Search

In sickle cell disease (SCD), the β6 Glu→Val substitution leads to sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) represents a promising therapeutic option for patients lacking a compatible donor. We previously designed a lentiviral vector (βAS3 LV) expressing a potent anti-sickling βAS3 globin and demonstrated its safety and efficacy in SCD patient cells (Weber et al., 2018). In vitro and in vivo preclinical studies demonstrated the safety and efficacy of a gene therapy (GT) protocol based on the efficient transduction of plerixafor-mobilized SCD HSPCs by βAS3 LV. Based on these results a Phase 1/2 clinical trial (NCT03964792) started in January 2020. Three homozygous SCD patients (P1, P2 and P3) were recruited. Before GT, patients suffered from severe SCD symptoms that were not controlled despite of a strict adherence to the treatment. Plerixafor-mobilized HSPCs were transduced achieving a VCN of 0.9±0.2 in liquid culture, 1.6±0.3 in BFU-E and 0.8±0.2 in CFU-GM. The patients were hypertransfused to reach HbS levels Here, for the 3 patients we report the follow-up of 18 (P1), 10 (P2) and 6 (P3) months. No drug-related severe adverse events were observed. Importantly, no sign of clonal hematopoiesis was detected by NGS sequencing performed on the graft before and after transduction. Despite of the similar VCN in the drug product, gene marking in peripheral blood mononuclear cells was variable and ranged between 0.2 and 0.6, as measured at the last follow-up (0.4 in P1, 0.6 in P2 and 0.2 in P3). These results pinpoint the difficulty to estimate the gene marking, self-renewal and engraftment potential of HSCs in the grafts. P1 and P2 clinically benefit from the GT treatment despite they presented non-severe vaso-occlusive crises (VOCs) not requiring hospitalization (two for P1 at month 6 and 14 and one in P2 at month 8 post-GT). Despite the rapid resolution of these episodes, P1 started hydroxyurea at month 10 and stopped it at month 16. P2 initiated a phlebotomy program to treat liver iron overload and decrease viscosity. Intravascular hemolysis improved in P1 and P2, as shown by a decrease in plasma hemoglobin and heme, and an increase in hemopexin (HPX). Interestingly, in P1 a mild worsening occurred at the time of VOC. In P1 and P2, total bilirubin, a marker of extravascular hemolysis, decreased post-GT. Deformability, measured using osmoscan, improved in P1 and to a lesser extent in P2; however, both still exhibited some degree of dehydration compared to normal RBCs (Fig. 1). Adhesion to thrombospondin decreased after GT with an increase during VOC in P1 or prior the VOC in P2 (Fig. 1). In both patients, markers of inflammation (e.g., C reactive protein) and reticulocyte counts decreased upon GT. An in vitro sickling assays was performed for P1 and showed significant improvement, reaching a frequency of sickling cells similar to SCD heterozygous carriers (from 88% before GT to 45% 6 months post-GT). The GT treatment was not effective in P3 due to the modest intake and rapid decline of gene modified cells. In P1 and P2, who were no longer on transfusions, HbAS3 levels correlated with the VCN (2.1 g/dl for P1 and 3.3 g/dl for P2) and the therapeutic Hb accounted for 21.5%, and 28.7% of the total hemoglobins, respectively. For P3, who remained transfusion-dependent, this frequency was Overall, these clinical data indicate a variable efficacy of the DREPAGLOBE GT treatment, which likely depends on the extent of gene marking achieved in HSCs in vivo and on the engraftment capability of genetically modified HSCs in SCD patients' bone marrow. Single-cell RNA-seq analysis of HSPCs and evaluation of the bone marrow niche in SCD patients will aid to define critical parameters for achieving successful outcomes in GT clinical trials for SCD. Figure 1 Figure 1. Disclosures Joseph: bluebird bio: Consultancy. El Nemer: Hemanext: Consultancy. Bartolucci: INNOVHEM: Other: Co-founder; AGIOS: Consultancy; GBT: Consultancy; Bluebird: Consultancy, Research Funding; Jazz Pharma: Other: Lecture fees; Fabre Foundation: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Steering committee, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy; Hemanext: Consultancy; Addmedica: Consultancy, Other: Lecture fees, Research Funding; Emmaus: Consultancy. Cavazzana: Smart Immune: Other: co-founder.

