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Patients with beta-thalassemia show 5-fold increase in agestandardized lethality due to SARS-CoV-2 infection, representing a high-risk population compared with age- and sex-matched healthy subjects.(1) Vaccination against SARS-CoV-2 is crucial to reduce mortality and morbidity of frail patients.(2) Up to now, limited data have been available on the responses of patients with beta-thalassemia immunized with anti-SARS-CoV-2 mRNA vaccines.(3)

Introduction: β-thalassemia major (TM) is one of the most prevalent inherited hemoglobinopathies in Pakistan. It has one of the highest prevalence of transfusion-dependent TM patients globally, with an estimated greater than 100,000 active cases. Each year, an estimated 5000-9000 new cases of TM are being diagnosed in the country. Blood transfusions (BT) are essential in the management of severe TM; it is critical to have a safe BT to reduce the risk of transfusion transmissible infections (TTIs). Frequent blood transfusions in these patients increase their risk of acquiring TTIs compared to the general population. In this systematic review, we aimed to identify the prevalence of TTIs in transfusion-dependent β -thalassemia major patients in Pakistan. Methods & Material: We performed a systematic literature search to identify studies related to the TTIs and transfusion-related infections in Pakistan from January 1, 2010, to January 31, 2020. The search was conducted using PubMed and PakMediNet (Largest medical database of Pakistan), with initial search retrieved 981 studies. Among these, 166 studies met the inclusion criteria. After further screening by reviewing the articles for relevance and availability of full-length articles, only 14 studies met the final criteria for qualitative synthesis. Results Analysis of 14 studies (n=3786) showed that the seroprevalence of Hepatitis B virus (HBV) of 3.13% (0.66 % to 7.4%) and Hepatitis C virus (HCV) of 26 % (5.56% to 68.2%). There were only two studies reported HIV seroprevalence of 0 % & 0.5% (n=6). The rate of seropositivity for HBV and HCV was directly related to the number of transfusions, higher ferritin levels, and older age groups. There was an increase in the HCV rate with the increasing age of patients. Thalassemia patients who were older than ten years of age had a greater HCV compared to those who were less than ten years of age, i.e., 22% vs. 8.4%, p:0.005, respectively. The mean age was higher in HCV reactive children than non-reactive children. A comparison of HCV in healthy donors vs. thalassemia patients showed a rate of 1.9% vs. 13.1% for T.M. patients. There was HCV infection rate of 74% in the group with greater than 100 BTs compared to 33 % in a group with fewer than 35 BTs. The rate of HCV increased to 75% for the patients who had more than 100 BTs. The majority of the patients were males (51% to 88%). The seroprevalence of TTIs was higher in males than in females (73.4% vs. 26.6%). On average, a single TM patient is exposed to at least 17 different donors annually, requiring 1-2 transfusions every month. The free BT is accessible only in 1 out of 4 thalassemia centers. The majority of patients either need to bring their donors or are dependent on an external source of financial aid as they could not afford the cost of BT treatment. More than half of thalassemia patients (57.2%) need to contact multiple BT centers to search for required blood products. About 42.1% of parents of TM patients did not know about TTIs, whereas 31.6% of them did not know about the bloodborne transmission of HBV and HCV. The majority of parents of TM children had a low income, with 75% of them having income less than 10,000 Pakistani rupees (PKR) per month. The prevalence of TTIs in TM patients was significantly higher (96% vs. 4%) compared to the patients requiring multiple transfusions due to other causes such as leukemia, aplastic anemia, and thrombocytopenia. Conclusion: Our data highlights that the prevalence of transfusion-transmitted infections, especially HCV, is alarmingly higher (26%) in the TM population than in the general population. This is because of a lack of resources, inadequate safety measures, and a fragmented blood transfusion system. These findings warrant the urgent need for better public health measures, safe blood transfusion practices, voluntary remunerated blood-based transfusions, and universal quality-assured donor screening. Without these positive interventions, the current transfusion system can lead to a further worsening of the situation. Large prospective multi-centered clinical trials are required to understand better the high prevalence of TTIs in patients with TM. Disclosures Anwer: Incyte, Seattle Genetics, Acetylon Pharmaceuticals, AbbVie Pharma, Astellas Pharma, Celegene, Millennium Pharmaceuticals.:Honoraria, Research Funding, Speakers Bureau.

Introduction We investigated the impact of β-thalassemia genotypes and disease genetic modifiers including HBA and KLF1 genotype and sentinel single-nucleotide polymorphism (SNP) genotypes at 3 major HbF quantitative trait loci (QTL) on clinical outcomes of TDT patients treated with beti-cel gene therapy in two phase 3 studies, HGB-207 (NCT02906202) and HGB-212 (NCT03207009). Methods HBA deletions and triplications were determined by gap-polymerase chain reactions. HBG2 (including rs7482144, Xmn1 site) and HBG1 promoters, HBA2, HBA1, and KLF1 underwent individual nucleotide sequencing. Multiplex amplification refractory mutation system (ARMS) tests were used to identify HbF QTL SNPs (rs10128556 in HBBP1; rs766432, rs1427407, rs10189857 in BCL11A; rs9399137, rs66650371 in HMIP). Thalassemia severity score (TSS) was calculated as defined by Danjou et al, Haematologica, 2015, considering gender, HBB and HBA genotypes, and 4 SNPs in HbF QTL (HBG2, BCL11A, HMIP). Correlative analyses were performed to assess relationships between genotype, presence/absence of non-HBB mutations (HBA2, HBA1, KLF1), presence/absence of HbF QTL SNPs (HBG2, BCL11A, HMIP), and TSS with the achievement of transfusion independence (TI; weighted average hemoglobin [Hb] ≥9 g/dL without red blood cell [RBC] transfusions for ≥12 months). Correlation coefficients used percentage bend correlation. Statistical significance threshold was p ≤ 0.05. Results As of 3 March 2020, 38 patients were treated in HGB-207 and HGB-212 (β0/β0 genotype n=9; non-β0/β0 genotype n=29 [β+/β+ n=8; β0/β+ n=15; βE/β0 n=6]). All patients were heterozygous or homozygous for mutations or SNPs that may modulate disease severity; 20 patients were homozygous for ≥1 mutation or SNP. Patients had the following alleles associated with higher HbF synthesis: HBG2 rs7482144 C>T (Xmn1 site), C/T n=9, T/T n=1; BCL11A rs1427407 G>T, G/T n=8; BCL11A rs10189857 A>G, A/G n=16, G/G n=18; HMIP rs9399137 T>C, T/C n=9, C/C n=2. Three patients were heterozygous for single α-globin gene deletion (-α/αα) and 2 were heterozygous for α-globin gene triplication (αα/ααα). Median TSS was 3.65 (min - max 0.4 - 8.1). TI was achieved by 23/27 (85%) evaluable patients; 4 patients with ≥ 12 months follow-up have been transfusion free for > 10 months but were not yet evaluable for TI (Figure). β-thalassemia genotype did not strongly correlate with TI (two-sided Fisher's Exact Test, p-value = 0.78). Month 12 median (min - max) peripheral blood vector copy number (PB VCN) was 1.5 (0.2 - 5.0) c/dg in TI or transfusion-free patients (n=27) and 0.2 (0.2 - 0.4) c/dg in patients who did not achieve TI (n=4). The transfusion-free patient (β0/β0) with the lowest month 12 PB VCN was homozygous for T/T at rs7482144 (HBG2Xmn1 site) and G/G at rs10189857 (BCL11A), and heterozygous for single α-globin gene deletion. Endogenous Hb (5.9 g/dL HbF + 0.2 g/dL HbA2) and gene therapy-derived HbAT87Q (4.4 g/dL) at month 12 enabled this patient to stop transfusions. Tests of association of SNPs and mutations with TI were not significant; no p-value < 0.31 (chi-squared test). As only 4 patients did not achieve TI, the power to detect an association was limited. Larger sample sizes are needed to determine if individual SNPs and mutations may have an impact on TI. In TI or transfusion-free patients (n=27), TSS correlated strongly with month 12 endogenous unsupported Hb (HbA + HbA2 + HbF + HbE without RBC transfusions for 60 days) (correlation coeff. = -0.76, p < 0.0001), but not with HbAT87Q (correlation coeff. = 0.26, p = 0.19) or unsupported total Hb (correlation coeff. = 0.32, p = 0.10). Beti-cel-related adverse events (AE) in >1 patient were abdominal pain (n=2 non-β0/β0; n=1 β0/β0), thrombocytopenia (n=3 non-b0/b0). Serious AEs in ≥3 patients post-infusion were thrombocytopenia (n=2 non-β0/β0; n=1 β0/β0), pyrexia (n=1 non-b0/b0; n=2 β0/β0), veno-occlusive disease (n=3 non-β0/β0). Summary Genetic characterization of TDT patients treated with beti-cel revealed diverse HBB and non-HBB mutations and polymorphisms that may influence disease severity. Higher PB VCN and HbAT87Q levels were associated with increased likelihood of TI. In instances of lower HbAT87Q, higher endogenous HbF might be a determinant in whether TI is achieved. Despite genetic heterogeneity, beti-cel enabled patients to achieve TI regardless of β-thalassemia genotype, TSS, disease genetic modifiers including HBA, and HbF QTL SNP genotypes. Disclosures Walters: Veevo Biomedicine: Consultancy; AllCells, Inc: Consultancy; Editas: Consultancy. Chui:bluebird bio, Inc.: Other: Payment for lab use for the sequencing analyses done for the studies. Lal:bluebird bio, Inc.: Research Funding; Agios Pharmaceuticals: Consultancy; Celgene, BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; La Jolla Pharmaceutical Company: Research Funding; Novartis: Research Funding; Protagonist Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Terumo Corporation: Research Funding; Chiesi USA: Consultancy; Insight Magnetics: Research Funding. Locatelli:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharmaceeutical: Speakers Bureau. Kwiatkowski:bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy; Apopharma: Research Funding; Bristol Myers Squibb: Consultancy; Imara: Consultancy; Celgene: Consultancy; Sangamo: Research Funding; Terumo Corp: Research Funding; Novartis: Research Funding. Porter:bluebird bio, Inc.: Consultancy, Honoraria; Vifor Pharmaceuticals: Honoraria; La Jolla Pharmaceuticals: Honoraria; Protagonist Therapeutics: Honoraria; Agios Pharmaceuticals: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Silence Therapeutics: Honoraria. Thuret:Apopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis pharma: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials; bluebird bio, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials. Kulozik:Novartis: Consultancy, Honoraria; bluebird bio, Inc.: Consultancy, Honoraria. Thrasher:4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership. Yannaki:bluebird bio, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Speakers Bureau; SANDOZ: Speakers Bureau; Gilead: Speakers Bureau. Yang:bluebird bio,Inc.: Current Employment, Current equity holder in publicly-traded company. Whitney:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Petrusich:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Colvin:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Thompson:BMS: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; CRISPR/Vertex: Research Funding; Biomarin: Research Funding; Baxalta: Research Funding.

