ALBERT

Description: In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or β hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.

Description: Background. Wiskott Aldrich Syndrome (WAS) is a rare primary immunodeficiency associated with thrombocytopenia, eczema, severe infectious and autoimmune complications, and lymphomas. Mismatched allogeneic hematopoietic stem cell transplantation (HSCT) is an alternative for patient lacking an HLA-matched donor but is associated with an increased frequency of complications. Moreover low lymphoid and myeloid chimerism is related to a higher rate of autoimmunity and thrombocytopenia. Recent gene therapy (GT) trials showed that gene-corrected autologous CD34+ cells infusion could be an appropriate therapeutic approach for these patients. It has been recently shown that B cell homeostasis is altered in WAS. As the B cell reconstitution participates to the restoration of immunological competence, a comprehensive study of this compartment after GT and the comparison with mismatched allogeneic HSCT is crucial. Objective. To perform a longitudinal study of B cell reconstitution in WAS patients after lentiviral vector-mediated GT, compared to mismatched allogeneic HSCT. Methods. Five patients (age 0.8-15.5 years) underwent GT at our center since 2011(follow-up 1.5-4.2 years) after near-myeloablative and immunosuppressive conditioning regimen with (n=3) or without (n=2) anti-CD20 administration. Patient 2 (P2) died 7 months after GT from a pre-existing infectious complication. Eleven patients undergoing mismatched allogeneic HSCT (age 0.6-10.9 years) at the same center were studied (follow-up 5.1-14.7 years). Longitudinal B cell assessment included B cell count before and after treatment, and the following subsets: switched memory (SM, CD19+ CD27+ IgD-), marginal zone (MZ, CD19+ CD27+ IgD+), naives (CD19+ CD27- IgD+), transitional (CD19+ CD27- IgD+ CD24high CD38high), circulating plasma cells (CD19+ CD27+ IgD- CD27high CD38high) and CD21low B cells (CD19+ CD21low CD38-), a subset abnormally expanded in WAS. Quantification of the B cell replication history was assessed through k-deleting recombination excision circles (KRECs). Analyses were compared to age-matched controls. WAS protein (WASP) expression and vector copy number (VCN) were measured in sorted B cells. Results. All alive GT patients show stable engraftment of functionally corrected lymphoid cells, without adverse events. Transduced B cells number and WASP expression increased progressively after GT. Absolute B cell count attained normal values in all the patients, and correlates with WASP expression and VCN in B cells. IgM levels are below normal ranges in four patients. P3 and P4 attained a B-cell phenotype within normal ranges; P3 discontinued intravenous immunoglobulin (IvIg) replacement. No expansion of CD21low B cells was observed. P1 and P5 (follow-up 18 months) present a variable defect in SM, naives and/or MZ B cells. P1 recently developed autoimmune manifestations; no significant changes were observed concomitantly. A defect in B cell lymphopoiesis was observed before GT as measured by KRECs analysis, normalizing after GT (P1, P3 and P4). Several complications were recorded in patients undergoing mismatched allogeneic HSCT, including dysimmunity, arthritis, developmental deficit and infections. Total B cell count normalized in eight patients, IgM levels were low in three. Among patients with available information, four still remain under IvIg replacement. Four patients developed a mixed lymphoid and myeloid chimerism, variably associated with low B cell count, low IgM and IvIg replacement. A complete B cell assessment for these patients is ongoing. Conclusions. B cell transgene expression is obtained after lentiviral vector-mediated GT in WAS patients and is associated with improved B cell lymphopoiesis. A correct B cell phenotype is observed in two patients who did not receive rituximab prior GT. The question whether this is related to the treatment will need a longer follow-up to be answered. Patients undergoing mismatched allogeneic HSCT present a higher frequency of complications. Although a higher proportion of these patients discontinued IvIg replacement, B cell reconstitution is not optimal. Analysis of patients in particular with mixed chimerism will provide important information in the setting of GT. The analysis of B cell reconstitution after GT and mismatched allogeneic HSCT deserves particular attention in the assessment of immunological reconstitution. Disclosures No relevant conflicts of interest to declare.

Description: Severe combined immunodeficiencies (SCID) are a heterogeneous group of inherited disorders characterized by a profound reduction or alteration of T lymphocyte function. They arise from a variety of molecular defects which affect T lymphocytes development and function. The number of infections prior hematopietic stem cells tansplantaton (HSCT), genotype, and the type of donor are described as prognostic factors for stem cell transplants. In this retrospective study, we included 30 pediatric patients suffering from SCID who underwent to CD34+-selected grafts between January 2008 to December 2017 in our center. Diagnosis of reticular dysgenesis, ADA deficiency, or leaky SCIDs and intra thymic deficiency were excluded. A mechanistic mathematical model of all available data was performed and provided a dynamic appreciation of immune reconstitution, while removing bias. T-cell populations were maintained through proliferation and loss model and thymic output have been integrated to the production function. This joint modeling approach aimed to predict rate and extent of T cell immune reconstitution over time (mainly CD3+ T cells, CD3+CD4+ helper T cells, and the CD3+CD4+ CD45RA+ cells). With a median follow-up time of 97.28 months [range 0.85; 131.54], there were 345 points of T cell phenotyping concentrations in total with a median of 12 samples per patient (range, 0 -35 samples) taken post-transplantation. In this data-set, 13 % of the patients (n= 4) died from infections. Time to reach half of the maximal T cell concentrations was estimated to 3.4 months, 95%CI [2.5 - 4.6]. In covariate analysis, genetic diagnosis (p= 0.0047) and conditioning regimen (p= 0.01) were found to be significant pre transplant covariates which impacted the trajectory of T cell concentration with time. This modeling approach appeared to be the best method to learn about the dynamic T cell reconstitution after transplant in patients suffering from SCID. In the context of new cell therapy approach for T cell depletion and in vitro thymic maturation, this mechanistic joint model can be used for the design and analysis of incoming clinical trials. Figure Disclosures Cavazzana: Smartimmune: Other: Founder of Smartimmune.

