Adenine base editor-mediated correction of the common and severe IVS1-110 (G>A) ß-thalassemia mutation.

ß-Thalassemia (BT) is one of the most common genetic diseases worldwide and is caused by mutations affecting ß-globin production. The only curative treatment is allogenic hematopoietic stem/progenitor cells (HSPCs) transplantation, an approach limited by compatible donor availability and immunological complications. Therefore, transplantation of autologous, genetically-modified HSPCs is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors are not equally effective in all patients and CRISPR/Cas9 nuclease-based strategies raise safety concerns. Thus, base editing strategies aiming to correct the genetic defect in patients' HSPCs could provide safe and effective treatment. Here, we developed a strategy to correct one of the most prevalent BT mutations (IVS1-110 [G>A]) using the SpRY-ABE8e base editor. RNA delivery of the base editing system was safe and led to ∼80% of gene correction in the HSPCs of patients with BT without causing dangerous double-strand DNA breaks. In HSPC-derived erythroid populations, this strategy was able to restore ß-globin production and correct inefficient erythropoiesis typically observed in BT both in vitro and in vivo. In conclusion, this proof-of-concept study paves the way for the development of a safe and effective autologous gene therapy approach for BT.

Hydroxyurea is associated with later onset of acute splenic sequestration crisis in sickle cell disease: Lessons from the European Sickle Cell Disease Cohort-Hydroxyurea (ESCORT-HU) study.

Acute splenic sequestration crisis (ASSC) is a potentially life-threatening complication of sickle cell disease (SCD), typically occurring in young patients under 5 years of age, with a median age at first episode of less than 2 years. Because a beneficial effect of hydroxyurea (HU) on spleen perfusion and splenic function has been suspected, we hypothesized that HU treatment might be associated with later onset of ASSC in patients with SCD. To investigate this hypothesis, we analyzed data from the ESCORT-HU study on a large cohort of patients with SCD receiving HU, enrolled between January 2009 and June 2017 with a follow-up of 7309 patient-years of observation. The median age at ASSC of the 14 patients who experienced a first episode of ASSC during the study period was 8.0 [IQR: 5.0-24.1] years. The median age at HU initiation was significantly lower in these 14 patients (4.8 [IQR: 3.3-18.7] years) compared to the 1664 patients without ASSC (19.9 [8.8-33.4] years, p = .0008). These findings suggest that ASSC may occur at an unusually late age in patients receiving HU, possibly reflecting longer preservation of spleen perfusion and function secondary to early initiation of HU. Further studies are needed to better characterize the effects of HU on spleen perfusion/function and on the occurrence of ASSC in patients with SCD (ClinicalTrials.gov identifier: NCT02516579; European registry ENCEPP/SDPP/10565).

Safety of coronavirus disease 2019 vaccines in 213 adult patients with sickle cell disease.

Given the lack of information about safety of the COVID-19 vaccines for sickle cell disease (SCD) patients, we sought to determine whether COVID-19 vaccine was associated with subsequent hospital admission for vaso-occlusive events (VOEs). We included 402 patients with SCD, including 88 regularly transfused. As of July 31, 2021, 213 (53.0%) of them had received a least one dose of COVID vaccine (Pfizer 93.0%). We showed similar risk of hospital admission for a VOE among vaccinated patients (whether transfused or not) and among a control group of non-vaccinated patients matched for age, sex and genotype.

A study of 28 pregnant women with sickle cell disease and COVID-19: elevated maternal and fetal morbidity rates.

Not available.

A new step in understanding stem cell mobilization in patients with Fanconi anemia: A bridge to gene therapy.

