Introduction: Gene therapy is a highly promising therapeutic strategy in sickle cell disease (SCD). The Phase 1/2 HGB-205 (NCT02151526) clinical study in France is evaluating the safety and efficacy of LentiGlobin gene therapy, which consists of autologous CD34+ cells transduced with a lentiviral vector encoding a human β-globin gene with a point mutation (T87Q) that confers anti-sickling properties. Data from the first successfully treated patient have been published (Ribeil et al, 2017 NEJM). In order to establish the effect of βAT87Q-globin production on red blood cell properties, we have analyzed membrane properties, hemolysis markers, morphology, hemoglobin content, and the extent of HbS polymerization. Methods: Whole blood samples were obtained from 3 patients with SCD (1204, 1207 and 1208) treated in HGB-205 during their clinical follow-up. HbS polymerization level was assessed by O2 dissociation and association curves and cell morphology. Membrane properties were evaluated by RBC density curves in phthalate gradient, deformability under increasing osmolality (LORRCA) and level of adherence to surfaces coated with thrombospondin (TSP), under increasing shear stress (from 0.5 to 5 dynes/cm²). Hemolytic level was determined by measurement of classical markers (LDH, bilirubin and haptoglobin). Hemoglobin contents of total RBCs and reticulocytes (CD71-positive cells sorted) were assessed by reverse-phase HPLC. Results were compared against untreated βSβS patients (n=11 for deformability assay, n=4 for adhesion assay) and healthy donors (n=10 for deformability assay, n=3 for adhesion assay). Results: As of May 29 2018, follow-up, total Hb and HbAT87Q contribution to total Hb for patients 1204, 1207 and 1208 were: 42, 18 and 15 months, 12.2, 8.4, and 10.4 g/dL, and 49.44, 7.77 and 26.99%, respectively. At approximately 30 months post-infusion patient 1204 developed vaso-occlusive pain following an episode of acute gastroenteritis, since then the patient has not had any vaso-occlusive episodes or acute chest syndrome (ACS). Patient 1207 had 2 episodes of ACS approximately 6 and 8 months after LentiGlobin gene therapy and has since been on chronic transfusions and hydroxyurea treatment; the patient subsequently experienced 1 vaso-occlusive pain episode. Patient 1208 has had no episodes of VOCs or ACS post LentiGlobin gene therapy. Dissociation and association of O2 curves for RBCs isolated from the 2 patients free of chronic transfusions (1204 and 1208) and performed 36 and 8 months post infusion, respectively, showed only a slight increase in P50 during re-oxygenation, indicating anti-sickling capability of transgenic HbAT87Q and low levels of HbS polymerization. Density curves showed an overall normal RBC hydration at multiple time points during follow-up, with dense cells contributing 0-4% compared to a mean (±SD) of 12.8% (±7.8) in untreated patients. The deformability of RBCs from the 2 patients (1204 and 1208) evaluated in HGB-205 study was lower than observed for healthy donors but higher than for untreated SCD patients. Under controlled shear stress, TSP adherence was consistently lower for RBCs isolated from the 2 patients (1204 and 1208) in HGB-205 compared to untreated patients with SCD. Slight intravascular hemolysis was observed for the 3 HGB-205 patients during follow-up, but the hemolytic levels improved compared to baseline. RP-HPLC analysis of total RBCs isolated at last visit showed an increase in βAT87Q and a decrease in βS in comparison to reticulocytes, indicating an improved survival of RBCs expressing more anti-sickling β-globin transgene (Table 1). Data on deformability, distribution of fetal Hb and additional adhesion markers will be presented. Conclusions: Our results suggest an improvement in RBC properties for 2 of 3 patients with SCD treated with LentiGlobin gene therapy in the HGB-205 clinical trial compared to non-treated patients with SCD, suggesting a promising potential of this treatment. Disclosures El Nemer: Imara: Research Funding. Negre:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Ribeil:Vitalaire: Research Funding; Bluebird Bio, inc.: Employment. Bartolucci:Addmedica: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; Fondation Fabre: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.
