ALBERT

Description: Background Transfusion-dependent β-thalassemia (TDT) is a severe genetic disease caused by impaired β-globin production, leading to severe anemia, lifelong transfusion dependence with iron overload and serious comorbidities. Gene therapy (GT) offers a potentially transformative option for these patients. LentiGlobin GT contains autologous CD34+ hematopoietic stem cells (HSCs) transduced ex vivo with the BB305 lentiviral vector (LVV) encoding β-globin with a T87Q substitution. The safety and efficacy of LentiGlobin in patients with TDT was assessed in the phase 1/2 Northstar study in which 8/10 patients with non-β0/β0 genotypes and 3/8 patients with a β0/β0 genotype stopped transfusions. A refined manufacturing process to improve drug product (DP) characteristics is being evaluated in the studies presented here. Methods Northstar-2 (HGB-207; NCT02906202) and Northstar-3 (HGB-212; NCT03207009) are ongoing, international, single-arm, phase 3 studies in patients with TDT (≥ 100 mL/kg/yr of red blood cells [RBCs] or ≥ 8 RBC transfusions/yr) and non-β0/β0 genotypes or a β0/β0 genotype, respectively. HSCs were collected by apheresis after G-CSF and plerixafor mobilization. CD34+ HSCs were transduced with the BB305 LVV using a refined manufacturing process. Patients received single-agent, myeloablative busulfan conditioning and transduced cells were infused. The primary endpoint in Northstar-2 is the proportion of patients achieving transfusion independence (TI, weighted average hemoglobin [Hb] ≥ 9g/dL without RBC transfusions for ≥ 12 months continuously) and in Northstar-3 is the proportion of patients achieving transfusion reduction (≥ 60% reduction in transfused RBC volume post-DP infusion compared to pre-DP infusion). Patients were evaluated for engraftment, DP and peripheral blood vector copy number (VCN), GT-derived Hb (HbAT87Q), adverse events (AEs), vector integration, and evidence of replication competent lentivirus (RCL). Patients are followed for 2 years and offered participation in a long-term follow-up study. Results Eleven patients (median age 20 [min - max: 12 - 24] years) with TDT and non-β0/β0 genotypes (5 β+/β0, 4 βE/β0, 2 β+/β+) have been treated in Northstar-2 as of May 15, 2018 with a median follow-up of 8.5 (min - max: 0.3 - 16.2) months. DPs had a median cell dose of 7.4 x 106 (min - max: 5.0 - 19.4 x 106) CD34+ cells/kg, median VCN of 3.4 (min - max: 2.4 - 5.6) copies/diploid genome (c/dg) and a median of 82% (min - max: 53 - 90%) CD34+ cells were transduced. Median time to neutrophil and platelet engraftment was 21.5 (min - max: 16 - 28) and 44.5 (min - max: 34 - 84) days, respectively, in 10 patients; 1 patient was not yet evaluable. Serious AEs after DP infusion included 2 events of grade 4 liver veno-occlusive disease treated with defibrotide and 1 event each of hypotension, hypoxia, sepsis, and transfusion reaction, all resolved. Only 1 AE (grade 1 abdominal pain) was related to LentiGlobin. There were no deaths or graft failure and no evidence of vector-mediated RCL or clonal dominance. Of 8 patients with ≥ 6 months follow-up, 7 have stopped RBC transfusions. At last study visit, peripheral blood VCN was 1.1 - 5.0 c/dg and total Hb was 11.1 - 13.3 g/dL of which 7.6 - 10.2 g/dL (68 - 92%) was contributed by HbAT87Q. Median Hb at month 6 was 11.9 (min - max: 11.2 - 13.3) g/dL. The first treated patient achieved TI. The additional patient with ≥ 6 months follow-up had no transfusions for 11 months, however had a peripheral blood VCN of 0.2 c/dg and resumed transfusions due to symptomatic anemia. Bone marrow assessment of dyserythropoesis and data with longer follow-up will be presented. Two patients, 26- and 7- years old, have been treated in Northstar-3. Both had 2 DP lots manufactured with DP VCNs of 2.9/3.3 and 3.4/3.9 c/dg and 82%/85% and 78%/78% CD34+ cells were transduced, respectively. Both successfully engrafted. Additional data for these patients will be presented. Summary Seven of 8 patients with TDT and non-β0/β0 genotypes produced sufficient HbAT87Q to stop chronic transfusions following LentiGlobin GT in Northstar-2. The safety profile appears consistent with busulfan myeloablative conditioning with no grade ≥ 3 DP-related AEs. Initial results show DP characteristics in Northstar-3 are consistent with those in