ALBERT

Description: Introduction: Fetal hemoglobin (HbF) expression is a major modulator of sickle cell disease (SCD) severity by decreasing the HbS polymerization. However, the distribution of HbF in red blood cells (RBC) is heterogeneous in SCD patients. In the hypothesis of an HbF "threshold" in RBCs for inhibiting the HbS polymerization, accurate measurement of the HbF content in each red blood cell (HbF/RBC) is mandatory. To this purpose we developed a new and accurate method allowing the direct measurement of HbF content per RBC. Thanks to it, as a proof of concept, we analyzed HbF distribution and content in RBC from SCD patients before and after 6 months of treatment by hydroxyurea (HU). To determine if a threshold of HbF/RBC could modulate SCD, we analyzed the associations between the %RBC reaching different thresholds of HbF (in picograms), and biological parameters and the incidence of severe VOC. Methods: 14 SCD (βS/βS or βS/β0) patients were included to study HbF distribution during HU for a period of 6 months. RBCs were collected during each outpatient visit (Week 0, Week 2, Week 4, Week 12, and ≥ Week 24). HbF content was measured in RBCs using an anti-Human-HbF antibody by flow cytometry. Normalized RBC fluorescence intensity was then converted in picograms of HbF/RBC by using the linear association between mean HbF content and mean RBC fluorescence obtained from subjects presenting homogeneous HbF distribution (patients with hereditary persistence of HbF (HPFH), or β-Thalassemia or δβ-Thalassemia). Quantitative analysis were performed before and during HU treatment to characterize the response by comparing percentages of RBC classes based on different ranges of HbF/RBC during HU treatment. We therefore analyzed the associations between HbF/RBC thresholds (%RBC containing at least 2, 4, 6, 8, 10 or 20 pg HbF) and biological parameters before and ≥ 6 months of HU treatment. Finally HbF/RBC thresholds at Week 0 were compared to the incidence of hospitalized VOC within 3 years before W0, and HbF/RBC thresholds at Week 24 were compared to the incidence of hospitalized VOC within 3 years after W24 at a stable dose. Results: After 6 months of HU, mean %HbF, assessed by HPLC, raised from 6.16% (±3.5) to 15.2% (±8.7) (mean ± standard deviation). Quantitative analysis of HbF/RBC revealed a statistically significant decrease between D0 and ≥M6 of 24% of RBCs containing less than 2 pg (p = 0.0015) and a 2-fold increase of RBCs containing between 2 and 4 pg (p = 0.0025) (Friedman test). For biological parameters we observed an increase in mean %HbF, MCV and MCH and a decrease in RBC count significantly associated (p

Description: In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the question of the capacity of cultured RBCs (cRBCs) to survive in humans. By using a culture protocol permitting erythroid differentiation from peripheral CD34+ HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen, and expression of blood group antigens. We then demonstrated in the nonobese diabetes/severe combined immunodeficiency mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 1010 cRBCs generated under good manufacturing practice conditions and labeled with 51Cr. The level of these cells in the circulation 26 days after injection was between 41% and 63%, which compares favorably with the reported half-life of 28 ± 2 days for native RBCs. Their survival in vivo testifies globally to their quality and functionality. These data establish the proof of principle for transfusion of in vitro–generated RBCs and path the way toward new developments in transfusion medicine. This study is registered at http://www.clinicaltrials.gov as NCT0929266.

Description: 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.