Background LentiGlobin gene therapy contains autologous CD34+ hematopoietic stem cells (HSCs) transduced with the BB305 lentiviral vector (LVV), encoding human β-globin with a T87Q substitution. This substitution confers anti-sickling properties to the gene therapy-derived hemoglobin (HbAT87Q) and allows for its quantification in transduced HSCs. The proof of concept for LentiGlobin gene therapy in patients with transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) was established in the recently completed HGB-205 study (NCT02151526). Herein, we provide the safety and efficacy outcomes and long-term follow-up data for all 7 treated patients, 4 with TDT and 3 with SCD. Methods Patients 5−35 years old with TDT (≥ 100 mL/kg of packed red blood cells [pRBCs]/year) or severe SCD (e.g., ≥ 2 acute chest syndromes [ACS] or ≥ 2 vaso-occlusive crises in the preceding year or the year before regular transfusions) were enrolled. CD34+ HSCs were obtained by mobilization and apheresis in patients with TDT or by bone marrow harvest in patients with SCD. Following collection, cells were transduced with the BB305 LVV. Patients underwent busulfan myeloablative conditioning and were infused with transduced cells. Patients were monitored for engraftment, adverse events (AEs), HbAT87Q levels, and other hematologic and clinical parameters. After 2 years in HGB-205, patients transitioned into the long-term follow-up study, LTF-303 (NCT02633943). Summary statistics are shown as median (min-max). Results As of June 2019, patients with TDT (n=4) and SCD (n=3) had a median follow-up of 49.6 (40.5-60.6) and 28.5 (25.5-52.5) months, respectively. Table 1 shows patient and drug product characteristics and several key efficacy outcomes. All patients achieved HSC engraftment. LentiGlobin safety profile was consistent with busulfan myeloablative conditioning and, in case of SCD, with the underlying disease state. The most common non-hematologic Grade ≥ 3 AEs post-LentiGlobin gene therapy (≥ 2 patients) for patients with TDT were stomatitis (n=4) and increased aspartate aminotransferase (n=2), and for patients with SCD were ACS (n=2) and vaso-occlusive pain (n=2). In all 4 patients with TDT, total Hb and HbAT87Q levels remained generally stable up to 5 years post-LentiGlobin infusion. Three of 4 patients achieved transfusion independence (TI; defined as weighted average Hb ≥ 9g/dL without pRBC transfusions for ≥ 12 months), for an ongoing duration of 56.3 (38.2-57.6) months. Weighted average total Hb during TI was 11.4 (10.5-13.0) g/dL. One patient has been off transfusions for 37.5 months and had total Hb of 7.7 g/dL, which was below the ≥ 9 g/dL requirement to meet the protocol definition of TI. At last visit, HbAT87Q levels in these 4 patients ranged from 6.2-11.2 g/dL, which contributed 73.8-86.8% of the total Hb. The first patient treated with LentiGlobin for SCD experienced one vaso-occlusive pain episode, which developed at 30 months after LentiGlobin gene therapy following a case of acute gastroenteritis with fever and dehydration. The second SCD patient had 2 serious AEs (SAEs) of ACS approximately 6 and 8 months after LentiGlobin gene therapy. The patient resumed chronic pRBC transfusions and hydroxyurea treatment and subsequently experienced 2 SAEs of vaso-occlusive pain; no additional SAEs of vaso-occlusive pain or ACS were reported during the last 16 months of follow-up after LentiGlobin infusion. The third SCD patient had no episodes of vaso-occlusive pain or ACS during 25.5 months of follow-up post-LentiGlobin gene therapy as of the data cut-off. Two patients with SCD who have been off chronic pRBC transfusions, showed improvement in hemolysis markers post-LentiGlobin treatment and stabilization of HbAT87Q expression at approximately 6 months post-LentiGlobin infusion. Total Hb levels for patients with SCD at last visit were 13.0 g/dL (patient 1), 9.4 g/dL (patient 2), and 9.8 g/dL (patient 3), with corresponding HbAT87Q contributions of 47.9%, 7.9%, and 25.8%, respectively. Summary With up to 5 years of follow-up, treatment with LentiGlobin gene therapy was well tolerated and resulted in improvement in hematologic parameters and disease-related symptoms. Further results from the completed study will be presented. Disclosures Hermine: Celgene: Research Funding; Novartis: Research Funding; AB science: Consultancy, Equity Ownership, Honoraria, Research Funding. Brousse:bluebird bio, Inc: Consultancy; AddMedica: Consultancy. El Nemer:Hemanext: Other: Other. Bartolucci:Novartis: Membership on an entity's Board of Directors or advisory committees; AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; HEMANEXT: Membership on an entity's Board of Directors or advisory committees. Asmal:bluebird bio, Inc: Employment, Equity Ownership. Whitney:bluebird bio, Inc: Employment, Equity Ownership. Gayron:bluebird bio, Inc: Employment, Equity Ownership. Huang:bluebird bio, Inc.: Employment, Equity Ownership. de Montalembert:AddMedica: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Ribeil:bluebird bio, Inc: Employment, Equity Ownership. Cavazzana:SmartImmune: Other: Founder.