β-Thalassemia and sickle cell disease (SCD) are the most prevalent monogenic diseases. These disorders are caused by quantitative or qualitative defects in the production of adult hemoglobin. Gene therapy is a potential treatment option for patients lacking an allogenic compatible hematopoietic stem cell (HSC) donor. New-generation lentiviral vectors (LVs) carrying a β-globin-like gene have revolutionized this field by allowing effective HSC transduction, with no evidence of genotoxicity to date. Several clinical trials with different types of vector are underway worldwide; the initial results are encouraging with regard to the sustained production of therapeutic hemoglobin, improved biological parameters, a lower transfusion requirement, and better quality of life. Long-term follow-up studies will confirm the safety of LV-based gene therapy. The optimization of patient conditioning, HSC harvesting, and HSC transduction has further improved the therapeutic potential of this approach. Novel LV-based strategies for reactivating endogenous fetal hemoglobin (HbF) are also promising, because elevated HbF levels can reduce the severity of both β-thalassemia and SCD. Lastly, genome-editing approaches designed to correct the disease-causing mutation or reactivate HbF are currently under investigation. Here, we discuss the clinical outcomes of current LV-based gene addition trials and the promising advantages of novel alternative therapeutic strategies.

There is a Blood Commentary on this article in this issue.

Introduction: With the emergence of the Coronavirus Disease 2019 (COVID-19) as a global pandemic came the concern that it would adversely affect individuals with comorbid conditions. In sickle cell disease (SCD), the apprehension was largely driven by concerns that the disease causes chronic inflammation, organ dysfunction and a prothrombotic state, especially during a vaso-occlusive crisis. Our assumption and hypothesis was that SCD pathology would be exacerbated by SARS CoV-2 infection leading to higher rates of acute chest syndrome (ACS), venous thromboembolism (VTE), more robust inflammatory responses requiring ICU care and increased death rates. Sickle cell trait (SCT) is frequently perceived as a benign condition; however, mounting evidence has shown that severe hypoxia (such as that seen in COVID-19 infection) can trigger sickle-related complication such as VTE, papillary necrosis and splenic infarcts and, in rare cases, ACS. In this retrospective analysis, we aimed to determine if sickle cell disorders (SCD & SCT) would confer poorer outcomes and/or increased mortality due to COVID-19 infection. Methods: Within the MedStar health care system there are 7,551 adult patients with a diagnosis of sickle cell disorder. We performed a retrospective chart review of all patients in the MedStar system with hemoglobinopathy and a lab-verified diagnosis of COVID-19 infection between March 1, 2020 and June 30, 2020. Sixty-one patients met our search criteria with 21 patients admitted to the hospital and 40 patients managed in the outpatient setting. Of the 21 patients admitted: 9 patients had sickle cell disease (Hb SS, Hb SC, Hb SB thalassemia) and 11 patients had sickle cell trait. One patient had HbC disease. Results: Results were analyzed by SCD genotype (Hb SS, Hb SC, Hb SB Thalassemia) versus the presence of a SCT. Due to the small sample size, statistical significance was not met in the variables. In the 21-patient cohort: 76.2% were female (60% of SCD and 90.9% of SCT) and 100% were African American. Median age was 46.5 years in the SCD group and 38 years in the SCT group. Length of stay was 10 days in the SCD group and 6 days in the SCT group. Mean peak white blood cell count and platelet count were significantly higher in SCD group (WBC: 15.1 k/uL vs 7.5 k/uL; p value 0.099. Platelets: 523 k/uL vs 185 k/uL; p value 0.082). Inflammatory markers were more elevated in the SCD cohort: mean Ferritin (915 ng/mL vs 520 ng/mL), mean D Dimer (2.96 mg/L vs 1.07 mg/L) and mean LDH (543 units/L vs 392 units/L). However, mean peak C-reactive protein (CRP) was higher in the SCT group (117.5 mg/L vs 67.7 mg/L). Admission to the intensive care unit (ICU) occurred in 11.1% with SCD & 36.4% with SCT and renal replacement therapy was required in 11.1% with SCD & 28.6% with SCT. Nasal cannula was required in 75% of patients (88.9% of SCD & 57.1% of SCT; p value 0.262) with only one SCT patient requiring intubation and mechanical ventilation whom later died. Two patients with Hb SS developed ACS, one of whom died. One patient with HbSC disease developed ACS and an acute cerebrovascular accident. No VTE or pulmonary embolisms were documented in this cohort. Therapeutic drugs used in the cohort varied, with 43.8% receiving antibiotics (other than Azithromycin), 37.5% receiving Azithromycin and 25% receiving Hydroxychloroquine. None of the patients received convalescent plasma, steroids, tociluzumab, sarilumab or remdesivir. Anticoagulation was used in 93.8% of the patients (13.3% of which were in therapeutic doses). Conclusions: Sickle cell disease diagnosis did not seem to influence response to COVID19 illness when compared to age-matched individuals with SCT. The number of patients with sickle cell disease admitted to the hospital after contracting SAR CoV2 infection was also remarkably low. These findings were, in general, unexpected. Although the inflammatory biomarkers were generally higher in the patients with SCD, more patients with SCT were admitted to the ICU and required renal replacement therapy. The major drawbacks of the study are the small sample size and missing data points (lack of labs, lack of documentation, transfer to another facility) that did not allow us to draw statistical conclusions about the differences in the patients with SCD versus SCT. We were also unable to determine the influence of SCD severity and pre-existing organ complications on outcomes in COVID 19 disease. Additional studies with larger cohorts is warranted. Disclosures No relevant conflicts of interest to declare.

The complex pathophysiology in β-thalassemia can translate to multiple morbidities that affect every organ system. Improved survival due to advances in management means that patients are exposed to the harmful effects of ineffective erythropoiesis, anemia, and iron overload for a longer duration, and we started seeing new or more frequent complications in adult compared with younger patients. In this article, we highlight particular aspects of managing adult patients with β-thalassemia, using our own experience in treating such patients. We cover both transfusion-dependent and nontransfusion-dependent forms of the disease and tackle specific morbidities of highest interest.

Hepcidin agonists are a new class of compounds that regulate blood iron levels, limit iron absorption, and could improve the treatment of hemochromatosis, β-thalassemia, polycythemia vera, and other disorders in which disrupted iron homeostasis causes or contributes to disease. Hepcidin agonists also have the potential to prevent severe complications of siderophilic infections in patients with iron overload or chronic liver disease. This review highlights the preclinical studies that support the development of hepcidin agonists for the treatment of these disorders.

Thalassemias are a heterogeneous group of red blood cell disorders, considered a major cause of morbidity and mortality among genetic diseases. However, there is still no universally available cure for thalassemias. The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoiesis. In β-thalassemia, β-globin synthesis is reduced causing α-globin accumulation. Unpaired globin chains, with heme attached to them, accumulate in thalassemic erythroblasts causing oxidative stress and the premature cell death. We hypothesize that in β-thalassemia heme oxygenase (HO) 1 could play a pathogenic role in the development of anemia and ineffective erythropoiesis. To test this hypothesis, we exploited a mouse model of β-thalassemia intermedia, Th3/+. We observed that HO inhibition using tin protoporphyrin IX (SnPP) decreased heme-iron recycling in the liver and ameliorated anemia in the Th3/+ mice. SnPP administration led to a decrease in erythropoietin and increase in hepcidin serum levels, changes that were accompanied by an alleviation of ineffective erythropoiesis in Th3/+ mice. Additionally, the bone marrow from Th3/+ mice treated with SnPP exhibited decreased heme catabolism and diminished iron release as well as reduced apoptosis. Our results indicate that the iron released from heme because of HO activity contributes to the pathophysiology of thalassemia. Therefore, new therapies that suppress heme catabolism may be beneficial in ameliorating the anemia and ineffective erythropoiesis in thalassemias.

