ALBERT

Description: Sperm parameters are known to be impaired in men with sickle cell disease (SCD). Although treatment with hydroxyurea (HU) impacts sperm quality, sperm preservation is impossible before puberty. The present study's primary objective was to analyze and compare sperm parameters in male SCD patients exposed (or not) to HU before puberty. Twenty-six sperm samples from 15 patients (median (range) age: 17 (16-23)) treated with HU during childhood were compared with 46 samples from 23 HU-naïve patients (median (range) age: 20 (16-24)). The median (range) age at HU initiation was 6 years (1-14), the median duration of HU treatment was 4 years (0.5-10) and the mean ± standard deviation dose of HU was 22.4 ± 3.7 mg/kg/day. Although we observed substantial quantitative and qualitative semen abnormalities in all patients, there were no significant differences in semen volume, sperm concentration, total sperm count, and spermatozoa motility, morphology and vitality between the HU-exposed and HU-naïve groups. At the time of the semen analysis, respectively 100% and 52% of the patients in the HU-exposed and HU-naïve groups were on transfusion therapy. Hydroxyurea's specific effect on spermatogenesis in very young infants and the putative value of transfusion for reversing HU's toxicity warrant further investigation.

Description: Background β-globin gene transfer into hematopoietic stem cells (HSCs) could reduce or eliminate sickle cell disease (SCD)-related manifestations. LentiGlobin for SCD gene therapy contains autologous CD34+ cells transduced with the BB305 lentiviral vector (LVV), encoding a human β-globin gene with the anti-sickling T87Q mutation (βA-T87Q). The safety and efficacy of LentiGlobin for SCD is being evaluated in the ongoing Phase 1/2 HGB-206 Study (NCT02140554). The initial 7 patients (Group A) were treated with LentiGlobin made from bone marrow harvested HSCs. The protocol was modified to improve HbAT87Q production by including pre-harvest red blood cell (RBC) transfusions, increasing the total busulfan exposure, and using a refined LentiGlobin manufacturing process (Group B, n=2). An additional modification was made for Group C patients where HSC collection by plerixafor mobilization followed by apheresis was instituted. Data from these Group C patients are discussed here. Results from patients in Groups A and B are reported separately. Methods Patients (≥ 18 years) with severe SCD (including those with recurrent vaso-occlusive crisis [VOC] and acute chest syndrome [ACS]) were screened for eligibility. Patients received 240 µg/kg of plerixafor 4-6 hours prior to HSC collection via apheresis. CD34+ cells were transduced with BB305 LVV. Patients underwent myeloablative busulfan conditioning and subsequent LentiGlobin drug product (DP) infusion. Patients were monitored for adverse events (AEs), engraftment, vector copy number (VCN), total hemoglobin (Hb) and HbAT87Q expression, hemolysis markers, and SCD clinical manifestations. Data are presented as median (min-max). Results: As of 7 March 2019, 19 Group C patients, aged 26 (18-36) years, had initiated mobilization/apheresis and 13 patients were treated with LentiGlobin for SCD gene therapy. Median DP VCN, % transduced cells, and CD34+ cell dose in the 13 treated patients were: 3.8 (2.8-5.6) copies/diploid genome (c/dg), 80 (71-88) %, and 4.5 (3.0-8.0) x 106 CD34+ cells/kg, respectively. The median follow-up