Description: BCL11A regulates the fetal-adult hemoglobin switch by repressing expression at the gamma (γ)-globin locus (Sankaran et al., Science, 2008), and thus it represents an appealing therapeutic target for sickle cell disease (SCD). BCH-BB694 is a lentiviral vector (LVV) encoding a shRNA targeting BCL11A embedded in a microRNA scaffold (shmiR) allowing erythroid-specific knockdown to induce γ-globin expression and concomitantly and coordinately repress β-sickle globin expression (Brendel et al. JCI, 2016). In a pilot and feasibility gene therapy study we are evaluating the safety of infusion of BCH-BB694-transduced autologous CD34+ cells in patients with severe SCD. The study is an IND enabled and IRB approved open label, non-randomized, single center trial (NCT 03282656). We report here data from the full adult cohort which has completed enrollment with 〉 6 months of follow up in all patients. The adult cohort included three patients 〉/= 18 years old. Autologous CD34+ cells were collected by plerixafor mobilization and then transduced ex vivo with the BCH-BB694 shmiR lentiviral vector. Cell doses and vector copy number (VCN) are shown in the Table. After testing and release, gene modified cells were infused into subjects who had received busulfan conditioning. There were no Grade 3 or 4 AEs associated with mobilization, collection or infusion. All three adults (age 21-26 years old) demonstrated neutrophil engraftment on day +22 with adverse events consistent with busulfan conditioning. These patients are now 7, 9, and 17 months post infusion. One subject resumed red cell transfusions at 3 months due to pre-existing moyamoya using a pre-defined conservative trigger value of 40% sickle Hb in whole blood and will be detailed separately. There have been no adverse events related to the gene therapy product. VCN has been stable in bone marrow (BM) and peripheral blood (PB) in all cell lineages during the length of the study, with the latest time point studied at 15 months (BCL002) and ranged from 0.45-2.85 copies per cell in erythroid progenitor cells. BCL11A protein levels evaluated by immunoblot in subject BCL002 at 30 days (PB) and 6 months (BM) post-infusion showed highly effective and selective knockdown of BCL11A in erythroid progenitors with no reduction in BCL11A expression in B lymphoid cells. The number of HbF-containing cells (F cells) was assessed by flow cytometry and the kinetics of F cell production was remarkably similar in all subjects. The two untransfused subjects (BCL002 and BCL004) produced 70% F-cells in PB at 3 and 5 months, which has remained stable until the last point assayed (15 months and 7.5 months, respectively) (table). Calculated average HbF per F cell was 〉10pg in all subjects (table) and quantitative single cell HbF flow analysis showed the majority of F cells had 〉4pg F/cell, a level that is believed to prevent sickling under physiological oxygen saturation (Rakotoson et al., ASH 2017). In both untransfused subjects, total Hb remained stable with evidence of reduced hemolysis by reticulocyte count (slightly elevated) and LDH (normal in one subject, slightly elevated in the other). At the 3-month timepoint before re-starting transfusions, the subject with moyamoya (BCL003) had a pre-transfusion Hb of 11 g/dL with 76% of non-transfused cells containing on average 17pg F/F cell. For all subjects, we estimated the fraction of RBCs containing significant Hb sickle polymers and the amount of polymer in each sickled RBC at physiologic oxygen tension (where 50% of monomeric hemoglobin was oxygen saturated, or the P50) (Di Caprio et al. PNAS 2019, in press). The results for all 3 subjects in this adult cohort showed fewer RBCs with significant Hb polymer than two hydroxyurea-responsive treated comparators and significantly less Hb polymer per sickled RBC than a third highly responsive hydroxyurea-treated comparator. In conclusion, these data demonstrate successful and sustained engraftment in three adult patients treated with LVV-delivered shmiR technology targeting BCL11A. Early results suggest an acceptable safety profile, validation of BCL11A as effective target for HbF induction in humans with high numbers of F cells in circulation containing high levels of HbF per F cell, and mitigation of cellular pathology of SCD. Disclosures Achebe: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacosmos: Membership on an entity's Board of Directors or advisory committees; Fulcrum Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Bartolucci:Novartis: Membership on an entity's Board of Directors or advisory committees; 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. Heeney:AstraZeneca: Research Funding; Micelle Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Research Funding; Ironwood / Cyclerion: Research Funding; Vertex / Crisper Therapeutics: Other: Data Safety Monitoring Board. Higgins:Sanofi: Consultancy, Research Funding. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Wood:Sanofi: Consultancy, Research Funding. Williams:Alerion Biosciences: Other: Co-founder; Novartis: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, Patents & Royalties: Potential for future royalty/milestone income, X-SCID., Research Funding; bluebird bio: Patents & Royalties: Licensed certain IP relevant to hemoglobinopathies to bluebird bio. Received payment in the past bluebird bio through a BCH institutional licensing agreement and there is a potential for future royalty/milestone income from this agreement., Research Funding.