The hallmarks of β-thalassemia (BT) include ineffective erythropoiesis (IE), splenomegaly and iron overload (IO). Recent studies have pointed to iron restriction (IR) to improve both anemia and IO in BT (Rivella, Blood). The decreased iron-uptake by early erythroid cells reduces hemichrome toxicity and prevents premature RBC hemolysis. One such IR therapy targets the matriptase-2 (Tmprss6) gene using antisense oligonucleotides (T-ASO). Our group has previously shown that treatment of Hbb th3/+(th3/+) mice (a mouse model for BT-intermedia) with T-ASO improved anemia, lengthened red blood cell (RBC) lifespan, reduced levels of erythroferrone (ERFE), hemichromes and reactive oxygen species, and ameliorated splenomegaly (Casu et al. Blood). Another novel therapeutic approach to improve anemia targets the Transforming Growth Factor (TGF)-β pathway to increase erythroid maturation. Luspatercept, a TGF-β trap-ligand, gained FDA approval in 2019 to treat transfusion dependent BT patients (Cappellini and Taher, Blood Adv). In mouse models of BT, its murine analog (RAP-536) was found to promote EPO-independent maturation of late-stage erythroid cells, and resulted in increased RBC parameters in a dose-dependent manner (Surgani, et al. Nat Med). In this work we treated th3/+ mice with an agent analogous to murine Luspatercept (RAP-GRL) in combination with the iron restriction (IR) drug T-ASO, (RAP-GRL+T-ASO) with the goal of targeting distinct morbidities associated with BT. To test our RAP-GRL construct, primary fibroblasts were transduced with an adenovirus containing the RAP-GRL sequence (FB Ad5RAP-GRL) and used to deliver RAP-GRL to mice. As a second strategy, RAP-GRL was expressed in a mammalian cell line and purified. Wild-type (WT) or th3/+ mice were subcutaneously (s.c.) implanted with 1x10 6 FB Ad5RAP-GRL or injected s.c. with 10mg/kg of RAP-GRL and monitored by complete blood counts. Implantation of FB Ad5RAP-GRL ortreatment with purified RAP-GRL increased RBC parameters in both WT and th3/+ mice (n=3-9, 2-4-month-old females and males). In the first combination therapy experiment we implanted FB Ad5RAP-GRL s.c. and delivered T-ASO via intraperitoneal (i.p.) injection in th3/+ mice. RBC parameters were increased in all treatment groups except controls after 6 weeks. The RAP-GRL+ T-ASO group displayed the most pronounced increase in RBC parameters with a mean increase in RBC of 3.067±0.73 10 6 cells/µL, Hb of 3.02±0.77 g/dL, and Hct of 5.88±2.36 % (Table 1). Additionally, we also treated th3/+ mice with two different doses of protein purified RAP-GRL in combination with T-ASO (Table 1). The best results using the protein purified RAP-GRL were achieved in the RAP-GRL+T-ASO group that was treated with two weekly 10mg/kg s.c. injections of RAP-GRL and two weekly 5mg/kg i.p. injections of T-ASO (Group 2) for 6 weeks. Flow cytometry analysis using CD71, TER119, and CD44 antibodies showed improvements in the bone marrow (BM) and spleen (SPL) of all treatment groups compared to controls. Additionally, ROS levels and splenomegaly were also greatly reduced in all T-ASO and RAP-GRL+T-ASO treated groups compared to controls. Serum assessment of T-ASO and RAP-GRL+T-ASO treated animals showed decreased levels of iron and transferrin saturations with a simultaneous increase in hepcidin levels. ERFE levels were decreased in all T-ASO and RAP-GRL+T-ASO groups, however, erythropoietin (EPO) levels were increased only in the RAP-GRL and RAP-GRL+T-ASO cohorts of Group 2. Additionally, although EPO was elevated in all RAP-GRL treated animals of Group 2, only the RAP-GRL+T-ASO group had reduced ERFE. This result is in agreements with our findings of decreased early (ERFE-producing) erythroid progenitors in the BM and SPL of RAP-GRL+T-ASO treated mice. This finding also suggests that higher doses of RAP-GRL may result in elevated EPO. Luspatercept, through heightened iron consumption, may increases EPO synthesis in the kidney via activation of the transcription factor HIF2-α, which can be stabilized not only by hypoxia, but also by iron deficiency. In conclusion our results provide pre-clinical support for combining IR and TFG-β trap-ligands in the treatment of BT. Our data shows that IR, in conjunction with the enhancing erythroid maturation action of Luspatercept (and potential activation of EPO), may offer an additive and more effective therapeutic strategy for BT patients. Figure 1 Figure 1. Disclosures Guo: Ionis Pharmaceuticals, Inc.: Current Employment. Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.

Introduction : In Greece, official data confirm that blood supply is consistently declining, primarily because of decreased voluntary blood donations. At this rate, it is unclear whether there will be sufficient red blood cell (RBC) units to meet future demand for patients with β-thalassemia. We modeled future blood demand in patients with transfusion-dependent β-thalassemia (TDT) over the next 15 years to assess whether such a demand can be met. Methods: A discrete time, stochastic, individual-based model, was developed to assess future blood needs of patients with TDT in Greece. The model adopted a cohort approach and patients enter the model shortly after birth and exit at death. The model was seeded with data from the National Registry for Hemoglobinopathies in Greece (NRHG; patient population) and data from the National Blood Centre (RBC units) (Table). To quantify the decrease in TDT incidence, a linear regression model was fitted to the incident cases as described by Voskaridou (Voskaridou E, et al. Ann Hematol 2019;98:55-66). The calculated annual reduction rate was then applied to the observed thalassemia major (TM) and thalassemia intermedia (TI) incident cases for 2012 (Voskaridou E, et al. Ann Hematol 2012;91:1451-1458). The model assumes the reduction would continue through to 2020, from which time the incidence curve would be constant. Results: The model estimates a decrease in both target population, primarily due to the implementation of the national prenatal screening program and TM related mortality, (9.2%, 18.6%, and 26.0% in 2025, 2030, and 2034, respectively) and in demand for RBC units (100,665, 89,428 and 80,565, respectively) over the next 15 years. After accounting for any blood donated, based on latest (2017) data as projected to 2020, Greece is expected to be short of 56,716 RBC units to meet needs of patients with TDT in 2020. By 2025, 2030, and 2034, cumulative required RBC units for patients with TDT are estimated to be 307,150, 501,698, and 617,074, respectively. Assuming RBC units are divided among patients ≥ 18 years and those < 18 years in line with the population mix in the NRHG, cumulative RBC units required for adult patients with TDT are estimated to be 292,837, 464,337, and 554,083 by 2025, 2030, and 2034, respectively. Conclusions: Though blood demand for patients with TDT is expected to decrease in line with a decrease in the patient population, it will remain a challenge for the healthcare system in Greece to meet the demand, primarily due to a continuing shortage of available RBC units from voluntary donations. This annual deficit is estimated to accumulate to over 600,000 units over the next 15 years. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background Early and effective iron chelation has improved life expectancy and decreased disease complications for people with transfusion dependent thalassemia (TDT) and Sickle Cell Disease (SCD). Fertility challenges and pregnancy complications have historically limited reproductive options in this group, however improved disease management has made subfertility a chronic disease complication requiring attention. Despite this, there are very few reports on rates of conception and pregnancy outcomes in this population. Methods A 20 year retrospective analysis (1997 - 2017) was performed to evaluate fertility outcomes in women with TDT and SCD at an Australian referral centre. Patients with TDT and SCD who tried to conceive during the study period were included. Use of assisted reproductive technologies (ART), as well as pregnancy outcomes and neonatal and maternal complications were assessed. Results Eleven women with TDT and 3 with SCD tried to conceive during the study period. Median age at conception was 28 years (range 21-35). A total of 28 pregnancies and 25 live births were reported, including 2 spontaneous early pregnancy losses, a termination for anencephaly and a reduction of triplets. There was 1 multiple gestation in the cohort. At least 1 live birth occurred in 13 of the 14 women (93%). Spontaneous conception was reported in 9 women, of whom 8 had at least one resulting live birth, with a total of 15 live births from spontaneous conception. Of 5 women who were unable to conceive spontaneously, four had a diagnosis of hypogonadotrophic hypogonadism (HH) - two conceived following ovarian stimulation (OS), one required in vitro fertilization (IVF), and one did not pursue IVF following unsuccessful OS. The cause of subfertility was unknown in one patient, who conceived with IVF following failed OS. Three women who had an initial spontaneous conception required assisted reproductive technology (ART) for subsequent pregnancies, with no cause for subfertility identified. Mean ferritin at conception was 2911 mmol/L (range 164 to 8697mmol/L), and there was no association between ferritin at conception and need for ART. A trend was observed between increasing age and use of ART. Nine of the thirteen (69%) women who achieved pregnancy underwent Cesarean section for their first delivery. Prematurity (birth prior to 37 weeks' gestation) occurred in 5 (20%) of live births. Intrauterine growth restriction (IUGR) evidenced by birth weight Conclusions Our data is the first analysis of fertility and pregnancy outcomes in Australian patients with TDT or SCD. Publications in this area are limited, and primarily report on pregnancy outcomes without capturing failure to conceive. Our findings are encouraging, with high conception rates achieved, with the use of ART where needed. Ferritin level did not predict difficulty with spontaneous conception and few of the women (29%) had HH, despite many having significant hyperferritinemia. Overall, 48% of live births resulted from ART, despite 58% of these patients not having a diagnosis of HH. This indicates that pituitary iron deposition with resultant HH alone does not adequately explain subfertility in this population. Our data also highlight the importance of affordable ART access for this patient population despite the clinical gains achieved with effective chelation therapy. Pregnancies were largely uncomplicated with excellent maternal and foetal outcomes. A high rate of IUGR was observed, supporting classification of pregnancy in this population as high risk. Rates of Cesarean section for first delivery were more than double the Australian average, likely in part due to high IUGR rates. Neonatal complications and PPH occurred at general population rates. Guidelines around pregnancy management in this population abound, however large prospective studies are needed to identify those at risk of sub- and infertility, even in the era of effective chelation. Disclosures No relevant conflicts of interest to declare.