was 9.0 (1.0-15.2) months. Twelve patients achieved neutrophil and platelet engraftments at a median of 19 (15-24) days and 28 (19-136) days, respectively. As of the data cut-off, engraftment was not yet evaluable in 1 patient at 1-month post-infusion. All patients stopped red blood cell (RBC) transfusions within about 3 months post-LentiGlobin gene therapy. Median total hemoglobin (Hb) and Hb fractions in patients at various time points are shown in Figure 1. Median HbS levels were at or below 50% in all patients with at least 6 months follow-up. The median total Hb at last visit in 8 patients with at least 6 months of follow-up, was 11.5 (10.2-15.0) g/dL, with a corresponding HbAT87Q median contribution of 5.3 (4.5-8.8) g/dL and a median HbS 5.7 (4.8-8.0) g/dL. Of these 8 patients, 6 had a history of VOCs or ACS. The median annualized VOC+ACS rate in these patients was 5.3 (3-14) pre-treatment and decreased to 0 (0-2) post-treatment. One Grade 2 VOC was observed 3.5 months post-treatment. No ACS or serious VOCs were observed in Group C patients' post- treatment. Lactate dehydrogenase, reticulocyte count, and total bilirubin at last visit post-LentiGlobin infusion were 225.0 (130.0-337.0) U/L, 150.0 (42.1-283.0) 109/L, 22.2 (3.42-39.3) µmol/L, respectively, trending towards normalization. The most common non-hematologic Grade ≥ 3 AEs were febrile neutropenia (n=10) and stomatitis (n=7) post-DP infusion. Serious AEs were reported in 6 patients post-LentiGlobin treatment, most common being nausea and vomiting. To date, there have been no DP-related AEs or graft failure, vector-mediated replication competent lentivirus detected, or clonal dominance reported. Longer follow-up and additional patient data will be presented. Summary The safety profile of LentiGlobin gene therapy for SCD remains consistent with single-agent busulfan conditioning and underlying disease. Patients in HGB-206 Group C experienced high-level, sustained expression of gene-therapy derived hemoglobin, with median HbS levels reduced to ~50% and median total Hb levels of 11.5 g/dL at 6 months. The cessation of clinical complications (no ACS or serious VOCs) and decreased hemolysis suggest a strong therapeutic effect after LentiGlobin gene therapy in patients with SCD. Disclosures Kanter: Peerview: Honoraria; NHLBI: Membership on an entity's Board of Directors or advisory committees; Rockpointe: Honoraria; SCDAA: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Imara: Consultancy; Jeffries: Consultancy; Modus: Consultancy; Guidepoint Global: Consultancy; GLG: Consultancy; Cowen: Consultancy; bluebird bio, Inc: Consultancy; Medscape: Honoraria; Sangamo: Consultancy. Kwiatkowski:Terumo: Research Funding; Novartis: Research Funding; Apopharma: Research Funding; Imara: Consultancy; Celgene: Consultancy; bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy. Schmidt:German Cancer Research Center, Heidelberg, Germany: Employment; GeneWerk GmbH, Heidelberg, Gemrany: Equity Ownership. Miller:bluebird bio, Inc.: Employment, Equity Ownership. Pierciey:bluebird bio, Inc.: Employment, Equity Ownership. Huang:bluebird bio, Inc.: Employment, Equity Ownership. Ribeil:bluebird bio, Inc.: Employment, Equity Ownership. Thompson:Baxalta: Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; bluebird bio, Inc.: Consultancy, Research Funding. Walters:AllCells, Inc: Consultancy; TruCode: Consultancy; Editas Medicine: Consultancy.