Introduction. Runt-related transcription factor 1 (RUNX1) is a transcription factor critical for normal and leukemic hematopoiesis. Three main isoforms, RUNX1 i-1a, i-1b and i-1c, are produced through alternative splicing of RUNX1. Unlike i-1b and i-1c, i-1a has a Runt homology domain (RHD) but lacks the transactivation domain (TAD), and inhibits transcriptional activity of -1b by competing with higher affinity for target gene sequences. Overexpression of i-1a in cell lines inhibits terminal myeloid differentiation. The i-1a is expressed at high levels in patients (pts) with acute myeloid leukemia (AML). Mutations of RUNX1 are common in CMML and MDS, whereas incidence and clinical correlates of RUNX1 mutations in MPNs are poorly known. Aim: To analyze the spectrum and clinical correlates of RUNX1 genetic abnormalities (RUNX1-abn), and the expression of RUNX-1a isoform, in a large series of pts with MPNs. Methods. 686 MPN pts (50 PV, 165 ET, 154 overt-PMF, 143 prefibrotic-PMF, 74 PPV-MF and 100 PET-MF, diagnosed according to 2016 WHO) were included in the study; of these, 625 were analyzed in chronic phase (CP) and 61 at leukemic transformation (LT; of a total of 106 LTs, 58%), including 29 cases with paired samples at diagnosis and LT. Established methods were used for JAK2, MPL and CALR genotyping. A high molecular risk (HMR) category was defined according to Vannucchi A, [Leukemia 2013]. Ion Torrent NGS platform was used to genotype all RUNX1 coding sequence. In 21/29 paired samples, a long reads genome sequencing by Oxford Nanopore technology (ONT) was performed. Copy number variation (CNV) analysis was performed with NanoGLADIATOR package [Magi A, Genome Biol 2019]. Differential expression of RUNX1 mRNA isoform1a and 1b was analyzed by qRT-PCR. Nonparametric Wilcoxon rank-sum test, Kaplan-Meier estimates and log-rank test were used as appropriate. Results. Somatic RUNX1 mutations were identified in 31 pts (4.5%): 14/625 in CP (2.2%) and 17/61 in LT (27.9%; P1 of these genes in 16/21 (76%) pts at LT vs 2/16 (12.5%) of CP pts. After median follow-up of 4.9y (range 0.1-36.2y) 273 pts died (39.8%). 106 pts (15.4%) progressed to AML after median of 4.7y (0.1-36.2y) from diagnosis. Incidence of LT differed significantly among diseases: 3 pts in PV (5.9%), 17 in ET (10.1%), 39 in overt-PMF (23.5%), 12 in prefibrotic-PMF (8.3%), 11 in PPV-MF (14.1%) and 24 in PET-MF (22.6%). RUNX1-abn were detected, respectively, in 100%, 53.0%, 23.1%, 41.7%, 36.4% and 12.5% of cases at either CP and/or LT. Leukemia-free survival (LFS) was shorter in pts harboring RUNX1-abn in CP compared to RUNX1-WT pt (2.8y vs NR; P Expression levels of i-1a and -1b were measured in 34 CP and 24 LT pts. Isoform-1a was significantly elevated in LT pts (mean 7.51±10.1) compared to CP (1.12±1.75; P=.009) (Fig 1B; lines represent the i-1a level rise from CP to LT in 4 paired samples). The i-1a expression levels were irrespective of RUNX1 mutated (mean 7.6±11.9) or unmutated (mean 9.2±6.3) status in LT pts (P=0.8). No difference of i-1b levels in CP (mean 0.9±0.7) versus LP (mean 1.6±1.4; P=0.08) was observed. The expression of i-1a was not dependent on gender, age, leukocyte or platelet count, hemoglobin and percentage of blast cells. Conclusions. These findings indicate that most MPN pts with LT have involvement of RUNX1 pathway through different mechanisms (mutations, CNV, altered isoform expression, genomic abnormalities of RUNX1 target genes), suggesting that a "RUNX1pathy" may represent a common event during leukemogenesis in MPNs. Figure 1 Disclosures Vannucchi: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

Background. Progression to acute myeloid leukemia (sAML) occurs in 20% of myelofibrosis (MF) and 10% of polycythemia vera (PV) or essential thrombocythemia (ET). sAML has dismal outcome with median survival of Aim. To study clonal heterogeneity and clonal progression in the leukemic transformation of MPN we performed whole exome (WES) and trascriptome (RNAseq) sequencing of paired samples (chronic (CP)/blast phase(BP). Patients and Methods. WES and RNAseq was done on CD34+ or blast cells of 12 paired samples from 5 PMF, 3 PET-MF, 1 PPV-MF, 3 ET pts who transformed to sAML. In addition, a myeloid neoplasm-relevant 34-gene panel was used for target NGS sequencing of paired samples plus an additional 23 samples collected from MPN pts at leukemic phase (15 PMF, 3 PET-MF, 3 ET, 2 PV). Results. In the entire series, 20 pts (57%) were JAK2 V617F mutated