifically address the role of HbF in cell survival, we used a CRISPR-Cas9 approach to mimic the effect of hereditary persistence of fetal hemoglobin (HPFH). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1) (Weber, Frati, et al. 2020). As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control. These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions, clearly demonstrating the positive and selective effect of HbF on SCD cell survival (Figure 2). In summary, our study shows that HbF has a dual beneficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing ineffective erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction. Disclosures No relevant conflicts of interest to declare.

International audience

The capacity to undergo substantial deformation is a defining characteristic of the red blood cell (RBC), facilitating transit through the splenic interendothelial slits and microvasculature. Establishment of this remarkable property occurs during a process of reticulocyte maturation that begins with egress through micron-wide pores in the bone marrow and is completed within the circulation. The requirement to undertake repeated cycles of deformation necessitates that both reticulocytes and erythrocytes regulate membrane-cytoskeletal protein interactions in order to maintain cellular stability. In the absence of transcriptional activity, modulation of these interactions in RBCs is likely to be achieved primarily through specific protein posttranslational modifications, which at present remain undefined. In this study, we use high-throughput methods to define the processes that underlie the response to deformation and shear stress in both reticulocytes and erythrocytes. Through combination of a bead-based microsphiltration assay with phosphoproteomics we describe posttranslational modification of RBC proteins associated with deformation. Using microsphiltration and microfluidic biochip-based assays, we explore the effect of inhibiting kinases identified using this dataset. We demonstrate roles for GSK3 and Lyn in capillary transit and maintenance of membrane stability following deformation and show that combined inhibition of these kinases significantly decreases reticulocyte capacity to undergo repeated deformation. Finally, we derive a comprehensive and integrative phosphoproteomic dataset that provides a valuable resource for further mechanistic dissection of the molecular pathways that underlie the RBC’s response to mechanical stimuli and for the study of reticulocyte maturation.

Spleen dysfunction is central to morbidity and mortality in children with sickle cell anemia (SCA). The initiation and determinants of spleen injury, including acute splenic sequestration (ASS) have not been established. We investigated splenic function longitudinally in a cohort of 57 infants with SCA enrolled at 3 to 6 months of age and followed up to 24 months of age and explored the respective contribution of decreased red blood cell (RBC) deformability and increased RBC adhesion on splenic injury, including ASS. Spleen function was evaluated by sequential 99mTc heated RBC spleen scintigraphy and high-throughput quantification of RBCs with Howell-Jolly bodies (HJBs). At 6 and 18 months of age, spleen filtration function was decreased in 32% and 50% of infants, respectively, whereas the median %HJB-RBCs rose significantly (from 0.3% to 0.74%). An excellent correlation was established between %HJB-RBCs and spleen scintigraphy results. RBC adhesion to laminin and endothelial cells increased with time. Adhesion to endothelial cells negatively correlated with splenic function. Irreversibly sickled cells (ISCs), used as a surrogate marker of impaired deformability, were detected at enrollment and increased significantly at 18 months. %ISCs correlated positively with %HJB-RBCs and negatively with splenic uptake, indicating a relationship between their presence in the circulation and spleen dysfunction. In the subgroup of 8 infants who subsequently experienced ASS, %ISCs at enrollment were significantly higher compared with the asymptomatic group, suggesting a major role of impaired deformability in ASS. Higher levels of %HJB-RBCs were observed after the occurrence of ASS, demonstrating its negative impact on splenic function.

Sickle cell disease is an autosomal recessive genetic red cell disorder with a worldwide distribution. Growing evidence suggests a possible involvement of complement activation in the severity of clinical complications of sickle cell disease. In this study we found activation of the alternative complement pathway with microvascular deposition of C5b-9 on skin biopsies from patients with sickle cell disease. There was also deposition of C3b on sickle red cell membranes, which is promoted locally by the exposure of phosphatidylserine. In addition, we showed for the first time a peculiar "stop-and-go" motion of sickle cell red blood cells on tumor factor-α-activated vascular endothelial surfaces. Using the C3b/iC3b binding plasma protein factor Has an inhibitor of C3b cell-cell interactions, we found that factor H and its domains 19-20 prevent the adhesion of sickle red cells to the endothelium, normalizing speed transition times of red cells. We documented that factor H acts by preventing the adhesion of sickle red cells to P-selectin and/or the Mac-1 receptor (CD11b/CD18), supporting the activation of the alternative pathway of complement as an additional mechanism in the pathogenesis of acute sickle cell related vaso-occlusive crises. Our data provide a rationale for further investigation of the potential contribution of factor H and other modulators of the alternative complement pathway with potential implications for the treatment of sickle cell disease.