Northstar-2. Additional data from Northstar-3 will determine the impact of HbAT87Q production on transfusion reduction in patients without endogenous β-globin production. Disclosures Locatelli: bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Walters:AllCells Inc.: Other: Medical Director; ViaCord Processing Lab: Other: Medical Director; bluebird bio: Research Funding; Sangamo Therapeutics: Consultancy. Kwiatkowski:Terumo: Research Funding; Apopharma: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding; bluebird bio: Consultancy, Honoraria, Research Funding. Porter:Agios: Honoraria; Cerus: Honoraria; Novartis: Consultancy. Thuret:Addmedica: Research Funding; bluebird bio: Research Funding; Novartis: Research Funding. Kulozik:bluebird bio: Consultancy, Honoraria. Lal:Terumo Corporation: Research Funding; Celgene Corporation: Research Funding; Insight Magnetics: Research Funding; Bluebird Bio: Research Funding; La Jolla Pharmaceutical Company: Consultancy, Research Funding; Novartis: Research Funding. Thrasher:Orchard Therapeutics: Consultancy, Equity Ownership; Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Elliot:bluebird bio: Employment, Equity Ownership. Tao:bluebird bio: Employment, Equity Ownership. Asmal:bluebird bio: Employment, Equity Ownership. Thompson:Amgen: Research Funding; Baxalta/Shire: Research Funding; La Jolla Pharmaceutical: Research Funding; Novartis: Research Funding; bluebird bio: Consultancy, Research Funding; Celgene: Research Funding; Biomarin: Research Funding.

Description: Abstract 2155 Despite progress in chelation regimens, excess iron still results in deleterious effect on the reproductive system of thalassemia major (TM) patients; subsequently compromised fertility is common in TM men and women. We have recently assessed the reproductive potential and its relation to iron overload in TM women as measured by the current methods for ovarian reserve testing (ORT) demonstrating a reduced ovarian antral follicle count (AFC) and corresponding low anti mullerian hormone (AMH) levels (Blood PMID 21757620). In the current study we further explored a possible link between iron overload and oxidative stress on fertility potential in thalassemia. Increase in reactive oxygen species (ROS) production and decrease in antioxidant defense mechanisms are thought to be major causes for accelerated follicle aging over time. Excess redox active non-protein bound iron acts as potent catalyst for highly deleterious ROS, causing oxidative damage to plasma lipids, DNA and proteins, and is therefore likely to further accelerate follicle aging and loss of reproductive potential. However, such an effect in TM women has not been demonstrated. Extensive oxidation of intracellular Glutathione (GSH) affects the structural integrity of cells. The ratio of GSH to its oxidized disulfide (GSH/GSSG) has been shown to be a sensitive measure of oxidative stress in biological systems and was noted to be compromised in thalassemia. We studied 26 TM women (median age 28y); obtaining total non-transferrin bound iron (NTBI) and its low-molecular component, labile plasma iron (LPI). Liver iron concentration (LIC) was measured by superconducting quantum interference device (SQUID) biosusceptometer system. Fourteen women had a trans-vaginal ultrasound (TVUS) for assessment of AFC and for ovarian volume measurements. Plasma GSH, GSSG and GSH precursors were obtained from the TM women and from 12 age-matched healthy non-smoking women, all fasting, were assayed by liquid chromatography–electrospray positive ionization–tandem mass spectrometry (LC-MS/MS) as previously described. GSH concentration was ∼25% lower in TM compared to normal controls, mostly due to low concentration of free GSH; 3.88±2.8 (median 2.9) and 5.74±1.4 (median 5.86), respectively. This resulted in a plasma GSH/GSSG ratio drop by 45% from the control ratio of 79.8 to a ratio of 43.4 in the thalassemia cohort (p=0.04). Plasma GSSG levels remained similar in both groups; 0.1±0.07μM in controls and 0.15±0.1μM in thalassemia women. TM women had an increase in precursors of GSH compare to controls (Table 1), suggesting that key metabolic pathways to augment synthesis of GSH may be up-regulated as a compensatory response to increased oxidative stress. GSH/GSSG, an oxidative measure, significantly correlated with AFC and with ovarian volume (R=0.57, p