Introduction: A recent systematic literature review (Betts M, et al . Clin Ther 2020;42:322-337) perceived that in the past 2 decades, advancements in the treatment and management of β-thalassemia have resulted in improvements in iron control. However, clinical complications, including alloimmunization, iron overload, and resultant cardiac and liver disease, remain a challenge. While red blood cell (RBC) transfusions are used to treat anemia in patients with β-thalassemia, those who are transfusion dependent reported poorer health-related quality of life (HRQoL) compared with the US general population. The literature misses a holistic understanding of the humanistic, psychosocial, and psychological burden of transfusion dependency in β-thalassemia in terms of the impact of disease and treatment on patients' daily lives, distress arising from the possibility of adverse events, and comorbidities. Methods: First, concept elicitation qualitative interviews (n=3) and cognitive pre-test interviews (n=3) were conducted with patients from the Cooley's Anemia Foundation (CAF) to test and revise a 30-minute web-based patient survey questionnaire for clarity and relevance. Following, a larger cross-sectional study included adult patients with β-thalassemia in the USA identified by the CAF. Patients were recruited via email invitation or social media, and invited by CAF to take the survey, at which point informed consent was obtained. Inclusion criteria included ≥18 years of age, self-reported physician diagnosis of β-thalassemia, and ≥1 RBC transfusion in the past 6 months. Survey instruments included the Functional Assessment of Cancer Therapy-Anemia (FACT-An), Patient Health Questionnaire-9 (PHQ-9), Generalized Anxiety Disorder-7 (GAD-7), ad hoc questions around patient treatment experience, side effects resulting from β-thalassemia treatment, and psychological burden due to treatment. All results were reported descriptively, with frequency counts and percentages for categorical/ordinal data and mean and standard deviations (SD) for continuous data. Results: Overall, 100 patients in the USA completed the survey; 65% were female, average age was 36.0 (SD=10.4) years, 42% were Asian/Pacific Islander, and 33% reported a household income of USD 100,000 or more. On average, patients had been diagnosed with β-thalassemia for 34.4 (SD=10.3) years and received 9.6 (SD=4.3) RBC transfusions in the past 6 months. Among all patients, 70% reported moderate to extremely high burden of RBC transfusions, 81% reported experiencing iron overload due to RBC transfusion, and 42% reported not being as social with friends/family due to the time needed for RBC transfusions and the associated recovery time. Over 50% of patients reported mild to severe depression (52%) via PHQ-9 and anxiety (53%) via GAD-7 symptoms over the previous 2 weeks. The FACT-An (0-188), where higher scores indicate better outcomes, average score was 132 (SD=33.5), while fatigue symptoms (0-52) average score was 33.3 (SD=12.3), and anemia symptoms (0-28) average score was 20.4 (SD=5.5) in the past 7 days. A week prior to receiving an RBC transfusion, 18% of patients reported their overall health as "very well" versus 52% 1 week after an RBC transfusion. Conclusions: Although upon RBC transfusion HRQoL temporarily improves, overall, these results demonstrated patients with transfusion-dependent β-thalassemia experienced poor health prior to RBC transfusion, mild to severe depression and anxiety, increased psychological burden, and suboptimal HRQoL. These findings contribute to the understanding of the humanistic and psychological burden of RBC transfusion dependency in patients with β-thalassemia and suggest that new treatment options that can improve outcomes in this population are needed. Disclosures Price: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Gupta: Bristol Myers Squibb: Consultancy, Research Funding; Kantar Health: Current Employment. Perkowski: Kantar Health: Current Employment; Bristol Myers Squibb: Consultancy, Research Funding. Costantino: Kantar Health: Current Employment; Bristol Myers Squibb: Consultancy, Research Funding. Inyart: Kantar Health: Current Employment; Bristol Myers Squibb: Consultancy, Research Funding. Ashka: Kantar Health: Current Employment; Bristol Myers Squibb: Consultancy, Research Funding. Clapp: Kantar Health: Current Employment; Bristol Myers Squibb: Consultancy, Research Funding. Knoth: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

β-hemoglobinopathies are caused by mutations affecting the adult hemoglobin production. In sickle cell disease (SCD), the β6 Glu→Val substitution leads to sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. In β-thalassemia, reduced β-globin production leads to precipitation of uncoupled α-chains causing ineffective erythropoiesis and the production of poorly hemoglobinized RBCs. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. The clinical severity of β-hemoglobinopathies is alleviated by the co-inheritance of mutations causing hereditary persistence of fetal Hb (HPFH). HPFH mutations clustering 200 nucleotides upstream of the TSS of the fetal γ-globin (HBG) genes either disrupt the binding site (BS) of the fetal Hb (HbF) repressor LRF or generate a de novo BS for the KLF1 activator. To reactivate γ-globin expression, nuclease-based approaches have been explored. However, nucleases generate double-strand breaks (DSBs), raising safety concerns for clinical applications. Base editing (BE) allows the introduction of point mutations without generating DSBs. In this study, we designed BE systems to introduce a variety of HPFH or HPFH-like mutations in the -200 region of the HBG promoters. First, we screened in erythroid cell lines known and novel BEs, and we selected combinations of BEs and guide RNAs that edit alternative bases of the -200 region. We then developed a clinically-relevant protocol based on RNA-transfection to deliver the BE system to HSPCs. The expression profile of genes activated by RNA stimuli revealed no immune response in HSPCs. A progenitor assay indicated no alteration in the growth and multilineage differentiation of edited HSPCs. We applied this protocol to SCD and β-thalassemia HSPCs, achieving editing efficiencies up to ~70% of the HBG promoters. In RBCs differentiated from edited SCD HSPCs, RT-qPCR, HPLC and flow cytometry showed a potent γ-globin reactivation with a high frequency of HbF + cells and a concomitant decrease in the HbS content/cell. Importantly, the pathological RBC sickling phenotype was corrected in the samples derived from edited HSPCs. Similarly, in β-thalassemia samples, RT-qPCR and HPLC analyses showed strong γ-globin induction and decrease of the α-globin precipitates. HbF expression rescued the delay in erythroid differentiation and ineffective erythropoiesis characterizing β-thalassemia, as demonstrated by the increased RBC enucleation rate and the reduced apoptosis and oxidative stress. We then compared BE strategies that either disrupt the LRF BS or create a de novo KLF1 BS in single colonies derived from erythroid progenitors. Generation of the KLF1 BS was associated with higher levels of HbF compared to the LRF BS disruption. These results suggest that eviction of the LRF repressor is sufficient to reactivate HBG genes, but recruitment of an activator is more effective to achieve high levels of gene expression. HbF expression induced by both LRF BS disruption and KLF1 BS generation was sufficient to rescue the SCD cell phenotype, but higher HbF levels - achieved only through KLF1 BS generation - were necessary to fully correct the β-thalassemia phenotype. In the majority of cases, we detected no DSB-induced insertions, deletions, or large genomic rearrangements in base-edited samples. Accordingly, DSB-induced DNA damage response (DDR) was absent in base-edited HSPCs, as measured by evaluating the expression of p21, a readout of p53-induced DDR. DNA off-target activity was assessed by GUIDE-seq and targeted sequencing of the potential off-target sites in edited HSPCs, while RNA off-target activity was evaluated by RNA-seq in HSPCs. Finally, BE-treated HSPCs were transplanted in immunodeficient mice to evaluate the engraftment and differentiation capability of edited HSCs. We detected good frequencies of human cells with up to ~60% of edited promoters in the peripheral blood of transplanted mice. In conclusion, we developed a clinically-relevant strategy to perform efficient BE in the HBG promoters that led to therapeutically-relevant HbF levels and rescued both the SCD and β-thalassemia phenotypes, thus providing sufficient proof of efficacy and safety to enable the clinical development of base-edited HSPCs for the therapy of β-hemoglobinopathies. Disclosures El Nemer: Hemanext: Consultancy.

63rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) -- DEC 11-14, 2021 -- Atlanta, GA, [No Abstract Available], Amer Soc Hematol

Introduction: β-thalassemia is a genetic blood disorder marked by the inability to produce hemoglobin. Diagnosed primarily in childhood, transfusion-dependent β-thalassemia (TDT) is the most severe form of this disorder and results in chronic anemia, requiring frequent red blood cell (RBC) transfusions to maintain hemoglobin levels. While previous studies have shown that health-related quality of life (HRQoL) is adversely affected in patients with TDT, the issues associated with the transfusion process itself in these patients (eg, side effects and time requirements) have not always been examined. These issues are the focus of this study. Methods: Following exploratory qualitative interviews with a small sample of patients identified by the Cooley's Anemia Foundation (CAF), a 30-minute web-based survey was developed to assess HRQoL variables in TDT. Subsequently, CAF identified patients in the US who were ≥ 18 years of age, had a self-reported physician diagnosis of β-thalassemia, and had received ≥ 1 RBC transfusion in the past 6 months, and invited them to participate in the study. Following informed consent, patients completed a number of validated and ad hoc questions about their treatment, side effects, psychological burden, time requirements for a transfusion, and caregiver information. Data were collected in May 2021. Descriptive results are presented as frequencies, means, and standard deviations (SD). Results: A total of 100 patients were identified and recruited by CAF and completed the survey. Of these, 35 (35%) were male, with a mean age of 36.0 (SD = 10.4) years. Patients had been diagnosed with TDT for a mean of 34.4 years (SD = 10.3) and had received a mean of 9.6 (SD = 4.3) RBC transfusions in the previous 6 months. In addition to receiving RBC transfusions (100%), other treatments ever received for TDT included iron chelation therapy (94%), surgery (47%), and stem cell/bone marrow transplantation (5%). Regarding RBC transfusions, 66% of patients reported requiring them more than once per month, 31% each month, and 3% every other month. Common patient-reported side effects of RBC transfusions were iron overload (81%), rashes/hives (74%), fatigue (61%), and pain/bruising at the injection site (52%). Additionally, 86% of patients reported feeling fatigued leading up to RBC transfusions, 75% reported that treatments for TDT had become routine, and 64% accepted the routine. Time required for an RBC transfusion was estimated at a mean of 7.4 hours (SD = 11.3) including transfusion preparation time. Travel and wait times were estimated as requiring an additional 7.1 hours. Other time requirements included frequent outpatient visits and frequent laboratory tests, averaging 3.9 visits and 6.1 visits in a 6-month period, respectively. Overall, 13% of patients reported having a caregiver. Caregiver activities included travel to and from appointments (77%), help with household duties (77%), and assistance in treatment decisions (62%). Conclusions: The results of this survey suggest there is a substantial time and side-effect burden associated with transfusion and treatment among patients diagnosed with TDT. This includes considerable time devoted to preparing for and receiving transfusions, transfusion-related side effects, frequent laboratory and outpatient appointments, and help from a caregiver. Medical treatments that would decrease the dependency on transfusions in TDT could decrease this burden and improve overall quality of life. Disclosures Gupta: Kantar Health: Current Employment; Bristol Myers Squibb: Consultancy, Research Funding. Costantino: Kantar Health: Current Employment; Bristol Myers Squibb: Consultancy, Research Funding. Perkowski: Kantar Health: Current Employment; Bristol Myers Squibb: Consultancy, Research Funding. Inyart: Kantar Health: Current Employment; Bristol Myers Squibb: Consultancy, Research Funding. Ashka: Kantar Health: Current Employment; Bristol Myers Squibb: Consultancy, Research Funding. Clapp: Kantar Health: Current Employment; Bristol Myers Squibb: Consultancy, Research Funding. Price: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Knoth: Bristol Myers Squibb: Current Employment.

