Tolerance Induction To FIX Padua With AAV Liver Gene Transfer In Inhibitor-Prone Hemophilia B Dogs

Abstract Emerging data from early phase clinical studies of AAV gene therapy for hemophilia B (HB) (factor IX [FIX] deficiency) show sustained expression of therapeutic levels of FIX and phenotypic improvement. However, the safety and efficacy of in vivo gene therapy is limited by the vector dose. Recently, we reported a naturally occurring, hyperfunctional FIX (FIX Padua) caused by a single amino acid change of arginine 338 to leucine that exhibits an 8-fold increase in specific activity in humans (N Engl J Med 2009), making it a potential candidate for HB gene therapy with reduced vector doses. However, to take advantage of FIX Padua for HB gene therapy, it is critical to first define the risk of immunogenicity of this variant in preclinical models of severe HB. We have previously shown that delivery of AAV-cFIX-Padua to skeletal muscle in HB dogs with a missense mutation in the canine (c) F9 gene resulted in no anti-FIX neutralizing antibodies (inhibitors), non-neutralizing antibodies (IgG) or FIX-specific T-cell response (Blood 2012). While promising, these dogs express FIX RNA and have a pre-existing tolerance to cFIX due to the nature of their mutation, and so do not represent the most rigorous model for immunogenicity studies. Here, we tested the efficacy and immunogenicity of cFIX Padua in a severe HB dog colony with an early stop codon mutation. This mutation results in no FIX RNA transcript, and the dogs are prone to develop cFIX inhibitors upon exposure to protein concentrates. Three dogs were infused peripherally with a liver-specific AAV8-cFIX-Padua at two different doses, and monitored for cFIX antigen and activity levels and inhibitors. The first dog, which received 3 x 1012 vg/kg, showed average plateaued expression levels of 3.98 ± 1.44% antigen and 24.5 ± 4.1% activity, with no development of anti-cFIX inhibitors or IgG antibodies. Whole blood clotting time (WBCT) and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >20 months (ongoing observations). A second dog was treated with a lower dose of 1 x 1012 vg/kg and showed average plateaued expression levels of 2.41 ± 0.05% antigen and 22.0 ± 0.4% activity, with no development of anti-cFIX inhibitors or IgG antibodies. WBCT and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >3 months. An additional dog, upon previous exposure to recombinant human (h) FIX protein, had developed inhibitors that cross-reacted with cFIX. This immune response was ongoing at the time of vector administration (3 x 1012 vg/kg). Anti-cFIX antibodies peaked at day 14 post-AAV, with 4.7 BUs and 3643 ng/mL IgG2, but dropped to undetectable levels by day 70. There was a concurrent rise in cFIX Padua expression levels, suggesting successful tolerization to the cFIX Padua. Antigen levels plateaued at 14.6 ± 4.3% and activity at 51.7 ± 23.5%, with ongoing normalization of WBCT and aPTTs for >18 months. In all three dogs, cholesterol, albumin and total protein were within normal limits with no clinical or laboratory evidence of nephrotic syndrome (a potential complication in FIX inhibitor patients that have undergone immune tolerance induction with frequent FIX protein injections). The safety of FIX Padua was further confirmed using a mouse model of HB. Mice (n=8-12/group) were treated with 5 x 1010 vg/kg liver-directed AAV8-hFIX-WT or AAV8-hFIX-Padua, resulting in expression levels of 1076 ± 343 ng/mL (21.5 ± 6.9% antigen, 67.5 ± 10.1% activity) and 797 ± 255 ng/mL (15.9 ± 5.1% antigen, 274.8 ± 73.8% activity), respectively. In cross-over experiments, the mice were then were immunologically challenged 10-15 weeks after gene delivery with 100 ug/kg of the reciprocal recombinant protein (ie mice expressing hFIX Padua were challenged with hFIX WT, and visa versa). Challenges were administered subcutaneously alone or with adjuvant (CFA) weekly for 4 weeks. In no instance did mice develop antibodies to either FIX, suggesting that tolerance was successfully induced in all cases. Together, these date show that FIX Padua shows no increase in immunogenicity compared to FIX WT and is capable not only of preventing inhibitor formation, but also of eradicating pre-existing inhibitory antibodies to FIX in an inhibitor-prone HB dog model. Thus, FIX Padua is an attractive transgene that will allow for decreased vector doses in human HB gene therapy, improving the safety profile of AAV liver gene therapy without increased immunogenicity. Disclosures: High: Alnylam Pharmaceuticals: Consultancy; BioMarin: Consultancy; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; Genzyme, Inc.: Membership on an entity’s Board of Directors or advisory committees; Intrexon: Consultancy; Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Shire : Consultancy; Benitec: Consultancy.

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Immune Tolerance Induction in Severe Hemophilia a Patients Using Wilate®, a Von Willebrand Factor/Factor VIII Concentrate

Blood ◽

10.1182/blood.v124.21.5057.5057 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5057-5057

Author(s):

John K. Wu ◽

Celina Woo ◽

Erica Crilly

Keyword(s):