Description: Abstract 5174 The U.S. Government's “2008 Physical Activity Guidelines for Americans” recommends children perform at least 60 minutes of moderate to vigorous intensity physical activities (PA) a day while the recommendation for adults is 30 minutes per day at least 5 days a week. As fewer Americans meet these guidelines, it is unknown where patients with thalassemia (Thal) stand. Based on anecdotal evidence, it is assumed that patients with Thal have decreased PA due to their severe anemia and cardiomyopathies secondary to iron overload. However, there is a paucity of objective data on physical activity patterns in Thal. The primary objective of this prospective observational study is to examine PA patterns in a small group of representative patients with Thal using a 3-dimensional accelerometer, compared to age-matched national reference data. Additionally we will explore the relationship between PA and body composition. Methods: Nine pediatric (4 M; 13.8 ± 1.7 yrs) and 10 adult (6M, 31.0 ± 8.4 yrs) patients were provided an Actigraph GT3X accelerometer to wear for 7 days on 2 separate occasions, 3 months apart. Partial results from the baseline measurement will be presented here. Accelerometer output, defined as counts or records of acceleration, was valid if the patient wore the Actigraph for 〉10 hrs/day for at least 3 weekdays and 1 weekend day. Based on published criteria using mean counts per minute, time spent in sedentary, moderate and vigorous physical activity intensity was compared to National Health and Nutrition Examination Survey (NHANES) data from 2003–2004 and to the 2008 PA guidelines. Whole body lean and fat mass was assessed by Dual Energy X-ray Absorptiometry. Lean mass index (LMI = kg/m2) Z-score was calculated and compared to national averages from the NHANES study. Results: Preliminary results show that Thal are less active than their healthy counterparts. Adults with Thal spend less time in vigorous intensity activities compared to the pediatric patients (p

Description: The α-globin Constant Spring (CS) mutation (α142 STOP → Gln; TAA → CAA) is the most prevalent non-deletional thalassemia in south East Asia and southern China. DNA diagnosis of Hb H Constant Spring (Hb H CS; 2 α-gene deletions and 1 CS mutation) is often required because it can be missed by electrophoresis. The clinical phenotype of Hb H CS is more severe than classical Hb H disease. We sought to characterize the unique hematological and clinical features of Hb H CS patients, especially compared to those with Hb H disease, who were identified through the Thalassemia Clinical Research Network (TCRN). A total of 836 patients enrolled in the TCRN registry in Canada and the U.S. (2001 to 2005) were screened for this analysis. Genotyping of 836 thalassemia patients identified 106/836 (12.7%) with Hb H and 46/836 (5.5%) with Hb H CS. 2 had other non-deletional mutations. Among Hb H CS patients, 48% were female; mean age was 13±11 years. Among patients who had spleens, splenomegaly was more prevalent in Hb H CS than Hb H patients (16% vs 1%, p=0.001). Among Hb H CS patients who had splenomegaly, average spleen span was 3.67 ± 2.25cm. 13% of the Hb H CS and 2% of the Hb H patients had their spleen removed (p=0.005). Mean Hb level was higher in the splenectomized Hb H CS patients than in the non-splenectomized Hb H CS patients (9.85 ± 2.25 g/dL vs. 8.25 ± 0.76 g/dL, p=0.006). Post-splenectomy portal vein thrombosis was reported in 1 Hb H CS patient. 7.5% (3/40) of non-splenectomized Hb H CS patients had bacteremia or infections requiring intravenous antibiotics. 8.7% (4/46) of Hb H CS patients underwent cholecystectomy. 24% of HbH CS and none of the Hb H patients were placed on regular transfusions (〉8/year) and chelation therapy. Mean age of initiation of transfusions was 3.5±1.3 years (range 2–5 years). Mean ferritin level was higher in the non-transfused Hb H CS patients than in the Hb H patients (369.9 ± 409.9 ng/ml vs 175.9 ± 304.2 ng/ml, p=0.01), suggesting increased gastrointestinal iron absorption in Hb H CS. In 5 transfused Hb H CS patients, liver iron concentration was obtained showing elevated levels (27.2 ± 13.9 μgm/gm dry wt). Growth delay was more apparent in the Hb H CS patients (n=19) compared to 20 Hb H patients (Mean height Z score: −1.34 ± 0.98 for Hb H CS vs −0.82 ± 1.15 for Hb H (p=0.16) and mean weight Z score: −1.15 ± 0.88 for Hb H CS vs. −0.83 ± 1.61 for Hb H (p=0.47)). Bone density scans in Hb H CS patients revealed a higher prevalence of low bone mass than that detected in Hb H patients: mean L1-L4 spine Z/T-score of −1.60 ± 0.86 vs. −0.93 ± 0.80 (p=0.02). 