International audience; In this paper we demonstrate the capability to detect red blood cells mechanical disorders, in particular the sickle cell disease, using the electrical signature of the cell transit within a microfluidic restriction mimicking the blood capillaries.

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult β-globin chain. In β-thalassemia, the reduced (β+) or absent (β0) production of adult 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 red blood cell (RBC) sickling, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) reduces the clinical severity of β-hemoglobinopathies. HPFH mutations in the promoter of the two γ-globin genes, HBG1 and HBG2 disrupt the binding sites (BS) for transcriptional repressors (e.g., BCL11A and LRF). Recently, we demonstrated that CRISPR/Cas9-mediated disruption of the LRF BS in the HBG promoters via non-homologous end joining and microhomology-mediated end joining (MMEJ) repair mechanisms mimics the effect of HPFH mutations by impairing the LRF binding and re-activating the γ-globin expression (Weber, Frati et al., Science Advances, 2020). Efficient editing of the LRF BS (≥ 3 γ-globin promoters in >70% of SCD hematopoietic stem/progenitor cells (HSPCs)) resulted in a robust HbF reactivation and a concomitant reduction in βS-globin levels recapitulating the phenotype of asymptomatic SCD-HPFH patients. RBCs derived from edited HSPCs displayed HbF levels sufficient to correct the SCD cell phenotype. Similarly, LRF BS targeting in β0-thalassemic cells results in HbF reactivation potentially correcting the α/β-like globin imbalance. Xenotransplantation of human HSPCs edited using several gRNAs targeting the LRF BS showed a robust engraftment of edited cells that were capable to differentiate into multiple lineages. HBG editing in engrafted cells ranged from 26% to 76% with a decrease (-33%) of editing events compared to the input HSPCs, partially due to a reduced occurrence of MMEJ-mediated events in hematopoietic stem cells (HSCs). Erythroid progenitors (BFU-E) obtained ex vivo from engrafted human cells, showed a relevant γ-globin expression (~40% of the total β-like chain) despite of the reduction in the number of edited promoters per BFU-E after transplantation. Moreover, mature RBCs ex vivo differentiated from edited human cells ensure therapeutically relevant HbF levels. Sequencing of top-scoring off-targets identified by GUIDE-seq showed a relatively high off-target activity within an intergenic site devoid of known regulatory elements both in vitro and in vivo in primary human cells treated with one of the gRNAs targeting the LRF BS. Although the occurrence of this off-target event in repopulating cells suggests that it has no detrimental effect on HSC engraftment and multilineage differentiation, we tested high fidelity Cas9 variants to reduce off-target activity in primary HSPCs. Finally, we used CAST-Seq assay to evaluate the potential chromosomal rearrangements in edited primary human cells in vitro and in vivo. To minimize the potential genotoxicity associated to Cas9 nuclease-mediated double-strand breaks (DSBs) in the genome, we explored the base editing system to introduce C>T point mutations in the LRF BS without generating DSBs. The absence of the canonical SpyCas9 NGG PAM close to the LRF BS, prompted us to use known and novel base editors containing non-NGG Cas9 variants that allowed the editing of up to 6 out of 8 cytosines of the LRF BS in erythroid cell lines and in primary HSPCs from SCD patients. These C>T conversions include not only known HPFH mutations but also mutations that can further impair LRF binding. In the majority of cases, we detected no insertions or deletions in base-edited samples, as compared to nuclease-edited samples. The 4.9-kb deletion that can be generated upon cleavage of the two identical HBG promoters by the Cas9 nuclease was barely detected in base-edited samples. Importantly, disruption of the LRF BS by non-NGG enzymes led to HbF de-repression, without affecting erythroid differentiation. This work identifies the LRF BS as an effective and safe therapeutic target for the treatment of β-hemoglobinopathies. Disclosures Casini: Alia Therapeutics: Current Employment, Current equity holder in publicly-traded company. Thrasher:Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

International audience; In this paper, a micro-device dedicated to the analysis of the Red Blood Cell (RBC) deformability is proposed. It is based on PDMS microfluidic channels mimicking the flowing in the capillary vascular network. The RBC transit time within the capillary is electrically sensed using embedded electrodes on glass associated to current blockage analysis. To get reusable device, a specific PDMS coated by parylen has been developed. In this way, the PDMS porosity to gaz is drastically reduced which permits to used depressurization for both controlling the biological sample flow and the non-permanent bonding of the device for reusable capability.

