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Fanconi anemia (FA), a genetic DNA repair disorder characterized by marrow failure and cancer susceptibility. In FA mice, metformin improves blood counts and delays tumor development. We conducted a single institution study of metformin in nondiabetic patients with FA to determine feasibility and tolerability of metformin treatment and to assess for improvement in blood counts. Fourteen of 15 patients with at least 1 cytopenia (hemoglobin < 10 g/dL; platelet count < 100 000 cells/µL; or an absolute neutrophil count < 1000 cells/µL) were eligible to receive metformin for 6 months. Median patient age was 9.4 years (range 6.0-26.5 ). Thirteen of 14 subjects (93%) tolerated maximal dosing for age; 1 subject had dose reduction for grade 2 gastrointestinal symptoms. No subjects developed hypoglycemia or metabolic acidosis. No subjects had dose interruptions caused by toxicity, and no grade 3 or higher adverse events attributed to metformin were observed. Hematologic response based on modified Myelodysplastic Syndrome International Working Group criteria was observed in 4 of 13 evaluable patients (30.8%; 90% confidence interval, 11.3-57.3). Median time to response was 84.5 days (range 71-128 days). Responses were noted in neutrophils (n = 3), platelets (n = 1), and red blood cells (n = 1). No subjects met criteria for disease progression or relapse during treatment. Correlative studies explored potential mechanisms of metformin activity in FA. Plasma proteomics showed reduction in inflammatory pathways with metformin. Metformin is safe and tolerable in nondiabetic patients with FA and may provide therapeutic benefit. This trial was registered at as #NCT03398824.

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells1,2. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34+ hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4–1.8 copies per neutrophil) and the persistence of 16–46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients. Initial results from phase I/II lentiviral gene therapy trials provide early evidence supporting its safety and efficacy in treating patients with X-linked chronic granulomatous disease.

Novel therapies for sickle cell disease (SCD) based on genetically engineered autologous hematopoietic stem and progenitor cells (HSPCs) are critically dependent on a safe and effective strategy for cell procurement. We sought to assess the safety and efficacy of plerixafor when used in transfused patients with SCD for HSC mobilization. Six adult patients with SCD were recruited to receive a single dose of plerixafor, tested at lower than standard (180 µg/kg) and standard (240 µg/kg) doses, followed by CD34+ cell monitoring in peripheral blood and apheresis collection. The procedures were safe and well-tolerated. Mobilization was successful, with higher peripheral CD34+ cell counts in the standard vs the low-dose group. Among our 6 donors, we improved apheresis cell collection results by using a deep collection interface and starting apheresis within 4 hours after plerixafor administration. In the subjects who received a single standard dose of plerixafor and followed the optimized collection protocol, yields of up to 24.5 × 106 CD34+ cells/kg were achieved. Interestingly, the collected CD34+ cells were enriched in immunophenotypically defined long-term HSCs and early progenitors. Thus, we demonstrate that plerixafor can be employed safely in patients with SCD to obtain sufficient HSCs for potential use in gene therapy.

Targeting the BCL11A erythroid enhancer by gene editing is a promising approach to fetal hemoglobin induction for beta-hemoglobinopathies. HbF levels vary widely among individuals, suggesting potential heterogeneity in HbF responses after therapeutic intervention. We hypothesize that maximizing both gene edit frequency and HbF induction potential could promote consistently favorable clinical outcomes. Here we compared CRISPR-Cas9 endonuclease editing of the BCL11A +58 enhancer with alternative gene modification approaches, including +55 erythroid enhancer editing alone or in combination with the +58 enhancer, as well as editing targeting the HBG1/2 promoter -115 BCL11A binding site and transduction by an shRNA knocking down the BCL11A transcript in erythroid precursors. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in the most potent HbF induction (52.4%±6.3%) of tested approaches (BCL11A +58 editing alone, 29.1%±3.9%; BCL11A +55 editing alone, 34.8±5.1%; HBG1/2 promoter editing, 34.1% ±5.4%; shmiR-BCL11A, 32.2%±4.4%; mock, 7.6%±3.4%). Based on assays in bulk and single cell derived erythroid cultures and xenografted immunodeficient mice, we found that disruption of core half E-box/GATA motifs at both the +58 and +55 enhancers was associated with greatest HbF induction, whether by small indels, interstitial 3.1 kb deletion, or 3.1 kb inversion. Rare gene edited clones with alleles that only partially disrupted these motifs were associated with intermediate HbF induction phenotypes. Combined editing of BCL11A +58 and +55 enhancers was compatible with HSC self-renewal in primary and secondary xenotransplant, with intact lymphoid, myeloid and erythroid repopulation. We conducted gene-edited cell product manufacturing process development and developed conditions using a MaxCyte electroporation instrument achieving mean 97.3±1.8% gene edits and 88.9%±6.4% viability 24 hours after electroporation in 3 engineering runs at clinical scale. We obtained similar results at small-scale with plerixafor-mobilized HSPCs from sickle cell disease (SCD) donors or G-CSF mobilized PBMCs from transfusion-dependent beta-thalassemia (TDT) donors, including 94.2%±4.4%, 99.5%±0.3% and 91.8%±6.3% of gene edits in engrafting cells from NBSGW 16 week mouse bone marrow of healthy, SCD and TDT donors respectively. Off-target analyses by pooled amplicon sequencing of 601 candidate off-target sites for the +58 and +55 targeting sgRNAs, nominated by a range of computational (CRISPRme) and experimental (GUIDE-seq and ONE-seq) methods, did not identify reference genome off-target edits at a sensitivity of 0.1% allele frequency. We evaluated +58/+55 enhancer combined targeting in nonhuman primates by performing ribonucleoprotein (RNP) electroporation in rhesus macaque mobilized peripheral blood CD34+ HSPCs with autologous re-infusion following busulfan myeloablation. We observed highly efficient gene edit frequency (85.2%, 88.8% and 84.9%) and durable HbF induction (26.4%, 57.5%, and 45.9% F-cells and 12.7%, 41.9%, and 28% gamma-globin) in the peripheral blood in 3 animals at most recent recorded time point post infusion (127, 78, and 54 weeks respectively). Single colony analyses and bulk ddPCR and unidirectional sequencing demonstrated that the long-term engrafting cells displayed a similar distribution of indels, 3.1 kb deletions, and 3.1 kb inversions as the input cell products. Erythroid stress due to hydroxyurea treatment, with or without phlebotomy, was associated with substantially augmented HbF responses (to 75.9%, 88.2%, and 57.8% F-cells and 47.9%, 68%, and 35.7% gamma-globin). No hematologic or other toxicities attributable to gene editing were observed. Together these results suggest that combined BCL11A +58 and +55 erythroid enhancer editing produces highly efficient on-target allelic disruption, erythroid-specific BCL11A downregulation, heightened HbF induction capacity compared to alternative approaches, preserved long-term multilineage engraftment potential by both human xenotransplant and rhesus autotransplant assays, and absence of evident genotoxicity, under clinically relevant SpCas9 RNP electroporation conditions. Disclosures No relevant conflicts of interest to declare.

CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to predict and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and β-thalassemia (Frangoul et al. NEJM 2021). We find that the top predicted off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF) = 4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels (~9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. This report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We expect that variant-aware off-target assessment will be required for therapeutic genome editing efforts going forward, including both ongoing and future clinical trials, and we provide a powerful approach for comprehensive off-target prediction. CRISPRme is available at crisprme.di.univr.it. Disclosures No relevant conflicts of interest to declare.