40% (4/10) of adult female Hb H CS patients had 1 or more successful pregnancies, some requiring transfusion support through pregnancy. Patients with Hb H CS have a severe phenotype of α-thalassemia. They have moderately severe anemia, which sometimes requires regular transfusions, splenectomy, or both. Patients with Hb H CS commonly have iron overload, growth delay, and reduced bone mass. Early diagnosis in the neonatal period, regular monitoring, and appropriate treatment considerations for initiation of regular transfusions are key.

Description: Background : Reduced bone mineral density (BMD) has been reported in adults and children with sickle cell anemia (SCA). Dual energy x-ray absorptiometry (DXA) is routinely used for measuring BMD because of less radiation exposure and lower cost. However, changes in vertebral body shape, marrow hyperplasia and bone infarction due to SCA may affect the evaluation of BMD with DXA. Hence, we compared DXA with quantitative computerized tomography (QCT), which measures true volumetric density, and may be less influenced by bone changes. Methods : The study enrolled children between 9–19 years of age with SCA, and one or more severe manifestations: 〉2 hospital admissions/year, growth failure, avascular necrosis, or regular red cell transfusions for sickle cell-related complications. BMD of lumbar spine was determined by performing DXA of lumbar spine (Hologic Delphi-A, Bedford, MA). The apparent volumetric bone mineral density (BMAD) was calculated from bone mineral content, and compared to age, sex and ethnicity-matched reference data. BMD of the lumbar spine was also measured by QCT (Mindways Software, San Francisco, CA), and compared to age and sex-appropriate reference data. Results : The study has enrolled 25 patients (13 females and 12 males), of which 16 were younger than 14 years. In 6 children the height was 6 months, including 10 who had been transfused for 〉2 years. Calcium intake, assessed by a standardized questionnaire, was less than recommended dietary allowance in 13 patients. The z-score for BMAD determined by DXA was 〉 −1.0 in 8, between −1.0 and −2.0 in 5, and 〈 −2.0 in 12 patients. The z-score for lumbar spine by QCT was 〉 −1.0 in 20, between −1.0 and −2.0 in 1 and 〈 −2.0 in 4 patients. DXA-derived BMD (areal density) and BMAD (apparent volumetric density) z-scores did not differ significantly (p=0.16). On the other hand, the paired values of z-scores by DXA (BMAD) and QCT were significantly different (p=.002). When z-scores were categorized as greater or less than −1.0, the results were concordant in 13 (both DXA and QCT normal in 8, and both DXA and QCT abnormal in 5), and discordant in 12 cases (abnormal DXA with normal QCT in every case). Among patients in discordant group, 9/12 had been on regular red cell transfusions for 〉6 months, compared to 4/13 with concordant results (p=.047). There was no difference in the serum ferritin values between the two groups (p=.685). No significant difference in the prevalence of low BMAD z-scores was detected between groups based upon age, calcium intake, or growth failure. Five out of the 12 patients with BMAD z-score 〈 −2.0 were not on regular transfusion program. Conclusions : Almost half of the children with SCA had BMD below −2 standard deviations compared to age-matched controls. Low BMD was observed in chronically transfused as well as non-transfused children. In comparison, 16% of the patients were classified as low BMD (z 〈 −2.0) by QCT. The paired DXA/QCT results were discordant in half of the sample, with patients on regular transfusions for 〉6 months more likely to have normal QCT results. It is likely that the reduction in marrow hyperplasia following initiation of regular transfusions may disproportionately affect the trabecular BMD measured by QCT. Longitudinal evaluation of BMD in patients starting on transfusion program could help to define the effect of transfusions on measures of BMD in SCA.