In order to identify very early prognostic factors that can provide insights into subsequent clinical complications, we performed a comprehensive longitudinal multi-center cohort study on 57 infants with sickle cell anemia (55 SS; 2 Sβ°) during the first 2 years of life (ClinicalTrials.gov: NCT01207037). Time to first occurrence of a severe clinical event-acute splenic sequestration (ASS), vaso-occlusive (VOC) event requiring hospitalization, transfusion requirement, conditional/ abnormal cerebral velocities, or death-was used as a composite endpoint. Infants were recruited at a mean age of 4.4 ±1 months. Median follow-up was 19.4 months. During the study period, 38.6% of infants experienced ≥1 severe event: 14% ASS, 22.8% ≥ 1 VOC (median age: 13.4 and 12.8 months, respectively) and 33.3% required transfusion. Of note, 77% of the cohort was hospitalized, with febrile illness being the leading cause for admission. Univariate analysis of various biomarkers measured at enrollment showed that fetal hemoglobin (HbF) was the strongest prognostic factor of subsequent severe outcome. Other biomarkers measured at enrolment including absolute neutrophil or reticulocyte counts, expression of erythroid adhesion markers, % of dense red cells, cellular deformability or ϒ-globin genetic variants, failed to be associated with severe clinical outcome. Multivariate analysis demonstrated that higher Hb concentration and HbF level are two independent protective factors (adjusted HRs (95% CI) 0.27 (0.11-0.73) and 0.16 (0.06-0.43), respectively). These findings imply that early measurement of HbF and Hb levels can identify infants at high risk for subsequent severe complications, who might maximally benefit from early disease modifying treatments.

Reticulocytes produced in the bone marrow undergo maturation in the bloodstream to give rise to erythrocytes. Although the proteome of circulating red cells has been the subject of several reports, the cellular populations used for these studies were never completely devoid of reticulocytes. In our current study, we used highly purified erythrocyte and reticulocyte populations to quantify the absolute expression levels of the proteins in each cell population. Erythrocytes and reticulocytes were purified in a multistep process involving cellulose chromatography, Percoll gradient centrifugation, and fluorescence cell sorting after thiazole orange labeling. Proteins were analyzed by mass spectrometry from whole cells and erythrocyte plasma membrane (ghosts), leading to the identification and quantification of 2077 proteins, including 654 that were reticulocyte-specific. Absolute quantifications of these proteins were made using the mean corpuscular hemoglobin content of the cells as a standard. For each protein, we calculated the percentage loss during the terminal stages of reticulocyte maturation and the percentage of association with the plasma membrane. In addition, we used modified adenosine triphosphate and adenosine diphosphate molecules that enable the transfer of a biotin molecule to the catalytic sites of kinases to isolate active kinases in the erythrocytes and determined the absolute expression of 75 protein kinases and the modification of their expression during reticulocyte maturation. Our findings represent the first absolute quantification of proteins that are specifically expressed in normal erythrocytes with no detectable contamination by reticulocytes. Our findings thus represent a reference database for the future proteomic analysis of pathological erythrocytes.

Thromboembolic events are the main cause of mortality in BCR-ABL1-negative myeloproliferative neoplasms (MPNs) but their underlying mechanisms are largely unrecognized. The Janus kinase 2 (JAK2)V617F mutation is the most frequent genetic alteration leading to MPN. Usually found in haematopoietic progenitors and stem cells, this mutation has also been described in endothelial cells (ECs) of MPN patients. In this study, we have questioned the impact of the JAK2V617F mutation on EC phenotype and functions. We developed an induced pluripotent stem cells strategy to compare JAK2 mutant and wild-type ECs. Transcriptomic assays showed that several genes and pathways involved in inflammation, cell adhesion and thrombotic events were over-represented in JAK2V617F ECs and expression levels of von Willebrand factor and P-selectin (CD62P) proteins were increased. Finally, we found that leucocytes from MPN patients adhere more tightly to JAK2V617F ECs. Our results show that JAK2V617F ECs have a pro-inflammatory and pro-thrombotic phenotype and were functionally pro-adherent.