Background: CD37 is a tetraspanin molecule expressed in B-cell and some T-cell lymphomas. We designed a Chimeric Antigen Receptor (CAR) targeting CD37 and with 4-1BB and CD3z intracellular signaling domains (CART37). Preclinical studies indicated comparable anti-tumor activity to CD19-directed CAR-T cells against B-cell lymphomas, promising activity against CD37 + T-cell lymphomas, and no evidence of off-tumor activity (PMID: 30089630). The lentiviral vector used in the clinical study includes a truncated EGFR reporter gene. NCT04136275 is a Phase 1, single-site, open-label, dose-escalation trial enrolling subjects with lymphoma who have received ≥ 2 prior regimens, and whose tumor cells express CD37. Methods: We developed a clinical assay to assess CD37 expression on patient tumor samples using flow cytometry and IHC. Peripheral blood mononuclear cells are collected via leukapheresis and manufactured using the CliniMACS Prodigy®. Following release testing, fresh or cryopreserved cell product is infused. Subjects undergo lymphodepletion with fludarabine and cyclophosphamide, then receive CART37 as a single infusion. Planned starting dose was 100 x 10 6 CAR + T cells, with options to dose-escalate to 300 x 10 6 CAR + T cells or dose de-escalate to 30 x 10 6 CAR + T cells in the event of dose-limiting toxicities (DLT) using a 3+3 design. The primary outcome measure is incidence of adverse events (AEs), including DLTs. Additional outcome measures are clinical response, progression-free and overall survival; correlative studies focus on quantification and persistence of CAR + cells in blood, residual tumor, and cytokine modulation. Results: As of July 13, 2021, 4 subjects (ages 35-70 years) have received CART37. Subjects had a median of 5.5 prior lines of systemic therapy. Two subjects had primary refractory double-hit high-grade B cell lymphoma (HGBCL) that had relapsed after commercial CD19 CAR-T; one subject had cutaneous T cell lymphoma relapsed after extracorporeal photopheresis, alemtuzumab, total skin electron beam radiation, allogeneic hematopoietic stem cell transplant (HSCT), brentuximab, and donor lymphocyte infusion, and one subject had Hodgkin's lymphoma refractory to six prior regimens, including chemotherapy, brentuximab vedotin, nivolumab and everolimus. All subjects were infused in the DL1 cohort, but one subject (with cutaneous T cell lymphoma) received only 19 x 10 6 CAR + due to limited ex vivo expansion. Three subjects developed low-grade CRS and ICANS, and one subject developed refractory Grade 3 CRS and Grade 3 ICANS which resolved with medical management. One patient with HGBCL developed CD19 neg and CD37 neg progressive disease prior to the day 28 evaluation. The three other subjects demonstrated deep responses (2 CR, 1 PR that converted to CR) as best response. Two subjects are alive 208 and 272 days from CAR37 infusion. All subjects had detectable expansion of CART37 by flow cytometry and molecular assays. Two subjects (who received ≥ 100 x 10 6 CART37 had robust expansion and developed prolonged pancytopenia with marrow aplasia; cetuximab infusion decreased detection of truncated EGFR on circulating T cells but had no impact on vector copy number. Both subjects underwent allogeneic HSCT after conditioning with flu/cy/TBI(400) and post-transplant cyclophosphamide (PTCy) based GVHD prevention and successfully engrafted, and had no detectable CART37 after HSCT. Conclusions: In this initial cohort, CART37 infusion resulted in CRS or ICANs as is common for CAR T cell products. Bone marrow aplasia was unexpected and was observed in two subjects who received at least 100 x 10 6 CART37; this was successfully rescued with allogeneic HSCT. Three of four subjects had deep clinical responses in heavily pretreated, refractory disease of diverse lymphoma subtypes. The protocol is open to enrollment with dose de-escalation to 30 x10 6 CART37 and has been amended to require identification of a potential donor prior to treatment in the case that rescue allogeneic HSCT is needed. CART37 has a potential role in enabling allogeneic transplantation in patients with relapsed or refractory hematologic malignancies. Disclosures Frigault: Takeda: Consultancy; Editas: Consultancy; BMS: Consultancy; Novartis: Consultancy, Research Funding; Iovance: Consultancy; Arcellx: Consultancy; Kite: Consultancy, Research Funding. Chen: Gamida: Consultancy; Incyte: Consultancy. Wehrli: CSL Behring: Patents & Royalties; Nestle: Current equity holder in publicly-traded company; Novartis: Current equity holder in publicly-traded company. Spitzer: Bluebird Bio: Consultancy; Jazz Pharmaceuticals: Consultancy; Qihan Bio: Consultancy; Syneos Health: Consultancy. Preffer: Cytek Biosciences: Other: Unspecified Relationship. Shaw: Orchard Therapeutics, Ltd: Current equity holder in publicly-traded company. Nikiforow: Kite/Gilead: Other: Ad hoc advisory boards; Novartis: Other: Ad hoc advisory boards; Iovance: Other: Ad hoc advisory boards; GlaxoSmithKline (GSK): Other: Ad hoc advisory boards. Ritz: Amgen: Research Funding; Equillium: Research Funding; Kite/Gilead: Research Funding; Avrobio: Membership on an entity's Board of Directors or advisory committees; Akron: Consultancy; Biotech: Consultancy; Blackstone Life Sciences Advisor: Consultancy; Clade Therapeutics, Garuda Therapeutics: Consultancy; Immunitas Therapeutic: Consultancy; LifeVault Bio: Consultancy; Novartis: Consultancy; Rheos Medicines: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy. Maus: Novartis: Consultancy; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Kite Pharma: Consultancy, Research Funding; GSK: Consultancy; Intellia: Consultancy; In8bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; Cabaletta Bio (SAB): Consultancy; BMS: Consultancy; Bayer: Consultancy; Atara: Consultancy; AstraZeneca: Consultancy; Astellas: Consultancy; Arcellx: Consultancy; Agenus: Consultancy; Adaptimmune: Consultancy; WindMIL: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company.

Fanconi anemia (FA), a genetic disorder affecting DNA repair, is characterized by bone marrow failure and cancer susceptibility. In FA mouse models, metformin (N,N-dimethylguanide) a biguanide metabolic agent, improves blood counts and delays tumor development. Given these findings, we conducted a single institution pilot study of metformin in non-diabetic patients with FA to assess feasibility, tolerability, and impact of metformin on hematologic response as defined by modified MDS International Working Group (IWG) criteria (Table 1). Fourteen of 15 patients with at least 1 cytopenia (hemoglobin Figure 1 Figure 1. Disclosures Pollard: Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees. Furutani: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Esrick: bluebird bio: Consultancy. Nakano: Novartis: Consultancy. Thompson: Celgene: Consultancy, Research Funding; bluebird bio: Consultancy, Research Funding; Biomarin: Research Funding; Vertex: Research Funding; Graphite Bio: Research Funding; Baxalta: Research Funding; CRISPR Therapeutics: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Consultancy; Beam Therapeutics: Consultancy; Global Blood Therapeutics: Current equity holder in publicly-traded company. OffLabel Disclosure: metformin- use in Fanconi anemia in an effort to improve hematopoiesis

Recent advances in clonal stem cell tracking strategies have enabled interrogation of unperturbed human hematopoiesis. Whole genome sequencing (WGS) can be used to map the clonal dynamics of hematopoietic stem and progenitor cells (HSPCs) by employing spontaneous somatic mutations as unique clonal tags (Lee-Six et al., Nature, 2018). These tags allow for retrospective analysis of individual stem cell clones and the construction of phylogenetic trees mapping out stem cell relatedness, with mutations being acquired in a near-linear fashion over the course of an individual's life. The unprecedented level of information obtained in these studies is particularly well-suited to understanding genomic changes in gene therapy trials aimed at curing diseases such as sickle cell disease (SCD). In addition to mapping relatedness between stem cells, sequencing data can be used to better define mutational signatures for HSPC clones that have been successfully gene-modified as well as those that lack an integrated copy of the therapeutic vector. Given this method's ability to identify low frequency mutations in individual HSPC clones, mutations with extremely low variant allele frequencies can be detected much more readily than through traditional bulk sequencing approaches, something that is particularly relevant given recent safety concerns in some SCD gene therapy trials. In this study, we have mapped the clonal dynamics of HSPCs obtained from pre- and post-gene therapy samples from 4 SCD patients who have undergone autologous gene therapy performed using a BCL11A shmiR lentivirus vector (NCT 03282656, 12-36 months follow-up). HSPCs from mobilized peripheral blood (pre-gene therapy), bone marrow aspirates (both pre- and post-gene therapy) or unmobilized peripheral blood (post-gene therapy) were expanded as single clones and 1508 individual colonies were then sequenced using WGS to an average sequencing depth of 12.3x. Initial results indicate that the mean mutation burden per cell in a pre-gene therapy sample is elevated for some patients compared to what would be expected based on patient age in similar studies. In pre-gene therapy samples, the structure of the phylogenetic trees appeared to be highly polyclonal, indicating that there were no significant clonal expansion events prior to gene therapy. In one patient where we undertook extensive profiling, approximately 15-20 excess mutations per HSPC were observed across the entire genome 24 months after transplantation, presumably acquired as a consequence of gene therapy and/or reconstitution post-transplantation, which is equivalent to approximately one year of normal ageing without a transplantation intervention. However, no clonal expansions or driver mutations were identified at this 24 month follow-up timepoint, suggesting that no strong selective advantage or pre-leukemic events were present prior to or following the gene therapy protocol. Extending this approach to a wider range and larger number of patients will allow for comprehensive mapping of the genomic landscape and clonal evolution of stem cells in sickle cell patients and will also set the stage for improved assessment of safety and potential leukemia-initiating events in the context of gene therapy. Disclosures Esrick: bluebird bio: Consultancy. Williams: bluebird bio: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Analysis Advisory Board, Patents & Royalties: BCH licensed certain IP relevant to hemoglobinopathies to bluebird bio. The current license includes the potential for future royalty/milestone income. Bluebird has indicated they will not pursue this as a clinical program and BCH is negotiating return of, Research Funding; BioMarin: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Advisory Board; Beam Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Emerging Therapy Solutions: Membership on an entity's Board of Directors or advisory committees, Other: Chief Scientific Chair; Geneception: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Alerion Biosciences: Other: Co-founder (now licensed to Avro Bio, potential for future milestones/royalties); Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Steering Committee, Novartis ETB115E2201 (eltrombopag in aplastic anemia). Advisory fees donated to NAPAAC.; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Membership on a safety advisory board (SAB): SAB position ended 05/20/2021. Co-founder , Patents & Royalties: Potential for future royalty/milestone income, X-SCID. Provided GMP vector for clinical trial, Research Funding. Campbell: Mu Genomics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Kent: STRM.bio: Research Funding.

