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Wiskott Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency associated with thrombocytopenia, eczema, infectious, autoimmune complications, and lymphomas. Patients lacking an HLA-matched donor may benefit from an alternative therapeutic approach based on the infusion of autologous gene corrected CD34+ cells. We previously reported a non-randomised, open-label, phase 1/2 clinical study applying a lentiviral vector based gene therapy (GT) protocol in 7 paediatric patients with severe WAS (score ≥ 3/5) (S. Hacein-Bey Abina et al, JAMA 2015). One patient died 7 months after GT because of pre-existing severe opportunistic infections, as reported. Two additional patients have been treated since that initial report, with a follow-up of at least 4 years. We here present a comprehensive long-term study on 8 patients with a follow-up from 4 to 9 years (median 7.6). The safety and efficacy of the approach is thoroughly investigated, with a particular focus on the correction of thrombocytopenia and auto-immunity. A stable engraftment of genetically and functionally corrected lymphoid and myeloid cells was reached in all patients, with no serious treatment-associated adverse events or concerning clonal expansion. Corrected lymphoid cells displayed a selective advantage over time with increasing vector copy number (VCN) level. In turn, this led to (i) sustained expression of WAS protein (WASp) in the patients' cells and (ii) clinical resolution of severe eczema and susceptibility to recurrent infections. In line with these results, T-cell function was restored after GT, as shown by the recovery of immune synapse assembly and the normalization of naïve T cell numbers. The T-cell compartment was also reconstituted in the patient treated at the age of 30 years, suggesting that GT for WAS is a treatment option in adult patients. In parallel with the robustness of T-cell reconstitution a normalized B-cell compartment was observed after GT, as shown in particular by increasing levels of WASp + switched memory B cells over time and the age-matched levels of KRECs. Five patients out of 8 were able to discontinue Ig replacement therapy while achieving normal post-vaccination antibody titers. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. After GT, a few autoimmune manifestations were observed: the persistence of lower extremity vasculitis (P2, very severe prior to GT), the new occurrence of nephrotic syndrome (P9), and the presence of anti-platelet antibodies (P2, P4, P7). The levels of circulating autoantibodies detected before GT (including ANA and vasculitis-related autoantibodies) normalized after treatment. Following GT, platelets were found to express sub-normal levels of WASp and to only partially augment their size. Platelet function studies indicated a partial correction of the platelet compartment achieved by GT, which may be sufficient to prevent occurrence of the hemorrhagic symptoms typical of WAS. Our results suggest that lentiviral GT provides sustained clinical benefits for patients with WAS. Overall clinical remission was observed in our patients despite very severe disease scores before GT. More efficacious and more reliable transduction protocols and conditioning regimen are likely to further improve outcomes, particularly with regard to platelet recovery, where the advantages of intrinsic correction are less apparent. Disclosures Booth: Orchard Therapeutics: Consultancy, Honoraria; SOBI: Consultancy, Honoraria; Takeda: Honoraria; GSK: Honoraria; Rocket Pharmaceuticals, Inc.: Consultancy. Thrasher: Orchard Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cavazzana: Smart Immune: Other: co-founder.

β-Thalassemia and sickle cell disease (SCD) are the most prevalent monogenic diseases. These disorders are caused by quantitative or qualitative defects in the production of adult hemoglobin. Gene therapy is a potential treatment option for patients lacking an allogenic compatible hematopoietic stem cell (HSC) donor. New-generation lentiviral vectors (LVs) carrying a β-globin-like gene have revolutionized this field by allowing effective HSC transduction, with no evidence of genotoxicity to date. Several clinical trials with different types of vector are underway worldwide; the initial results are encouraging with regard to the sustained production of therapeutic hemoglobin, improved biological parameters, a lower transfusion requirement, and better quality of life. Long-term follow-up studies will confirm the safety of LV-based gene therapy. The optimization of patient conditioning, HSC harvesting, and HSC transduction has further improved the therapeutic potential of this approach. Novel LV-based strategies for reactivating endogenous fetal hemoglobin (HbF) are also promising, because elevated HbF levels can reduce the severity of both β-thalassemia and SCD. Lastly, genome-editing approaches designed to correct the disease-causing mutation or reactivate HbF are currently under investigation. Here, we discuss the clinical outcomes of current LV-based gene addition trials and the promising advantages of novel alternative therapeutic strategies.