Background: Iron overload is inevitable but a manageable complication of frequent blood transfusion, the effectiveness and safety of two iron chelation therapy in young children with transfusion-dependent B thalassemia (TDB-T) using either deferiprone (DFP) or deferasirox (DFX) were well studied in clinical trials. The primary objectives were; reporting efficacy and frequency of all adverse events (AEs) as compared with published data. Secondary objective was reporting deviation from guidelines, The analysis of clinical data is the first step to design new strategies Methodology: An investigator initiated 2-year multicenter prospective study enrolling over 6 months period; 302 children with TDB-T from 11 centers; keeping pre-transfusion Hb >8gm/dl. When serum ferritin (SF) reached around 1000 ng/ml; they had received according to availability; either DFP liquid solution in a dose; 75 mg/kg/day (n=62) or DFX (n=240); (dispersed tablet; 25% or film-coated tablet 75% in a dose; 20-25 mg/kg/d or its equivalent). CBC was done biweekly in DFP and pre-transfusion in DFX group. Liver transaminases, serum creatinine and (SF) were done /3 months. Changes in growth velocity over time were documented. Results: 66% were males, age at enrolment was; (11-19 and 14-26 months) in DFP and DFX respectively; they had received (6-8) and (7-11) transfusions prior to chelation in both groups respectively in addition to (16-21) during the study. 20% of patients did not achieve the target pre-transfusion Hb level. At initiation of DFP; SF median; 960 (IQR; 122 ng/ml) while those on DFX had SF; median 1070 (IQR 138 ng/ml) both groups had comparable transfusion iron input during the study. At 24 months of regular transfusion-chelation; SF < 1000 ng/ml was noticed in 65% and 60% of the patients respectively with good growth velocity in children using either chelator. Unnecessary over-dosing of DFX > 40 mg/kg was observed in 5%, meanwhile 3.2% had received DFP in a dose >75-100 mg/kg while SF was 33% from baseline on 2 successive visits was observed in 5% in DFX without reducing the dose or re-check. No unexpected, serious severe adverse events were reported in both groups. Poorly controlled patients with SF > 1500 ng /ml at 12 or 24 months were 12, 8% in DFP group; in contrast to 15%, 10% in DFX group respectively; with no statistical difference; (compliance was comparable). The total DFP and DFX discontinuation ratio was 5% in each group. Conclusion: infants and young children with TDB-T naïve to chelation were put on mono-therapy at younger age than reported before. Both chelators were effective, well tolerated and with no severe safety concerns; however deviation from guidelines was observed as; unnecessary over-dosing of DFX and under-reporting of AEs in both groups in real life practice. Disclosures Hamdy: ApoPharma: Honoraria; Amgen: Honoraria; Bayer: Honoraria; Novartis: Honoraria; NovoNordisk: Honoraria; Roche: Honoraria; Takeda: Honoraria.

Introduction Beta thalassemia is an inherited disorder characterised by ineffective erythropoiesis leading to anemia and secondary iron overload. Neutrophils are the first line of innate immune defence against infection is dysfunctional in thalassemia patients. Iron is required for the oxidative response of neutrophils to allow the production of reactive oxygen species (ROS). However, the role of iron contributing to the neutrophil dysfunction is unclear. This is the first study to characterise the neutrophil iron metabolism in β-thalassemia and its association with oxidative burst capacity, phagocytosis, and systemic iron homeostasis. Method Sixteen thalassemia patients and fourteen healthy individuals were recruited in the department of Haematology, Christian Medical College, Vellore, India. Neutrophils were purified from human whole blood collected in EDTA tube, using neutrophil magnetic isolation kit (Miltenyi Biotec). Purified population (>95%) was confirmed by the presence of the surface marker CD62L evaluated using flow cytometry. Haematological parameters were analysed according to standard methods. Serum ferritin, iron, soluble transferrin receptor were measured using immunoassay. Serum hepcidin was measured using ELISA. Neutrophil RNA was isolated using trizol method and was reverse transcribed into complementary DNA using QIAGEN kit. The relative quantification of iron related genes were measured using real-time PCR. In oxidative burst assay, neutrophils were incubated with dihydrorhodamine 123 (DHR) and stimulated with Phorbol 12-Myristate 13-Acetate (PMA). Respiratory burst of the cell was analysed by flow cytometry. Phagocytosis and acidification capacity of human neutrophils were quantified using the pHrodo Green Staphylococcus aureus BioParticles kit (Thermo Fisher). Acquisition was performed using the Beckman Coulter(Navios) flow cytometer and analysed using kaluza software. Statistical analysis was performed using SPSS software. Results We investigated a cohort of β thalassemia Major (n=5), intermedia (n=6) and sickle beta thalassemia (n=5) patients who were on regular iron chelation therapy. The demographic and biochemical parameters are tabulated in Table 1. Serum iron, ferritin levels and transferrin saturation were significantly increased in thalassemia cohort as compared to healthy donors (Fig1a). There was no significant association between ferritin and hepcidin levels. The percentage of neutrophils in thalassemia was significantly reduced incomparision to healthy donors (p=0.032). Oxidative burst capacity of neutrophils from thalassemia major and intermedia patients were significantly decreased (p=0.002) compared to healthy donors upon stimulation with PMA (Fig1b). Neutrophil phagocytosis capacity indicated by the relative amounts of phagocytized fluorescein S.aureus particles were significantly lower in thalassemia patients compared with controls, after 15min/30min incubation (Fig1c). The recognition capacity of neutrophils towards bioparticles significantly decreased at 45-minute incubation in patients (p=0.017) (Fig1d). Serum iron overload and transferrin saturation had negative association with neutrophil phagocytosis capacity (r=-0.714; p=0.045 & r=-0.857; p=0.014), respectively. Neutrophil iron related gene expression was analysed and found significantly lower expression of FPN 1A (FPN1 containing iron regulatory element (IRE)), DMT1B without IRE region and IRP2 respectively (p=0.029, p=0.029 & p=0.016) (Fig1e). FPN1B without IRE region was upregulated in thalassemia patients (p=0.016). Serum ferritin had positive correlation with FPN1B (r=0.786; p=0.036). Soluble transferrin receptor had negative association with DMT1A (containing IRE region) and IRP2 respectively (r=-0.900; p=0.037 and r=-0.750; p=0.052). Aberrant neutrophil function was found in all thalassemia patients. Although oxidative burst activity was decreased in thalassemia major and intermedia patients, sickle β-thalassemia patients had normal burst activity. Systemic iron status had inverse correlation with phagocytosis capacity of neutrophils. Dysregulation of iron transporters in neutrophils was indicated by decreased expression of DMT1 and augmented FPN1B expression, despite systemic iron overload. These findings have to be explored in a larger cohort to elucidate the clinical significance. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction. Pancreatic iron deposition is a common finding in thalassemia major, being detected in more than one third of patients undergoing their first T2* Magnetic Resonance Imaging scan (MRI) for this purpose. However, no longitudinal studies on pancreatic iron are available in literature. Aim: The aim of this multicenter study was to evaluate the changes in pancreatic iron overload in TM patients enrolled in the Extension-Myocardial Iron Overload in Thalassemia (E-MIOT) Network who performed a baseline and a follow-up (FU) MRI scan at 18 months. Methods. We considered 416 TM patients (37.77±10.46 years; 220 females) consecutively enrolled. Iron overload was quantified by the T2* technique. T2* measurements were performed over pancreatic head, body and tail and global value was the mean. Results. Pancreatic iron overload (global pancreas T2* A significant inverse association was detected between % change in global pancreas T2*and baseline global pancreas T2* values (R=-0.369; P Changes (%) in global pancreas T2* were not associated with baseline serum ferritin levels or MRI liver iron concentration (LIC) values but were inversely correlated with % changes in serum ferritin levels (R=-0.199; P At baseline MRI, 169 patients showed an alteration of glucidic metabolism: 32 had impaired fasting glucose, 65 impaired glucose tolerance, and 72 diabetes mellitus. These patients showed significantly higher % changes in global pancreas T2* than patients with a normal glucidic metabolism (33.06±79.48% vs 11.93±59.47%; P=0.003). Conclusions. Our data showed that it is difficult to remove the iron from the pancreas and higher improvements were detected in more heavily loaded patients, with alterations of glucidic metabolism. The reduction in pancreatic iron was paralleled by a decrease in hepatic and cardiac iron. Figure 1 Figure 1. Disclosures Pepe: Bayer S.p.A.: Other: no profit support; Chiesi Farmaceutici S.p.A: Other: no profit support. Maggio: Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Celgene Corp: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Introduction: Patients with transfusion-dependent β-thalassemia (TDT) have increased morbidity and mortality and reduced health-related quality of life (HRQoL) compared with the general population owing to the mental, physical, and time demands of chronic transfusions, iron chelation, and treatment. Betibeglogene autotemcel (beti-cel) is a one-time ex vivo gene therapy for TDT that enables stable production of functional adult hemoglobin (Hb) sufficient for transfusion independence (TI). In 2 ongoing, fully enrolled, phase 3 studies, HGB-207 (Northstar-2; NCT02906202) and HGB-212 (Northstar-3; NCT03207009), 32/36 (89%) evaluable patients achieved TI (weighted average Hb ≥9 g/dL without packed red blood cell transfusions for ≥12 mo after drug product infusion) with a median (min-max) weighted average Hb of 11.52 (9.3-13.7) g/dL during TI (as of March 9, 2021). In the current analysis, we evaluated the effect of beti-cel on HRQoL in 12 adults and 18 pediatric/adolescent patients with TDT enrolled in phase 3 studies who achieved TI. Methods: CD34+ cells were mobilized using granulocyte-colony stimulating factor and plerixafor, collected via apheresis, transduced with BB305 lentiviral vector, and infused back into patients after single-agent, pharmacokinetic-adjusted, busulfan-based myeloablation. HRQoL over time was assessed using the Pediatric Quality of Life Inventory (PedsQL; range, 0-100) for pediatric and adolescent patients ( Results: Forty-one patients were infused with beti-cel (HGB-207, n=23; HGB-212, n=18). Age at informed consent was 4-34 years; most patients (27/41, 66%) were Conclusions: Beti-cel is potentially curative in patients with TDT through the achievement of TI and near-normal Hb levels. Beti-cel addresses the disease pathophysiology at a genetic level and allows patients to achieve TI. Pediatric and adult patients with TDT who achieved TI after receiving beti-cel in phase 3 studies showed early and sustained improvements in HRQoL, despite relatively high HRQoL with supportive care at baseline. Figure 1 Figure 1. Disclosures Kwiatkowski: bluebird bio,Inc.: Consultancy, Research Funding; Celgene: Consultancy; Sangamo: Research Funding; Apopharma: Research Funding; Bristol Myers Squibb: Consultancy; Imara: Consultancy, Research Funding; Agios: Consultancy; Silence Therapeutics: Consultancy; Bioverativ: Research Funding. Locatelli: Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyl: Honoraria; bluebird bio, Inc.: Consultancy; 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. Walters: AllCells, Inc: Consultancy; BioLabs, Inc: Consultancy; Vertex pharmaceuticals: Consultancy; Ensoma, Inc.: Consultancy. Porter: Silence Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene (BMS): 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; La Jolla Pharmaceuticals: Honoraria; Protagonism: Honoraria; Vifor: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Honoraria. Yannaki: Gilead: Speakers Bureau; SANDOZ: Speakers Bureau; bluebird bio, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Speakers Bureau. Kulozik: bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BioMedX: Consultancy, Honoraria. Thrasher: Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Thuret: bluebird bio, Inc.: Other: investigators for clinical trials, participation on scientific/medical advisory board; Apopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: investigators for clinical trials, participation on scientific/medical advisory board; Celgene: Other: investigators for clinical trials, participation on scientific/medical advisory board. Lal: Insight Magnetics: Research Funding; Protagonist Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; La Jolla Pharmaceutical Company: Research Funding; Chiesi: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; bluebird bio, Inc.: Research Funding; Agios Pharmaceuticals: Consultancy; Terumo Corporations: Research Funding. Guo: bluebird bio, Inc.: Current Employment, Other: Employee, Ownership Interest and Salary. Colvin: bluebird bio, Inc.: Current Employment, Other: Employee, Ownership Interest and Salary. Gruppioni: bluebird bio, Inc.: Current Employment, Other: Employee, Ownership Interest and Salary. Thompson: Baxalta: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; Biomarin: Research Funding; Celgene/BMS: Consultancy, Research Funding; CRISPR Therapeutics: Research Funding; Vertex: Research Funding; Editas: Research Funding; Graphite Bio: Research Funding; Novartis: Research Funding; Agios: Consultancy; Beam: Consultancy; Global Blood Therapeutics: Current equity holder in publicly-traded company.