NH and EE equally contributed. ADW and PB co-signed. The expression of fetal hemoglobin (HbF) is one of the main targets of sickle cell disease treatment, as it inhibits the polymerization of hemoglobin S. The hypothesis of an inhibitory threshold of HbF per red blood cell (RBC) has been suggested, 1 although not well defined, as the overall percentage of HbF does not reflect the heterogeneous distribution of HbF per cell. Likewise, the qualitative analysis of RBCs containing HbF, called F cells, is neither reproducible nor clinically interpretable, due to low expression. 2 We have developed a technique for measuring the amount of HbF per cell, to determine thresholds of HbF expression per RBC correlated with clinical and biological effects. 2 Among genes controlling its expression, BCL11A has a major repressive effect on the expression of gamma globin/HbF during the fetal to adult hemoglobin switch. Post-transcriptional silencing of BCL11A, using lentivirus expression of a shRNA embedded in a microRNA architecture (shmiR) to re-activate γ-globin expression, is safe and demonstrates high levels of %HbF in a pilot clinical study (NCT 03282656). 3 Here, we show the quantitative measurement of HbF per RBC and reticulocyte. Methods: During patient follow-up, HbF quantification per single cell RBC was performed using a fluorescent HbF antibody. 2 Addition of an anti-CD71 fluorescent antibody allowed selection of reticulocyte sub-populations for determining their HbF content. Fold-increase in percentage of RBC versus percentage of reticulocyte were calculated. Kinetics of HbF/RBC and HbF/Reticulocyte were modeled using mixed effects polynomial linear regression to account for the correlation between repeated data over time. Results: With a median follow-up of 15 months [12-20] after gene transfer, figure 1 shows the mathematical modeling of single-RBC HbF measurement representing RBC percentage containing at least 2, 4, 6, 8 and 10 pg of HbF. Percentage of RBC above each threshold was higher compared to 14 hydroxyurea treated patients for 6 months. Figure 2 shows fold increase between reticulocytes and RBCs with same thresholds of HbF/cell. For low thresholds, RBCs were found in same percentage as reticulocytes whereas RBCs containing increasing levels of HbF were found in higher percentage than reticulocytes, until 6pg/cell showing a clear selective advantage for red cells with a threshold ≥ 6pg/cell of HbF. Figure 3 shows different kinetics of HbF increase according to two different transduction strategies with 2 enhancers in patients 2-4 compared to one enhancer in patients 6-8. Conclusion: BCL11A down-regulation in six clinical trial subjects was associated with an in vivo selection process RBCs with ≥ 6pg HbF per cell attained with different engraftment kinetics, depending on transduction processes, and ultimately stable high level and broadly distributed HbF. 1 Steinberg MH, Chui DH, Dover GJ, Sebastiani P, Alsultan A. Fetal hemoglobin in sickle cell anemia: a glass half full? Blood. 2014 Jan 23;123(4):481-5. 2 Hebert N, Rakotoson MG, Bodivit G, et al. Individual red blood cell fetal hemoglobin quantification allows to determine protective thresholds in sickle cell disease. Am. J. Hematol. 3 Esrick EB, Lehmann LE, Biffi A, et al. Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease. N. Engl. J. Med. 2021;384(3):205-215. Figure 1 Figure 1. Disclosures Esrick: bluebird bio: Consultancy. Audureau: GBT: Honoraria. Higgins: Sebia, Inc.: Honoraria; Danaher Diagnostics: Consultancy. Williams: BioMarin: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Advisory Board; Geneception: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Emerging Therapy Solutions: Membership on an entity's Board of Directors or advisory committees, Other: Chief Scientific Chair; Beam Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Alerion Biosciences: Other: Co-founder (now licensed to Avro Bio, potential for future milestones/royalties); Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Steering Committee, Novartis ETB115E2201 (eltrombopag in aplastic anemia). Advisory fees donated to NAPAAC.; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Membership on a safety advisory board (SAB): SAB position ended 05/20/2021. Co-founder , Patents & Royalties: Potential for future royalty/milestone income, X-SCID. Provided GMP vector for clinical trial, Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Analysis Advisory Board, Patents & Royalties: BCH licensed certain IP relevant to hemoglobinopathies to bluebird bio. The current license includes the potential for future royalty/milestone income. Bluebird has indicated they will not pursue this as a clinical program and BCH is negotiating return of, Research Funding. Bartolucci: AGIOS: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Steering committee, Research Funding; Jazz Pharma: Other: Lecture fees; Emmaus: Consultancy; Addmedica: Consultancy, Other: Lecture fees, Research Funding; INNOVHEM: Other: Co-founder; Hemanext: Consultancy; GBT: Consultancy; Bluebird: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy; Fabre Foundation: Research Funding.

Introduction Fetal hemoglobin (HbF) induction is a successful strategy for treating sickle cell disease. Higher levels of total HbF% are associated with better clinical outcomes, and a more uniform distribution of HbF across individual red blood cells (RBCs) is predicted to lead to better outcomes by inhibiting hemoglobin (Hb) polymerization in a higher fraction of individual RBCs. 1 Post-transcriptional silencing of BCL11A using lentivirus expression of a shRNA embedded in a microRNA architecture (shmiR) to re-activate γ-globin expression has been safe and demonstrated high levels of total HbF expression broadly distributed in red cells in a pilot clinical study (NCT 03282656). 2 We assessed the effects of BCL11A down-regulation on Hb polymer formation at the single-RBC level in 7 subjects in this trial (BCL) using novel assays of single-RBC Hb polymer content levels and single-RBC HbF. Methods Hb polymer was detected in individual RBCs using a previously published microfluidic system. 3 The system detects Hb polymer based on RBC morphology and on the principle that Hb polymer lowers RBC oxygen saturation in proportion to polymer concentration. 4,5 Single-RBC HbF mass was measured by flow cytometry using a previously published protocol. 6 These two measurements of distributions of single-RBC HbF mass and single-RBC Hb polymer were then integrated at the cell-population level to estimate the threshold of single-RBC HbF needed to inhibit formation of a detectable level of polymer as function of oxygen tension. Blood samples from BCL subjects (HbF% range 23 - 44, after stabilizing post-therapy) were compared to 14 hydroxyurea (HU) responsive patients (mean HbF% = 17, range 6 - 32), including 3 highly-responsive patients (HbF% = 25, 27, 31) as well as 4 individuals with sickle cell trait (AS). Results At 3.7% oxygen tension typical of the human microcirculation 7 and some regions of the central nervous system, 7 the typical BCL subject had detectable levels of Hb polymer in 52% (median) of RBCs vs. 66% for HU (p = 0.02) and 6% for AS (Figure 1A). At lower oxygen tensions of 1.7% typical of the bone marrow 7 or kidney medulla 7 BCL subjects had a polymerized RBC fraction of 68%, compared to 79% for HU (p Conclusions BCL11A down-regulation in seven clinical trial subjects is associated with favorable distributions of single-RBC polymer at multiple physiologic oxygen tensions compared to HU. Future study is needed to determine whether these favorable Hb polymer distributions are simply a result of higher total HbF or also due to lower single-RBC HbF thresholds for polymer inhibition. References 1. Steinberg MH, Chui DHK, Dover GJ, Sebastiani P, Alsultan A. Fetal hemoglobin in sickle cell anemia: a glass half full? Blood. 2014;123(4):481-485. 2. Esrick EB, Lehmann LE, Biffi A, et al. Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease. N. Engl. J. Med. 2021;384(3):205-215. 3. Di Caprio G, Schonbrun E, Gonçalves BP, et al. High-throughput assessment of hemoglobin polymer in single red blood cells from sickle cell patients under controlled oxygen tension. Proc. Natl. Acad. Sci. U. S. A. 2019;116(50):25236-25242. 4. Benesch RE, Edalji R, Kwong S, Benesch R. Oxygen affinity as an index of hemoglobin S polymerization: A new micromethod. Anal. Biochem. 1978;89(1):162-173. 5. Fabry ME, Desrosiers L, Suzuka SM. Direct intracellular measurement of deoxygenated hemoglobin S solubility. Blood. 2001;98(3):883-884. 6. Hebert N, Rakotoson MG, Bodivit G, et al. Individual red blood cell fetal hemoglobin quantification allows to determine protective thresholds in sickle cell disease. Am. J. Hematol. 2020;95(11):1235-1245. 7. Keeley TP, Mann GE. Defining physiological normoxia for improved translation of cell physiology to animal models and humans. Physiol. Rev. 2019;99(1):161-234. Figure 1 Figure 1. Disclosures Esrick: bluebird bio: Consultancy. Bartolucci: INNOVHEM: Other: Co-founder; F. Hoffmann-La Roche Ltd: Consultancy; Bluebird: Consultancy, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Steering committee, Research Funding; GBT: Consultancy; Emmaus: Consultancy; Hemanext: Consultancy; AGIOS: Consultancy; Jazz Pharma: Other: Lecture fees; Fabre Foundation: Research Funding; Addmedica: Consultancy, Other: Lecture fees, Research Funding. Williams: Emerging Therapy Solutions: Membership on an entity's Board of Directors or advisory committees, Other: Chief Scientific Chair; Geneception: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; BioMarin: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Advisory Board; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Steering Committee, Novartis ETB115E2201 (eltrombopag in aplastic anemia). Advisory fees donated to NAPAAC.; Alerion Biosciences: Other: Co-founder (now licensed to Avro Bio, potential for future milestones/royalties); Beam Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Membership on a safety advisory board (SAB): SAB position ended 05/20/2021. Co-founder , Patents & Royalties: Potential for future royalty/milestone income, X-SCID. Provided GMP vector for clinical trial, Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Analysis Advisory Board, Patents & Royalties: BCH licensed certain IP relevant to hemoglobinopathies to bluebird bio. The current license includes the potential for future royalty/milestone income. Bluebird has indicated they will not pursue this as a clinical program and BCH is negotiating return of, Research Funding. Higgins: Danaher Diagnostics: Consultancy; Sebia, Inc.: Honoraria.