Description: Abstract 2021 Poster Board I-1043 Therapeutic regimens that combine two chelators have the potential to improve iron excretion while avoiding toxicities associated with high doses of single agents. This pilot study was designed to explore the safety of the combined treatment with deferasirox (DFX) and deferoxamine (DFO) in individuals with transfusion-dependent thalassemia and iron overload who had failed standard therapy. Subjects underwent baseline evaluation of liver iron, cardiac iron, cardiac function and target organ damage, and were enrolled in 3 groups (n=5 in each group) - Group B: adults with liver iron concentration (LIC) 〉15 mg/g dry weight, group A: adults with LIC 〉5 and 5 mg/g dry weight plus iron related organ dysfunction. The duration of therapy was 52 weeks, with DFX (20-30 mg/Kg) given daily and (DFO 35-50 mg/Kg/infusion) given for 3-5 days/week (groups A and C), or 5-7 days/week (group B). Subjects were monitored for hepatic and renal toxicity, visual or auditory changes, and development of new symptoms. Changes in serum ferritin, LIC (ferritometer), cardiac function (MRI) and myocardial iron (MRI T2*) were monitored. We have enrolled 14 subjects (target 15 subjects) with a median follow up of 29 weeks (range 18-52). The mean daily dose of DFO was 16, 33, and 17 mg/Kg/day for groups A, B and C, respectively, at the start of the study. The corresponding mean DFX dose was 21, 25 and 22 mg/Kg/day. At the end of 26 weeks, the mean LIC (mg/g dry wt) in evaluable patients declined from 13.0 (3.9-21.7) to 10.6 (0.60-18.3, p=0.015), and the mean ferritin (ng/mL) fell from 2631 (1000-5230) to 2158 (319-5845, p=n.s.). Cardiac evaluation revealed that mean MRI T2* (msec) improved from 22.7 (6.7-32.6) to 25.5 (10.7-38.1) and the mean LVEF (%) from 63.4 (47.5-68.5) to 64.3 (53.4-72.2), but these changes were not statistically significant. No subject developed evidence of significant myocardial iron (T2*

Description: Abstract 4260 Background: Alpha thalassemia disorders are rapidly increasing in North America. This has resulted in proposals for universal newborn screening (NBS) for hemoglobin H disease. However, the institution of routine newborn screening and construction of guidelines for early intervention requires longitudinal clinical data before setting national goals. Since 1995, California has performed universal screening for alpha thalassemia disorders. The longitudinal follow up of data from patients with hemoglobin H disorders diagnosed in the asymptomatic period provides essential information needed for formulating public health policy. Methods: Hemoglobin H disorders were diagnosed by high performance liquid chromatography with multiplex GAP-PCR assay to determine deletional hemoglobin H disease (deletion of 3 α globin genes, HbH) and the non-deletional hemoglobin H Constant Spring (α0 thalassemia with Constant Spring mutation, HCS). Longitudinal clinical data for all patients from the Northern California Thalassemia Center were analyzed. Ethnicity, growth data, clinic visits, hospitalizations, complications including splenectomy, transfusion, and iron overload were monitored. Quantitative liver iron concentration was determined by ferritometer. Results: 86 patients predominantly diagnosed through NBS were longitudinally followed. Out of these, 60 (70%) had HbH, 23 (27%) had HCS and 3 (3%) had other forms of hemoglobin H disease. The parental ethnicity in HbH was 79% Asian, 6% Hispanic, and 15% African-American (in one or both parents). All patients with HCS were of Asian ethnicity. Longitudinal data for hemoglobin revealed that anemia was more severe in HCS at all ages (p