at CP, 8 CALR (23%; 6 type-1 and 2 type-2) and 7 (8.6%) MPL mut, while 4 (11.4%) were triple-negative. JAK2 variant allele frequency (VAF) declined by ≥50% (n=3; 15%) or became undetectable (n=4; 20%) whereas 3 heterozygous pts (15%) became homozygous at the time of BP. One of the 8 CALR mut cases halved its VAF, whereas no meaningful VAF changes were observed in MPL mut pts. The most frequent mutated genes detected by NGS at BP, other than driver mutations, were ASXL1 (51.4%), RUNX1 (37.1%), TET2 (17%), SRSF2 (16%), IDH1 (14%), TP53 (14%), NRAS (14%), FLT3 (14%), U2AF1 (11%) and KRAS (11%); three (8%) cases were mutated for EZH2DNMT3A, CBL and PTPN11, 2 cases for IDH2, SH2B3, SF3B1 IKZF1, SETBP1 and ZRSR2, and 1 case for ABL1, ATV6, BRAF and ARID1A. There were also 5 CEBPA-mutated pts, 3 of which, unlike none of 27 CEBPA-mutated de novo AML, showed an in-frame 6-bp duplication polymorphism (p. P196_197insHP) reported in 20-40% of all de novo AML and By WES analysis, on average 60.000 variants per paired sample unique to the BP compared with CP were identified. However, no recurrent abnormalities were found in the 12 paired samples outside the above listed mutations found by target sequencing. For RNAseq analysis, transcripts were annotated according to UCSC hg19 for a total of 25,369 genes. We found large transcriptional differences between CP and BP with 129 transcripts differentially expressed (23 up- and 106 down-regulated) and 4,155 isoforms (2,120 up- and 2,035 down-regulated). Among the most abnormally expressed transcripts we selected 8 genes (5 down-regulated: LCN2, PDGFB, PRTG, CRISP3, PF4; 3 up-regulated CDKN2, SH2D1A and LIN28) for validation by QRT-PCR based on the extent of differential expression. PF4 and CDKN2 were confirmed to be down-regulated and over-expressed in BP, respectively (P Conclusions. Comprehensive comparative analysis of genomic and RNA abnormalities acquired in the transition from CP to BP in MPN has not been previously reported. Our data indicate that BP is associated with significant changes in mutation complexity and RNA expression, overall affecting different intracellular pathways whose further characterization might help to identifying potential targets for therapy. Disclosures No relevant conflicts of interest to declare.

Background The hemoglobin (Hb) response to the activin receptor type IIA ligand trap ACE-011 (Sotatercept) in non-transfusion-dependent thalassemia (NTDT), and the transfusion requirement response in TDT are described, but the mechanisms of action, clinical predictors and markers of response, are unclear. In principle, ACE-011 may act on early and/or late erythroblasts to decrease ineffective erythropoiesis (IE), both in healthy subjects and in thalassemia. As erythropoiesis intimately links to iron metabolism, changes in markers of iron metabolism relative to those of erythropoiesis may inform the mechanisms and developmental stage at which ACE-011 acts. Methods Markers of IE and iron metabolism in 46 thalassemia patients (30 NTDT, 16 TDT) were taken before and during a median follow up of 722.5 days, (IQR 830.5, range 152-1427) of escalating doses of ACE-011 (3-weekly 0.1 to 1.0 mg/kg s.c. injections), depending on the protocol, as part of the approved study (Cappellini, et al. 2018 under review). Markers of erythropoiesis included Hb, Reticulocytes (Ret), soluble transferrin receptor 1 (sTfR), growth differentiation factor 11 and 15 (GDF11, GDF15), erythroferrone (ERFE). Markers of iron metabolism included plasma hepcidin, serum ferritin (SF), transferrin saturation (TSAT), and non-transferrin-bound iron (NTBI). Data were analyzed using longitudinal multilevel model for change (LMMC) on STATA (Version 14) as generalized linear mixed model with random and fixed effects to account for repeated measures and highly complex temporal data structure. Final LMMCs were built to explain the change in Hb from baseline and the behavior of key biomarkers. Independent variables were entered into the models based on the study design (predictors from design) and theoretical background (predictors of interest and control variables). P value Results Predictors of response. In NTDT, Hb response associated positively with dose and duration of exposure (both at p Biomarker changes with time In NTDT, significant changes with time on study were increases in Hb, sTfR, ERFE and Ret and decreases in hepcidin, bilirubin, and NTBI. GDF15 showed no change. In TDT, GDF15, sTfR and ERFE