Risk Factors ◽

High Risk ◽

Board Of Directors ◽

Neutralizing Antibodies ◽

Hemophilia A ◽

Tolerance Induction ◽

Advisory Committees ◽

High Risk Factors ◽

Risk Patients ◽

Inhibitor Titer

Abstract Introduction: To prevent and treat bleeding episodes, hemophilia A patients undergo factor VIII (FVIII) replacement therapy with either recombinant FVIII (r-FVIII) or plasma-derived FVIII (pd-FVIII). Approximately 32% of patients develop neutralizing antibodies to the FVIII protein rendering FVIII therapy ineffective (Gouw et al. N Engl J Med. 2013; 368:231-239). Development of these neutralizing antibodies, or ‘inhibitors’ is the most serious and challenging complication in the treatment of hemophilia A patients and represents the highest economic burden for a chronic disease (Gringeri et al. Blood 2003; 102:2358-2363). The standard approach for eradicating inhibitors is to tolerize the patient to FVIII through prolonged, daily exposure to high-dose FVIII – a process termed immune tolerance induction (ITI). Although costly, ITI can successfully eliminate inhibitors in a large proportion of low-risk patients, however there are several risk factors that reduce the likelihood of ITI success. For high-risk patients, the choice of drug product (r-FVIII versus pd-FVIII) may be particularly important. Several small studies have demonstrated the superiority of pd-FVIII for ITI, as these products contain the FVIII stabilizing protein, von Willebrand Factor (VWF) (Gringeri et al., Haemophilia 2007; 13:373-379). Wilate® (Octapharma) is a high-purity pd-FVIII complex that contains the two proteins (VWF and FVIII) in a 1:1 ratio. In this retrospective study, we present the patient history and outcome for four severe hemophilia A patients that have undergone ITI with Wilate®. Patients and Methods. All patients presented with severe FVIII deficiency (<1%). Only 1/4 patients had undergone and failed a previous ITI attempt using a rFVIII product. The Wilate ITI dosing regimen ranged from 90 U/Kg to 100 U/Kg daily. Three of the four patients presented with high-risk factors for poor ITI outcome: 1 patient had 1, 1 patient had 3 and 1 patient had 5 poor prognostic factors. The definition of ITI success and failure were consistent with the definitions described in the consensus recommendations of the 2006 International ITI workshop (DiMichele et al. Haemophilia 2007;13 Suppl. 1: 1-22). Complete success (CS) was defined as having an undetectable inhibitor titer, FVIII recovery ≥66% and half-life ≥6 hours; partial success (PS) was defined as having a clinical response allowing for commencement of FVIII therapy associated with an inhibitor titer of <5 BU/mL (and with FVIII recovery <66% and/or half-life <6 hours). Results. ITI with Wilate was found to be well tolerated, with no reported drug-related adverse events. Although 75% of the patients presented with high-risk factors for poor ITI outcome, Wilate ITI reduced inhibitor levels below 5 BU/mL for 3 out of 4 patients. The clinical outcome of Wilate ITI correlated with the presence and number of high-risk factors. Patient 1 did not respond to Wilate, and was considered a failure. This patient had 3 high risk factors for poor ITI outcome including FVIII gene mutation type, peak historical titer >50, and inhibitor titer at ITI onset > 10 BU/mL. Of the three patients that responded to Wilate ITI, patient 4 experienced a CS to Wilate ITI in 4 months. For the remaining 2 patients, Wilate ITI is currently ongoing. Despite the presence of high risk factors, these two patients have thus far achieved a PS with inhibitor levels stable for several months at or below 1 BU/mL. One of these patients (patient 2) presented with 5 high-risk factors, including having failed a previous ITI attempt. The frequency of bleeding episodes were significantly reduced upon commencement of Wilate ITI, allowing for the eventual cessation of FEIBA prophylaxis. Conclusion. Despite the presence of high-risk factors, 3 out of 4 patients (75%) experienced a clinical response to Wilate ITI. The patients experienced improved quality of life. Wilate ITI resulted in a significant reduction in bleed episodes in a high-risk patient. The type of FVIII product to use during ITI has been debated, however, some have observed a higher success rate when plasma-derived FVIII products containing VWF are used. From our experience, we conclude that Wilate for ITI in patients with inhibitors is effective and produces high rates of ITI success in high-risk patients. Disclosures Wu: Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxter: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees; Biogen Idec: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; Octapharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees. Woo:Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Crilly:Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxter: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Octapharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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ZFN Mediated Targeting Of Albumin “Safe Harbor” Results In Therapeutic Levels Of Human Factor VIII In a Mouse Model Of Hemophilia A

Blood ◽

10.1182/blood.v122.21.720.720 ◽

2013 ◽

Vol 122 (21) ◽

pp. 720-720

Author(s):

Xavier M Anguela ◽

Rajiv Sharma ◽

Yannick Doyon ◽

Thomas Wechsler ◽

David E Paschon ◽

...

Keyword(s):