Cell therapy products have become more sophisticated both in type and manufacturing complexity (1-3). As clinicians' and researchers' understanding of disease and cell mechanisms of action continue to grow, cell therapy products are being increasingly used or considered for use in clinical trials (4-12). The safety and efficacy of cell therapy products have been reported within individual clinical trials and through review articles (13-19). These publications are often limited to an individual cell type and also differ by how the data are being collected and reported. The Health Resources and Services Administration (HRSA) has provided funds to study the use of cell therapy in the United States with a goal of building a database similar to that established and maintained by the bone marrow transplant community for years. The European Union has pursued a similar goal. Despite these efforts, there is limited published information that describes cell therapy product administration across both cell types and indications. To this end, The National Institutes of Health, National Heart, Lung and Blood Institute (NHLBI) Production Assistance for Cellular Therapies (PACT) program has collected data over the past 10 years on the administration of cell therapy products manufactured within the program. The PACT program was formed in 2003 through funding from the NHLBI. The program provides clinical cell therapy product manufacturing support to investigators wishing to transition a novel cell therapy from the developmental stage to clinical applications within the purview of the NHLBI. PACT is not responsible for directly monitoring the clinical trials of investigators receiving PACT-manufactured products. PACT has required collection of standardized information on product manufacturing, transport, receipt, administration, and adverse reactions with product administration. The purpose of this data collection is 1) to monitor administration of PACT cell therapy products, and 2) to build a product administration database to identify trends or safety concerns that may be associated with cell therapy product administrations. All clinical trials where PACT has provided manufacturing support were approved by local ethics committees.

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

Severe congenital neutropenia (SCN) is a life-threatening disorder of insufficient granulocytes. Lifelong granulocyte colony-stimulating factor (G-CSF) injections are the mainstay of treatment, yet there remains a high risk of myelodysplastic syndrome and acute myeloid leukemia. The most common etiology of SCN is germline ELANE mutation. These dominantly acting mutations preserve expression but alter the structure of the neutrophil elastase protein product resulting in altered protein folding and/or trafficking with excess cell death at the promyelocyte/myelocyte stage of maturation. Recent advances in gene editing technologies have enabled targeted genetic modification of hematopoietic stem cells (HSCs); nonetheless genetic repair of specific disease-associated mutations remains challenging. We hypothesized that introduction of premature termination codons (PTCs) by nuclease-mediated frameshift mutations within early exons of ELANE could constitute a universal, highly efficient, simple therapeutic approach for ELANE-associated SCN. We predicted that the PTCs would trigger nonsense mediated decay (NMD) of the mutant transcript resulting in its loss of expression and thus bypassing neutrophil precursor cell death and consequent neutropenia. The mild phenotype observed in the Papillon-Lefevre syndrome, characterized by combined serine protease deficiency, suggests that isolated neutrophil elastase deficiency would not result in clinically significant immunodeficiency. We delivered 3xNLS-SpCas9 and ELANE targeting sgRNA as ribonucleoprotein (RNP) complexes to primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) and conducted in vitro neutrophil maturation culture. Introducing indels at exon 2 of ELANE efficiently triggered NMD. Edited cells were fully competent for neutrophil maturation similar to neutral locus targeted control cells. Using three human donors, we found that ELANE exon 2 edited HSPCs produced similar human bone marrow (BM) chimerism as unedited cells in NBSGW recipient mice 16 weeks following infusion. We found similar lymphoid, erythroid, and myeloid engraftment including similar fraction of human neutrophils (13.4% of total human BM cells in unedited and 13.7% in ELANE exon 2 edited, despite 97.3% on-target indel frequency and 84.3% reduction in ELANE expression in the latter). Using CD34+ HSPCs from four ELANE mutant SCN patient donors, we demonstrated that exon 2 targeting RNPs achieve highly efficient editing exceeding 95% indel frequency, trigger ELANE transcript decay, and rescue promyelocyte stage maturation arrest. In contrast to these ameliorating early exon frameshifts, naturally occurring SCN-associated frameshifts affect late exons of ELANE, suggesting that these mutations might escape NMD. Indeed we found that targeting ELANE exon 5 in HSPCs resulted in robust indels (93.5%), preserving ELANE expression but resulting in cell death at the promyelocyte/myelocyte stages of development, recapitulating an SCN phenotype. To our surprise, we found that only -1 frameshifts and not -2 frameshifts induced by gene editing with NHEJ repair led to the SCN-like phenotype, although we noted that all 23 reported naturally occurring SCN-associated ELANE frameshift mutations result from -1 but not -2 bp frameshifts. Using xenograft of NBSGW recipients, we found that an RNP complex leading to efficient -1 frame indels in ELANE exon 5 produced profound neutrophil maturation block, with reduction from 13.4% neutrophils in controls to 0.5% neutrophils in ELANE exon 5 targeted recipients, with otherwise indistinguishable human monocyte, lymphoid, and erythroid reconstitution as compared to controls. This dramatic phenotype contrasts with mice engineered to express SCN-associated Elane mutations that do not exhibit neutropenia, indicating species differences in granulopoiesis. Together these results support the development of ELANE early exon targeting as a highly efficient universal therapy for ELANE mutant SCN, feasible with existing gene editing technology. Moreover, by late exon ELANE gene editing we have developed a robust new model of SCN using primary human HSPCs that recapitulates neutropenia in vivo following xenotransplant, refines the molecular genetics of mutant ELANE induced neutrophil maturation arrest, and offers opportunities to explore novel therapeutic approaches. Disclosures Newburger: TransCytos LLC: Consultancy; X4 Pharmaceuticals: Consultancy, Honoraria.

