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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.

Key Points IUT enables rapid immune reconstitution and avoids many clinical and economic problems; however, the indication is still limited. IUT may be a treatment option in select cases, eg, fetuses exposed to a significant infectious risk, where a matched sibling donor exists.

Sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT) are the most prevalent monogenic disorders worldwide. Trial HGB-205 ( NCT02151526 ) aimed at evaluating gene therapy by autologous CD34

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.

Although studies of mixed chimerism following hematopoietic stem cell transplantation in patients with sickle cell disease (SCD) may provide insights into the engraftment needed to correct the disease and into immunological reconstitution, an extensive multilineage analysis is lacking. We analyzed chimerism simultaneously in peripheral erythroid and granulomonocytic precursors/progenitors, highly purified B and T lymphocytes, monocytes, granulocytes and red blood cells (RBC). Thirty-four patients with mixed chimerism and ≥12 months of follow-up were included. A selective advantage of donor RBC and their progenitors/precursors led to full chimerism in mature RBC (despite partial engraftment of other lineages), and resulted in the clinical control of the disease. Six patients with donor chimerism 10 g/dL) and three with AS donors (hemoglobin

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Gene therapy for SCID and CID usually leads to a more efficient T-cell reconstitution as compared to HSCT from a mismatched related donor. However, as observed in ADA, SCID X1 and WAS gene therapy trials, T cell recovery is sometimes slow and associated with morbidity and mortality due to viral infections. In order to shorten post-transplant immunodeficiency and to reduce the frequency of these complications, one possible strategy is to transplant gene-corrected T-cell committed precursors. We have recently set up a protocol for transplanting in vitro-committed T-cell precursors able to seed the thymus and generate a wave of mature and polyclonal T-cells significantly faster than is usually observed. This protocol is based on a 3 to 7 day-culture of CD34+ cells in the presence of immobilized Notch ligand Delta-like-4 (DL-4), an adhesion molecule and a combination of cytokines; this culture system enables the in vitro generation of large amounts of T-cell precursor cells that (i) display the phenotypic and molecular signatures (i.e. gene expression pattern and TCR rearrangement pattern) of early thymic progenitor cells and (ii) have a high T-lymphopoietic potential in vitro and in vivo when transferred into NOD/SCPD/γc knock-out (NSG) mice (Reimann C, Stem Cells, 2012). The goal of the present project was to combine exposure to DL-4 and gene therapy approaches (DL-4/GT). For this purpose, gene correction of CD34+ cells from SCID-X1 patients was performed on DL-4 coated plates and the ex vivo transduced cells were either seeded on OP9/DL-1 culture or injected into NSG mice to determine their in vitro and in vivo capacity to generate mature and diverse T cells. In the DL-4/GT condition, the frequency and number of CD7+CD5+commited T-cell precursors was increased as compared to GT condition alone. Experiments to assess if the DL-4/GT condition accelerate T cell differentiation of gene corrected cells are currently ongoing. Altogether, our results suggest that a short exposure to the Notch ligand DL-4 might be an useful tool to generate large amounts of corrected T-cell precursors.

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.

Abstract 4350 Introduction: Transplantation of T cell depleted (TCD) HSC transplantation has been associated with:1) an increased risk of infectious complications due to a very late immune reconstitution, 2) a non negligible risk of Graft Versus Host Disease (GVHD) requiring immunosuppressive therapy, and 3) an increased risk of graft rejection. It has been demonstrated that GVHD in murine models is mostly mediated by naïve T cells. Memory T cells have a reduced capacity to induce GVHD while preserving the anti-infectious capacity (Anderson BE et al., 2003). Removing CD45RA cells from donor lymphocytes could reduce infectious complications without induction of GVHD. This procedure was evaluated in two patients presenting multiple infections and treated with mismatch HSC transplantation. Methods: Post transplant immune reconstitution has been compared between two groups. Group 1: 7 patients (1 ostepetrosis, 1 Fanconi anemia and 5 Severe Combined Immuno Deficiency) transplanted with TCD HSC (age: 3 months-11 years, sex ratio F/M: 4/3). Group 2: 2 patients (1 ORAI1 deficiency and 1 MHC class II deficiency) transplanted with TCD HSC and CD45RA depleted cells of the CD34 negative fraction (age: 8 and 23 months, 1 female and 1 male). All patients had myeloablative conditioning regimen. CD34+ cell selection and CD45RA cell depletion procedures were performed using the Clini Macs system (Miltenyi Biotec). Group 1 received a median of 15.3 × 106CD34+ cells/kg with less than 5000 T lymphocytes/kg. Group 2 received respectively 8.8 and 12.3×106 CD34+ cells/kg with less than 5000 T lymphocytes/kg in HSC transplant and 0.9 and 9.2×106/kg CD45RO+ T cells. The thresholds of 100 CD4+ T lymphocytes and 50 CD8+ T lymphocytes per microliter at three months post transplantation, shown to allow sufficient protection against infectious complications (Hakki et al. 2003), were used in our analysis. Results: No significant difference in GVHD incidence was shown between the two groups since only 2/7 patients presented moderate GVHD in group 1 and no GHVD in group 2. Engrafment for both kind of pathology in group 2 was also remarkable Immune reconstitution of CD4+ and CD8+ T lymphocytes was earlier in group 2 as at one month we detected CD4+ T lymphocytes (430 and 24/μl) and CD8+ T lymphocytes (520 and 40/μl) respectively for patient 1 and 2. Whereas in group 1 no T lymphocytes were detected before two months post transplant. The number of CD4+ and CD8+ T lymphocytes at three months post transplantation was considerably increased in group 2 (CD4+: 609 and 190/μl; CD8+: 2088 and 95/μl) versus group 1 (CD4+: 14/μl; CD8+: 0.4/μl). Patient 1 in group 2 presented CMV reactivation at day 10 post transplant (87650 copies/ml, threshold 500) and was able to clear this infection at day 37 concomitantly to an increased CMV tetramer positive cells percentage (Tetramers at day 37/tetramers at day 10: 433 fold increase). Conclusion: The two patients treated with T-cell depleted haematopoietic stem cells (HSC) transplantation and add back of CD45RA negative DLI showed good engraftment, earlier and enhanced immune reconstitution without GVHD. Moreover, one patient developed specific and efficient anti-CMV response probably due to an expansion of the injected CD45RO T cells. These interesting preliminary results should be confirmed by a clinical trial. Disclosures: No relevant conflicts of interest to declare.