Factor Viii ◽

Board Of Directors ◽

Genome Editing ◽

Hemophilia A ◽

Safe Harbor ◽

Review Committee ◽

Advisory Committees ◽

Grant Review ◽

Dual Expression

Abstract Genome editing utilizing engineered zinc finger nucleases (ZFNs) is a promising approach to achieve long-term expression of therapeutic genes in vivo. We have previously demonstrated in vivotargeting of the endogenous murine albumin locus as a “safe harbor” for high levels of protein production, resulting in sufficient Factor IX to correct the disease phenotype in hemophilia B mice. Targeted insertion of the donor sequence into the genome offers multiple advantages. First, we are able to exploit the high transcriptional activity of the native albumin enhancer/promoter. Second, by obviating the need for these regulatory elements within the donor, we expand the effective carrying capacity of adeno-associated viral (AAV) vectors to enable delivery of larger transgenes that may not package efficiently, such as coagulation factor 8. B-Domain Deleted Factor VIII (BDD-F8) cDNA is approximately 4.4kb. Inclusion of required enhancer/promoter elements results in a construct that exceeds the ideal packaging limitations of rAAV vectors. However, since these regions are not required for our gene editing approach the promoterless hBDD-F8 donor remained below the AAV packaging capacity. Importantly, intravenous delivery of 5e11 vg of AAV8-mAlb-ZFN and 5e11 vg of AAV8-BDD-F8-Donor to hemophilia A mice resulted in 54.6% (±4.1%) FVIII activity in the blood 2 weeks following administration - confirming the potential of the albumin locus to express high levels of the targeted transgene. To further optimize in vivo ZFN-based genome editing with a view toward the ultimate clinical use of this technology, we sought methods to further increase ZFN potency (and thus limit the dose of AAV necessary for function). Of several strategies pursued (e.g. codon optimization and inclusion of a intron in the expression cassette) the most successful was to deploy separate vectors expressing each individual ZFN rather than a single vector encoding a dual expression cassette carrying both ZFNs separated by a 2A fusion peptide. Using next generation sequencing (Illumina’s MiSeq) technology to quantify insertions and deletions indicative of DNA cleavage and repair, we observed a >3-fold increase in ZFN potency in vivo by transitioning from the dual expression vector to two individual ZFN vectors at equivalent total vector doses. Given the encouraging results obtained in mice, we next sought to examine the effectiveness of targeting the albumin locus in non-human primates (NHPs). Importantly, a single intravenous co-injection of two individual AAV vectors encoding each of the NHP targeted albumin-specific ZFNs resulted in persistent levels of gene modification in liver biopsies from treated Rhesus macaques - demonstrating successful in vivocleavage in a large animal model. These data support the use of ZFN technology in the targeting of endogenous loci with large therapeutic transgenes that are not ideally suited for episomal AAV based expression (such as F.VIII). Together our results support the further investigation of genome editing at the albumin locus as a novel method for in vivo protein replacement. Disclosures: Doyon: Sangamo BioSciences, Inc.: Employment. Wechsler:Sangamo BioSciences, Inc.: Employment. Paschon:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment. High:Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Intrexon: Consultancy; Genzyme, Inc.: Membership on an entity’s Board of Directors or advisory committees; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; BioMarin: Consultancy; Alnylam Pharmaceuticals: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Shire : Consultancy; Benitec: Consultancy.

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Strategies for Selection of AAV Vectors for Administration to Liver: Studies in Nonhuman Primates

Blood ◽

10.1182/blood.v128.22.2316.2316 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2316-2316 ◽

Cited By ~ 1

Author(s):

Lili Wang ◽

James M Wilson ◽

Roberto Calcedo ◽

Peter Bell ◽

Zhenning He ◽

...

Keyword(s):

Gene Therapy ◽

Clinical Trials ◽

Gene Transfer ◽

Board Of Directors ◽

Research Funding ◽

Nonhuman Primates ◽

Hemophilia B ◽

Advisory Committees ◽

Equity Ownership ◽

Liver Gene

Abstract Vectors based on adeno-associated virus (AAV) have demonstrated early promise in clinical trials, including published reports in patients with hemophilia B where therapeutic levels of factor IX have been achieved using AAV serotype 8, a member of the clade E family. With the success of these hemophilia B clinical trials utilizing AAV8, numerous alternative AAV capsid types that target the liver have begun to enter clinical testing across multiple disease indications. Specific properties of the various AAV capsids should be taken into account such as overall efficiency of hepatocyte gene transfer, differential gene transfer across the porto-central axis within the liver, durability of gene expression and the potential for vector re-administration. In this study, we evaluated these aspects of AAV-directed liver gene transfer in rhesus macaques across various capsid serotypes. Animals were injected with vectors expressing the secreted reporter gene rhesus bhCG and produced using different capsids [AAV5, AAV3b and two clade E vectors (AAVrh10 and AAV8)]. Nonhuman primates (NHPs) injected with clade E vectors expressing bhCG were administered 3 months later with AAV5 or AAV3b vectors expressing rhesus derived AFP. The key findings were: 1) in naïve animals, clade E vectors demonstrated the highest levels of periportal gene transfer with AAV5 vectors having the lowest levels of periportal gene transfer; 2) AAVrh10 and AAV5 elicited higher levels of neutralizing antibodies (NAb) than AAV8 and AAV3b; 3) significant animal-to-animal variation in transgene expression was noted with AAV3b in seronegative animals; and 4) within the short time frame tested NAb elicited from AAVrh10 appears to have inhibited subsequent in vivo transduction with the serologically distinct AAV3b serotype; prior exposure to AAV8 did not interfere with AAV3b transduction. These studies highlight the influence that capsids can play in efficiency and immunogenicity of AAV vectors for liver gene therapy. Disclosures Wilson: Dimension Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Solid Gene Therapy: Consultancy, Membership on an entity's Board of Directors or advisory committees; REGENXBIO: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Kattenhorn:Dimension Therapeutics: Employment. Wadsworth:Dimension Therapeutics: Employment, Equity Ownership.

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Clonal Tracking By Whole Genome Sequencing Permits Comprehensive Mapping of the Genomic Landscape in Pre- and Post-Gene Therapy Sickle Cell Patients

Blood ◽

10.1182/blood-2021-152262 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 559-559

Author(s):

Alyssa H. Cull ◽

Michael Spencer Chapman ◽

Marioara Ciuculescu ◽

Emily Mitchell ◽

Myriam Armant ◽

...

Keyword(s):

Gene Therapy ◽

Stem Cell ◽

Board Of Directors ◽

Sickle Cell ◽

Research Funding ◽

Phylogenetic Trees ◽

Insertion Site ◽

Advisory Board ◽

Advisory Committees ◽

Genomic Landscape

Abstract 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.

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Northstar-2: Updated Safety and Efficacy Analysis of Lentiglobin Gene Therapy in Patients with Transfusion-Dependent β-Thalassemia and Non-β0/β0 Genotypes

Blood ◽

10.1182/blood-2019-126046 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3543-3543 ◽

Cited By ~ 1

Author(s):

Alexis A. Thompson ◽

Mark C. Walters ◽

Janet L. Kwiatkowski ◽

Suradej Hongeng ◽

John B. Porter ◽

...

