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lovo-cel (bb1111; LentiGlobin for sickle cell disease [SCD]) gene therapy (GT) comprises autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector encoding a modified β-globin gene (βA-T87Q ) to produce anti-sickling hemoglobin (HbAT87Q ). The efficacy and safety of lovo-cel for SCD are being evaluated in the ongoing phase 1/2 HGB-206 study (ClinicalTrials.gov: NCT02140554). The treatment process evolved over time, using learnings from outcomes in the initial patients to optimize lovo-cel's benefit-risk profile. Following modest expression of HbAT87Q in the initial patients (Group A, n = 7), alterations were made to the treatment process for patients subsequently enrolled in Group B (n = 2, patients B1 and B2), including improvements to cell collection and lovo-cel manufacturing. After 6 months, median Group A peripheral blood vector copy number (≥0.08 c/dg) and HbAT87Q levels (≥0.46 g/dL) were inadequate for substantial clinical effect but stable and sustained over 5.5 years; both markedly improved in Group B (patient B1: ≥0.53 c/dg and ≥2.69 g/dL; patient B2: ≥2.14 c/dg and ≥6.40 g/dL, respectively) and generated improved biologic and clinical efficacy in Group B, including higher total hemoglobin and decreased hemolysis. The safety of the lovo-cel for SCD treatment regimen largely reflected the known side effects of HSPC collection, busulfan conditioning regimen, and underlying SCD; acute myeloid leukemia was observed in two patients in Group A and deemed unlikely related to insertional oncogenesis. Changes made during development of the lovo-cel treatment process were associated with improved outcomes and provide lessons for future SCD GT studies.

BackgroundDonor availability and transplantation-related risks limit the broad use of allogeneic hematopoietic-cell transplantation in patients with transfusion-dependent β-thalassemia. After previously establishing that lentiviral transfer of a marked β-globin (βA-T87Q) gene could substitute for long-term red-cell transfusions in a patient with β-thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion-dependent β-thalassemia.MethodsIn two phase 1-2 studies, we obtained mobilized autologous CD34+ cells from 22 patients (12 to 35 years of age) with transfusion-dependent β-thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q). The cells were then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, vector integration, and levels of replication-competent lentivirus. Efficacy assessments included levels of total hemoglobin and HbAT87Q, transfusion requirements, and average vector copy number.ResultsAt a median of 26 months (range, 15 to 42) after infusion of the gene-modified cells, all but 1 of the 13 patients who had a non-β0/β0 genotype had stopped receiving red-cell transfusions; the levels of HbAT87Q ranged from 3.4 to 10.0 g per deciliter, and the levels of total hemoglobin ranged from 8.2 to 13.7 g per deciliter. Correction of biologic markers of dyserythropoiesis was achieved in evaluated patients with hemoglobin levels near normal ranges. In 9 patients with a β0/β0 genotype or two copies of the IVS1-110 mutation, the median annualized transfusion volume was decreased by 73%, and red-cell transfusions were discontinued in 3 patients. Treatment-related adverse events were typical of those associated with autologous stem-cell transplantation. No clonal dominance related to vector integration was observed.ConclusionsGene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long-term red-cell transfusions in 22 patients with severe β-thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB-204 and HGB-205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526 .).

Introduction Granulomatosis with polyangiitis (GPA) is a systemic autoimmune disease characterized by granulomatous inflammation and necrotizing vasculitis (1). GPA is associated with anti-neutrophil cytoplasmic antibodies (ANCAs) directed against the neutrophil- [...], Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating [CD4.sup.+] T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology.

To investigate the hypothesis that HSP70 can be sequestrated in the cytoplasm of mature β-TM erythroblasts by binding to free α-globin chains of haemoglobin or aggregates, we analysed its subcellular [...], β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates (1,2). Despite extensive knowledge of the molecular defects causing β-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage (3-6). We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation (7). Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage (8). The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation (9). Here we show in vitro that during the maturation of human β-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GAT A-1 mutant restores terminal maturation of β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-TM.

The pathophysiology of ineffective erythropoiesis in b-thalassemia is poorly understood. We report that RAP-011, an activin receptor IIA (ActRIIA) ligand trap, improved ineffective erythropoiesis, corrected anemia and limited iron overload [...]

Abstract Background Sickle cell disease (SCD) leads to tissue hypoxia resulting in chronic organ dysfunction including SCD associated nephropathy. The goal of our study was to determine the best equation to estimate glomerular filtration rate (GFR) in SCD adult patients. Methods We conducted a prospective observational cohort study. Since 2007, all adult SCD patients in steady state, followed in two medical departments, have had their GFR measured using iohexol plasma clearance (gold standard). The Cockcroft-Gault, MDRD-v4, CKP-EPI and finally, MDRD and CKD-EPI equations without adjustment for ethnicity were tested to estimate GFR from serum creatinine. Estimated GFRs were compared to measured GFRs according to the graphical Bland and Altman method. Results Sixty-four SCD patients (16 men, median age 27.5 years [range 18.0-67.5], 41 with SS-genotype were studied. They were Sub-Saharan Africa and French West Indies natives and predominantly lean (median body mass index: 22 kg/m2 [16-33]). Hyperfiltration (defined as measured GFR >110 mL/min/1.73 m2) was detected in 53.1% of patients. Urinary albumin/creatinine ratio was higher in patients with hyperfiltration than in patients with normal GFR (4.05 mg/mmol [0.14-60] versus 0.4 mg/mmol [0.7-81], p = 0.01). The CKD-EPI equation without adjustment for ethnicity had both the lowest bias and the greatest precision. Differences between estimated GFRs using the CKP-EPI equation and measured GFRs decreased with increasing GFR values, whereas it increased with the Cockcroft-Gault and MDRD-v4 equations. Conclusions We confirm that SCD patients have a high rate of glomerular hyperfiltration, which is frequently associated with microalbuminuria or macroalbuminuria. In non-Afro-American SCD patients, the best method for estimating GFR from serum creatinine is the CKD-EPI equation without adjustment for ethnicity. This equation is particularly accurate to estimate high GFR values, including glomerular hyperfiltration, and thus should be recommended to screen SCD adult patients at high risk for SCD nephropathy.