Description: Hydroxycarbamide (HU) is a myelosuppressive drug marketed since 1968 for the treatment of hematological cancer, and authorized since 2007 in Europe as orphan medicinal product for the prevention of recurrent vaso-occlusive crises including acute chest syndrome in adults and children older than 2 years with sickle cell disease (SCD). ESCORT-HU (European Sickle Cell Disease Cohort – Hydroxyurea) is a multicenter prospective non interventional study implemented in Europe to collect more information about the safety profile of HU and morbi-mortality in SCD patients treated with HU. The study responds to EMA (European Medicines Agency) request and has been approved by the Ethical of Necker Enfants Malades Hospital (Paris, France).The ongoing study involves the largest number so far of patients with SCD treated with HU. Primary endpoints of ESCORT HU are to determine frequency of adverse events, and possible consequent changes of HU treatment. Secondary endpoints are to evaluate morbi-mortality of the disease although in the absence of control group. From June 2008 to June 2014, 483 patients (255 females; 228 males) were enrolled from 3 European countries, Greece (24%), Germany (19%), and France (56%). 67% patients were adults, median aged 37.35 yrs (17-83.5) and 33% were children, median aged 11.06 yrs (2.6-16.9). genotypes were HbS/HbS in 71.4% cases, and compound heterozygotous HbS/β-thalassemia in 22.8 % (Table 1). 137 (28.4%) patients experienced 421 events (Table 2). 132 (32.2%) of these events may be attributed to HU. The safety profile is roughly similar in children and adults. As expected the most frequent side effects were firstly blood disorders (n=86 events, 42.4%) such as neutropenia or thrombocytopenia. In all cases, these cytopenias were rapidly resolved with the transitory stop of HU. 71 events related to skin and subcutaneous tissue disorders were observed, mostly cutaneous dryness, skin reactions, alopecia and nails or skin pigmentation; 4 patients had a leg ulcer (34.8%). Most of these events are ongoing or stabilized despit the decrease of HU. No secondary cancer has been reported until now. Even if HU is an old drug with a relatively well-known safety profile, some uncertainties remain in terms of long-term safety as well as tolerance in the youngest people. The main interest of ESCORT HU is to offer the possibility of safety surveillance of hydroxycarbamide in European sickle cell patients. Table 1 Demographic data Adults Children 〈 17 years old Total Number of patients 322 (67%) 161 (33%) 483 Females/Males 183/139 72/89 255/228 Median age (yrs) (range) 37.35 (17-83.5) 11.06 (2.6-16.9) 28.58 Genotype SS 206 (64%) 139 (86.3%) 345 (71.4%) SC 1 (0.3%) 3 (1.86%) 4 (0.8%) Sβ0 51 (15.8%) 11 (6.8%) 62 (12.8%) Sβ+ 46 (14.2%) 2 (1.2%) 48 (9.9%) Other 18 (5.5%) 6 (3.7%) 24 (4.9%) Treatment with HU before enrollment in ESCORT HU No of pts 232 83 315 (65%) Median duration (range) of HU treatment before ESCORT HU 8.2 yrs (0.5 ans-24 yrs) 3. 1 yrs ( 71 days – 8.9 yrs) 6.85 (71 days-24 years)] HU ESCORT Daily mean dose (mg/kg/d) 16.11 ± 4.79 19.63 ± 4.69 17.32 ± 4.94 Abstract 564. Table 2 The most frequent events of hydroxycarbamide in the two populations of SCD patients ADULTS CHILDREN No ofGerman(%) No of adults No ofEpisodes(%) No of children Total(% /411) Events Related to HU treatment (Siklos®)(%**) Blood and lymphatic system disorders (%) 32 (17.7) 22 54 (31.03) 28 86 (20.9) 56 (65.1) Skin and subcutaneous tissue disorders (%) 42 (23.2) 28 29 (16.7) 19 71 (17.3) 46 (64.8) Nervous system disorders Headache (24), Dizziness/vertigo (14), 32 (17.7) 23 12(6.9) 10 44 (10.7) 11 (25) Gastrointestinal disorders Nausea (14), diarrhea (8), other (14) 20 (11) 17 23 (13.2) 16 43 (10.4) 7 (16.3) Metabolic and nutrition disorders: vit D deficiency (17), weight gain (5) 13 (7) 11 18 (18.3) 18 36 (8.75) 4 (11.1) Fever 11 (6) 10 12(6.9) 7 23 (5.6) 1 (4.3) Cardiac disorders (hypertension, bradycardia, chest pain, cardiomegaly) 4 4 2 2 6 1 (16.6) General disorders : fatigue 5 5 0 0 5 0 Hepatobiliary disorders 2 2 0 0 2 0 Neoplasms benign, malignant and unspecified (incl. cysts and polyps) Harmatoma, benign vulvar sebaceous cyst 2 2 0 0 2 0 Renal & urinary disorders 2 2 0 0 2 0 Reproductive system and breast disorders 3 3 0 0 3 0 Other 13 13 21 14 34 6 (17.1%) 181 80 /181(24.8%) 174 57 / 174(35.4%) 411 132/411 (32.2%) ** compared to the total number of “system organ class” events Disclosures No relevant conflicts of interest to declare.