Keyword(s):

Gene Therapy ◽

Clinical Trials ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Adult Patients ◽

Employment Equity ◽

Advisory Committees ◽

Transfusion Independence ◽

Equity Ownership

Background Transfusion-dependent β-thalassemia (TDT) is treated with regular, lifelong red blood cell (RBC) transfusions and despite iron-chelating therapy, carries a risk of serious organ damage from iron overload and other complications. Transplantation with autologous CD34+ cells encoding a βA-T87Q-globin gene (LentiGlobin for β-thalassemia) is being evaluated in patients with TDT. Interim results are presented here from the ongoing, international, single-arm, phase 3 Northstar-2 study (HGB-207; NCT02906202) of LentiGlobin gene therapy in pediatric, adolescent, and adult patients with TDT (defined by receiving ≥100 mL/kg/yr of RBCs or ≥8 RBC transfusions/yr) and non-β0/β0 genotypes. Methods Patients undergo hematopoietic stem cell (HSC) mobilization with G-CSF and plerixafor. Following apheresis, CD34+ cells are transduced with BB305 lentiviral vector and infused into patients after pharmacokinetic-adjusted, single-agent busulfan myeloablation. The primary efficacy endpoint is transfusion independence (TI; weighted average hemoglobin [Hb] ≥9 g/dL without RBC transfusions for ≥12 months). HSC engraftment, βA-T87Q-globin expression, Hb levels, detection of replication competent lentivirus (RCL), and adverse events (AE) are also assessed. Patients are followed for 2 years and offered participation in a long-term follow-up study. Summary statistics are presented as median (min - max). Results Twenty patients were treated in Northstar-2 as of 13 December 2018 and have been followed for a median of 8.1 (0.5 - 22.2) months. At enrollment, median age was 16 (8 - 34) years; 5 patients were <12 years of age. Median drug product cell dose was 8.0 (5.0 - 19.9) x106 cells/kg and vector copy number was 3.2 (1.9 - 5.6) copies/diploid genome. Time to neutrophil and platelet engraftment in the 18/20 and 15/20 evaluable patients was 22.5 (13 - 32) and 45 (20 - 84) days, respectively. Non-hematologic grade ≥3 AEs in ≥3 patients after LentiGlobin infusion included stomatitis (n=12), febrile neutropenia (n=6), pyrexia (n=4), epistaxis (n=3), and veno-occlusive liver disease (n=3). One serious AE of grade 3 thrombocytopenia was considered possibly related to LentiGlobin. No patient died, had graft failure, or had detection of RCL. No insertional oncogenesis has been observed. Gene therapy-derived HbAT87Q stabilized approximately 6 months after infusion. In adolescent and adult patients treated with LentiGlobin, median HbAT87Q at Months 6, 12 and 18 was 9.5 (n=11), 9.2 (n=8), and 9.5 (n=3) g/dL, respectively. The median total Hb without transfusions at Months 6, 12, and 18 were 11.9 (n=11), 12.4 (n=8), 12.3 (n=2) g/dL, respectively. At Month 6, 91% (10/11) of patients had total Hb of >11 g/dL without transfusions. Five adolescent and adult patients were evaluable for the primary endpoint of transfusion independence, 4 (80%) of whom achieved TI. The median weighted average Hb during TI was 12.4 (11.5 - 12.6) g/dL which compared favorably to pre-transfusion nadir Hb levels before enrollment (median 9.1 g/dL [7.5 - 10.0 g/dL]). At time of analysis, the median duration of TI was 13.6 (12.0 - 18.2) months. One patient who did not achieve TI stopped transfusions for 11.4 months but resumed transfusions due to recurrent anemia. This patient had a 71.4% reduction in RBC transfusion volume from Month 6 to Month 18 compared to baseline. Marrow cellularity and myeloid:erythroid (M:E) ratios were evaluated in 8 adolescent and adult patients with ≥12 months follow-up to assess the effect of LentiGlobin treatment on dyserythropoiesis. Seven of 8 patients had improved marrow M:E ratios at Month 12 (0.63 - 1.90) compared with baseline (0.14 - 0.48). In patients who stopped transfusions, soluble transferrin receptor levels were reduced by a median of 72% (58% - 78%) at Month 12 (n=6). Updated outcomes in adolescents and adults and outcomes in pediatric patients will be reported. Summary In this update of the Northstar-2 study of LentiGlobin gene therapy in patients with TDT and non-β0/β0 genotypes, transfusion independence was observed in 4/5 evaluable adolescent and adults and 10/11 treated patients had total Hb of >11 g/dL without transfusion support 6 months after LentiGlobin infusion. HbAT87Q stabilized approximately 6 months after treatment and patients who stopped RBC transfusions had improved erythropoiesis. A safety profile consistent with busulfan conditioning was observed after LentiGlobin gene therapy. Disclosures Thompson: bluebird bio, Inc.: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Baxalta: Research Funding. Walters:TruCode: Consultancy; AllCells, Inc: Consultancy; Editas Medicine: Consultancy. Kwiatkowski:bluebird bio, Inc.: Consultancy, Research Funding; Terumo: Research Funding; Celgene: Consultancy; Agios: Consultancy; Imara: Consultancy; Apopharma: Research Funding; Novartis: Research Funding. Porter:Protagonism: Honoraria; Celgene: Consultancy, Honoraria; Bluebird bio: Consultancy, Honoraria; Agios: Consultancy, Honoraria; La Jolla: Honoraria; Vifor: Honoraria; Silence therapeutics: Honoraria. Thrasher:Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 4BIOCapital: Membership on an entity's Board of Directors or advisory committees. Thuret:BlueBird bio: Other: investigators for clinical trials, participation on scientific/medical advisory board; Celgene: Other: investigators for clinical trials, participation on scientific/medical advisory board; Novartis: Other: investigators for clinical trials, participation on scientific/medical advisory board; Apopharma: Consultancy. Elliot:bluebird bio, Inc.: Employment, Equity Ownership. Tao:bluebird bio, Inc.: Employment, Equity Ownership. Colvin:bluebird bio, Inc.: Employment, Equity Ownership. Locatelli:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Miltenyi: Honoraria.