Background Sickle cell disease (SCD) and thalassemia are inherited hemoglobinopathies characterized by lifelong anemia. In SCD, a single mutation in the β-globin gene results in sickle hemoglobin (HbS) that polymerizes upon deoxygenation, causing red blood cells (RBCs) to sickle leading to a variety of complications. In thalassemia, mutation(s) in α- or β-globin genes result in reduced or absent adult Hb causing ineffective erythropoiesis and downstream complications. The resultant anemias are exacerbated by impaired RBC function due to decreased ATP content. Supportive care and agents like hydroxyurea are used most in SCD, with a subset of patients (pts) on regular transfusions. Regular or episodic transfusions, with their own set of complications, are the mainstay of treatment for thalassemias. Etavopivat, a potent, selective, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) increases ATP and decreases 2,3 diphosphoglycerate (2,3-DPG). In a Phase 1 study, etavopivat 300‒600 mg once daily in pts with SCD not regularly transfused was well-tolerated, improved hematological markers, decreased hemolysis, and improved markers of RBC functional health (Brown et al. EHA 2021 # EP1202). Etavopivat 200 and 400 mg once daily (dose levels predicted to provide the desired pharmacodynamic response profiles) is being evaluated in a Phase 2/3 study of pts with SCD who are not on chronic transfusions (The Hibiscus Study, NCT04624659). Herein we describe the design of a Phase 2, open-label, multicenter study (NCT04987489) evaluating the efficacy and safety of etavopivat in pts with: SCD on chronic transfusions (Cohort A), transfusion-dependent thalassemia (Cohort B), and non-transfusion-dependent thalassemia (Cohort C). Study Design and Methods Up to 20 pts aged 12-65 years will be enrolled in each of the three cohorts described above. Pts will receive etavopivat 400 mg once daily for 48-wks (Figure). In cohorts A and B, pts must have received ≥6 RBC units in the 24 wks before the first dose of etavopivat without a >35-day transfusion-free period during that period and be on iron chelation therapy for >3 months before enrollment. Additionally, pts in Cohort A should have received ≥24 monthly transfusions for the prevention or treatment of primary stroke. Pts in Cohort C should have a Hb ≤10 g/dL. Key exclusion criteria include significant infection, hepatic/renal dysfunction, history of malignancy/cardiac/pulmonary disease, a drug malabsorption disorder, prior/concomitant therapies ≤3 months before the first dose (eg, chronic systemic glucocorticoids, new chelation therapy). Baseline assessments will include medical, disease, transfusion, and medication histories. Transfusions received during the study (every ~3-5 wks) will be recorded and include Hb values before and ≥15 minutes after transfusion, number of RBC units, and volume of packed RBCs. If a pt has an increase in pre-transfusion Hb of ≥1.0 g/dL compared with their baseline pre-transfusion Hb, the Investigator may delay transfusion by 1 wk or reduce the number of RBC units transfused. In pts with SCD, RBC exchange may also be performed. The primary endpoint for Cohorts A/B is erythroid response defined as the proportion of pts with ≥20% reduction in transfusions over a continuous 12-wk treatment period versus baseline, and for Cohort C is Hb response at Wk 12 defined as an increase in Hb of ≥1.0 g/dL from baseline. For Cohorts A/B, secondary and exploratory endpoints include the proportion of pts with a reduction in transfusions over 12 wks of ≥33% and ≥50%, respectively, and a reduction in transfusions over 12, 24, and 48 wks; and for Cohort C, Hb response at Wks 24 and 48, and changes from baseline in Hb over 12, 24, and 48 wks. The following additional endpoints will be assessed for all cohorts: changes from baseline in quality of life as assessed via the Short Form Health Survey and Patient-Reported Outcome Measurement Information System Fatigue Scale; changes from baseline in the levels of serum ferritin at 12, 24, and 48 wks; liver iron at 48 wks; 2,3-DPG and ATP; pharmacokinetics; and safety. All primary endpoints will be analyzed using a 1-sided test at α=0.025. Summary Etavopivat is a novel, investigational, PKR activator designed to improve the functional health of RBCs. This Phase 2 study will assess the safety of etavopivat and its impact on Hb levels and transfusion burden in pts with SCD or thalassemia. Figure 1 Figure 1. Disclosures Lal: Chiesi: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; bluebird bio, Inc.: Research Funding; Protagonist Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharmaceuticals: Consultancy; Insight Magnetics: Research Funding; Novartis: Research Funding; Terumo Corporations: Research Funding; La Jolla Pharmaceutical Company: Research Funding. Brown: Novo Nordisk: Consultancy; Novartis: Consultancy, Research Funding; Imara: Consultancy, Research Funding; Forma Therapeutics: Research Funding; Pfizer: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding. Coates: Apo Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Chiesi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Forma Pharma: Consultancy; Sangamo: Consultancy; UpToDate: Patents & Royalties; Vifor Pharma: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kalfa: Agios Pharmaceuticals, Inc.: Other: Steering Committee, Research Funding; FORMA Therapeutics, Inc: Research Funding. Kwiatkowski: Bioverativ: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; CRISPR: Research Funding; Imara: Consultancy, Research Funding; Silence Therapeutics: Consultancy; Sangamo: Research Funding; bluebird bio: Consultancy, Research Funding; Chiesi: Research Funding; Vertex: Research Funding. Brevard: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Potter: Forma Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company; Bristol Myers Squibb: Ended employment in the past 24 months. Wood: Forma Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Sheth: Bluebird bio: Consultancy; CRISPR: Consultancy; Bristol Myers Squibb: Consultancy, Research Funding; Imara: Research Funding; Chiesi: Consultancy; Agios: Consultancy; Dispersol: Research Funding.