Description: The JAKV617F mutation is responsible for the majority of breakpoint cluster region (BCR)/Abelson (ABL)–negative myeloproliferative disorders. Ongoing clinical trials of Janus kinase 2 (JAK2) inhibitors in myeloproliferative disorder patients use small molecules targeting both wild-type and mutated JAK2. To selectively target malignant cells, we developed JAK2V617F-specific small interfering RNAs or short hairpin RNAs. Expression of these RNAs in cell lines or CD34+ cells from patients reduced JAK2V617F-driven autonomous cell proliferation. Mechanisms of inhibition involved selective JAK2V617F protein down-regulation, and consequently, decrease in signal transducer and activator of transcription 5 phosphorylation, cell-cycle progression, and cell survival. However, the addition of high concentrations of cytokines to cell lines or erythropoietin to patient cells greatly reduced growth inhibition. Similarly, the efficacy of a JAK2 small molecule inhibitor on cell line and patient cell proliferation dose dependently decreased with the addition of cytokines. Our results demonstrate that it is possible to specifically target JAK2V617F by RNA interference (RNAi) strategies. In addition, cytokines partially reverse the inhibition induced by both RNAi and small molecule approaches. This strongly suggests that patient cytokine levels in current JAK2 inhibitor clinical trials modulate the outcome of these therapies.

Description: Background LentiGlobin for SCD gene therapy contains autologous CD34+ hematopoietic stem cells (HSCs) transduced with the BB305 lentiviral vector (LVV) encoding β-globin with an anti-sickling substitution (T87Q). Its safety and efficacy are being studied in the ongoing multi-center Phase 1/2 HGB-206 trial (NCT02140554). Initial patients received LentiGlobin drug product (DP) using bone marrow-harvested (BMH) CD34+ HSCs transduced with the BB305 LVV under original manufacturing. All patients had successful engraftment but levels of gene therapy-derived hemoglobin (HbAT87Q) were lower than expected. Despite this, hemolysis markers and the annualized rate of vaso-occlusive crisis (VOCs) plus acute chest syndrome (ACS) were reduced post-infusion. Here, we provide an update on these patients and explore factors that may contribute to the clinical benefit. Data on patients treated more recently are presented separately. Methods Adults with SCD-related complications (previously described) were enrolled in HGB-206. All patients received myeloablative busulfan conditioning before DP infusion. The initial group (Group A; N=7) was treated with DP from BMH HSCs using the original LentiGlobin manufacturing process. The protocol was amended, and 2 patients were subsequently treated in Group B with DP from BMH HSCs. Patient 1 had DP made with original and refined manufacturing processes and patient 2 had DP made only with the refined process. Patients were followed for 2 years in HGB-206 and offered participation in the LTF-303 study for long-term follow-up. Adverse events (AEs), Hb fractions, and additional laboratory and clinical parameters were monitored. Results As of 7 March 2019, the median follow-up post-DP infusion was 35.8 (min-max: 29.8-44.5) months in Group A; it was 17.2 and 20.2 months for Group B patients 1 and 2. There was full hematological recovery with no graft failure. The safety profile of LentiGlobin post-DP infusion was consistent with myeloablative busulfan conditioning and underlying SCD. No cases of Grade ≥ 3 DP-related AEs, veno-occlusive liver disease, vector-mediated