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Aberrant Expression of the MLL5, BAALC, ID1, and WT1 Genes Is Associated with Higher Induction Mortality and Poorer Overall Survival in Acute Promyelocytic Leukemia Patients Treated with ATRA and Anthracycline-Based Chemotherapy: An International Consortium On Acute Promyelocytic Leukemia Study

Blood ◽

10.1182/blood.v120.21.1407.1407 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1407-1407

Author(s):

Antonio R Lucena-Araujo ◽

Rafael Henriques Jacomo ◽

Haesook T Kim ◽

Raul A Melo ◽

Rosane Bittencourt ◽

...

Keyword(s):

Gene Expression ◽

Overall Survival ◽

Board Of Directors ◽

Acute Promyelocytic Leukemia ◽

Outcome Prediction ◽

Promyelocytic Leukemia ◽

Advisory Committees ◽

Aberrant Expression ◽

Expression Levels ◽

Gene Expression Levels

Abstract Abstract 1407 Background: Aberrant expression of MLL5, BAALC, ID1, and WT1 genes is frequently associated with inferior outcome in cytogenetically normal acute myeloid leukemia patients (Damm et al. Blood 2011; 117(17):4561–8). The expression levels of these genes vary in patients with acute promyelocytic leukemia (APL), but the clinical significance of these findings remains unclear. Objective: (1) to determine if the gene expression levels of MLL5, BAALC, ID1, and WT1 are associated with clinical outcome of APL patients treated with ATRA and anthracycline-based chemotherapy, (2) to generate an integrative score (IS) based on these potential prognostic factors and clinical parameters and (3) to use this score for outcome prediction in APL. Design and Methods: One hundred and fifty APL patients (age, 15–73y) from seven different Brazilian institutions and treated according to the IC-APL protocol were included. The treatment schedule was identical to the PETHEMA-LPA 2005, except for the replacement of idarubicin by daunorubicin; ATRA treatment was initiated immediately in all cases in which the diagnosis of APL was suspected based on morphology. Gene expression profile was analyzed by Real-time PCR. Integer weights for the IS were derived from Cox proportional hazard model, using overall survival (OS) as outcome parameter. Hazard ratios (HR) for OS were calculated for each variable separately (Table 1). Variables with P<0.05 in univariate analyses were included in the model. Variables considered for the model inclusion consisted in 2 clinical (WBC counts, albumin levels) and 5 molecular markers (FLT3-ITD status and gene expression levels of MLL5, BAALC, ID1, and WT1). Other candidates, such as age, platelet count, gender, ECOG performance status, PML breakpoint and FAB subtype were not significant and not included in the score. The HR were converted to integer weights according to the following: variables with HR < 1 were excluded from analyses; variables with HR 3 1 and < 1.5 were assigned a weight of 1; variables with HR 3 1.5 and < 2.5 were assigned a weight of 2; variables with HR 3 2.5 were assigned a weight of 3. The final score was the sum of these integer weights. Based on maximally selected rank statistics, the scores were grouped into 3 risk-groups: 0–5 (low-IS), 6–9 (intermediate-IS), and > 9 (high-IS). Results: The integrative weights of variables analyzed are summarized in Table 1. The IS was modeled in 137 patients (median score: 6; range, 1–17). According to PETHEMA-GIMEMA relapse risk criteria, 22%, 23% and 70% of patients assigned in the low-IS (n=46), intermediate-IS (n=57) and high-IS (n=34) groups were deemed high-risk of relapse (P<0.001). Overall, 118 (86%) patients achieved CR; the remaining 19 patients (14%) experienced early death due to hemorrhage (n=12), therapy-related infection (n=6) and differentiation syndrome (n=1). Induction mortality was significantly higher in the high-IS group (low: 2%; intermediate: 15%; high: 26%) (P=0.001). CR was achieved in the low-, intermediate-, and high-IS group in 98%, 84%, and 73% of the patients, respectively (P=0.007). With a follow-up of 24 months among survivors, patients assigned in the high-IS group had a lower 2-y OS rate (63%) compared with those in the intermediate- (80%) and low-IS groups (97%; P<0.001). Eight relapses were recorded. The IS was not predictive of relapses (P=0.351). Conclusions: Our results suggest that MLL5, BAALC, ID1, and WT1 expression levels are associated with clinical outcome and that the IS may become a useful tool for outcome prediction in APL. Disclosures: Lo-Coco: Cephalon: Speakers Bureau; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees. Löwenberg:Skyline Diagnostics: Membership on an entity's Board of Directors or advisory committees.

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Natural History Of Inhibitor Recurrence Following Successful Immune Tolerance Induction

Blood ◽

10.1182/blood.v122.21.1106.1106 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1106-1106 ◽

Cited By ~ 2

Author(s):

Ana G. Antun ◽

Paul Monahan ◽

Marilyn J. Manco-Johnson ◽

Michael Callaghan ◽

Guy Young ◽

...