HbE/β-thalassemia is the commonest form of severe β-thalassemia, and comprises approximately 50% of all cases worldwide. HbE/β-thalassemia is caused by the HbE codon 26 G>A mutation on one allele and any severe β 0-thalassemia mutation on the other. These mutations lead to a reduction in β-globin production, resulting in a relative excess in α-globin chains that go on to cause ineffective erythropoiesis. Importantly, individuals with a mutation on one, but not two, alleles have β-thalassemia trait, a carrier state with a normal phenotype. Recent gene therapy and gene editing approaches have been developed to treat β-thalassemia but do not directly repair the causative mutation in-situ. Gene replacement approaches rely on lentiviral vector-based sequence insertion or homology directed repair (HDR). HbF induction strategies also rely on non-homologous end joining (NHEJ) targeting of enhancers in-trans. These approaches, whilst variably successful, are associated with potential safety concerns. Adenine base editors (ABEs) potentially circumvent these problems by directly repairing pathogenic variants in-situ through deamination. ABEs catalyse A-T to G-C conversions through targeting with a Cas9-nickase and single-guide RNA (sgRNA). Conversion of the HbE codon to normal through base editing is an attractive strategy to recapitulate the phenotypically normal β-thalassemia trait state without potentially harmful double-strand breaks or random vector insertions (Figure 1A). ABEs are able to convert the HbE codon (AAG, lys) to wild-type (GAG, glu), but also to GGG (gly) or AGG (arg). GGG at codon 26 is found in a naturally occurring hemoglobin, Hb Aubenas. Heterozygotes have normal red cell indices and are phenotypically normal. We electroporated the latest generation of ABE8 editors (ABE8e, ABE8.13 and ABE8 V106W) as mRNA into WT CD34+ hematopoietic stem and progenitor cells (HSPCs) with sgRNAs targeting the middle A of the WT GAG codon. These had similar editing efficiencies although ABE8 V106W had marginally higher on-target efficiency. V106W has been evolved to have a favourable off-target profile. V106W mRNA/sgRNA was electroporated into 3 different severe HbE/β-thalassemia donor HSPCs. The HbE codon was converted to WT with a mean 28.7% efficiency, to Hb Aubenas 48.6% and to an undescribed AGG codon 2.1%. The mean conversion from HbE to a normal or normal variant was 78.7±8.7% (Figure 1B). The indel rate from inadvertent on-target Cas9 cleavage was below 0.5%. Edited cells did not show any perturbations in erythroid differentiation as assessed by Immunophenotyping and cellular morphology. In differentiated erythroid cells, RT-qPCR showed a mean fall in the α/β mRNA ratio to 0.65±0.08 (unedited patient cells normalised to 1, n=5, Figure 1C), indicating a reduction in the relative excess α-globin gene expression. Protein analysis by CE-HPLC showed a 3.6-fold reduction in HbE levels (SD±1.3) and a 13.5-fold increase in HbA/Hb Aubenas (SD±2.4, Figure 1C and D). To prove that base editing using mRNA was possible in long-term HSCs, CD34+ cells from 4 WT cord blood donors were edited using ABEmax. Mice were culled after 16 weeks, and human cells were collected and transplanted into 7 secondary mice, which were also culled after 16 weeks. Each secondary mouse showed the presence of hCD45+ cells, indicating engraftment of LT-HSCs. All secondary replicates showed editing, with a mean editing efficiency of 34.5% (initial editing 46.3%). In both rounds of mice, there was robust lymphoid and myeloid engraftment and expected levels of erythroid engraftment for the NSG model in bone marrow and spleen. Potential off-target effects were assessed in-vitro by CIRCLE-seq in triplicate. These sites were assessed by targeted oligonucleotide capture of DNA from mRNA edited patient cells to detect in-vivo editing. Together these data provide robust evidence for base editing as an effective and safe therapeutic strategy for HbE/β-thalassemia. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Ineffective erythropoiesis (IE) is a crucial hallmark of β-Thalassemia (β-Thal) and sets the goal for treatment of both Transfusion-Dependent (TDT) and Non Transfusion-Dependent Thalassemia (NTDT) patients. The Growth Differentiation Factor (GDF) group has a relevant role in the molecular regulation of IE. Specifically, GDF11 contributes to the inhibition of RBC maturation and it is targeted by activin traps, such as luspatercept. However, its role is still debated; i.e., in a mouse model of β-Thal, the absence of GDF11 alone is not sufficient to mitigate IE (Guerra A et al., Blood, 2019). GDF15 increases from early until late phases of erythroid differentiation and negatively regulates erythroid cell development in-vitro, modulating maturation and apoptosis. (Ranjbaran R et a.l, Exp Cell Res, 2020). A few clinical studies found elevated serum GDF15 levels in β-Thal (Tanno T et al., Nat Med, 2007; Huang Y et al., Int J Med Sci, 2019), but data are sparse and a clear correlation with the severity of the pathology is still missing. Methods: We run an observational study at our institution. At routine checks, patients were asked to consent to a specific blood sample for GDF15, whereas hemoglobin (Hb), serum erythropoietin (Epo), ferritin (Ftn), iron, and transferrin saturation (TSat) were measured as part of clinical practice. In a small subset of patients, a consent for serial sampling was added. Serum GDF15 was measured by ELISA (DuoSet DY957, R&D Systems). Demographics were collected from clinical records. Statistical analysis was performed using Statistica 10 (Statsoft). Results: GDF15 levels were measured in 458 individuals: 267 TDT, 77 NTDT, 45 β-Thal trait carriers (BTC), and 69 healthy (H) subjects. Median (IQR) levels of GDF15 were significantly different among diagnoses (P Discussion: GDF15 levels correlated with the severity of β-Thal phenotype, showing a 26-fold (TDT), a 6-fold (NTDT) and a 2-fold (BTC) increase compared to controls. In TDT patients, higher GDF15 levels correlated with lower Hb and higher Epo, which are typically observed as a result of IE in thalassemia. In addition, GDF15 correlated with markers of altered iron metabolism, such as TSat and serum iron. In individual patients, GDF15 showed strong and consistent variation with treatment. GDF15 was also associated with quantitative markers of disease in NTDT and BTC patients. This is to our knowledge the larger sample of patients carrying β-thal mutations in which GDF15 levels were measured and correlated with the severity of the disease. These results show that GDF15 may be a suitable and useful quantitative marker of IE. Figure 1 Figure 1. Disclosures Piga: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acceleron: Research Funding. Longo: Bristol Myers Squibb: Honoraria; BlueBird Bio: Honoraria.

β-thalassemia is an autosomal recessive disorder due to mutations in the β-globin gene, that leads to defective production of hemoglobin (Hb) and red blood cells (RBC). The main features of the disease are anemia, ineffective erythropoiesis and iron overload. Patients affected by the most severe form of β-thalassemia are transfusion-dependent (TDT) and require lifelong blood transfusions and iron chelation, symptomatic treatments that affect the quality of life. The only curative option, unfortunately limited by the insufficient number of HLA-matched donors, is allogenic bone marrow (BM) transplantation. Other recently approved treatments (i.e. Luspatercept and gene therapy) are only partially effective and/or suitable for selected patients. Thus, the identification of novel therapeutic approaches is a clinical need. Transferrin receptor 2 (TFR2) contributes to the transcriptional activation of hepcidin, the master regulator of iron homeostasis, in the liver and is a brake of Erythropoietin signaling in erythroid cells, thus balancing RBC production and iron availability. We have recently proved that BM Tfr2 deletion enhances erythropoiesis in wild-type mice (Nai et al., Blood 2015) and ameliorates anemia in non-TDT mice models, both alone (Artuso et al., Blood 2018) and in combination with iron-restricting agents (Casu, Pettinato et al., Blood 2020). Here we aim at investigating whether Tfr2 targeting might be beneficial also for TDT. To this purpose we generated TDT mice (Hbb th1/th2; Casu et al., Haematologica 2020) with heterozygous (Tfr2 BMhetero/Hbb th1/th2) and homozygous (Tfr2 BMKO/Hbb th1/th2) BM Tfr2 deletion by transplantation of Hbb th1/th2, Tfr2-hetero/Hbb th1/th2 and Tfr2-ko/Hbb th1/th2 fetal liver cells (FLT) from day E14.5 embryos into lethally irradiated wild-type mice. BM Tfr2 deficient mice have increased RBC count and Hb levels and decreased percentage of reticulocytes with a gene dosage effect 8 weeks after FLT, before the onset of transfusion-dependance. At this time-point, complete BM Tfr2 deletion ameliorates ineffective erythropoiesis, decreasing the percentage of polychromatic erythroblasts and increasing orthochromatic erythroblasts and mature RBCs both in the BM and in the spleen. The improved anemia was also accompanied by a partial correction of two debilitating TDT complications, iron overload and cardiomegaly. The beneficial effect of Tfr2 deletion was maintained over time. Indeed, Hbb th1/th2 mice became transfusion-dependent 14 weeks after FLT, when Hb levels drop below 5.5 g/dL, requiring transfusions of an average 108.75±56.87μL of blood/animal/week. On the contrary, animals with both heterozygous and homozygous BM Tfr2 deletion are still non-transfusion-dependent at 20 weeks, maintaining Hb levels above 7 and 9 g/dL respectively. Overall, our results prove that, despite the persistence of the genetic defect, hematopoietic Tfr2 deletion ameliorates anemia, ineffective erythropoiesis and secondary complications also in the most severe form of β-thalassemia. This improvement is associated to a substantial increase in transfusion-free survival of both Tfr2 BMhetero/Hbb th1/th2 and Tfr2 BMKO/Hbb th1/th2 mice, which are transfusion-independent 6 weeks after the time of blood transfusion requirement in Hbb th1/th2 animals. The difference of blood transfusions needs will be evaluated over time for at least 10 additional weeks. Our findings demonstrate that TFR2 targeting represents a new promising therapeutic opportunity for the management of β-thalassemia, worth to be tested both as a monotherapy and in association with available treatments. Disclosures Rivella: Incyte: Consultancy; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forma Theraputics: Consultancy; Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy.