Severe Combined Immunodeficiencies (SCID) are defined by a complete absence of T lymphocytes in the blood and lymphoid organs, with variable defects in other WBC subsets depending on the gene defect. From a clinical perspective SCIDs are characterized by early development of life-threatening infections accounting for early death if untreated. The treatment of choice is allogeneic HSCT with very high success rates if a HLA identical sibling (MRD) or unrelated donor (MUD) is used. However, due to the scarcity of matched-related donors, SCID can benefit from haploidentical HSCT. In contrast to the continuous improvement of HLA compatible donor transplantations, no significant improvements have been obtained over the last twenty years for haploidentical HSCT. The profound immunodeficiency during the first months following haploidentical HSCT exposes patients to opportunistic viral, bacterial and fungal infections, which account for approximately 30-40% of the transplant related mortality (TRM). The rapid restoration of the T-cell compartment is the main aim of stem cell therapy in this setting. To this end we have recently set up a phase I/II clinical trial (ClinicalTrials.gov Identifier: NCT03879876) aiming to accelerate the immune reconstitution by injection of ex vivo generated Human T lymphoid progenitors (ProTcell TM) following haploidentical HSCT. T cell progenitors in this trial are generated in vitro within 7 days from mobilized peripheral blood (mPB) CD34 + hematopoietic stem and precursor cells (HSPCs) using our Notch ligand Delta-like 4 GMP culture platform so called SMART Immune's SMART101 product. This open-label, non-randomized study evaluates safety and efficacy of the SMART101 injection following CD34 + selected, haploidentical HSCT in SCID patients and is designed as a dose-escalation study comprising 6 doses of the SMART101 product obtained from the patient's haploidentical stem cell graft. The aim of this protocol is to define the highest efficacy dose without any toxicity. The conditioning regimen is based on Busulfan and Fludarabine according to IE-WP/EBMT guidelines with upfront administration of ATG to prevent graft rejection. Tight monitoring of ATG serum levels is applied in order to assure injection of SMART101 when ATG is below the lymphotoxicity threshold. Here we report the results of the first two SCID patients. P1 presented a homozygous Artemis deficiency. At diagnosis he had an ALC of 341/µl, with complete absence of T cells (CD3 + < 4/µl, CD4 + < 1/µl, CD8 + < 2/µl) and B cells (CD19 + 0/µl). NK cells were present in the normal range for age (CD16 +CD56 + 331/µl). In the absence of an HLA compatible donor, the patient`s father was chosen as haploidentical stem cell donor. P1 received upfront ATG (5 mg /kg total dose), Busulfan (AUC of 16058 microM.min) and Fludarabine (160 mg/m²). He received Defibrotide prophylaxis from D0 until D+21, as well as Ursodeoxycholic acid until D+80. The CD34 + immunoselected graft contained 1.04 x 10 8 nucleated cells/kg with 24.15 x10 6 CD34 + cells/kg and 4000 CD3 + cells/kg on D0. After ATG monitoring 0.12x10 6 Smart101 cells were administered at D+14 post- HSCT. In the follow-up P1 didn't develop any acute or chronic SAEs, no acute or chronic GVHD, and no infection. He was discharged at D+121 post HSCT. The day +100 post transplantation CD4 + cell count/microliter exceeded 10 times the CD4 + count of our historical cohort of RAG1/2 or Artemis deficient patients transplanted with haploidentical HSCT alone following the same conditioning regimen. At 6 months post HSCT this difference remains important (851 versus 300 CD4 + cells/µl); Ig replacement therapy could be stopped as early as 9 months post transplantation and vaccinations have been started. At last follow up; almost 14 months post HSCT P1 is alive and well. P2 had an undefined molecular SCID diagnosis. She has been treated with the same conditioning regimen and received the second dose of 0.2x10 6 CD7 + cells, but unfortunately died from severe VOD emphasizing the need to replace chemotherapy with less toxic myeloablative agents. The preliminary results obtained after injection of Human T lymphoid progenitors in P1 are encouraging. While deserving confirmation in larger numbers of patients they could represent an important step forward in improving the outcome of haploidentical HSCT for SCID. Disclosures Cavazzana: Smart Immune: Other: co-founder.

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.

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.

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.

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.

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.

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.