increased, whereas hepcidin decreased. The Hb change was insignificant in TDT (as expected per protocol) and was dose-independent, however the mean 41% reduction in transfusion iron load rate (ILR) was dose dependent, implying an equivalent net production of Hb in TDT (Table 1). Other significant relationships Absolute hepcidin level in NTDT and TDT was negatively predicted by ERFE (p Interpretation and conclusions NTDT patients allow a cleaner interpretation of biomarker changes as these are confounded in TDT by increased bone marrow stress from less transfusion. In NTDT, sTfR and ERFE (total erythropoiesis) increase on study while GDF15 (IE) and EPO do not. Thus Hb gain may result from increased effectiveness of late stage erythropoiesis (sTfR+ and ERFE+) possibly from decreased apoptosis and more rapid maturation (Carrancio et al, BJH 2014) in this compartment. We speculate that increased survival of those erythroid progenitor cells expressing TfR (and hence sTfR) and ERFE (hence increased ERFE) also explains the observed hepcidin reduction. Despite exposure to lower hepcidin, there is no iron loading: NTBI falls against stable SF in NTDT, consistent with shunting of iron into the sink of effective erythropoiesis. The reduction in GDF11 in vivo, not previously reported for ACE-011, is also consistent with increased apoptosis of the early progenitor pool and with improved erythroblast differentiation relative to proliferation as suggested in murine β-thalassemia treated with ACE-011 (Dussiot, et al. Nat Med 2014). Disclosures Garbowski: Vifor: Consultancy. Hermine:Erythec: Research Funding; AB Science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene Corporation: Research Funding; Hybrigenics: Research Funding; Novartis: Research Funding. Cappellini:Sanofi/Genzyme: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Vifor: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Origa:Bluebird Bio: Consultancy; Novartis: Honoraria; Cerus Corporation: Research Funding; Apopharma: Honoraria. Forni:Celgene: Research Funding; Novartis: Other: travel expenses, Research Funding; Shire: Research Funding; Roche: Consultancy; Apopharma: Other: DSM Board. Voskaridou:Acceleron: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corp: Membership on an entity's Board of Directors or advisory committees, Research Funding. Galactéros:Addmedica: Other: grant; Novartis: Other: grant. Taher:Novartis: Consultancy, Honoraria, Research Funding; La Jolla Pharmaceutical: Research Funding; Protagonist Therapeutics: Consultancy; Celgene Corp.: Research Funding; Ionis Pharmaceuticals: Consultancy. Ribeil:bluebird bio: Consultancy; Vitalaire: Other: grant; Addmedica: Other: grant; Cydan: Consultancy; Novartis: Consultancy. Laadem:Celgene: Employment, Equity Ownership. Miteva:Celgene Corporation: Employment, Other: grants. Zou:Celgene Corporation: Employment, Equity Ownership. Zinger:Celgene Corporation: Employment. Schwickart:Celgene Corporation: Employment, Equity Ownership. Sung:Celgene Corporation: Employment, Equity Ownership. Porter:Novartis: Consultancy; Cerus: Honoraria; Agios: Honoraria.

Endothelial cells (EC) were extracted through a lectin-based method from bone marrow of 57 patients with active multiple myeloma (MM) and compared with their healthy quiescent counterpart, human umbilical vein EC (HUVEC). MMECs exhibit specific antigens that indicate ongoing angiogenesis and embryo vasculogenesis; solid intercellular connections, hence stability of MM neovessels; and frequent interactions with plasma cells, hence tumor dissemination. They show heterogeneous antigen expression, hence existence of subsets. Their main genetic markers are indicative of a vascular phase. They show intrinsic angiogenic ability, because they rapidly form a capillary network in vitro, and extrinsic ability, because they generate numerous new vessels in vivo. They vividly secrete growth and invasive factors for plasma cells. They signal through kinases mandatory for development of neovascularization. Ultrastructurally, they are abnormal and show metabolic activation, like tumor ECs. Thalidomide heavily interferes with their functions. Vasculogenesis and angiogenesis might contribute to the MM vascular tree and progression, in the form of growth, invasion, and dissemination. In view of the heterogeneity of the antigenic phenotype of MMECs, a mixture (or a sequence) of antiangiogenic agents coupled with thalidomide would seem plausible for the biologic management of MM.