Resolvins (Rvs), endogenous lipid mediators, play a key role in the resolution of inflammation. Sickle cell disease (SCD), a genetic disorder of hemoglobin, is characterized by inflammatory and vaso-occlusive pathologies. We document altered proresolving events following hypoxia/reperfusion in humanized SCD mice. We demonstrate novel protective actions of 17R-resolvin D1 (17R-RvD1; 7S, 8R, 17R-trihydroxy-4Z, 9E, 11E, 13Z, 15E, 19Z-docosahexaenoic acid) in reducing ex vivo human SCD blood leukocyte recruitment by microvascular endothelial cells and in vivo neutrophil adhesion and transmigration. In SCD mice exposed to hypoxia/reoxygenation, oral administration of 17R-RvD1 reduces systemic/local inflammation and vascular dysfunction in lung and kidney. The mechanism of action of 17R-RvD1 involves (1) enhancement of SCD erythrocytes and polymorphonuclear leukocyte efferocytosis, (2) blunting of NF-κB activation, and (3) a reduction in inflammatory cytokines, vascular activation markers, and E-selectin expression. Thus, 17R-RvD1 might represent a new therapeutic strategy for the inflammatory vasculopathy of SCD.

International audience; Naturally occurring, large deletions in the beta-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and beta-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the delta- and beta-globin genes and a putative gamma-delta intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in gamma-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the beta-globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of gamma-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of beta-hemoglobinopathies.

The human red blood cell is a biconcave disc of 6-8 × 2 μm that is highly elastic. This capacity to deform enables it to stretch while circulating through narrow capillaries to ensure its main function of gas exchange. Red cell shape and deformability are altered in membrane disorders because of defects in skeletal or membrane proteins affecting protein-protein interactions. Red cell properties are also altered in other pathologies such as sickle cell disease. Sickle cell disease is a genetic hereditary disorder caused by a single point mutation in the β-globin gene generating sickle haemoglobin (HbS). Hypoxia drives HbS polymerisation that is responsible for red cell sickling and reduced deformability. The main clinical features of sickle cell disease are vaso-occlusive crises and haemolytic anaemia. Foetal haemoglobin (HbF) inhibits HbS polymerisation and positively impacts red cell survival in the circulation but the mechanism through which it exerts this action is not fully characterized. In this study, we designed a microfluidic biochip mimicking the dimensions of human capillaries to measure the impact of repeated mechanical stress on the survival of red cells at the single cell scale under controlled pressure. We show that mechanical stress is a critical parameter underlying intravascular haemolysis in sickle cell disease and that high intracellular levels of HbF protect against lysis. The biochip is a promising tool to address red cell deformability in pathological situations and to screen for molecules positively impacting this parameter in order to improve red cell survival in the circulation.

The protein toxin Cytotoxic Necrotizing Factor 1 (CNF1) is a major virulence factor of pathogenic Escherichia coli strains. It belongs to a family of single chain AB-toxins, which enter mammalian cells by receptor-mediated endocytosis. Recently, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as a cellular receptor for CNF1. Here, we identified the Ig-like domain 2 of Lu/BCAM as main interaction site of the toxin by direct protein-protein interaction and competition studies. Using surface plasmon resonance, we showed a high affinity CNF-Lu/BCAM interaction with a KD of 2.8 nM. Furthermore, we performed small-angle X-ray scattering to define the molecular envelope of the Lu/BCAM-CNF1 complex, suggesting a 6:1 ratio of Lu/BCAM to CNF1 in the receptor-toxin complex. This study leads to a deeper understanding of the interaction between CNF1 and Lu/BCAM, and presents novel opportunities for the development of future anti-toxin strategies.

Red blood cells (RBCs) are deformable and flow through vessels narrower than their own size. Their deformability is most stringently challenged when they cross micrometer-wide slits in the spleen. In several inherited or acquired RBC disorders, blockade of small vessels by stiff RBCs can trigger organ damage, but a functional spleen is expected to clear these abnormal RBCs from the circulation before they induce such complications. We analyzed flow behavior of RBCs in a microfluidic chip that replicates the mechanical constraints imposed on RBCs as they cross the human spleen. Polymer microchannels obtained by soft lithography with a hydraulic diameter of 25 μm drove flow into mechanical filtering units where RBCs flew either slowly through 5- to 2-μm-wide slits or rapidly along 10-μm-wide channels, these parallel paths mimicking the splenic microcirculation. Stiff heated RBCs accumulated in narrow slits seven times more frequently than normal RBCs infused simultaneously. Stage-dependent retention of Plasmodium falciparum-infected RBCs was also observed in these slits. We also analyzed RBCs from patients with hereditary spherocytosis and observed retention for those having the most altered mechanical properties as determined by ektacytometry. Thus, in keeping with previous observations in vivo and ex vivo, the chip successfully discriminated poorly deformable RBCs based on their distinct mechanical properties and on the intensity of the cell alteration. Applications to the exploration of the pathogenesis of malaria, hereditary spherocytosis, sickle cell disease and other RBC disorders are envisioned.Am. J. Hematol. 90:339–345, 2015. © 2015 Wiley Periodicals, Inc.