Background Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, infections, autoimmunity and lymphoma. Gene therapy (GT) using autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplant for patients who lack well matched donors, avoiding graft-versus-host-disease. An initial experience with gene therapy using a γ-retroviral vector showed correction of hematological defects in 9/10 patients, but was aggravated by development of leukemia in 7 of them. We report the outcomes of a phase I/II clinical trial in which 5 WAS patients underwent GT using a self-inactivating lentiviral (SIN-LV) vector expressing the human WAS cDNA under the control of a 1.6kB fragment of the human WAS promoter. Subjects and Methods Five patients with severe WAS (clinical score 3-5) were enrolled at a median age of 1.8 years (1.4 - 8 years) at a single pediatric tertiary care center. WAS protein (WASP) was absent or markedly decreased in 2 and 3 subjects, respectively. Purified CD34+ cells from mobilized peripheral blood (n = 4) or both mobilized peripheral blood and bone marrow (n = 1) were transduced ex-vivo with the SIN-LV vector and re-infused after conditioning with busulfan (target AUC of 70-80 mg*h/L) and fludarabine (120mg/m2). The median dose of CD34+ cells infused was 9.8 x 106 cells/kg (6.3 - 24.9 x 106 cells/kg) with a mean vector copy number (VCN) of 1.7 copies/cell in CD34+ cells (0.54 - 3.37). In addition to eczema, thrombocytopenia and WAS-related infections in all patients, two subjects also had autoimmunity pre-GT, manifested as skin vasculitis and autoimmune cytopenias. Results All 5 subjects were alive and well at median follow-up of 4.8 years (2.5 - 5.9 years). Multi-lineage vector gene marking was sustained over time. All subjects had improvement or resolution of eczema and none have had any intercurrent severe infectious events. WASP expression measured by flow cytometry in T cells was increased over baseline in all patients, but remained below normal levels and correlated with VCN and cell dose received. Proliferation of T cells in response to anti-CD3, which was initially defective in 4/5 patients, improved post-GT. Humoral immune deficiency was also ameliorated, as evidenced by independence from Ig replacement and vaccine responses in those tested. All subjects remained platelet transfusion-free and none have had severe bleeding events. Platelet levels increased to >50 x 103 cells/uL in two patients with a VCN ≥2 in transduced stem cells and myeloid VCN ~1 copy/cell in neutrophils; the other 3 subjects sustained platelet counts No severe GT-related adverse events have occurred to date. Replication-competent lentivirus was not detected. Analysis of integration site distributions in five subjects showed reconstitution to be highly polyclonal, with no clones expanded to >20% of the transgene-marked cell population. To date, there have been no malignancies reported, either related to GT or WAS itself. Conclusion In summary, our data confirm and extend the safety and efficacy of GT in correcting disease manifestations associated with WAS, as seen in other studies using SIN-LV. Higher VCN in the drug product and in transduced stem cells correlated with better reconstitution of platelets and myeloid function. In contrast to other groups, we found in our study that patients with poor lymphocyte reconstitution post-GT may be at risk of ongoing autoimmunity despite high-level gene marking. Disclosures London: ArQule, Inc: Consultancy; United Therapeutics: Consultancy. Despotovic:Novartis: Research Funding; Amgen: Research Funding; Dova: Honoraria. Forbes:Takeda: Consultancy. Galy:Genethon: Employment. Williams:Novartis: Membership on an entity's Board of Directors or advisory committees; bluebird bio: Other: License of certain IP relevant to hemoglobinopathies. Potential for future royalty/milestone income. Received payment in past through BCH institutional licensing agreement., Research Funding; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, potential for future royalty/milestone income, Research Funding; Alerion Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder. OffLabel Disclosure: CliniMACS technology for CD34+ cell selection

Currently, there is a growing need for culturing hematopoietic stem/progenitor cells (HSPCs) in vitro for various clinical applications including gene therapy. Compared with cord blood (CB) CD34+ HSPCs, it is more challenging to maintain or expand CD34+ peripheral blood mobilized stem/progenitor cells (PBSCs) ex vivo. To fill this knowledge gap, we have systematically surveyed 466 small-molecule drug compounds for their potential in cytokine-dependent expansion of human CD34+CD90+ HSPCs. We found that epigenetic modifiers, especially histone deacetylase inhibitors (HDACis), could preferentially maintain and expand these cells. In particular, treatment of CD34+ PBSCs with a single dose of HDACi trichostatin A (TSA) at a concentration of 50 nmol/L ex vivo yielded the greatest expansion (11.7-fold) of CD34+CD90+ cells when compared with the control (dimethyl sulfoxide [DMSO] plus cytokines) group. Additionally, TSA-treated PBSC CD34+ cells had a statistically significant higher engraftment rate than the control-treated group in xenotransplantation experiments. Mechanistically, TSA treatment was associated with increased expression of HSPC-related genes such as GATA2 and SALL4. Furthermore, TSA-mediated CD34+CD90+ expansion was reduced by downregulation of SALL4 but not GATA2. Overall, we have developed a robust, short-term (5-day), PBSC ex vivo maintenance/expansion culture technique and found that the HDACi–TSA/SALL4 axis is important for the biological process.

Posttransplant lymphoproliferative disorders (PTLDs) are potentially fatal, EBVdriven B-cell malignancies that develop in immunocompromised solid organ or hematopoietic stem cell recipients. In PTLD, the expression of EBV proteins, including latent membrane protein 1 (LMP1) and LMP2A, viral immune evasion strategies, and impaired host immune surveillance foster the proliferation of EBV-transformed B cells. Current PTLD treatment strategies include reduction of immunosuppression, which increases the risk of graft rejection, anti-CD20 treatment, combination chemotherapy, and administration of EBV-specific cytotoxic T cells. In the present study, we report that EBV-transformed lymphoblastoid Bcell lines (LCLs) and primary PTLDs overexpress galectin-1 (Gal1), a carbohydratebinding lectin that induces tolerogenic dendritic cells and triggers the selective apoptosis of CD4 Th1 and Th17 cells and cytotoxic T cells. In transcriptional reporter assays, LMP2A and LMP1 each increased Gal1-driven luciferase expression, and the combination of LMP2A and LMP1 was additive. In addition, small interfering RNA (siRNA)–mediated depletion of LMP2A decreased Gal1 protein abundance in EBV-transformed LCLs. Gal1 expression in LCLs was dependent on both activating protein 1 (AP-1) and PI3K. A newly developed neutralizing Gal1 mAb selectively inhibited Gal1-mediated apoptosis of EBV-specific CD8 T cells. Given the tolerogenic and immunosuppressive function of Gal1, antibody-mediated Gal1 neutralization may represent a novel immunotherapeutic strategy for PTLD and other Gal1-expressing tumors. Fil: Ouyang, Ying. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Juszczynski, Przemyslaw. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Rodig, Scott J.. Brigham and Women’s Hospital. Department of Pathology; Estados Unidos Fil: Green, Michael R.. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: O´ Donnell, Evan. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Currie, Treeve. Brigham and Women’s Hospital. Department of Pathology; Estados Unidos Fil: Armant, Myriam. Immune Disease Institute/Harvard Medical School. Center for Human Cell Therapy; Estados Unidos Fil: Takeyama, Kunihiko. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Monti, Stefano. Broad Institute; Estados Unidos Fil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Ritz, Jerome. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Kutok, Jeffery L.. Brigham and Women’s Hospital. Department of Pathology; Estados Unidos Fil: Shipp, Margaret A.. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos

Autologous gene therapy (GT) for beta-hemoglobinopathies has demonstrated encouraging early safety and efficacy using addition of a sickling-resistant globin gene to stem cells. Another therapeutic strategy for sickle cell disease (SCD) is erythroid-specific inactivation of BCL11A, which is a validated repressor of gamma globin expression (Sankaran et al. Science 2011). This approach has the distinct advantage of simultaneously inducing fetal hemoglobin (HbF) while coordinately decreasing sickle hemoglobin (HbS). Since hemoglobin (Hb) polymerization in sickle red cells is highly dependent on the intracellular concentration of HbS and is strongly inhibited by HbF, effective BCL11A repression should prevent the sickling phenotype within red cells. We have shown that erythroid-specific expression of microRNA-adapted shRNAs (shRNAmiR) targeting BCL11A effectively induces HbF in human erythroid cells derived from transduced HSCs, largely attenuating the hematologic effects of SCD in a murine model while avoiding negative effects in HSCs and B lymphocytes (Brendel et al. JCI 2016). Here we report the initial results of a pilot clinical study utilizing a shRNAmiR lentiviral vector (LVV) targeting BCL11A for autologous GT in SCD patients. Transduction of hematopoietic cells with GMP lentiviral vector (BCH_BB-LCRshRNA(miR)) expressing the shRNAmiR for BCL11A in an erythroid-specific fashion showed no toxicity in engraftment and genotoxicity assays, efficient transduction rates of 80-95% of CD34+-derived erythroid colony forming cells from healthy donors and SCD patients, and >95% of transduced erythroid colonies demonstrating HbF levels of 50-95% of total Hb. Transduction at clinical scale with plerixafor mobilized CD34+ cells from three SCD donors yielded vector copies of 3.7 - 5.2/cell. Patients with severe SCD were screened for eligibility according to an IND enabled, IRB-approved investigator-initiated protocol. The first cohort included patients ≥ 18 years old. After at least 3 months of protocol-required transfusions, autologous CD34+ cells were collected by plerixafor mobilization and apheresis, and then transduced under GMP conditions with the BCH_BB-LCRshRNA(miR) vector. As of July 28, 2018, 3 patients representing the adult cohort had undergone a total of 3 (n=2) or 4 (n=1) days of mobilization. Mean single-day apheresis yields were 3.2 (range 1.5 - 6.8) x 106 CD34+ cells/kg. No Grade 3 or 4 AEs were attributed to mobilization and collection, although one subject developed an incidentally-discovered line-associated atrial clot and pulmonary embolism. Transduced cell products for these 3 patients have cell doses of 3.3 - 6.7 x 106 CD34+ cells/kg, VCN of 3.3 - 5.1 copies per cell and >95% vector-positive CD34+-derived colonies. One subject (BCL002), who had been regularly transfused for 17 years, has undergone infusion of gene-modified cells after myeloablative busulfan conditioning and achieved neutrophil engraftment after 22 days. Post-infusion follow-up is 78 days. At the time of the last analysis 76 days after GT and 64 days after last RBC transfusion (Table 1) subject BCL002 had a sustained Hb of >10 g/dL and, compared to pre-GT, there was a notable absence of irreversibly sickled cells on peripheral smear and a persistently low absolute reticulocyte count consistent with markedly reduced hemolysis. Hb electrophoresis showed 23.3% HbF, 51.8% HbS and 22.3% HbA (from residual transfused cells) with a HbF/(HbF+HbS) ratio of 29.7%. At day 76, the number of F cells had risen to 59.7% with 12pg HbF/F cell. In flow-sorted immature erythroid cells γ-globin mRNA was >80% of total β-like globins in the graft-derived population and BCL11A protein was reduced by ~90%. Adverse events observed from the start of conditioning until latest follow-up were consistent with myeloablative conditioning, and there have been no product-related adverse events and no SCD-related complications. These early results show: (1) feasibility of enrollment, cell procurement, and GMP manufacturing of gene modified CD34+ cells in 3 adult SCD patients; (2) the first proof of principle demonstrating shRNAmiR-based gene knockdown in humans, and (3) successful rapid induction of HbF in the first patient infused, with marked attenuation of hemolysis in the early phase of autologous reconstitution. Based on the trajectory of increasing HbF/(HbF+HbS), near full suppression of the SCD phenotype is expected. Disclosures Esrick: Bluebird Bio: Honoraria. Negre:bluebird bio: Other: Spouse employed by bluebird Bio. Dansereau:Bluebird Bio: Consultancy. Braunewell:Bluebird Bio: Employment, Equity Ownership. Christiansen:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Nikiforow:Kite Pharma: Consultancy. Achebe:Luitpold Pharmaceutical: Consultancy; AMAG Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Syros pharmaceuticals: Consultancy. Negre:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Heeney:Sancilio Pharmaceuticals: Consultancy, Research Funding; Ironwood: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Vertex/Crisper: Other: Data Monitoring Committee; Pfizer: Research Funding; Astra Zeneca: Consultancy, Research Funding. Williams:Bluebird Bio: Research Funding.

Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells (HSCs) for use in allogeneic transplantation. Key advantages of UCB are rapid availability and less stringent requirements for HLA matching. However, UCB contains an inherently limited HSC count, which is associated with delayed time to engraftment, high graft failure rates, and early mortality. 16,16-Dimethyl prostaglandin E2 (dmPGE2) was previously identified to be a critical regulator of HSC homeostasis, and we hypothesized that brief ex vivo modulation with dmPGE2 could improve patient outcomes by increasing the "effective dose" of HSCs. Molecular profiling approaches were used to determine the optimal ex vivo modulation conditions (temperature, time, concentration, and media) for use in the clinical setting. A phase 1 trial was performed to evaluate the safety and therapeutic potential of ex vivo modulation of a single UCB unit using dmPGE2 before reduced-intensity, double UCB transplantation. Results from this study demonstrated clear safety with durable, multilineage engraftment of dmPGE2-treated UCB units. We observed encouraging trends in efficacy, with accelerated neutrophil recovery (17.5 vs 21 days, P = .045), coupled with preferential, long-term engraftment of the dmPGE2-treated UCB unit in 10 of 12 treated participants.

The X-linked disorder Wiskott-Aldrich Syndrome (WAS), characterized by thrombocytopenia, eczema, recurrent infections, autoimmunity and cancer, is typically treated with allogeneic transplantation. Somatic gene therapy (GT) using autologous CD34+ cells is a promising treatment alternative for high-risk patients lacking matched allogeneic donors, as it avoids graft versus host disease. GT using a γ-retroviral vector with intact viral enhancers was efficacious but had a high rate of leukemia due to insertional oncogenesis. We report here preliminary results of 4 WAS patients who underwent GT using a self-inactivating lentiviral (SIN-LV) vector, in which viral enhancers have been deleted and the human WAS cDNA is controlled by a 1.6kB fragment of the human WAS promoter (w1.6_hWASP_WPRE SIN-LV). WASP expression per cell induced by this vector was lower than in normal cells when examined in murine models and human trials. We hypothesized that the SIN-LV would readily correct immune abnormalities due to selective advantage for WAS protein (WASP) expressing T cells whereas correction of myeloid and platelet abnormalities would require high vector copy number (VCN) in the infused cells. Patients with severe WAS (clinical scores 3-5) were enrolled at a median age of 32 months (17 months-8 years). Patients 1 and 3 had detectable but low WASP expression. Patients 2 and 4 carried mutations that abrogated WASP expression but had evidence of somatic reversion in T and/or NK cells. CliniMACS purified CD34+ mobilized peripheral blood or bone marrow cells were transduced with the vector and infused after busulfan (12-15mg/kg) and fludarabine (120mg/m2) conditioning. CD34+ cell doses ranged from 6.3-24.91 x 106 cells/kg. VCN of the infused cells was variable (3.37, 1.34, 0.54, 1.01 copies/cell). Busulfan exposure was myeloablative or near-myeloablative in patients 1, 3, and 4 (81.2, 77.2, 84.5 mg*h/L) and submyeloablative in patient 2 (48.8 mg*h/L). All patients are alive with median follow-up of 13.5 months (9-24 months). All patients had improvement in eczema, remain platelet transfusion independent and have had no severe bleeding events. WASP expression in T cells was increased post-GT over baseline. Selective advantage for WASP expressing T cells was apparent in patients 1, 2 and 4, who had higher VCN in T cells at 6 months post-GT (0.93-2.21) than in B (0.48-1.7) or myeloid (0.13-0.89) cells. The presence of revertants in patients 2 and 4 did not appear to interfere with T cell reconstitution. In contrast patient 3 who had the highest WASP expression at baseline and the lowest VCN of infused cells (0.5 copies/cell), had the lowest VCN in T cells at 8 months (0.1 copies/cell). Defective T cell proliferation in response to anti-CD3 stimulation, characteristic of WAS, was improved post-GT. Next generation sequencing of T cell receptors in sorted naïve, memory and regulatory T cells revealed profound abnormalities of diversity, decreased entropy and marked clonal expansions pre-GT; most of these improved at 6 months post-GT. Cytoskeletal function in myeloid cells was highly abnormal pre-GT, as shown by absence of podosome formation in monocyte-derived dendritic cells (0-1% vs. 61% in controls). Podosome formation at 6 months post GT was improved but subnormal (4-40%). Only patient 1, who received the highest cell dose, the highest VCN, and myeloablative busulfan exposure had robust platelet reconstitution (pre-GT 24 versus 110 x 109/L 6 months post-GT) and high level gene marking in myeloid cells 0.89 copies/cell. At the same timepoint, patients 2, 3 and 4 had platelet counts of 20-30 x 109/L and correspondingly lower VCN in myeloid cells (0.13-0.27 copies/cell). No severe adverse events related to GT have occurred to date, with relatively short follow-up. Integration site analysis of sorted cells showed highly polyclonal reconstitution, with distributions of integration acceptor sites as expected for the lentiviral vector backbone. In summary, GT using a SIN-LV that induces expression of WASP at levels below wild type improved the clinical and laboratory manifestations of WAS, with better reconstitution in patients receiving cells with high VCN. Disclosures Off Label Use: Off-label use of CliniMACS purified CD34+ cells.