Keyword(s):

Board Of Directors ◽

Immune Tolerance ◽

Half Life ◽

Research Funding ◽

Tolerance Induction ◽

Advisory Committees ◽

Immune Tolerance Induction ◽

Baxter Healthcare ◽

Inhibitor Titer

Abstract Introduction The formation of Factor VIII (FVIII) inhibitory antibodies is a major complication of hemophilia A. Currently immune tolerance induction (ITI) is successful in up to 70% of patients. Outside of the International Immune Tolerance Registry, where 6 of 128 patients had a recurrent inhibitor between 1 and 6 years, little is known about the probability of inhibitor recurrence following successful ITI. Objective To determine the probability of inhibitor recurrence and the influence of adherence to post-ITI prophylaxis on inhibitor recurrence following successful ITI. Methods All persons with hemophilia A (FVIII level < 50%) who completed ITI (defined as inhibitor titer <0.6 BU/ml) between 1/1/1998 and 8/15/2010 at 12 U.S. Hemophilia Treatment Centers were identified. Demographic and clinical characteristics were obtained through review of subject medical records and included age at start of ITI, race, ethnicity, hemophilia severity, peak inhibitor titer prior to the start of ITI and ITI regimen. For those subjects where tolerance was confirmed with measurement of FVIII half-life > 6 hours and/or FVIII recovery > 66% in addition to inhibitor titer < 0.6 BU/ml, information was also collected on post-ITI prophylaxis regimen, adherence to post-ITI prophylaxis, and the presence of a recurrent inhibitor titer (≥ 0.6 BU/ml) or last inhibitor titer prior to 8/15/2011. Adherence during the 6 months prior to inhibitor recurrence or last inhibitor titer was determined by review of pharmacy and infusion logs compared with prescribed treatment regimen. Follow-up time started when the subject was considered tolerized (normalized half-life or recovery if half-life not performed) and ended at the time of inhibitor recurrence or the last recorded inhibitor titer. Estimates of the probability of remaining inhibitor-free at 1, 3 and 5 years were calculated with the Kaplan-Meier method. The association between adherence (completing >80% of prescribed infusions vs. < 80% of prescribed infusions) and inhibitor recurrence was assessed using the chi-square test. Results Eighty-three male subjects were enrolled. The median age at start of ITI was 3.3 years (range: 0.08 - 39). The majority of the subjects were white (73%) and non-Hispanic (73.5%). Seventy-one (85.6%) had severe hemophilia. The median peak inhibitor titer was 8.5 BU/ml (range: 0.6 - 950). Four subjects (5%) had a prior unsuccessful course of ITI. FVIII alone was used in 85% of subjects. Sixty-seven (80.7%) met criteria for tolerance and 64 had follow-up data available, with a median follow up time of 3.4 years (range: 0.08-12.4). Forty-four subjects (68.7%) remained tolerant without a recurrent inhibitor titer after a median 4.7 years (range: 0.25-12.4) of follow-up. Twenty subjects (31.3%) had at least one inhibitor titer ≥ 0.6 BU/ml after a median of 1.6 years (range 0.08-5.7). The probability of recurrent inhibitor at 1 year is 0.15 (95% CI: [0.05, 0.20]); at 3 years is 0.30 (95% CI: [0.2, 0.4]) and 5 years is 0.35 (95% CI: [0.2, 0.5]) (Figure 1). Four subjects discontinued post-ITI prophylaxis anywhere from 6 months to greater than 6 years after tolerance was achieved, of whom 2 (50%) developed a recurrent inhibitor. Of those that remained on post-ITI prophylaxis, 41 subjects (64.1%) were adherent (took >80% of prescribed infusions) to their post-ITI prophylaxis regimen, of whom 13 (31.7%) developed a recurrent inhibitor. Twenty-three (35.9%) who were non-adherent (took <80% of the prescribed infusions) of which 7 (30.4%) subjects developed a recurrent inhibitor; no statistically significant association was found between adherence and inhibitor-free status (p=0.92). Conclusion ITI is currently the most effective treatment to eradicate FVIII inhibitors, however 5 years after completion, 30-35% of patients will have at least one inhibitor titer ≥ 0.6 BU/ml. A recurrent inhibitor is unlikely after 5 years. Adherence to post-ITI prophylaxis does not appear to be a major driver of inhibitor recurrence. It is imperative to elucidate the factors that influence the durability of successful ITI to improve quality of life and cost of treatment in these patients. Disclosures: Monahan: Baxter: Consultancy, Honoraria, Research Funding, travel support, travel support Other; Bayer: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Novo Nordisk: Consultancy, Honoraria, Research Funding; Pfizer: Honoraria; Prolor Biotech: Research Funding; Asklepios: Consultancy, Research Funding, travel support Other. Manco-Johnson:Eisai: Research Funding; Novo Nordisk: Membership on an entity’s Board of Directors or advisory committees; Biogen Idec: Membership on an entity’s Board of Directors or advisory committees; Baxter BioScience: Membership on an entity’s Board of Directors or advisory committees; CSL Behring: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bayer HealthCare: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Carpenter:Novo Nordisk: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Grifols: Honoraria, Research Funding. Kruse-Jarres:Bayer HealthCare: Consultancy; Biogen IDEC: Consultancy; Grifols: Consultancy; Kedrion: Consultancy; Novo Nordisk: Consultancy; Baxter Healthcare: Consultancy. Ragni:Novo Nordisk: Research Funding; Merck: Research Funding; CSL Behring: Research Funding; Bayer: Research Funding; Baxter: Research Funding; Tacere Benitec: Consultancy; Smith Kline Glaxo: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Biogen Idec: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding. Kempton:Novo Nordisk: Research Funding; Baxter Healthcare: Membership on an entity’s Board of Directors or advisory committees.