Introduction: Patients (pts) with non-transfusion-dependent β-thalassemia (NTDT) suffer from chronic anemia due to ineffective erythropoiesis. Low hemoglobin (Hb) levels in pts with anemia are associated with development of clinical complications and poor quality of life (QoL). There are no approved treatments for NTDT-associated anemia. Luspatercept has been shown to increase Hb levels in pts with NTDT in the BEYOND study (Taher AT, et al. HemaSphere 2021;5[Suppl 2];Abstract S101). This Hb increase was correlated with improvement in the NTDT Patient-Reported Outcomes (NTDT-PRO) Tiredness/Weakness (T/W) domain. Here we report the effect of luspatercept on red blood cell transfusion (RBCT) burden and QoL outcomes in pts with NTDT in the BEYOND study. Methods: Eligible pts with Hb ≤ 10 g/dL were randomized to luspatercept (1.0-1.25 mg/kg) or placebo (PBO) subcutaneously for ≥ 48 weeks (wk). The disease-specific instrument NTDT-PRO, measuring chronic anemia symptoms in pts with NTDT, was administered to pts as a daily diary starting on the 7 days prior to Dose 1, Day 1, until wk 24, and thereafter over the 7 days prior to dosing at every other dosing visit. The 36-item Short Form Health Survey Version 2 (SF-36 v2) and Functional Assessment of Chronic Illness Therapy-Fatigue, Fatigue subscale (FACIT-F FS) were completed by the pts at screening and at every other dosing visit thereafter starting with day 1, dose 1. Mean change from baseline in NTDT-PRO T/W and Shortness of Breath (SoB) domain scores and in FACIT-F FS score over a continuous 12-wk interval during wk 13 to 24 and wk 37 to 48 was assessed using analysis of covariance (ANCOVA) with treatment group and other relevant covariates in the model. The SF-36 v2 physical component summary (PCS) and mental component summary (MCS) mean change from baseline at wk 24 and wk 48 were analyzed using a mixed-effect repeated measures model with intercept and time as the random effect. The model included change from baseline scores up to wk 48 as the outcome, treatment group assignment, time, baseline scores, randomization stratification factor(s), time-by-baseline score, and time-by-treatment group assignment terms. Descriptive statistics were used to ascertain the number of RBC units, RBCT events, reason for RBCT, and time to first RBCT event. Results: Of 145 pts, 96 were randomized to luspatercept and 49 to PBO. NTDT-PRO analyses were conducted in the intention-to-treat population. The QoL-evaluable populations for FACIT-F FS and SF-36 v2 analyses (luspatercept vs PBO) were: 94 vs 47 and 92 vs 46 pts, respectively. Baseline domain scores of the QoL measures were comparable between the 2 treatment arms. Mean change from baseline (least squares mean [LSM]) in NTDT-PRO T/W domain score (wk 13-24) was −0.68 vs −0.20 for luspatercept vs PBO, respectively (LSM difference [LSMD] −0.48; 95% confidence interval [CI] −1.03 to 0.08; P = 0.0924). Improvement was more pronounced in the luspatercept arm during wk 37-48 (Table). Within the NTDT-PRO SoB domain, mean change from baseline (wk 13-24) was −0.46 vs 0.02 for luspatercept vs PBO, respectively (LSMD −0.49; 95% CI −1.02 to 0.04; P = 0.0721) and during wk 37-48 was −0.59 vs 0.47 for luspatercept vs PBO, respectively (LSMD −1.07; 95% CI −1.80 to −0.33; P = 0.0047) (Table). Favorable QoL improvements were also observed in the luspatercept arm vs PBO and increased over time for both FACIT-F FS and SF-36 v2 assessments (Table). During wk 1-48, 79/96 (82.3%) luspatercept vs 22/49 (44.9%) PBO pts were RBCT-free (P < 0.0001); 5/13 (38.5%) and 1/7 (14.3%) pts treated with luspatercept and PBO, respectively, who received RBCTs at baseline, became RBCT-free during wk 1-48. Among all pts who received RBCTs during wk 1-48, pts receiving luspatercept required fewer RBCTs compared with pts receiving PBO (median 1.0 RBCTs [range 1.0-6.0] vs 2.0 [1.0-7.0], respectively), and fewer RBC units (median 2.0 units [range 1.0-8.0] vs 2.5 [1.0-13.0], respectively). Time to first RBCT (median [range] days) was longer for pts receiving luspatercept vs PBO (336 [2.0-336.0] vs 260.0 [9.0-336.0], respectively) and, in both arms, the main reasons for receiving RBCTs were anemia and surgery. Conclusions: In pts with NTDT, luspatercept improved RBCT burden and QoL compared with PBO. The improvement was more pronounced over time and consistent across 3 QoL-measuring instruments. Most pts receiving luspatercept remained RBCT-free during wk 1-48 of treatment. Figure 1 Figure 1. Disclosures Kattamis: Novartis: Consultancy, Honoraria, Research Funding; Chiesi: Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding; CRISPR/Vertex: Consultancy, Honoraria; VIFOR: Consultancy; IONIS: Consultancy; Agios Pharmaceuticals: Consultancy; Amgen: Consultancy. Viprakasit: Ionis Pharmaceuticals,: Consultancy, Research Funding; Protagonist Therapeutics: Consultancy, Research Funding; La Jolla Pharmaceuticals: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Vifor Pharma: Consultancy, Research Funding. Cappellini: IONIS Pharmaceuticals: Consultancy; Protagonist Therapeutics: Research Funding; La Jolla: Research Funding; Vifor: Consultancy; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; CRISPR Therapeutics: Research Funding. Voskaridou: NOVARTIS: Research Funding; IMARA: Research Funding; BMS: Consultancy, Research Funding; GENESIS: Consultancy, Research Funding; PROTAGONIST: Research Funding; ADDMEDICA: Consultancy, Research Funding. Piga: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acceleron: Research Funding. Porter: Celgene (BMS): 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; Silence Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vifor: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Honoraria; Protagonism: Honoraria; La Jolla Pharmaceuticals: Honoraria. Coates: UpToDate: Patents & Royalties; Sangamo: Consultancy; Forma Pharma: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Apo Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Chiesi: Consultancy, 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; Vifor Pharma: Consultancy. Forni: Bluebirdbio: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Shetty: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Miteva: BMS: Current Employment. Esposito: Bristol Myers Squibb: Current Employment. Kuo: Bristol Myers Squibb: Current Employment. Guo: Gilead: Consultancy; Janssen: Consultancy; UCB: Consultancy; Daiichi Sankyo: Consultancy; EMD Serono: Consultancy; Bristol Myers Squibb: Consultancy; Evidera: Current Employment. Pelligra: Evidera: Current Employment; Bristol Myers Squibb: Research Funding. Lord-Bessen: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Yucel: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Taher: Novartis: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Vifor Pharma: Consultancy, Research Funding; Agios Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy, Research Funding.

Allogeneic stem cell transplantation (SCT) is a curative treatment for beta-thalassemia major. The Pesaro risk stratification classifies patients with thalassemia into three groups (class I, II, and III) to predict the transplant-related mortality (TRM) and graft rejection after myeloablative conditioning with busulfan and cyclophosphamide. However, in developing countries, where inadequate chelation therapy prior to transplant is common, most patients would fall under the class III category. There is heterogeneity in the clinical outcomes in this group. Hence, our center introduced a further refinement in this risk stratification, identifying a subset of "class III high risk" as those with age greater than or equal to 7 years and liver size > 5cm (Mathews V et al. BBMT 2007). Subsequently, we showed that the use of a conditioning regimen containing thiotepa, treosulfan, and fludarabine (TTF) along with a peripheral blood stem cell graft led to improved outcomes in this subset with a reduction in early TRM from 46% (with busulfan-cyclophosphamide) to 13% (Mathews V et al. PLoS One 2013). There is still a need to identify predictors of poor clinical outcomes to optimize further the clinical outcomes of these class III high-risk thalassemia patients. Endothelial activation and stress index (EASIX) is a simple biomarker calculated using lactate dehydrogenase, creatinine, and platelet counts. EASIX has been shown to be predictive of overall survival in various settings like GVHD following reduced-intensity transplants for malignancies in adults, veno-occlusive disease, and nonrelapse mortality following SCT (Luft T et al. Lancet Haem 2017; Jiang S et al. Haematologica 2021; Luft T et al. BMT 2020). Here, we evaluated the role of EASIX (measured before conditioning therapy) as a biomarker in predicting early TRM in patients with thalassemia major who have undergone SCT with a uniform conditioning regimen using TTF at our center. During the study period from January 2012 to December 2019, 281 patients with thalassemia major underwent SCT with a uniform TTF protocol at our center. The median age was nine years (range 1 to 25 years). One hundred and nine (38.8%) were females. As per Pesaro classification (with Vellore modification), three (1.1%) were class I, 34 (12.1%) were class II, 134 (47.7%) were class III low risk, and 110 (39.1%) were class III high risk. The stem cell donors were matched sibling (n=218, 77.6%), matched related non-sibling (n=23, 8.2%), or matched unrelated donors (n=40, 14.2%). The stem cell donor was HLA matched in all cases except 21 (7.5%), wherein there was a mismatch at one locus. Five (1.8%) had a bone marrow graft, while others had peripheral blood stem cell grafts. Thirty-eight (13.5%) patients had transplant-related mortality by day 100 (TRM100). The median follow-up of the cohort was 31 months (range 0 to 103 months). EASIX score pre-transplant was available for 184 (65.5%) patients. There was no difference in the rate of TRM100 in patients where EASIX was available compared to those where EASIX was not available (14.7% versus 11.3%, p 0.47). Also, there was no difference in overall mortality rate in patients where EASIX was available compared to those where EASIX was not available (21.2% versus 17.5%, p 0.53). Among patients with TRM100 vis-à-vis those who did not, the median EASIX score was significantly higher (1.09 versus 0.75, p 0.008). We then plotted a receiver operating characteristics (ROC) curve for predicting TRM100 using the EASIX score. The area under the curve was 0.661 (Figure 1a). A cut-off of 0.85 for the EASIX score had a 70.4% sensitivity and 62% specificity for predicting TRM100. The TRM100 for patients with EASIX above 0.85 was significantly higher than those with EASIX less than 0.85 (24.4% versus 7.5%, p 0.003). On multivariable logistic regression analysis, the factors independently predicting TRM100 were pre-transplant EASIX score, pre-transplant ferritin, unrelated donor source, and chimerism at day 60 (Figure 1b). In patients with thalassemia major undergoing SCT using a uniform TTF conditioning, EASIX score measured pre-transplant can identify patients at greater risk for Day100 TRM. Figure 1 Figure 1. Disclosures Mathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.