Description: Background LentiGlobin gene therapy contains autologous CD34+ hematopoietic stem cells (HSCs) transduced with the BB305 lentiviral vector (LVV), encoding human β-globin with a T87Q substitution. This substitution confers anti-sickling properties to the gene therapy-derived hemoglobin (HbAT87Q) and allows for its quantification in transduced HSCs. The proof of concept for LentiGlobin gene therapy in patients with transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) was established in the recently completed HGB-205 study (NCT02151526). Herein, we provide the safety and efficacy outcomes and long-term follow-up data for all 7 treated patients, 4 with TDT and 3 with SCD. Methods Patients 5−35 years old with TDT (≥ 100 mL/kg of packed red blood cells [pRBCs]/year) or severe SCD (e.g., ≥ 2 acute chest syndromes [ACS] or ≥ 2 vaso-occlusive crises in the preceding year or the year before regular transfusions) were enrolled. CD34+ HSCs were obtained by mobilization and apheresis in patients with TDT or by bone marrow harvest in patients with SCD. Following collection, cells were transduced with the BB305 LVV. Patients underwent busulfan myeloablative conditioning and were infused with transduced cells. Patients were monitored for engraftment, adverse events (AEs), HbAT87Q levels, and other hematologic and clinical parameters. After 2 years in HGB-205, patients transitioned into the long-term follow-up study, LTF-303 (NCT02633943). Summary statistics are shown as median (min-max). Results As of June 2019, patients with TDT (n=4) and SCD (n=3) had a median follow-up of 49.6 (40.5-60.6) and 28.5 (25.5-52.5) months, respectively. Table 1 shows patient and drug product characteristics and several key efficacy outcomes. All patients achieved HSC engraftment. LentiGlobin safety profile was consistent with busulfan myeloablative conditioning and, in case of SCD, with the underlying disease state. The most common non-hematologic Grade ≥ 3 AEs post-LentiGlobin gene therapy (≥ 2 patients) for patients with TDT were stomatitis (n=4) and increased aspartate aminotransferase (n=2), and for patients with SCD were ACS (n=2) and vaso-occlusive pain (n=2). In all 4 patients with TDT, total Hb and HbAT87Q levels remained generally stable up to 5 years post-LentiGlobin infusion. Three of 4 patients achieved transfusion independence (TI; defined as weighted average Hb ≥ 9g/dL without pRBC transfusions for ≥ 12 months), for an ongoing duration of 56.3 (38.2-57.6) months. Weighted average total Hb during TI was 11.4 (10.5-13.0) g/dL. One patient has been off transfusions for 37.5 months and had total Hb of 7.7 g/dL, which was below the ≥ 9 g/dL requirement to meet the protocol definition of TI. At last visit, HbAT87Q levels in these 4 patients ranged from 6.2-11.2 g/dL, which contributed 73.8-86.8% of the total Hb. The first patient treated with LentiGlobin for SCD experienced one vaso-occlusive pain episode, which developed at 30 months after LentiGlobin gene therapy following a case of acute gastroenteritis with fever and dehydration. The second SCD patient had 2 serious AEs (SAEs) of ACS approximately 6 and 8 months after LentiGlobin gene therapy. The patient resumed chronic pRBC transfusions and hydroxyurea treatment and subsequently experienced 2 SAEs of vaso-occlusive pain; no additional SAEs of vaso-occlusive pain or ACS were reported during the last 16 months of follow-up after LentiGlobin infusion. The third SCD patient had no episodes of vaso-occlusive pain or ACS during 25.5 months of follow-up post-LentiGlobin gene therapy as of the data cut-off. Two patients with SCD who have been off chronic pRBC transfusions, showed improvement in hemolysis markers post-LentiGlobin treatment and stabilization of HbAT87Q expression at approximately 6 