Introduction Hyperhemolysis has been well described in delayed hemolytic transfusion reaction (DHTR), which is one of the most serious complications of transfusion especially in patients with sickle cell disease (SCD). In the most severe cases, this life-threatening syndrome may reach 10% lysis of total red blood cells (RBCs) (Habibi A et. al. Am J Hematol 2016). DHTR is characterized by an acute anemia with increased plasma free Hb and free heme. Hyperhemolysis can also be found in other hemoglobinopathies and extracorporeal circulation procedures. Most in vitro studies on hyperhemolysis used only purified Hb or heme which did not allow to investigate all deleterious effects on the endothelium. We have developed a global and physiopathologic approach to assess the mechanism of hyperhemolysis-induced endothelial dysfunction. For this purpose, we created a microfluidic model reproducing hyperhemolysis observed in the most severe DHTR but it's also applicable for other pathogenesis of hyperhemolysis. Among the potential therapeutic approaches, carbon monoxide (CO) is beneficial on endothelial cells by its anti-inflammatory, antioxidant and vasodilator effect which reduce the toxicity of Hb and improve tissue perfusion (Motterlini R et.al. Nat. Rev. Drug Discov 2010). Here we investigated the therapeutic effects of a CO-releasing molecule, CORM-401, in hyperhemolysis-induced endothelial dysfunction. Materials and methods Flow culture Human Umbilical Vein Endothelial Cells (HUVECs) in fibronectin-coated µ-Slides were preconditioned for 4 hours under shear stress of 1dyn/cm² with sonicated RBCs of healthy donors (AA) reconstituted in serum of either AA or SCD patients (SS) at 7 g/L of free Hb. In negative control conditions, cells were pretreated similarly with either AA serum or culture medium. Cells were then collected for analyses of actin network, membrane markers and endothelial function. Treatment with CORM-401 or inactive CORM (iCORM) was performed at fixed concentration during the preconditioning and perfusion steps. ResultsHemolysis induces multiple endothelial damage and dysfunction After exposure to hemolysate, we noted a significant increase of subendothelial matrix exposition (fibronectin-coated surfaces) due to actin network reorganization and cells detachment. Immunofluorescence staining showed a moderate activation of HUVECs induced by hemolysate with membrane expression of adhesion molecules such as ICAM-1, E-Selectin and VCAM-1, but not VWF and P-Selectin. At a similar free Hb level, preconditioning with hemolysate without microparticles (MPs) had the lowest deleterious impact on HUVECs suggesting a role of MPs in hyperhemolysis. Perfusion of AA whole blood on HUVECs pretreated with hemolysate resulted in aggregation and activation of platelets in a GPIIbIIIa-dependent manner at injury sites. We also observed a significant increase of adhered RBCs (14-fold compared to control). Compared to whole blood RBC lysis, purified Hb induced similar subendothelial exposure and RBC adherence but did not lead to significant HUVECs activation. To study the DHTR in RBC transfusion of SCD patients, SS serum was used to perform hemolysis preconditioning of HUVECs. Adherence of RBCs was similar to condition using AA serum suggesting that endothelial damage in DHTR of SCD patients depend on hemolysate compositions and RBC MPs, rather than on patients' serum.Effects of CORM-401 on hemolysis-induced endothelial dysfunction Adding CORM-401 (50 to 100 µM) to the hemolysate produced 15% free COHb leading to a concentration-dependent decrease in hemolysis-induced HUVECs activation and RBC adherence (Figure 1 C, E). While no significant effects at the subendothelial exposure were observed, the aggregation of platelets at injury sites was decreased after CORM-401 treatment compared to controls (Figure 1 A, B, D), confirming the anti-platelet aggregation effect of this molecule (Chlopicki S et. al. Naunyn Schmiedebergs Arch Pharmacol 2012). Conclusion Our in vitro model enabled the reproduction of endothelial damage by hyperhemolysis and to determine the deleterious effects of each hemolysate component. The in vitro beneficial effects of CORM-401 were also demonstrated and an in vivo study on SCD mice is underway to explore the therapeutic effects of this molecule against hyperhemolysis-induced endothelial damage. Figure 1. Figure 1. Disclosures Bartolucci: Addmedica: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; Fondation Fabre: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.