Background The assessment of residual tumor cells persisting after therapy, or minimal residual disease (MRD), is a central component of accurate disease prognosis in acute lymphoblastic leukemia (ALL) (Pui et al, JCO 2011). MRD assessment has recently been shown to be useful for monitoring disease before and after stem cell transplantation and during salvage therapy for early detection of an imminent relapse (Bruggemann et al., SemOncol 2012). Allele-specific oligonucleotide (ASO)-PCR can be used to assess MRD; however, this technique requires preparation of clonotype-specific primers for each individual which is laborious and time-consuming. We recently demonstrated the utility of sequencing-based MRD assessment in lymphoid malignancies (Faham et al., Blood 2012). This quantitative approach, termed the LymphoSIGHT™ platform, relies on amplification and sequencing of immunoglobulin and T-cell receptor gene segments using consensus primers and can address some of the limitations associated with traditional MRD detection techniques. This sequencing platform has a sensitivity to detect one cancer cell per million leukocytes in peripheral blood and bone marrow samples. In this retrospective study, we evaluated the ability of the sequencing and ASO-PCR methods to detect MRD prior to clinical relapse in 17 patients with childhood ALL. Methods Using the sequencing assay, we analyzed bone marrow and/or peripheral blood samples collected at the diagnostic and relapse time points from 17 childhood ALL patients. Diagnostic and relapse samples were assessed for clonal rearrangements of immunoglobulin (IGH-VDJ, IGH-DJ, IGK) and T cell receptor (TRB, TRD, TRG) genes. Following identification of leukemia specific clonotype(s), we measured the corresponding MRD levels in 66 follow-up samples that were collected prior to clinical relapse. We analyzed the time from MRD positivity to clinical relapse for each patient using the sequencing and ASO-PCR methods. Results Sequencing detected the presence of a high-frequency clonal rearrangement of at least one receptor (“calibrating receptor”) in all the 17 childhood ALL patients; all patients had at least 2 calibrating receptors at diagnosis and/or relapse, 15 patients had at least 3 calibrating receptors, and 6 patients had 4 or more. The IGH-VDJ and TRG assays were the most frequent gene rearrangement: at least one IGH-VDJ and/or TRG clonal rearrangement was detected in 13 ALL patients. TRD was the third most informative receptor, with clonal rearrangements being detected in 11 patients. In the majority of patients, most leukemic clones identified at diagnosis were also present at high levels in the relapse sample. We analyzed the time from MRD positivity to clinical relapse in the patient cohort. In 2 patients (Figure 1, Patients G and P), we observed MRD positivity by sequencing 16 and 6.5 months prior to relapse, respectively. ASO-PCR also detected MRD positivity in Patient P at the 6.5 month time point. In 6 patients there was a sample collected within 1-3 months prior to relapse, and sequencing detected MRD in 4 of these patients (67%, Figure 1 Patients B, A, R, I), while ASO-PCR detected MRD in 2 of the 6 patients (33%, Figure 1 Patients B, R). Conclusions The clinical value of monitoring MRD by ASO-PCR for assessment of treatment response and outcome has been established in multiple lymphoid malignancies. This preliminary study provides further support for the sequencing-based MRD monitoring in ALL patients. This new approach offers improvements over ASO-PCR in the ability to monitor multiple clonal sequences and in the sensitivity to detect the leukemic cell. Thus, the sequencing method represents a new potential approach for MRD monitoring. However a larger number of patients must be analyzed to support its clinical application. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Pepin:Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership.

Naturally occurring, large deletions in the β-globin locus result in increased fetal hemoglobin (HbF) expression (HPFH, Hereditary Persistence of Fetal Hemoglobin), a condition that mitigates the clinical severity of Sickle Cell Disease (SCD) and β-thalassemia. Here, we integrated BCL11A and GATA1 transcription factor binding site analysis and HPFH mutational data to identify potential HbF silencers in the β-globin locus. Based on this analysis, we designed a CRISPR/Cas9 strategy to disrupt: (i) a 3.5-kb δγ-intergenic region that is specifically deleted in individuals with HPFH. This region contains BCL11A and GATA1 binding sites in adult, HbF-negative primary erythroblasts, thus representing a potential HbF silencer; (ii) a 7.2-kb region, encompassing the 3.5-kb δγ-intergenic element, which is the minimal naturally occurring deletion associated with HPFH ("Corfù" deletion; Chakalova et al., Blood, 2005); (iii) a 13.6-kb genomic region commonly deleted in HPFH, which includes the δ- and β-globin genes and putative intergenic HbF silencers. Targeted deletion or inversion of the 13.6-kb genomic region caused a substantial increase in γ-globin mRNA levels, re-activation of HbF synthesis and a concomitant decrease in HbA expression in adult human erythroid cells (HUDEP-2). Interestingly, deletion of the Corfù region or of the 3.5-kb putative HbF silencer, increased γ-globin gene transcription but failed to produce accumulation of γ-globin mRNA, suggesting a post-transcriptional regulation of γ-globin synthesis in the presence of an intact and active β-globin gene. We then tested re-activation of HbF synthesis in primary adult hematopoietic stem/progenitor cells differentiated towards the erythroid lineage in liquid and clonogenic cultures. Targeting the 13.6-kb genomic region resulted in a high proportion of γ-globin expressing primary erythroblasts, with HbF representing up to 50% of total hemoglobin. Cell morphology, erythroid marker profile, total hemoglobin levels and erythroid maturation were unaffected, consistent with the asymptomatic phenotype of adult HPFH carriers. These data suggest that this region could serve as target for therapeutic genome editing for HbF induction in β-hemoglobinopathies. Overall, this study contributes to the knowledge of the mechanisms underlying fetal to adult Hb switching, and provides clues for a genome editing approach to the treatment of SCD and β-thalassemia. Disclosures Cradick: CRISPR Therapeutics: Employment. Lundberg:CRISPR Therapeutics: Employment, Equity Ownership. Porteus:CRISPR Therapeutics: Consultancy, Equity Ownership. Mavilio:Adverum Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding.