BACKGROUND: Fanconi anemia (FA) is an inherited disorder characterized clinically by congenital abnormalities, progressive bone marrow failure (BMF), and a predisposition to malignancy. Gene therapy (GT) of FA, via the infusion of gene-corrected peripheral blood (PB) autologous hematopoietic stem cells (HSCs), may constitute a cure for BMF. GT bypasses the donor restrictions and adverse events associated with allogenic HSC transplantation. However, adequate harvesting of PB-HSCs is a crucial determinant of successful engraftment in gene therapy. Harvesting the low numbers of HSCs in patients with FA is particularly challenging. STUDY DESIGN AND METHODS: This open-label phase I/II trial evaluates the feasibility and safety of co-administration of G-CSF and plerixafor in patients with FA for the mobilization and harvesting of peripheral HSCs, intending to use them in a gene therapy trial. Patients with mutations in the FANCA gene received two subcutaneous injections of G-CSF (6 µg/kg × 2/d from D1 to D8. Plerixafor (0.24 mg/kg/d) was administered 2 h before apheresis (from D5 onward). RESULTS: CD34+ cells were mobilized for four patients quickly but transiently after the plerixafor injection. One patient had a CD34+ cell count of over 100/µl; the mobilization peaked 2 h after the injection and lasted for more than 9 h. There were no short-term adverse events associated with the mobilization or harvesting procedures. CONCLUSION: Our data in patients with FA show that the mobilization of HSCs with G-CSF and plerixafor is safe and more efficient in younger individuals without BMF.

Fetal hemoglobin rescues ineffective erythropoiesis in sickle cell disease.

While ineffective erythropoiesis has long been recognized as a key contributor to anemia in thalassemia, its role in anemia of sickle cell disease (SCD) has not been critically explored. Using in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. Modeling the bone marrow hypoxic environment, we found that hypoxia induces death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). Cell death was associated with cytoplasmic sequestration of heat shock protein 70 and was rescued by induction of HbF synthesis. Importantly, we document that in the bone marrow of SCD patients similar cell loss occurs during the final stages of terminal differentiation. Our study provides evidence for ineffective erythropoiesis in SCD and highlights an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. These findings imply that the beneficial effect on anemia of increased HbF levels is not only due to the increased life span of red cells but also a consequence of decreased ineffective erythropoiesis.

Real-Life experience with hydroxyurea in patients with sickle cell disease: Results from the prospective ESCORT-HU cohort study.

Several controlled studies have evidenced good efficacy and short-term and mid-term safety profiles for hydroxyurea (HU), which has become the cornerstone for prevention of sickle-cell disease (SCD)-related vaso-occlusive crises. However, there are few large-scale reports on its long-term use and certain caregivers and patients have concerns about its safety. Following the licensing of HU in Europe for children and adults with severe forms of SCD, ESCORT-HU was designed as a Phase IV observational cohort study. It included 1906 participants, of whom 55% were adults. The most common hemoglobin (Hb) genotypes were HbSS (84.7%) and HbSß+ (7.0%). The median duration of follow-up was 45 months, for a total of 7309 patient-years of observation. The dose of HU after 1 year was 20.6 mg/kg/d for children and 16.3 mg/kg/d for adults. There was a statistically significant decrease in the number of vaso-occlusive episodes lasting >48 h, acute chest syndrome episodes, hospitalizations, and the percentage of patients requiring blood transfusions within the first 12 months relative to the year before enrolment. Neutropenia and thrombocytopenia were the most commonly reported adverse effects. No new HU toxicity was identified. Overall, 125 pregnancies were reported in 101 women and no malformations were observed in the neonates. There were 12 pregnancies for partners of male patients treated with HU. One case of fatal myelodysplastic syndrome was reported, for which a causal association with HU could not be excluded. This cohort study of patients with SCD highlights the positive benefit-to-risk ratio of HU in children and adults.

Vascular access for optimal hematopoietic stem cell collection.