Introduction Sickle cell disease (SCD) is the most prevalent and severe monogenic disorder due to a mutation in the b-globin gene, responsible for the production of an abnormal hemoglobin (HbS) which polymerizes under hypoxia. Cerebral vasculopathy (CV) generally appearing during childhood, is responsible for ischemic stroke, making SCD the first etiology of stroke in children and young adults. To date, several biological and hemodynamical determinants have been identified in CV development such as severe anemia and/or high intracranial vascular flow velocities (> 200 cm/s). Chronic blood exchange transfusion decreases the risk of stroke in children having a pathological Doppler. However some patients still have a progressive impairment despite conventional treatment highlighting the need for new therapeutic strategies and a better understanding of the physiopathology. Therefore, by developing a 3D carotid model reproducing exactly vascular parameters of a SCD patient, we aim to: (i) determine the mechanisms of CV development in SCD, (ii) find new therapeutic approaches and (iii) predict the risk of progression of CV. Materials and methods Three-dimensional reconstructions of the internal carotid, middle cerebral and anterior cerebral artery from SCD patients were generated from magnetic resonance angiograms (MIMICS & 3Matics software, Materialise). We performed 3D simulations of the Navier-Stokes equations in patient specific geometries, including the state-of-the-art techniques of Computational Hemodynamics (multiscale coupling, backflow stabilization - FeLiSCe software) and other factors - such as the increase of the ejection fraction or the drop of peripheral resistances). Blood viscosity was based on a SCD cohort. Hemodynamic properties such as flow velocities (TMMV) and wall shear stress (WSS) in different areas of modelled carotid were then computed according to flow variations. Modelled carotid was obtained by 3D printing according to computer design (CATIA software). The next steps will consist in 1/importing doppler parameters from patients in a programmable pump for flow assays with blood mimicking fluid to measure TMMV and WSS at different areas in carotid, 2/incorporating resting or activated platelets in BMF to evaluate impact of high WSS on platelets degranulation, 3/developing a flow co-culture of smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs) on carotid wall. HUVECs and SMCs at different zones of the carotid undergoing high/low WSS and oscillatory flow will be analysed Preliminary results Our preliminary results suggest that the carotid inlet flow but not blood viscosity is responsible for the pathological intra cranial velocities (Figure 1A). At high carotid inlet flow, areas of high and low WSS appeared in children (Figure 1B), suggesting the existence of turbulent flow that could lead to arterial wall damages. Figure 2A shows a 3D printed carotid reproducing the exact SCD child's one. The material of artificial carotid is compatible with HUVECs culture (Figure 2B) and fluidic experiment at high inlet flow (Figure 2C). On Doppler ultrasonography, the velocities measured in different sections of carotid were comparable to patient's data and these velocities were modified according to variations of inlet flow values. Conclusions and perspectives By modification of input conditions, our 3D personalized model can predict high or low vascular velocities areas and will allow a better understanding of the pathophysiological processes involved at the interface between abnormal flow and cells on carotid wall. This innovative model could be a pertinent tool to evaluate individually effectiveness of new therapeutic strategies in SCD patients. Furthermore, this work may constitute a proof of concept that can be transposed to other diseases. Disclosures Verlhac: Addmedica, Paris: Other: Financial Support; Bluebird Bio: Consultancy. Bartolucci:AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; HEMANEXT: 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: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.
Background:Sickle cell disease (SCD) is one of the most prevalent inherited disorders worldwide. The ex vivo gene therapy phase 1/2 study HGB‐205 conducted in France, evaluates the treatment of SCD and TDT (Transfusion Dependent Thalassemia) with LentiGlobin Drug Product (DP) containing autologous CD34+ cells transduced with the BB305 lentiviral vector encoding a human β‐globin gene with a single substitution (βA‐T87Q).Aims:We evaluated the clinical impact and the production of gene therapy‐derived hemoglobin (HbAT87Q) in the 3 HGB‐205 SCD patients (1204, 1207 and 1208).Methods:As of May 2018, the 3 patients had a follow‐up of 42, 18 and 15 months respectively. HbS polymerization level was evaluated by O2 dissociation/association curves and cell morphology; membrane properties by red blood cells (RBCs) density curves, deformability under increasing osmolality and level of adherence to surfaces coated with thrombospondin (TSP). Hemoglobin contents