Description: Fanconi anemia (FA) is an inherited disorder, clinically characterized by congenital abnormalities, a fatal progressive bone marrow failure (BMF), and a predisposition to develop malignancies. Gene therapy by infusion of FA-corrected autologous hematopoietic stem cells (HSCs) may offer a potential alternative cure and to get around the problems of the Hematopoietic stem cell transplantation toxicity or the donor restriction. For gene therapy, an adequate number of HSC collected is a key point to a successful engraftment. However, the HSC collection in FA patients implies particular challenges because of their reduced BM stem cells numbers and implies a theorical risk of an inner depletion in stem cell reserve following collection.The main objective of this pilot study was to evaluate the feasibility and the safety of co-administration of G-CSF and plerixafor in patients with FA for the mobilization and collection of peripheral HSC for potential use in a GT trial. We present the results of this open-label phase I/II trial (N°EUDRACT 2014-005264-14) from 4 selected FANCA mutated patients (FA-A) with a weight 〉10 Kg and an age between 2 to 18 years old. A systematic combination of G-CSF (12μg/kg twice a day) plus plerixafor (Mozobil® 0.240 mg/kg/d ) was used to maximise the CD34+ cells mobilization. CD34+ cells and white blood cells (WBC) blood counts were monitored tightly along the mobilization protocol. No short-term adverse events linked to the mobilization and the collection procedures were observed. The combination of G-CSF and Plerixafor allowed crossing the PB mobilization threshold (≥5 CD34+cells/μL) for 2 patients. Interestingly, CD34+cells were mobilized quickly but transitionally after plerixafor injection. One patient mobilization had more than 100 CD34+cells μ/L with a early peak 2h after injection. The peak disappeared 11 hours after injection. We adapted the time of collection to the C34+ cells mobilization. No CD34+ blood cell rebound was observed after the apheresis was stopped. Our new datas suggest that mobilization of FA patients with G-CSF and plerixafor is safe. However, the age of the patient, a potential cytopenia or the lack of bone marrow progenitor cell may heavely compromise the collection. Nevertheless, the datas show a stable cytopenia despite the stimulation and collection of stem cells during the following months. This study underlines that a very cautious collection of stem cell in the Fanconi anemia to consider gene therapy is a necessity. These results also confirm that the kinetic of CD34+ cells mobilization is one of the key point to a successful stem cell harvesting for gene therapy trial. Disclosures Cavazzana: Smartimmune: Other: Founder of Smartimmune.

Description: Key Points SCID-X1 patients treated with gene therapy show faster T-cell development compared with patients treated with haploidentical HSCT.

Description: The historical model of hematopoiesis, mainly derived from murine studies, is based on the existence of a single hematopoietic stem cell (HSC) capable of generating all blood cell lineages. However this model has been challenged by the proposal of four types of murine HSC, which differ by their relative contribution to the myeloid and lymphoid lineages. Here we have used data from a gene therapy trial to treat Wiskott-Aldrich syndrome (WAS) to explore hematopoiesis in humans. In the trial, the therapeutic vector (lentivirus) integrates into the genome at unique positions in each hematopoietic stem and progenitor cell (HSPC) and is consequently transmitted to all its progeny. Thus hematopoietic ontogeny in humans can be inferred by tracking the appearance of unique integration sites in fractionated blood cell populations. This provides a unique opportunity to model the developmental complexity of the human haematological system. Considerable effort over the last 15 years has been devoted to optimizing retroviral integration sites (RIS) analysis using ligation mediated PCR (LM-PCR), combined with acoustic shearing and high-throughput Illumina sequencing. Acoustic shearing enables more precise quantification of RIS abundance through the enumeration of the various sizes of shear fragments (SonicAbundance) containing a given RIS, which correspond to individual ancestor HSPC and its blood progeny. In four WAS patients treated by gene therapy, we have sorted peripheral blood samples for 5 cell types: myeloid (granulocytes and monocytes) and lymphoid subpopulations (T, B and NK cells), and analysed their RIS profile. Each RIS corresponds to a particular stem/progenitor cell clone, with a particular pattern defined by its presence or absence in each of the 5 lineages. These data are then use to reconstruct aspects of the hematopoietic hierarchy. In order to face the challenging issue of cell sorting contamination we have been using a stringent sort precision mode and we treat residual contamination explicitly in downstream statistical models. Using these approaches, we have characterized up to tens of thousands RIS per patients with a follow up of 4 years. In order to minimize biases due to sparse sampling, we concentrate on the more abundant RIS clones that can be more easily caught and are therefore analysed more reliably. We showed that a significant fraction of RIS clones are detected in a single lineage, while other RIS clones are characterized by different levels of contribution to the myeloid and lymphoid lineages, highlighting the heterogeneity of human HSC. Clones contributing to all 5 lineages are readily recovered but this study also unravels a diversity of inferred hematopoietic programs with various potentials contributing to human blood homeostasis. Longitudinal analysis of clonal dynamics is ongoing, with preliminary results highlighting the maintenance of this heterogeneity of HSPC over time. These new findings provide unique data on human hematopoiesis based on gene corrected WAS patients. Disclosures No relevant conflicts of interest to declare.

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: 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.