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Lin28B/Let-7 Axis Regulates Multiple Myeloma Proliferation By Enhancing c-Myc and Ras Survival Pathways

Blood ◽

10.1182/blood.v122.21.273.273 ◽

2013 ◽

Vol 122 (21) ◽

pp. 273-273

Author(s):

Salomon Manier ◽

John T Powers ◽

Antonio Sacco ◽

Michaela R Reagan ◽

Michele Moschetta ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Advisory Board ◽

Loss Of Function ◽

Advisory Committees ◽

Expression Levels ◽

Gain And Loss

Abstract Background MicroRNAs (miRNAs) play a pivotal role in tumorigenesis, due to their ability to target mRNAs involved in the regulation of cell proliferation, survival and differentiation. Lin28B is an RNA binding protein that regulates Let-7 miRNA maturation. Lin28B and Let-7 have been described to act as oncogenes or tumor suppressor genes, respectively, as demonstrated both in solid cancer and hematologic malignancies. However, the role of the Lin28B/Let-7 axis in Multiple Myeloma (MM) has not been studied. Method Lin28B level expression in MM patients was studied using previously published gene expression profiling (GEP) datasets. Let-7 expression levels were assessed in CD138+ primary MM cells and bone marrow stromal cells (BMSCs) by using PCR, as well as in circulating exosomes using miRNA array (Nanostring® Technology). Exosomes were collected from both normal and MM peripheral blood, using ultracentrifugation; and further studied by using electron microscopy and immunogold labeling for the detection of CD63 and CD81. The knockdown of Lin28B was performed on MM cell lines (U266, MM.1S, MOLP-8) by using a lentiviral Lin28B shRNA. Gain- and loss-of function studies for Let-7 were performed using Let-7 mimic and anti-Let-7 transfection in MM cell lines (MM1S, U266) and primary BMSCs. Cell proliferation has been evaluated by using thymidine assays. Effects of Let-7 and Lin28B on signaling cascades have been evaluated by western blot. Results Two independent GEP datasets (GSE16558; GSE2658) were analyzed for Lin28B expression, showing a significantly higher level in MM patients compared to healthy controls. In addition, high Lin28B levels correlated with a shorter overall survival (p = 0.0226). We next found that the Let-7 family members are significantly down-regulated in MM primary cells, particularly Let-7a and b (5 fold change, p < 0.05), as demonstrated by using qRT-PCR. Similarly, miRNA arrays showed a lower expression of Let-7-related miRNAs in circulating exosomes obtained from MM patients compared to healthy individuals. We further dissected the functional relevance of Lin28B in MM cells, by performing Lin28 knockdown (KD) in MM cell lines (U266, MOLP-8). This led to a significant decrease in MM cell proliferation associated with G1 phase cell cycle arrest. This was supported by up-regulation of Let-7 and down-regulation of c-Myc, Ras and Cyclin D1 in Lin28 KD MM cells. To further prove that Lin28B-dependent effects on MM cells are mediated by Let7, we next showed that let-7 gain- and loss-of-function studies regulate MM cell proliferation and Myc expression. Lin28B regulation in MM cells is dependent on Let-7, as demonstrated by an increase of both cell proliferation and c-Myc expression after anti-Let-7 transfection in the Lin28B KD cells. We therefore studied the regulation of Let-7 in MM cells through the interaction with BMSCs. Let-7 expression levels were significantly lower in BMSCs obtained from MM patients compared to healthy donors. Interestingly, the Let-7 expression level in MM cells was increased after co-culture with Let-7 over-expressing BMSCs, associated with a decrease of both cell proliferation and c-Myc expression. This suggests a potential transfer of Let-7 from BMSCs to MM cells. Conclusion This work describes a new signaling pathway involving Lin28B, Let-7, Myc and Ras in MM. Let-7 expression in MM cells is also regulated through the interaction of MM cells with BMSCs, leading to cell proliferation and Myc regulation in MM. Interference with this pathway might offer therapeutic perspectives. Disclosures: Leleu: CELGENE: Honoraria; JANSSEN: Honoraria. Daley:Johnson and Johnson: Consultancy, Membership on an entity’s Board of Directors or advisory committees; MPM Capital: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Verastem: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Epizyme: Consultancy, Membership on an entity’s Board of Directors or advisory committees; iPierian: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Solasia, KK: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

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Eradication of neutralizing antibodies to factor VIII in canine hemophilia A after liver gene therapy

Blood ◽

10.1182/blood-2010-06-288001 ◽

2010 ◽

Vol 116 (26) ◽

pp. 5842-5848 ◽

Cited By ~ 100

Author(s):

Jonathan D. Finn ◽

Margareth C. Ozelo ◽

Denise E. Sabatino ◽

Helen W. G. Franck ◽

Elizabeth P. Merricks ◽

...

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Neutralizing Antibodies ◽

Viral Vectors ◽

Hemophilia A ◽

Major Complication ◽

Complete Disappearance ◽

Anamnestic Response ◽

Endogenous Expression ◽

Liver Gene

Abstract Inhibitory antibodies to factor VIII (FVIII) are a major complication in the treatment of hemophilia A, affecting approximately 20% to 30% of patients. Current treatment for inhibitors is based on long-term, daily injections of large amounts of FVIII protein. Liver-directed gene therapy has been used to induce antigen-specific tolerance, but there are no data in hemophilic animals with pre-existing inhibitors. To determine whether sustained endogenous expression of FVIII could eradicate inhibitors, we injected adeno-associated viral vectors encoding canine FVIII (cFVIII) in 2 strains of inhibitor hemophilia A dogs. In 3 dogs, a transient increase in inhibitor titers (up to 7 Bethesda Units [BU]) at 2 weeks was followed by continuous decline to complete disappearance within 4-5 weeks. Subsequently, an increase in cFVIII levels (1.5%-8%), a shortening of clotting times, and a reduction (> 90%) of bleeding episodes were observed. Immune tolerance was confirmed by lack of antibody formation after repeated challenges with cFVIII protein and normal protein half-life. A fourth dog exhibited a strong early anamnestic response (216 BU), with slow decline to 0.8 BU and cFVIII antigen detection by 18 months after vector delivery. These data suggest that liver gene therapy has the potential to eradicate inhibitors and could improve the outcomes of hemophilia A patients.

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Interim Results from a Phase 1/2 Clinical Study of Lentiglobin Gene Therapy for Severe Sickle Cell Disease

Blood ◽

10.1182/blood.v128.22.1176.1176 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1176-1176 ◽

Cited By ~ 19

Author(s):

Julie Kanter ◽

Mark C. Walters ◽

Matthew M. Hsieh ◽

Lakshmanan Krishnamurti ◽

Janet Kwiatkowski ◽

...