Introduction: β-globin gene transfer into hematopoietic stem cells (HSCs) has the potential to reduce or eliminate the symptoms of severe sickle cell disease (SCD) and reduce or eliminate transfusion requirements in transfusion-dependent β-thalassemia (TDT). LentiGlobin Drug Product (DP) contains autologous CD34+ cells transduced with the BB305 lentiviral vector, which encodes a human β-globin gene containing a single point mutation (AT87Q) designed to confer anti-sickling properties similar to those observed with γ-globin. We previously reported proof of concept for LentiGlobin DP treatment in severe SCD and early data from 4 treated patients with TDT. We now report 18 months of follow-up for the patient with SCD and between 9 and 30 months of follow-up for the 4 patients with TDT. Patients (5-35 years of age) with severe SCD (e.g. ≥2 acute chest syndrome episodes or ≥2 vaso-occlusive crises [VOC] in preceding year/in year prior to regular transfusions) or TDT (≥100mL/kg of packed red blood cells [RBCs] per year) were enrolled. Following mobilization and apheresis (for TDT) or bone marrow harvest (for SCD), autologous CD34+ cells were transduced with the BB305 lentiviral vector. Patients underwent myeloablative conditioning with busulfan prior to infusion of the transduced cells. After infusion, patients were monitored for hematologic engraftment, vector copy number (VCN), and HbAT87Q expression. Disease-specific assessments included transfusion requirements for TDT, or VOCs and hospitalizations for SCD. Safety assessments included adverse events (AEs) and integration site analysis. Results: As of July 2016, 1 patient with severe SCD (male; 13 years old) and 4 patients with TDT (2 male, 2 female; 16-19 years old) have received LentiGlobin DP in Study HGB-205. The median LentiGlobin DP cell dose was 8.9 (range 5.6-13.6) x 106 CD34+ cells/kg with a DP VCN of 1.2 (range: 0.8-2.0) vector copies/diploid genome. Median post-infusion follow-up as of July 6, 2016 is 20.8 months (range 9.5-31.3). All subjects successfully engrafted after receiving LentiGlobin DP, with a median time to neutrophil engraftment of 17 days (range 14-38 days). VCN in peripheral blood has remained generally consistent from Month 3 in all patients with a range of 0.2 to 3.4 at last measurement. The toxicity profile observed from start of conditioning to latest follow-up remains consistent with myeloablative conditioning with single-agent busulfan, with no DP-related ≥Grade 3 AEs or serious AEs and no evidence of clonal dominance reported to date. Three patients with TDT have β0/βE genotypes and 1 is homozygous for the severe β+ mutation IVS1 nt 110 G>A. The 2 patients who have completed the 2-year primary follow-up period (both β0/βE) have not required RBC transfusions for 31 and 28 months, with total Hb of 10.9 and 13.5 g/dL, and HbAT87Q expression of 7.7 and 10.1 g/dL, respectively, at most recent study visit. Iron chelation has been discontinued and phlebotomy initiated for 1 of the patients. The remaining patient with β0/βE genotype has 9 months of follow-up and has not required transfusions since 4 days post-LentiGlobin DP infusion, achieving a total Hb of 11.3 g/dL at last study visit. The patient with the severe IVS1 genotype has 12 months of follow-up and has been free of transfusions for 9 months, with a total Hb of 8.3 g/dL at last study visit. The patient with severe SCD, who prior to study enrollment received regular RBC transfusions, has experienced no clinical symptoms or complications of SCD in the 18 months since treatment, despite discontinuing transfusions 3 months after LentiGlobin DP infusion. Total Hb in this patient was 12.5 g/dL, with 6.6 g/dL HbAT87Q (53%) and 5.7 g/dL HbS (45%) at last study visit. Compared with values at screening, unconjugated bilirubin had dropped 78% (50 to 11 μmol/L), lactate dehydrogenase had dropped 54% (626 to 287 U/L), and reticulocyte count had dropped 45% (238x109/L to 132x109/L) by Month 18. Conclusions: Data from this ongoing Phase 1/2 clinical study suggest that treatment with LentiGlobin DP can result in sustained production of therapeutic HbAT87Q, which ameliorates the clinical and biochemical effects of severe SCD and TDT, with an acceptable safety profile. Gene therapy presents a potentially promising therapy for patients with severe hemoglobinopathies. Further follow-up and additional data from patients are needed to confirm the encouraging results seen to date in this study. Disclosures Ribeil: Bluebirdbio: Consultancy; Addmedica: Research Funding. Payen:bluebird bio: Patents & Royalties. Hermine:Alexion: Research Funding; Celgene: Research Funding; Novartis: Research Funding; AB science: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau. Asmal:bluebird bio: Employment, Equity Ownership. Joseney-Antoine:bluebird bio: Employment, Equity Ownership. De Montalembert:Addmedica: Research Funding; Novartis: Research Funding, Speakers Bureau. Leboulch:bluebird bio, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process.