replication competent lentivirus or clonal dominance were observed. Three years after LentiGlobin gene therapy, one Group A patient developed myelodysplastic syndrome, reported as unlikely related to LentiGlobin. The patient subsequently received an HLA-haploidentical donor transplant. The annualized rate of VOCs plus ACS from LentiGlobin infusion to last follow-up was reduced by a median of 89 (min-max 10-100) % compared to that in the 2-year interval before enrollment in the 8 patients who had a history of VOC and/or ACS. One Group A patient who had a stroke pre-DP infusion has not had any red blood cell (RBC) transfusions through ~3 years post-DP infusion and no stroke recurrence. Median HbAT87Q levels were 1.0 (min-max 0.7-2.8) g/dL for Group A and 3.4 g/dL for Group B patient 1 at last visit and were stable for up to 3.5 years follow-up (Figure 1A). In several patients, HbF levels increased post-LentiGlobin treatment, peaking at ~2-6 months, and remaining higher at last visit compared to 1-month post-DP infusion. At last visit, the median HbF level was 0.6 (min-max 0.1-1.8) g/dL for all Group A and 0.8 g/dL for Group B patient 1 (Figures 1B and 2). In these patients, the median fraction of anti-sickling Hb (HbAT87Q + HbF + HbA2) was 22.9 (min-max 14.2-29.8) % and 39.4%, respectively. Patient 2 in Group B, who had DP made entirely using refined manufacturing process, had no HbF but produced high levels of HbAT87Q (7.5 g/dL) that contributed to 53.7% of total Hb. Summary In the HGB-206 Group A patients, the modest expression of gene therapy-derived HbAT87Q is accompanied by an induction of HbF. Elevated HbF levels have been shown to be associated with reduced severity of SCD. The resulting 14-30% of anti-sickling Hb observed in Group A patients, while not likely to be curative, showed clinical benefit as suggested by a reduction in the annualized rate of VOC plus ACS. Further, Group A and B patients have maintained HbAT87Q production, demonstrating the durability of gene therapy-derived β-globin gene expression. There have been no Grade ≥ 3 DP-related AEs in LentiGlobin-treated patients with up to 3.5 years of follow-up. Longer follow-up will help determine whether the initial induction of HbF, as is common after myeloablation, will be sustained and continue to contribute to therapeutic anti-sickling Hb levels. Disclosures Walters: Editas Medicine: Consultancy; TruCode: Consultancy; AllCells, Inc: Consultancy. Kwiatkowski:Apopharma: Research Funding; Imara: Consultancy; Celgene: Consultancy; bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy; Terumo: Research Funding; Novartis: Research Funding. Schmidt:German Cancer Research Center, Heidelberg, Germany: Employment; GeneWerk GmbH, Heidelberg, Gemrany: Equity Ownership. Miller:bluebird bio, Inc.: Employment, Equity Ownership. Pierciey:bluebird bio, Inc.: Employment, Equity Ownership. Huang:bluebird bio, Inc.: Employment, Equity Ownership. Ribeil:bluebird bio, Inc: Employment, Equity Ownership. Kanter:Rockpointe: Honoraria; GLG: Consultancy; Guidepoint Global: Consultancy; Novartis: Consultancy, Honoraria; Imara: Consultancy; Sangamo: Consultancy, Honoraria; Modus: Consultancy, Honoraria; Medscape: Honoraria; Peerview: Honoraria; bluebird bio, Inc.: Consultancy; SCDAA: Membership on an entity's Board of Directors or advisory committees; NHLBI: Membership on an entity's Board of Directors or advisory committees; Jeffries: Consultancy; Cowen: Consultancy. Thompson:bluebird bio, Inc.: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Baxalta: Research Funding.