Abstract 1906 Reactivation of latent viruses such as cytomegalovirus (CMV) and adenovirus (AdV) is responsible for infections which may be life-threatening in HSCT recipients. In the post-transplantation period, severity and frequency of these infections depend on (a) the degree of donor-recipient HLA incompatibility and (b) the intensity of immunosuppressive therapy used to prevent immunological complications. Antiviral drugs may be partially effective, often toxic and cannot always control those viral infections.T cell immunity plays a major role in the control of viral infections. It has been demonstrated that the transfer of donor T lymphocytes specifically directed against viral antigens is capable of preventing, controlling and clearing viral infection (Feuchtinger T et al., 2004 and 2010). The present project aimed the evaluation of specific, cell-based immunity against CMV and AdV by injection of IFN-g-positive CD4+and CD8+ donor T lymphocytes isolated ex vivo after stimulation with viral peptides. Methods: Our protocol was designed for pediatric or adult patients treated by allogeneic HSCT and matching the following inclusion criteria: (1) biological and/or clinical symptoms of CMV and/or AdV infection 2) no response or contraindication to conventional antiviral treatment and (3) no or low grade pre-existing aGvHD at inclusion (≤ grade II) controlled by corticoids ( Donor IFN-g-positive T lymphocytes are isolated with the CliniMACS Cytokine Capture System (Miltenyi Biotech) after incubation with viral peptide pools. Primary evaluation criterion is the efficacy of the treatment on CMV viral load 21 days after the first injection. In the event of a negative or partial response and the absence of aGvHD, a second injection may be scheduled. Secondary evaluation criteria are (1) the occurrence of de novo aGvHD or aggravation of existing aGvHD, (2) the evolution of clinical symptoms potentially related to the infection, (3) the demonstration of biological in vivo expansion of injected T lymphocytes (as evidenced by the IFN-g secretion capacity and specific tetramer assays) and (4) for AdV infection, evaluation of efficacy (viral load, in vivo expansion of transfused lymphocytes, clinical symptoms) and the safety (occurrence of aGvHD) of this immunotherapy. Results: From September 2010 to July 2012, 9 patients were included: 3 male adults (46–54 years, 1 CLL, 1 CML and 1 AA, 2 geno- and 1 pheno-identical transplantation) and 6 children (age: 7–25 months, sex ratio F/M: 4/2, 4 FLH, 1 SCID and 1AA, 4 haplo, 1 geno- and 1 pheno-id transplantation). 4/9 patients were treated for CMV, 3/9 for AdV and 2/9 for CMV and AdV reactivation. 5/9 patients received 2 cytotoxic T lymphocytes (CTL) injections. Mean number of CD3 IFN-g positive cells injected was 4206/kg (1167–6000/kg) with 55% and 69% of CD4 and CD8 anti CMV-T cells and 56% and 61% of CD4 and CD8 anti AdV T cells respectively. Mean delay of first immunotherapy was 109 days (28–270) after transplantation. 2/9 patients were not evaluable due to early death ( Conclusion: The CliniMACS Cytokine Capture System allows the isolation of virus-specific T cells in a brief delay (24 hours) with a satisfactory enrichment of both CD4 and CD8 T cells. First results show efficacy of virus-specific T cells injection on viral load without signs of aGvHD in 5/6 evaluable patients. More patients need to be included in this trial in order to confirm these encouraging results. Disclosures: Cambouris: Miltenyi Biotec: Employment.