Factor XI deficiency (FXI-Def) is associated with mild, unpredictable bleeding tendency. FXI is believed to participate in a thrombin-dependent positive feed-back that leads to the formation of a second burst of thrombin, which, in turn, inhibits fibrinolysis mainly through TAFI activation. It is conceivable, therefore, that FXI-Def leads to the formation of clots that are more susceptible to lysis. Our study was undertaken to test this hypothesis. Eighteen subjects with FXI-Def (1-58%), and 17 matched controls were studied. All assays were performed on citrate-plasma stored at -80°C. The fibrinolytic resistance to t-PA (30 ng/ml) of clots generated by 0.5 pM tissue factor was lower in samples from FXI-Def subjects than in controls (lysis time: 61.1 ± 9.7 vs 74.9 ± 14.9 min, P=0.008). Generation of thrombin and TAFIa during fibrinolysis was markedly reduced in FXI-Def samples ( Disclosures Peyvandi: NovoNordisk: Research Funding, speaker's fee Other; CSL Behring: speaker's fee, speaker's fee Other; LFB: speker's fee, speker's fee Other; Grifols: speaker's fee, speaker's fee Other; Bayer: speaker's fee Other; Baxter: speaker's fee Other; Biotest: Research Funding; Kedrion biopharma: Research Funding. Colucci:Boehringer Ingelheim : Research Funding.

The prothrombin gene mutation G20210A is a common risk factor for thrombosis and is associated with increased prothrombin levels. However, the mechanism whereby hyperprothrombinemia predisposes to thrombosis remains unclear. Because thrombin is the physiologic activator of TAFI (thrombin activatable fibrinolysis inhibitor), the precursor of an antifibrinolytic carboxypeptidase (TAFIa), we evaluated the influence of hyperprothrombinemia on fibrinolysis. Thirty-two heterozygous carriers of the G20210A mutation and 30 noncarriers were studied. Plasma fibrinolytic factors and TAFI levels were similar in the 2 groups. Mean lysis time of tissue factor–induced plasma clots exposed to 25 ng/mL exogenous tissue-type plasminogen activator (t-PA) was significantly longer in 20210A carriers than in control donors. This difference disappeared on addition of a specific inhibitor of TAFIa. Determination of thrombin and TAFIa activity, generated during clot lysis, revealed that G20210A mutation was associated with a significant enhancement of late thrombin formation and an increase in TAFI activation. Plasma prothrombin level was highly significantly correlated with both clot lysis time and TAFI activation. The addition of purified prothrombin, but not of factors X or VIIa, to normal plasma caused a concentration-dependent, TAFI-mediated inhibition of fibrinolysis. These findings provide a new mechanism that might contribute to the thrombotic risk in prothrombin 20210A carriers.

Plasminogen activator inhibitors (PAIs) play a pivotal role in the control of fibrinolysis. The mechanisms regulating the plasma levels of PAI(s) are still unknown. We report here that the infusion of bovine thrombin (1 U/kg/min, over 60 minutes) in rabbits treated with 0.5 microgram/kg endotoxin (to induce an increase in circulating fast- acting PAI) causes a marked reduction of PAI (50% of preinfusion value), as indicated by functional assay and reverse fibrin autography. Moreover, blood clots prepared from samples obtained after thrombin infusion lysed faster than preinfusion clots when exposed, in vitro, to tissue plasminogen activator. Donor-receiver transfusion experiments showed that the half-life of circulating PAI activity was shorter in thrombin-infused rabbits than in controls (4.1 minutes versus 7.4 minutes), suggesting an accelerated clearance. As expected, thrombin infusion resulted also in activation of protein C (PC). The following observations suggest a close relationship between PC activation and PAI reduction. (1) Infusion of thrombin in rabbits made deficient in vitamin K-dependent plasma proteins by warfarin treatment did not result in modification of PAI activity. (2) Treatment of the latter animals with a barium citrate eluate (PE) of rabbit plasma restored both the anticoagulant and profibrinolytic response to thrombin. (3) Short infusion of thrombin-activated PE (containing activated PC, PCa), but not of unactivated PE, induced both anticoagulation and reduction of PAI activity. In vitro, incubation of PAI-rich rabbit serum with thrombin-activated PE and phospholipids resulted in a progressive disappearance of PAI activity with a t1/2 of 30 minutes. However, this slow inactivation rate does not fully explain the results obtained in vivo. Our data suggest that thrombin infusion in rabbits causes a reduction of circulating PAI activity and that activation of PC is the intermediary mechanism involved in this phenomenon.