Description: Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative syndrome characterised by the expression of the recombinant oncoprotein Bcr-Abl. Imatinib inhibits Bcr-Abl tyrosine kinase activity leading to apoptosis of leukemic cells sparing normal hematopoiesis. Several mechanisms of resistance to imatinib have been identified both in vitro and in vivo: Bcr-Abl mutations, an over-expression of the Bcr-Abl kinase itself or other tyrosine kinase bypass. To identify unknown mechanism, we used an imatinib resistant cell line (K562-R) generated from the erythroblastic cell line K562 (K562-S) (Blood, 2000; 93: 1070–1079) for which all described mechanisms of resistance have been previously invalidated. Previous results from a proteomic study identified some chaperon proteins such as heat shock proteins with an increased expression level in K562-R. One of them, the heat shock protein 70, Hsp70, has a 3 fold increase expression level in K562-R cells, results which have been confirmed by western-blot analysis. To characterise the role of Hsp 70 in imatinib resistance, we inhibit Hsp 70 expression by RNA silencing (siRNA) in K562-R cells and over-express it in K562-S cells. Inhibition of Hsp70 protein expression by siRNA decrease Hsp70 expression rapidly over 90% at day 4 which is associated with a significant reduction of viability (66 ± 6%, n = 5, p 〈 0.03). Over expression of Hsp 70 in K562-S cells induced a significant increase of resistance to imatinib since the addition of imatinib only increases mortality by 27 ± 5 % in comparison to 52 ± 4 % for K562-S cells (n = 4, p 〈 0.001). Detection of HSF-1 phosphorylation, the major transcription factor involved in Hsp 70 expression, did not show significant differences between K562-S and K562-R cells although over a 3 fold increase is detected in the mRNA level of Hsp 70 in K562-R cells by quantitative PCR. Furthermore, the comparison of Hsp70 expression in mononuclear cells of 7 CML patients before imatinib treatment and at the relapse time shows that Hsp 70 is increased in imatinib resistant patients suggesting it could also play a role in resistance in vivo. Present study confirmed that over expression of Hsp 70 in the cell line K562-R is involved in the mechanism of imatinib resistance in vitro. Moreover, the correlation between the increase of Hsp 70 in CML patient cells and resistance suggests it could be an interesting marker and potentially a therapeutic target.

Description: Abstract 247 Background: β-thalassemia is associated with ineffective erythropoiesis, accelerated erythroid differentiation and apoptosis resulting in anemia and iron overload. The molecular mechanism involved is still incompletely understood. Members of the TGF-β superfamily participate in both proliferation and differentiation of erythroid progenitors. However, the role of these molecules in models of ineffective erythropoiesis has not been addressed so far. RAP-011 is a ligand trap consisting of the extracellular domain of ActRIIA linked to mouse IgG1 Fc domain. We aimed to study the role of ActRIIA signaling in the ineffective erythropoiesis of β-thalassemia and to evaluate the therapeutic impact of RAP-011. Methods: Hbbth1/th1 mice (a model of β-thalassemia intermedia) were subcutaneously treated with RAP-011 (10mg/kg body weight) twice a week for 30–60 days and biological and biochemical parameters were followed. Results: RAP-011 treatment significantly increased hemoglobin levels, red blood cell counts, MCV, MCH and hematocrit with a concomitant decrease in bilirubin levels and reticulocyte counts (since 10 days of treatment and sustained until day 60 of follow up). Flow cytometry analysis showed that RAP-011 significantly decreased late basophilic and polychromatic erythroblast cell numbers in both bone marrow and spleen indicating that RAP-011 corrects ineffective erythropoiesis. We next evaluated the expression of putative ActRIIA ligand(s) in β-thalassemia. Increased expression of Growth Differentiation Factor 11 (GDF11) was observed in cultured erythroblasts and in spleen sections of thalassemic mice. RAP-011 treatment decreased these elevated GDF11 levels in both bone marrow and spleen. We further investigated how BMP/Activin signaling was involved in ineffective erythropoiesis. Anti-GDF11 antibodies, follistatin (activin and GDF11 antagonist) and dorsomorphin (a small molecule inhibitor of SMAD1/5/8 phosphorylation) reduced differentiation, induced FAS-L expression and apoptosis in erythroblasts both in vivo and in vitro whereas noggin (a BMP-2/4 antagonist) had no effect on erythroblast differentiation. Altogether, these data suggest that Activin/BMP signaling controls erythroblast differentiation and targeting BMP type II /activin type II receptors can decrease ineffective erythropoiesis of β-thalassemia. Summary: Sotatercept (a humanized version of RAP-011) is currently in phase II clinical trials for treatment of anemia in patients with Myeloma Bone Disease and End Stage Renal Disease and data from our non-clinical findings support a newly initiated β-thalassemia clinical trial. Our results suggest that sotatercept would be a potential therapeutic tool to improve anemia, increase hemoglobin levels and correct ineffective erythropoiesis and its side effects in β-thalassemic patients. Disclosures: Daniel: Celgene Corporation: Employment. Chopra:Celgene Corp: Employment, Equity Ownership. Sung:Celgene: Employment.