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.

The association of the SH3 (Src homology 3) domain of SFKs (Src family kinases) with protein partners bearing proline-rich motifs has been implicated in the regulation of SFK activity, and has been described as a possible mechanism of relocalization of SFKs to subcellular compartments. We demonstrate in the present study for the first time that p13, an accessory protein encoded by the HTLV-1 (human T-cell leukaemia virus type 1), binds the SH3 domain of SFKs via its C-terminal proline-rich motif, forming a stable heterodimer that translocates to mitochondria by virtue of its N-terminal mitochondrial localization signal. As a result, the activity of SFKs is dramatically enhanced, with a subsequent increase in mitochondrial tyrosine phosphorylation, and the recognized ability of p13 to insert itself into the inner mitochondrial membrane and to perturb the mitochondrial membrane potential is abolished. Overall, the present study, in addition to confirming that the catalytic activity of SFKs is modulated by interactors of their SH3 domain, leads us to hypothesize a general mechanism by which proteins bearing a proline-rich motif and a mitochondrial localization signal at the same time may act as carriers of SFKs into mitochondria, thus contributing to the regulation of mitochondrial functions under various pathophysiological conditions.

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative disorders characterized by an increased incidence of thrombo-hemorrhagic complications. The acquired somatic Janus kinase 2 (JAK2) V617F mutation is present in the majority of PV and ET patients. Because aberrant protein Tyr-phosphorylation has been associated with hematopoietic malignancies, the activity of the tyrosine kinases Src and JAK2 was analyzed in resting and thrombin-stimulated platelets from 13 PV and 42 ET patients. JAK2 was found inactive in healthy and pathological resting cells regardless of the V617F mutation. In addition, Src was inactive in all resting platelets, but in the pathological specimens it was present in a preactivated conformation as a consequence of anomalous dephosphorylation of its inhibitory phospho-Tyr527 residue, likely mediated by Src homology-2 domain-containing protein Tyr-phosphatase-2 (SHP-2), whose constitutive activity correlated with its recruitment to Src. Low thrombin concentration triggered a more rapid Src-signaling activation, higher [Ca2+]c increase, and aggregation in pathological platelets compared with controls. Thrombin-induced Src activation preceded JAK2 activation, which occurred simultaneously in normal and pathological platelets. Our results indicate that a constitutive Src kinase preactivation is implicated in platelet hypersensitivity and likely involved, at least partially, in the functional abnormalities of PV and ET platelets.

The dimerization and auto-transphosphorylation of platelet-derived growth factor receptor (PDGFR) upon engagement by platelet-derived growth factor (PDGF) activates signals promoting the mitogenic response of hepatic stellate cells (HSCs) due to liver injury, thus contributing to the development of hepatic fibrosis. We demonstrate that the tyrosine phosphatases Src homology 2 domain-containing phosphatase 1 and 2 (SHP-1 and SHP-2) act as crucial regulators of a complex signaling network orchestrated by PDGFR activation in a spatio-temporal manner with diverse and opposing functions in HSCs. In fact, silencing of either phosphatase shows that SHP-2 is committed to PDGFR-mediated cell proliferation, whereas SHP-1 dephosphorylates PDGFR hence abrogating the downstream signaling pathways that result in HSC activation. In this regard, SHP-1 as an off-switch of PDGFR signaling appears to emerge as a valuable molecular target to trigger as to prevent HSC proliferation and the fibrogenic effects of HSC activation. We show that boswellic acid, a multitarget compound with potent anti-inflammatory action, exerts an anti-proliferative effect on HSCs, as in other cell models, by upregulating SHP-1 with subsequent dephosphorylation of PDGFR-β and downregulation of PDGF-dependent signaling after PDGF stimulation. Moreover, the synergism resulting from the combined use of boswellic acid and imatinib, which directly inhibits PDGFR-β activity, on activated HSCs offers new perspectives for the development of therapeutic strategies that could implement molecules affecting diverse players of this molecular circuit, thus paving the way to multi-drug low-dose regimens for liver fibrosis.