Plasminogen activator inhibitors (PAIs) play a pivotal role in the control of fibrinolysis. The mechanisms regulating the plasma levels of PAI(s) are still unknown. We report here that the infusion of bovine thrombin (1 U/kg/min, over 60 minutes) in rabbits treated with 0.5 microgram/kg endotoxin (to induce an increase in circulating fast-acting PAI) causes a marked reduction of PAI (50% of preinfusion value), as indicated by functional assay and reverse fibrin autography. Moreover, blood clots prepared from samples obtained after thrombin infusion lysed faster than preinfusion clots when exposed, in vitro, to tissue plasminogen activator. Donor-receiver transfusion experiments showed that the half-life of circulating PAI activity was shorter in thrombin-infused rabbits than in controls (4.1 minutes versus 7.4 minutes), suggesting an accelerated clearance. As expected, thrombin infusion resulted also in activation of protein C (PC). The following observations suggest a close relationship between PC activation and PAI reduction. (1) Infusion of thrombin in rabbits made deficient in vitamin K-dependent plasma proteins by warfarin treatment did not result in modification of PAI activity. (2) Treatment of the latter animals with a barium citrate eluate (PE) of rabbit plasma restored both the anticoagulant and profibrinolytic response to thrombin. (3) Short infusion of thrombin-activated PE (containing activated PC, PCa), but not of unactivated PE, induced both anticoagulation and reduction of PAI activity. In vitro, incubation of PAI-rich rabbit serum with thrombin-activated PE and phospholipids resulted in a progressive disappearance of PAI activity with a t1/2 of 30 minutes. However, this slow inactivation rate does not fully explain the results obtained in vivo. Our data suggest that thrombin infusion in rabbits causes a reduction of circulating PAI activity and that activation of PC is the intermediary mechanism involved in this phenomenon.

It has been suggested that cancer cell procoagulant activity influences metastasis formation by promoting fibrin deposition around tumors. We have investigated the procoagulant activity of various tumor cell sublines with different metastatic capacity derived from metastatic nodules of a murine fibrosarcoma. All the cells tested possessed a marked thromboplastin-like activity; they were, however, heterogeneous as regards the degree of procoagulant activity; the two cell lines with virtually no metastatic capacity showed 6--8 times higher procoagulant activity than the cells from the parent line; in contrast, the procoagulant activity of the two sublines with higher metastatic capacity did not differ significantly from that of the parent line. These findings support the hypothesis that fibrin is part of a defence reaction against cancer cell invasiveness.

Intralipid, a fat emulsion widely used in parenteral nutrition, can produce marked functional changes of the mononuclear phagocyte system. We investigated the effect of Intralipid administration on the generation of procoagulant activity by rabbit mononuclear phagocytes. Two groups of ten rabbits given either a single infusion of Intralipid 10% or a similar volume of sterile saline were studied before and after infusion. Procoagulant activity was measured on isolated blood mononuclear cells after incubation with and without endotoxin, using a one-stage clotting assay. Cells from animals infused with Intralipid produced significantly more procoagulant activity than controls (P less than .01). Results were similar when freshly collected whole blood was incubated with and without endotoxin, and procoagulant activity was measured on subsequently isolated mononuclear cells (P less than .01). In addition, when rabbits were given a single injection of endotoxin, blood and spleen mononuclear cells harvested 50 to 60 minutes after the injection from animals pretreated with Intralipid expressed five to seven times more procoagulant activity than did cells from animals pretreated with saline. In all instances, procoagulant activity was identified as tissue factor. These findings suggest that Intralipid may cause functional changes in mononuclear phagocytes, resulting in increased production of tissue factor on incubation in short-term culture in vitro and in response to endotoxin in vivo.

This study investigated the procoagulant activity (PCA) of mononuclear phagocytes from rabbits bearing the V2 carcinoma. Macrophages harvested from either intraperitoneally or subcutaneously growing tumors were found to express a very strong procoagulant activity as compared with peritoneal macrophages and circulating mononuclear cells from the same animals. On the other hand, when incubated with or without endotoxin in short-term cultures, circulating mononuclear cells from tumor-bearing animals generated significantly more procoagulant activity than those from control animals. In all instances, procoagulant activity was identified as tissue factor by using assay systems with plasmas selectively deficient in the various clotting factors. These results indicate that, besides cancer cells, mononuclear phagocytes might also play an important role in the activation of blood coagulation and in the deposition of fibrin at the host-tumor interface.