Hematopoietic stem cells (HSCs) possess the unique capacity to self-renew and give rise to all types of mature cells within the blood and immune systems. Despite our progress in understanding the molecular factors that support the self-renewal and differentiation of the hematopoietic system in vivo, less is known on how to modulate the factors that govern the self-renewal of hematopoietic stem/progenitor cells (HSPCs) ex vivo. Unlike in the case of embryonic stem (ES) cells, expansion of CD34+ HSPC in culture in general is at the expense of loss of “stemness”. HSPCs can be collected from cord blood (CB), mobilized peripheral blood (PBSC), and rarely bone marrow (BM) at the present practice. Due to the limited CD34+ cell number in one single cord blood unit, much of the current efforts on developing technology of ex vivo expansion of HSPC uses cord blood as a source and is clinically applied to cord blood HSPC transplants. However, there are growing needs for expanding PBSCs for transplant-related practices such as HSPC expansion from poor autologous mobilizations, gene therapy or genome-editing via TALENs or CRISPR/Cas9. Developing a technology that would allow HSPC ex vivo expansion from both CB and PBSC sources is a key step towards this goal. Several groups have reported that ex vivo culture of CB CD34+ cells with HDAC inhibitors (HDACi) can lead to expansion of a CD34+CD90+ population, which is responsible for enhanced marrow-repopulating potential. In this study, we ask whether HDACi can have a similar effect on PBSC CD34+ cells. Furthermore, we have explored the mechanism(s) mediated by HDACi in CD34+CD90+ cell expansion. First we assessed a panel of HDACi to identify the most potent molecule for CD34+CD90+ cell expansion and selected trichostatin A (TSA) for future study. Next, TSA was added to the cytokines (SCF, Flt3 ligand, IL-3 and IL-6) to further characterize its potential in PBSC CD34+CD90+ cell expansion. We observed TSA treated CD34+ cultures with cytokines yielded 4.8 times greater numbers of CD34+CD90+ cells as compared to the cultures containing cytokines with DMSO solvent control. We next examined SCID repopulating ability (SRA) to evaluate the cultured CD34+CD90+ cells in vivo. We observed that mice transplanted with 3 million CD34+ cells treated with TSA had higher degree of human cell chimerism than those treated with DMSO at 8 weeks bone marrow and peripheral blood (32% vs 18%; p < 0.05), which was further confirmed by secondary transplantation. Furthermore, these cells were capable of differentiating into cells belonging to multiple hematopoietic lineages. To investigate the molecular mechanisms responsible for the expansion of functional HSCs and progenitors that were observed following TSA treatment, we analyzed the expression levels of several HSPC related genes, which were compared between CD34+ cells treated with TSA and DMSO. Significantly higher transcript levels were detected for GATA 2 (p < 0.05), HOXB4 (p < 0.05), HOXA9 (p < 0.05), and SALL4 (p < 0.05) by real time quantitative RT-PCR in TSA expanded cells as compared with controls. To evaluate whether these transcription factors can contribute to the expansion of CD34+CD90+ cells, GATA2, HOXB4 or SALL4 shRNAs were transfected into PBSC CD34+ cells, followed by culture with TSA. Among these transcription factors, knocking down SALL4 expression led to the most significant reduction of CD34+CD90+ cell numbers (33% of reduction). In addition, overexpression of SALL4 in PBSC CD34+ cells led to an increase of CD34+CD90+ cell numbers when compared to controls (p < 0.05). Overall, our study demonstrated a novel HDACi mediated ex vivo PBSC culture technology that leads to the expansion of CD34+CD90+ cells and an increase of the marrow repopulating potential of these cells. Both gain-of-function and loss-of-function studies support that SALL4 is a key transcription factor responsible for the process. Future study on the use of HDACi or other methods to increase SALL4 expression/function will be highly beneficial to ex vivo HSPC (CB and PBSC) expansion technology. Disclosures No relevant conflicts of interest to declare.

Abstract 164FN2 Somatic gene therapy for X-linked severe combined immunodeficiency (X-SCID) using a MLV-based gammaretroviral vector expressing the IL-2 receptor gamma chain (MFG-γc) resulted in excellent immunologic reconstitution but also in insertional oncogenesis. In 5/20 treated children, T cell leukemia developed, with insertional activation of LMO2 proto-oncogene in 4 of the 5. We reasoned that replacing the virus-derived promoter and enhancer elements with a weaker cellular promoter would result in improved safety yet retain efficacy. We therefore generated the pSRS11.EFS.IL2RG.pre* self-inactivating (SIN) gammaretroviral vector in which expression of γc is controlled by an intronless EF1-α promoter, in a MLV vector devoid of the LTR U3 (enhancer/promoter) region. In preclinical studies we determined ‘relative safety' using several surrogate assays. In a reporter assay, the pSRS11.EFS.IL2RG.pre* vector when inserted into the oncogenic LMO2 locus induced LMO2 expression 6–90-fold less than the parent MFG vector (MFG-γc). pSRS11.EFS.IL2RG.pre* had lower activity in a murine in vitro immortalization assay (0.2 clones per 10e5 cells, fitness score 0.00007), compared to MFG-γc (0.54 clones per 10e5 cells, fitness score 0.00025). Peripheral blood and bone marrow from C57BL6 mice transplanted with murine bone marrow transduced with pSRS11.EFS.IL2RG.pre* and followed in primary recipients over 4 months and in secondary recipients over 1 year in vivo showed no evidence of vector associated leukemias. Deep sequencing demonstrated 8 of 3621 insertions into the MDS-associated gene Evi1 in mice transplanted with MFG-γc-transduced cells while there were none (0 of 2690 insertions into Evi1) in mice transplanted with pSRS11.EFS.IL2RG.pre* vector transduced cells (P=0.025). In preclinical efficacy studies, circulating T and B lymphocytes were detectable in the peripheral blood of 7/7 γc-deficient mice transplanted with pSRS11.EFS.IL2RG.pre* transduced cells while 4/4 mice repopulated with SFFV-eGFP transduced cells remained alymphoid. Experimental animals were sacrificed approximately five months post-transplant for analysis of immune reconstitution. Flow cytometric analysis of the spleens and bone marrow revealed restoration of mature B220+IgM+ B cells and NK cell populations in all mice transplanted with pSRS11.EFS.IL2RG.pre* transduced cells. CD4+ and CD8+ T cells were also detected in both tissues and in thymi recovered from transplanted animals. T cells in these mice proliferated in response to mitogenic stimuli. Immunoglobulin subclasses IgG1 and IgG2a detected in the plasma from pSRS11.EFS.IL2RG.pre* reconstituted mice also indicated restored B cell function in these animals. Human preclinical studies also supported the correction of XSCID cellular defects using this vector. Based on these data, a multi-institutional phase I/II trial was initiated with the pSRS11.EFS.IL2RG.pre* vector using an identical clinical protocol as in the previous X-SCID trials, to determine efficacy and safety compared with the MFG-γc vector. The first patient was treated in December 2010. Six months post-gene therapy, he has attained CD3 T cell count of >800, normal proliferation to mitogens, and normal NK cell numbers. He has cleared medically-resistant oral ulcers, and a rotavirus infection acquired post-gene therapy. Nearly all of the circulating T cells (86%) and 41% of his NK cells express γc, albeit at modestly lower density than normal, as expected. However, the early kinetics of T cell reconstitution was comparable to several subjects treated with MFG-γc. These data suggest that the improved safety profile demonstrated with numerous surrogate preclinical studies is associated with efficacious transgene expression and functional immune recovery in the initial human patient treated. Disclosures: Off Label Use: CliniMACS for selection of CD34+ hematopoietic cells. Baum:Patent office: Patents & Royalties.

Abstract 1451 A systematic investigation was performed to optimize the treatment protocol for ex vivo incubation of human hematopoietic stem cells (HSCs) with 16,16-dimethyl prostaglandin E2 (FT1050) prior to transplantation. This protocol is part of an ongoing Phase Ib clinical trial of FT1050-enhanced double cord blood (CB) transplantation after reduced intensity conditioning. FT1050 has been previously shown in vertebrate models to improve the engraftment potential of HSCs from bone marrow (BM) and CB after a brief ex vivo treatment. In these models, treatment of BM or CB with FT1050 was performed for 1 to 2 hours at 4 °C, followed by a wash and subsequent cell infusion into the recipient (North et al. Nature 2007, Hoggatt et al. Blood 2009). Several groups have demonstrated that under these conditions, FT1050-treated cells have an engraftment advantage over vehicle treated cells. The objective of the current investigation was to identify a set of conditions that maximizes the biologic activity of FT1050. Genome-wide expression analysis and cAMP assays were used to optimize the ex vivo FT1050 treatment protocol with respect to concentration, time and temperature. Using this approach, hundreds of up- and down-regulated genes were identified in FT1050-treated CD34+ cells. These signature genes include upregulation of CXCR4, a known mediator of HSC homing via SDF-1a, and CREB, a key gene involved in cAMP signaling. Results from these experiments demonstrated that FT1050 concentrations above 10 μM did not result in increased levels of biologic activity. In terms of duration of incubation, cAMP activity reached maximal levels within 30 minutes of exposure while a 2 hour treatment period was necessary to maximize the changes in gene expression. Finally, the biologic activity of FT1050 was highly sensitive to temperature, with treatment of cells at 37 °C yielding larger changes in cAMP production and gene expression as compared to incubation of cells at 25 °C and 4 °C. The biological effects of FT1050 on subsets of CD34+ cells isolated from CB were also determined. Interestingly, the stem/progenitor subsets of CD34+ cells (Lin-CD34+CD38-CD90+CD45RA-, Lin-CD34+CD38-CD90-CD45RA- and Lin-CD34+) had a greater response to FT1050 relative to the lineage positive cells. The different conditions were also evaluated using CFU-C and 7-AAD assays. No evidence of adverse effects were observed. Based upon these findings, the ongoing clinical trial incorporates the optimized FT1050 ex vivo treatment protocol (10 μM for 120 minutes at 37 °C). Disclosures: Desponts: Fate Therapeutics, Inc.: Employment, Equity Ownership. Robbins:Fate Therapeutics, Inc.: Employment, Equity Ownership. Le:Fate Therapeutics, Inc.: Employment, Equity Ownership. Thies:Fate Therapeutics, Inc.: Employment, Equity Ownership. Mendlein:Fate Therapeutics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Grayson:Fate Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Multani:Fate Therapeutics, Inc.: Employment, Equity Ownership. Shoemaker:Fate Therapeutics: Employment, Equity Ownership.