Keyword(s):

Gene Therapy ◽

Board Of Directors ◽

Research Funding ◽

Phase 1 ◽

Globin Gene ◽

Advisory Committees ◽

Cell Dose ◽

Cd34 Cells ◽

Equity Ownership

Abstract β-globin gene transfer into hematopoietic stem cells (HSCs) has the potential to reduce or eliminate the symptoms and long-term complications of severe sickle cell disease (SCD). LentiGlobin Drug Product (DP) is a gene therapy product containing autologous CD34+ cells transduced with the BB305 lentiviral vector. BB305 encodes a human β-globin gene containing a single point mutation (AT87Q) designed to confer anti-sickling properties similar to those observed in fetal hemoglobin (γ-globin). In two ongoing studies, subjects with transfusion-dependent β-thalassemia (Studies HGB-204 and HGB-205) or SCD (Study HGB-205) receiving LentiGlobin DP have demonstrated sustained expression of 3-9 g/dL therapeutic hemoglobin (HbAT87Q) and have shown marked improvements in clinical symptoms 1 year post-treatment. Study HGB-206 is a multi-center, Phase 1/2 safety and efficacy study of LentiGlobin DP in adults with severe SCD. We previously (ASH 2015) presented results from 2 subjects, who had 3 and 6 months of follow-up after LentiGlobin treatment. We now present data from 7 treated subjects, 4 of whom have ≥6 months of follow-up data. Subjects (≥18 years of age) with severe SCD (history of recurrent vaso-occlusive crisis [VOC], acute chest syndrome, stroke, or tricuspid regurgitant jet velocity of >2.5 m/s) were screened for eligibility. Following bone marrow harvest (BMH), CD34+ cells were transduced with the BB305 vector. Subjects underwent myeloablative conditioning with busulfan prior to infusion of the transduced cells. Safety assessments include adverse events (AEs), integration site analysis (ISA) and surveillance for replication competent lentivirus (RCL). After infusion, subjects are monitored for hematologic engraftment, vector copy number (VCN), HbAT87Q expression, and other laboratory and clinical parameters. As of July 2016, 7 subjects with severe SCD (median age: 26 years, range 18-42 years) have received LentiGlobin DP in this study. All subjects successfully underwent BMH, with a median of 2 harvests required (range 1-4). Fifteen Grade 3 AEs in 5 subjects were attributed to BMH: pain (n=10), anemia (n=3) and VOC (n=2); all resolved with standard measures. Table 1 summarizes cell harvest, DP characteristics, and lab results. The median LentiGlobin DP cell dose was 2.1x10e6 CD34+ cells/kg (range 1.6-5.1) and DP VCN was 0.6 (0.3-1.3) copies/diploid genome. Median post-infusion follow-up as of July 2016 is 7.1 months (3.7-12.7 months). All subjects successfully engrafted after receiving LentiGlobin DP, with a median time to neutrophil engraftment of 22 days (17-29 days). The toxicity profile observed from start of conditioning to latest follow-up was consistent with myeloablative conditioning with single-agent busulfan. To date, there have been no DP-related ≥Grade 3 AEs or serious AEs, and no evidence of clonal dominance or RCL. The BB305 vector remains detectable at low levels in the peripheral blood of all subjects infused, with median VCN 0.08 (0.05-0.13, n=7) at last measurement. All subjects express HbAT87Q, with a median of 0.4g/dL (0.1-1.0 g/dL, n=7) at 3 months; most subjects demonstrated modest increases over time, and the 2 subjects with the longest follow-up expressed 0.31 and 1.2 g/dL HbAT87Q at 9 months. All 4 subjects with ≥6 months of follow-up experienced multiple VOCs in the 2 years prior to study entry (2-27.5 VOCs annually). Since LentiGlobin DP infusion, 3 of these 4 subjects have had fewer VOCs, although this trend may be confounded by the short follow-up, the effects of transplant conditioning, and/or post-transplant RBC transfusions. The decrease in VCN between DP and peripheral cells contrasts with previous reports of successful LentiGlobin gene therapy in ongoing studies HGB-204 and HGB-205. The relatively low in vivo VCN in this study appears to result in the lower HbAT87Q expression seen to date. We are exploring multiple hypotheses as to the etiology of the VCN drop between DP and peripheral blood, including the adverse impact of sickle marrow pathology on HSCs, the adequacy of myeloablation, and the magnitude of the transduced cell dose. We will provide an update on study data and ongoing efforts to increase in vivo VCN in patients with SCD, such as increasing the transduced cell dose through alternate HSC procurement methods or enhancing the DP VCN through manufacturing improvements. Disclosures Kanter: Novartis: Consultancy. Walters:Bayer HealthCare: Honoraria; AllCells, Inc./LeukoLab: Other: Medical Director ; ViaCord Processing Laboratory: Other: Medical Director ; Leerink Partners, LLC: Consultancy; Kiadis Pharma: Honoraria; bluebirdBio, Inc: Honoraria. Kwiatkowski:Ionis pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Shire Pharmaceuticals: Consultancy; Sideris Pharmaceuticals: Consultancy; Apopharma: Research Funding; Luitpold Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. von Kalle:bluebird bio: Consultancy; GeneWerk: Equity Ownership. Kuypers:Children's Hospital Oakland Research Institute: Employment; bluebird bio: Consultancy. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Joseney-Antoine:bluebird bio: Employment, Equity Ownership. Asmal:bluebird bio: Employment, Equity Ownership. Thompson:bluebird bio: Consultancy, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Amgen: Research Funding; Baxalta (now part of Shire): Research Funding; ApoPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mast: Research Funding; Eli Lily: Research Funding.

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