months post-LentiGlobin infusion. Total Hb levels for patients with SCD at last visit were 13.0 g/dL (patient 1), 9.4 g/dL (patient 2), and 9.8 g/dL (patient 3), with corresponding HbAT87Q contributions of 47.9%, 7.9%, and 25.8%, respectively. Summary With up to 5 years of follow-up, treatment with LentiGlobin gene therapy was well tolerated and resulted in improvement in hematologic parameters and disease-related symptoms. Further results from the completed study will be presented. Disclosures Hermine: Celgene: Research Funding; Novartis: Research Funding; AB science: Consultancy, Equity Ownership, Honoraria, Research Funding. Brousse:bluebird bio, Inc: Consultancy; AddMedica: Consultancy. El Nemer:Hemanext: Other: Other. Bartolucci:Novartis: Membership on an entity's Board of Directors or advisory committees; AddMedica: Honoraria, 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; Roche: Membership on an entity's Board of Directors or advisory committees; HEMANEXT: Membership on an entity's Board of Directors or advisory committees. Asmal:bluebird bio, Inc: Employment, Equity Ownership. Whitney:bluebird bio, Inc: Employment, Equity Ownership. Gayron:bluebird bio, Inc: Employment, Equity Ownership. Huang:bluebird bio, Inc.: Employment, Equity Ownership. de Montalembert:AddMedica: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Ribeil:bluebird bio, Inc: Employment, Equity Ownership. Cavazzana:SmartImmune: Other: Founder.

Description: Introduction: Hemolytic anemia combines 3 components to various extents: extravascular hemolysis, intravascular hemolysis and dyserythropoiesis. Global hemolysis in excess to defense lines induces oxidative and inflammatory syndromes and vascular damages in various organs. Therefore, accurate hemolysis biomarkers are required for a better evaluation of hemolytic disorders, such as sickle cell disease (SCD) or thalassemia syndromes and to evaluate the efficacy of various therapies. Accordingly, we have developed a new spectrophotometric method to measure and calculate several hemolysis biomarkers in plasma or serum including Hemoglobin in various forms (HbO2, HbCO, MetHb), Heme or Hemin bound to albumin or to hemopexin, total bilirubin and total hemopexin. Patients and Method : Blood samples were collected at steady-state for 77 SCD adults (mean age 39.8 ± 10.2 yrs, M/F ratio 0.64) and 23 beta thalassemia patients (mean age 42.7 ± 16 yrs, M/F ratio 0.91); SCD patients (SS or Sb0-Thalassemia) were either treated with Hydroxycarbamide (HU: 27) or not (NT: 50). For comparison, plasma samples from healthy volunteers (HV) were also analyzed. Continuous variables were expressed as means ± SD or medians [interquartile range], depending on their normal or asymmetric distributions. Categorical variables were expressed as numbers (%). Univariate analyses were done using Student's t-test or Mann-Whitney non-parametric test, depending on the distribution. Correlation were analyzed using a spearman test. The dosage methodology is based on the light absorption spectrophotometry of plasma samples using an appropriate mathematical conversion of the signal, reference spectra of the different species and some chemical modifications of the iron redox and ligation states. Results: The levels of plasma Hb were statistically higher in homozygous SCD patients compared to beta-thalassemia patients (p=0.001) and healthy volunteers, with median of 6.3 [3.4-11], 2.6 [1-5.4] and 1.7 [0.5-3] µM respectively (Table 1). Interestingly levels of plasma heme were higher in beta thalassemia patient compared to SCD patients (p=0.0001) and HV, with a mean of 1.05 [0.05-3], 10.5 [3.5-24] and ≤ 0.2 µM level of detection respectively (Table 1). A significant negative correlation was found between heme and hemopexin levels in both diseases (p