BACKGROUND: Autologous and allogeneic hematopoietic stem cell transplantation of cytokine-mobilized peripheral blood stem cells (PBSCs) is increasingly used to treat patients with hematologic disorders. Different types of vascular access have been exploited for the apheresis procedure, including peripheral veins (PV) and central venous catheter (CVC). In some cases, PV access is unavailable. There are few published data on the efficiency and quality of harvesting with different types of vascular access. This study brings out complications and morbidity of this procedure linked to these different access. METHODS: We performed a comparative, retrospective, single-center study of hematopoietic stem cell collection using these two types of vascular access. We compared the efficiency and complication rate for 617 adults apheresis sessions in 401 patients and healthy donors, for PBSC collection via PV or CVC between 2010 and 2016. The quality of the HSC product was evaluated in terms of the total CD34 + count and neutrophil contamination. RESULTS: The PV and CVC groups did not differ significantly in terms of the quality of the apheresis product, mean ± SD CD34 + cells collected in PV group was 383.1 ± 402.7 × 10e6 and 298.8 ± 372.7 × 10e6 and the level of neutrophil contamination was 21.0 ± 17.8% in the PV group and 20.6 ± 18.4% in the CVC group. The complication rate did not differ between the two groups. CONCLUSION: The type of vascular access for apheresis hematopoietic stem cell harvesting must be determined by trained staff. Successful harvesting can be performed via PV then CVC is not needed or not available.

Therapeutic plasma exchange for life-threatening pediatric disorders.

INTRODUCTION: Therapeutic plasma exchange (TPE) is acknowledged to be an effective treatment in life-threatening pediatric disorders. Apheresis for pediatric diseases has been poorly investigated, and most studies to date featured small numbers of patients and lacked control groups. The objective of the present study was to evaluate the tolerance of TPE in pediatric patients. MATERIALS AND METHODS: A retrospective cohort study via a web-based electronic case report form including pediatric patients referred for TPE between January 2005 and December 2014. RESULTS: A total of 78 patients (median [range] age: 9.8 [0.53-17.93]) and 731 TPE procedures were analyzed. The indications were antibody-mediated rejection (n = 33; 42%) and desensitization therapy (n = 5; 6%) after solid organ or hematopoietic stem cell transplantation, thrombotic microangiopathy (n = 17; 22%), pediatric inflammatory diseases (n = 16; 21%), kidney diseases (n = 6; 8%), and hyperviscosity syndrome (n = 1; 1%). On average, each patient underwent six procedures during the first session [range: 1-19]. In the 2 weeks following the start of a session, 72 patients (92%) presented a total of 311 adverse events (AEs) potentially related to TPE. The risk of AEs was not related to the indication for TPE, the intensity of care, venous access, plasma substitute use, or body weight. None of the deaths was related to the TPE. CONCLUSION: We studied one of the largest retrospective pediatric cohorts described to date. Our experience of TPE children's TPE feasibility concerned specific, life-threatening conditions and otherwise treatment-refractory diseases.

Plerixafor enables safe, rapid, efficient mobilization of hematopoietic stem cells in sickle cell disease patients after exchange transfusion.

Sickle cell disease is characterized by chronic anemia and vaso-occlusive crises, which eventually lead to multi-organ damage and premature death. Hematopoietic stem cell transplantation is the only curative treatment but it is limited by toxicity and poor availability of HLA-compatible donors. A gene therapy approach based on the autologous transplantation of lentiviral-corrected hematopoietic stem and progenitor cells was shown to be efficacious in one patient. However, alterations of the bone marrow environment and properties of the red blood cells hamper the harvesting and immunoselection of patients' stem cells from bone marrow. The use of Filgrastim to mobilize large numbers of hematopoietic stem and progenitor cells into the circulation has been associated with severe adverse events in sickle cell patients. Thus, broader application of the gene therapy approach requires the development of alternative mobilization methods. We set up a phase I/II clinical trial whose primary objective was to assess the safety of a single injection of Plerixafor in sickle cell patients undergoing red blood cell exchange to decrease the hemoglobin S level to below 30%. The secondary objective was to measure the efficiency of mobilization and isolation of hematopoietic stem and progenitor cells. No adverse events were observed. Large numbers of CD34+ cells were mobilized extremely quickly. Importantly, the mobilized cells contained high numbers of hematopoietic stem cells, expressed high levels of stemness genes, and engrafted very efficiently in immunodeficient mice. Thus, Plerixafor can be safely used to mobilize hematopoietic stem cells in sickle cell patients; this finding opens up new avenues for treatment approaches based on gene addition and genome editing. Clinicaltrials.gov identifier: NCT02212535.