Abstract 96 EBV-associated post-transplant lymphoproliferative disorders (PTLD) are potentially fatal EBV-driven B cell proliferations that develop in immunocompromised solid organ or bone marrow allograft recipients. Proliferation of EBV-infected B cells in PTLD is maintained by expression of EBV latent genes such as latent membrane protein 1 (LMP1) and LMP2A, viral immune evasion strategies, and impaired host immune surveillance. Management of early PTLD lesions is currently based on reduction or withdrawal of immunosuppression which increases the risk of graft rejection. For these reasons, alternative treatments that specifically boost host anti-EBV immune responses are needed. To identify novel T-cell inhibitory molecules induced by EBV and expressed in PTLDs, we compared the transcription profiles of a series of EBV-transformed lymphoblastoid B-cell lines (LCLs) and diffuse large B-cell lymphoma (DLBCL) cell lines. The LCLs overexpressed galectin-1 (Gal-1), a carbohydrate-binding lectin that induces tolerogenic dendritic cells and triggers the selective apoptosis of CD4+ Th1 and CD8+ cytotoxic T cells. Gal-1 protein expression was also uniformly high in LCLs and low or undetectable in DLBCL lines by western blotting. Immunohistochemical staining of primary tumors confirmed abundant Gal-1 expression in PTLD B-cells, whereas DLBCLs were negative. To elucidate the mechanisms responsible for Gal-1 overexpression in EBV-associated PTLD, we performed transcriptional reporter assays using cotransfected FLAG-tagged LMP1 and/or LMP2A and a luciferase reporter vector driven by Gal-1 promoter. LMP2A and LMP1 each increased Gal-1-driven luciferase expression 3- to 6-fold, respectively, and the combination of LMP2A and LMP1 was synergistic. In complementary experiments, siRNA-mediated depletion of LMP2A decreased Gal-1 protein abundance in EBV-transformed lymphoblastoid cells. The mechanism of LMP1 and LMP2A-induced Gal-1 expression in LCLs was both AP-1 and PI3K-dependent; the PI3K selective inhibitor, LY294002, markedly inhibited Gal-1 expression in these cells. Given the tolerogenic and immunosuppressive function of Gal-1 and the EBV-driven overexpression of the lectin in LCLs and primary PTLDs, we postulated that Gal-1 might be a targetable immune escape mechanism in EBV-associated malignancies. For these reasons, we assessed the neutralizing activity of a recently developed series of anti-Gal1 monoclonal antibodies. After confirming that these reagents selectively identified both recombinant and native Gal1, we asked whether certain Gal-1 monoclonal antibodies neutralized recombinant Gal-1-mediated apoptosis of EBV-specific polyclonal, largely CD8+, T-cells. Preincubation of human recombinant Gal-1 (hrGal-1) with Gal-1 monoclonal antibodies, but not with isotype-matched controls, selectively inhibited hrGal-1 induced apoptosis of EBV-specific CD8+ T-cells. Taken together, these studies indicate that EBV-transformed LCLs and primary EBV-associated PTLDs express Gal-1 in a LMP-1 and LMP2A-dependent manner. Given the acknowledged role of Gal-1 in mediating an immunopriviledged tumor microenvironment, the lectin likely inhibits the host anti-tumor response in these tumors. Antibody-mediated neutralization of secreted Gal-1 may represent a novel immunotherapeutic strategy in PTLD and other Gal1-expressing tumors. Disclosures: No relevant conflicts of interest to declare.

Abstract 369 Hematopoietic stem cells (HSCs) comprise the base of the entire hematopoietic system and alone possess the ability to both self-renew and differentiate into all mature blood lineages, thereby maintaining immune function, tissue perfusion and hematopoietic homeostasis. HSCs are therapeutically valuable for the treatment of hematological malignances, immunodeficiencies and bone marrow failure. Prostaglandin (PG) E2 has been shown to enhance HSC engraftment in allogeneic murine transplantation models by our lab (North et al., Nature 2007) and others (Hoggatt et al., Blood 2009); PGE2 was additionally found to influence the balance of apoptosis and proliferation in the HSC population via modulation of wnt activity (Goessling et al., Cell 2009), and to modify CXCR4-responsive homing to the hematopoietic niche following transplantation (Hoggatt et al., Blood 2009). In order to translate the therapeutic potential of a stabilized version of PGE2, dmPGE2, we sought to determine the safety and efficacy of ex vivo dmPGE2 exposure in human cord blood (hCB) stem cells. Compared to matched control cord samples, no significant negative impact on hCB cell viability was observed following dmPGE2 treatment (10?M for 1 hour) using either fresh or frozen cord blood units; of note, the CD34+ stem and progenitor compartment seemed particularly able to tolerate the treatment protocol. To determine whether dmPGE2 treatment was not only safe, but potentially valuable for preserving hCB cell viability, apoptosis was measured by FACS analysis for 7AAD and AnnexinV in pooled CD34-enriched (CD34+) hCB samples treated in parallel with 1?M dmPGE2 or the vehicle control (DMSO); at 6 and 9 hours post exposure, cells treated with dmPGE2 showed a significant reduction in apoptosis compared to controls. Cell proliferation assays confirmed results seen in prior murine studies and demonstrated that dmPGE2 not only suppressed apoptosis, but enhanced HSC self-renewal. To determine if dmPGE2 exposure altered the functional characteristics of human cord blood samples, in vitro culture assays were conducted; pooled CD34+ samples were exposed over a time series (12 and 30 mins, 1, 3, 6 and 12 hours) to the vehicle control and dmPGE2 (1?M) then plated at limiting dilutions (2000, 800, 320). A significant 2-fold enhancement in total colony number (p Disclosures: Goessling: Fate Therapeutics: Consultancy, Patents & Royalties. Zon:FATE Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Stemgent: Consultancy. North:Fate Therapeutics: Consultancy, Patents & Royalties.

Hematopoietic stem cells (HSCs) are commonly used in transplantation therapy to rescue the hematopoietic and immune systems following systemic chemotherapy or irradiation. However, some patients receive inadequate numbers of HSCs and this often results in delayed reconstitution of hematopoiesis and immune function and associated toxicities. We previously demonstrated that a stabilized derivative of prostaglandin (PG) E2 increases vertebrate HSCs both in vivo and in vitro. 16,16-dimethyl PGE2 (dmPGE2) significantly increased HSCs during zebrafish embryogenesis and in the adult marrow following injury. Incubation of murine embryonic stem cells with dmPGE2 during embryoid body differentiation resulted in a dose-dependent increase in hematopoietic colonies, demonstrating that the function of PGE2 in HSC regulation is conserved in mammals. Finally, ex vivo treatment of murine bone marrow with dmPGE2 resulted in a 2-fold increase in engrafting cells in a limiting dilution competitive repopulation assay. No negative effects on serial transplantability of HSCs were observed in these animal models. To investigate the therapeutic potential of PGE2 for the amplification of blood stem cells, we exposed human cord blood (hCB) cells to dmPGE2 in vitro and measured the effects on stem and progenitor populations both in vitro and in vivo. Red cell depleted umbilical cord blood specimens, cryopreserved for clinical use, were thawed and divided for parallel processing. Ex vivo treatment of hCB cells for 1 hour with dmPGE2 in dextran/albumin had no negative impact on absolute cell count or the viability and relative distribution of both CD45 and CD34 positive cells compared to vehicle treated control hCB cells. Significantly, hCB treated with dmPGE2 produced enhanced numbers of GM and GEMM colonies in methylcellose CFU-C assays compared to controls. Human CB cells treated ex vivo with dmPGE2 for 1 hour and transplanted at a dose of 20 million live CD45+ cells per recipient were capable of repopulating NOD/SCID mice after sublethal irradiation. In comparative studies at 6 weeks post transplantation, human CD34+ and CD45+ cells could be detected in the marrow (>2%) of dmPGE2 treated (4/8) and control treated (1/6) recipients. Long-term and competitive transplantation experiments to assess the effect of dmPGE2 treatment on functional HSCs are currently in progress. Our data suggests that treatment of human cord blood products with dmPGE2 will be both safe and effective in achieving expansion of hematopoietic stem cells for transplantation in the clinical setting. TE North and W Goessling contributed equally to this work.