Description: Introduction When monitoring bone health in patients with hemoglobinapathies, it is unknown if iron in surrounding tissues can lead to inaccuracies in the 2-dimensional assessment by Dual Energy X-ray Absorptiometry (DXA). Objective The aims of this study were: 1) to determine if the accuracy of lumbar spine assessment by DXA is affected by high liver iron concentration in patients with Sickle Cell Disease (SCD) or Thalassemia (Thal), 2) to test the effect of high tissue iron on vertebral Z-scores using phantoms, 3) to explore the ability to account for potential high-iron content effects when performing DXA examinations. Methods This study consisted of a retrospective chart review of data collected by the Children’s Hospital & Research Center Oakland, Bone Density Clinic and Iron Measurement Program. Data from both DXA and Super Conducting Quantum Interference Device (SQUID) examinations collected between 2002 and 2013 from were abstracted. Only those patients with a diagnosis of SCD or Thal, who had a DXA and SQUID measurement within the same year were divided into an iron overload group (liver iron concentration (LIC) 〉3,000 µg Fe/g wet) and low iron (LIC 3,000 ug/g wet tissue, it is important to consider the effects of iron contribution from the liver on the DXA spine scans and delete L1 and/or L2 from the total Z-score prior to making an interpretation. Failing to do so may under diagnose low bone mass in this at risk patient population. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4261 Osteoporosis in thalassemia is extremely common and preventive approaches are required to avoid serious complications in adults. Vitamin D deficiency, which contributes to suboptimal bone mineralization, is frequently observed in thalassemia despite routine prescription for supplementation with 400–800 IU vitamin D per day. Screening for vitamin D status was conducted in 71 patients with thalassemia, of which 52 were transfusion-dependent and 19 were transfusion-independent (including 11 patients with hemoglobin H or H Constant Spring disease). Baseline assessment of plasma 25-OH vitamin D revealed 57 (80.3%) patients were either deficient (

Description: Abstract 4269 Therapeutic regimens that combine two iron chelators may enhance chelation efficiency by improving access to different tissue iron stores and control of the toxic labile iron pool. The combination of two chelators can reduce toxicity through averting the need for high doses of a single drug, but it is essential to establish the safety such regimens. We therefore explored the combined use of deferasirox (DSX) and deferoxamine (DFO) in patients with transfusion-dependent thalassemia who had failed standard chelation therapy with single drug. Patients were eligible if the liver iron concentration (LIC) 〉15 mg/g dry liver-weight or if iron-induced end organ injury was present. Subjects were monitored for hepatic and renal toxicity, visual or auditory changes, and the development of new complications from iron overload. The ability of the combined therapy to control systemic iron burden (serum ferritin and LIC) and myocardial iron overload (MRI T2*) was evaluated. We also measured changes in plasma levels of non-transferrin bound iron (NTBI) and labile plasma iron (LPI). Fifteen subjects were enrolled in 3 groups: adults with LIC 15 mg/g (group B), and children 8–18 years with LIC 〉5 mg/g (group C). The duration of therapy was 52 weeks. DSX (20-30 mg/Kg) was administered daily and DFO (35-50 mg/Kg/infusion) was infused on 3–7 days/week (as 8–12 hour infusion) based upon the degree of iron overload present at baseline. At the initiation of the study, the mean daily dose of DFO was 16, 33, and 17 mg/Kg/day and mean DSX dose was 21, 25 and 22 mg/Kg/day for groups A, B and C, respectively. At the conclusion of the trial, the median LIC declined by 48% from 10.8 mg/g (3.9-34.8 mg/g) to 5.7 mg/g (1.0-24.0 mg/g, p=0.003). The median ferritin fell by 43% from 2030 ng/mL (1000-5230 ng/mL) to 1150 ng/mL (421-5260 ng/mL, p=0.008). Myocardial iron in the 3 subjects who had T2*