Transfusion-related adverse events are decreased in pregnant women with sickle cell disease by a change in policy from systematic transfusion to prophylactic oxygen therapy at home: A retrospective survey by the international sickle cell disease observatory.

Sickle cell disease (SCD) in pregnancy can be associated with adverse maternal and perinatal outcomes. Furthermore, complications of SCD can be aggravated by pregnancy. Optimal prenatal care aims to decrease the occurrence of maternal and fetal complications. A retrospective, French, two-center study compared two care strategies for pregnant women with SCD over two time periods. In the first study period (2005-2010), the women were systematically offered prophylactic transfusions. In the second study period (2011-2014), a targeted transfusion strategy was applied whenever possible, and home-based prophylactic nocturnal oxygen therapy was offered to all the pregnant women. The two periods did not differ significantly in terms of the incidence of vaso-occlusive events. Maternal mortality, perinatal mortality, and obstetric complication rates were also similar in the two periods, as was the incidence of post-transfusion complications (6.1% in 2005-2010 and 1.3% in 2011-2014, P = .15), although no de novo alloimmunizations or delayed hemolysis transfusion reactions were observed in the second period. The results of this preliminary, retrospective study indicate that targeted transfusion plus home-based prophylactic nocturnal oxygen therapy is safe and may decrease transfusion requirements and transfusion-associated complications.

Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition.

Cancer cells require glutamine to adapt to increased biosynthetic activity. The limiting step in intracellular glutamine catabolism involves its conversion to glutamate by glutaminase (GA). Different GA isoforms are encoded by the genes GLS1 and GLS2 in humans. Herein, we show that glutamine levels control mitochondrial oxidative phosphorylation (OXPHOS) in acute myeloid leukemia (AML) cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacologic GLS1 inhibition by the drug CB-839 can reduce OXPHOS, leading to leukemic cell proliferation arrest and apoptosis without causing cytotoxic activity against normal human CD34(+) progenitors. Strikingly, GLS1 knockdown dramatically inhibited AML development in NSG mice. The antileukemic activity of CB-839 was abrogated by both the expression of a hyperactive GAC(K320A) allele and the addition of the tricarboxyclic acid cycle product α-ketoglutarate, indicating the critical function of GLS1 in AML cell survival. Finally, glutaminolysis inhibition activated mitochondrial apoptosis and synergistically sensitized leukemic cells to priming with the BCL-2 inhibitor ABT-199. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy for AML.

A comprehensive view of pregnancy in patients with sickle cell disease in high-income countries: the need for robust data and further decline in morbidity and mortality.

Sickle cell disease is a major public health concern due to its prevalence and associated morbidities. In high-income countries, diagnosis and treatment advancements have extended patient's lives and enabled women to embrace motherhood. Although the provision of care in specialist centres has reduced maternal-fetal complication rates, the mortality rate among pregnant women with sickle cell disease remains disproportionately high. Complications arise from vaso-occlusive events, worsening organ damage, thrombotic risks, infections, and pregnancy-related issues, such as pre-eclampsia, premature birth, small-for-gestational-age, and pregnancy loss. Effective management during pregnancy includes preconception planning, genetic counselling, education, and collaborative care. There is no consensus on the overall approach to managing pregnant women with sickle cell disease; however, fostering a collaborative relationship between health-care professionals and researchers is crucial for advancing the understanding and management of this illness. The disparities in health-care outcomes associated with ethnicity and economic insecurity affect patients with sickle cell disease but have not been examined extensively. Hence, health-care personnel need sufficient training to address these issues alongside broader societal efforts to confront racism and discrimination. Comprehensive national and global action plans are required to address the multifaceted challenges of sickle cell disease.