of RBCs and reticulocytes were assessed by RP‐HPLC.Results:Two (1204 and 1208) of the 3 patients with SCD were free of any SCD‐related treatment, with stable peripheral blood vector copy numbers and total anti‐sickling haemoglobin (HbAT87Q + HbF) at 45–50% of total Hb. Both patients have less‐common SCD genotypes: 1204 has a βS/βS genotype with an alpha‐thalassemia trait and 1208 has a β0/βS genotype. At approximately 30 months post‐infusion, patient 1204 developed vaso‐occlusive pain following an episode of acute gastroenteritis; since then the patient has not had any vaso‐occlusive episodes or acute chest syndrome (ACS). Patient 1208 had no episodes of vaso‐occlusive crisis or ACS post LentiGlobin gene therapy. Patient 1207 had 2 episodes of ACS approximately 6 and 8 months after LentiGlobin gene therapy and re‐started chronic transfusions and hydroxyurea treatment; the patient subsequently experienced 1 Grade ≥3 vaso‐occlusive pain episode. Total Hb and % HbAT87Q contribution to total Hb for patients 1204, 1207 and 1208 at last visit were: 12.2, 8.4, and 10.2 g/dL, and 50.4, 4.4 and 26.4%, respectively. Dissociation and association of O2 curves for RBCs isolated from the 2 patients free of chronic transfusions (1204 and 1208) and performed 36‐ and 8‐months post infusion, respectively, showed only a slight increase in P50 during re‐oxygenation, indicating anti‐sickling capability of transgenic HbAT87Q and low levels of HbS polymerization. Density curves showed an overall normal RBC hydration at multiple time points during follow‐up, with dense cells contributing 0–4% compared to a mean (±SD) of 12.8% (±7.8) in untreated patients. The deformability of RBCs from the 2 patients (1204 and 1208) was lower than observed for healthy donors but higher than for untreated SCD patients. Under controlled shear stress, thrombospondin adherence was consistently lower for RBCs isolated from the 2 patients (1204 and 1208) compared to untreated patients with SCD. Slight intravascular hemolysis was observed for the 3 HGB‐205 patients during follow‐up, but the hemolytic levels improved compared to baseline. RP‐HPLC analysis of total RBCs isolated at last visit showed an increase in βA‐T87Q and a decrease in βS in comparison to reticulocytes, indicating an improved survival of RBCs expressing more anti‐sickling β‐globin transgene. A longer follow‐up and data on deformability, distribution of fetal Hb and additional adhesion markers will be presented.Summary/Conclusion:Further investigations are needed to better define the sickle cell disease phenotype which could most benefit from a gene therapy approach.
In sickle cell disease (SCD), the red blood cells carry a mutated form of hemoglobin (HbS) leading to altered shape and deformability. The mutation causes abnormal hemorheological properties, mechanical hemolysis, and adhesion. The chronic vascular inflammation observed in SCD and hemolysis-related endothelium activation may trigger the vaso-occlusion of blood vessels. The prothrombotic and pro-inflammatory LIGHT/TNFSF14 is a tumor necrosis factor (TNF)-superfamily cytokine implicated in various inflammatory diseases. It is expressed by various immune cells and is considered an actor in T cell-mediated immunity and immune cell recruitment. LIGHT has also been shown to activate endothelial cells (ECs) strongly. LIGHT levels are high in the plasma of SCD patients, and platelets are a major source of its circulating form. We studied a cohort of 82 homozygous adult patients with SCD (n=108 samples) to determine whether LIGHT levels were linked to the clinical state of patients included in the Basal steady state or during an Acute crisis. Soluble LIGHT levels were high in the plasma of SCD patients during acute phases of the disease, particularly during painful occlusive crises. LIGHT levels were associated with Hb levels and inflammatory markers (mainly interferon- alpha and tumor necrosis factor alpha, specifically in acute SCD patients). Our findings confirm that LIGHT is a strong activator of cultured ECs, inducing a type II inflammatory cytokine profile and the expression of adhesion molecules. Using a physiological flow adhesion test on biochips, we showed that the LIGHT-induced activation of ECs led to the adhesion of both sickle platelets (but not their AA counterparts), and in a less extend sickle RBCs to activated HUVECs, potentially constituting the first step in vaso-occlusion. Indeed, the pretreatment of HUVECs with neutralizing polyclonal Abs against LIGHT, but not the non-specific counterpart, showed a reversal of both the inflammation process activated by LIGHT treatment and platelet adhesion to endothelial cells. Soluble LIGHT appears to be a promising therapeutic target for preventing adverse occlusive events in SCD through the blockade of its receptor, to prevent the adhesion of blood cell components to the endothelium. Future studies should consider whether soluble LIGHT contributes to other clinical complications in SCD.
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