gene therapies

2026-05-27 | Successful gene therapy for transfusion-dependent α-thalassemia: a case report.

Alpha-thalassemia is a severe inherited hemoglobin disorder with limited curative options for transfusion-dependent patients lacking suitable donors. We report the first-in-human application of lentiviral vector-mediated α-globin gene therapy in a 14-year-old girl with transfusion-dependent Hemoglobin H disease (--SEA/αCSα). Autologous CD34+ hematopoietic stem and progenitor cells were mobilized, collected, and genetically modified ex vivo using the lentiviral vector (Lenti-HBA) to restore α-globin expression, then reinfused following busulfan conditioning. The patient achieved engraftment within three weeks and started to be free of transfusions from the fourth week onward. In the last year of follow-up (M12-M24), the average hemoglobin level remained around 90 g/L without transfusion support, with HbA accounting for >85% of total hemoglobin. Vector copy numbers were stable, and no evidence of clonal dominance, insertional mutagenesis, or replication-competent lentivirus was detected. Adverse events were limited to expected busulfan-related toxicities, including Grade 2 menstrual irregularity and ovarian failure (SAE). Despite cessation of chelation therapy, iron overload progressed minimally and no cardiac iron deposition occurred. Immune recovery, growth and development were preserved. This case demonstrates the feasibility, safety, and durable efficacy of lentiviral α-globin gene therapy as a potential curative treatment for transfusion-dependent α-thalassemia. This trial was registered at www.clinicaltrials.gov as #NCT05851105.

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2025-11-26 | Revisiting hemoglobin constant spring: molecular insights, pathophysiological mechanisms, and clinical perspectives.

BACKGROUND: Hemoglobin Constant Spring (Hb CS) is an α-globin variant predominantly found in Southeast Asian populations. Compound heterozygosity of Hb CS with α-thalassemia results in a spectrum of chronic hemolytic anemia. Recent advances in cellular therapy, particularly gene editing approaches currently under in-vitro investigation, offer potential new avenues for treatment in patients with severe phenotypes. MAIN BODY: This review discusses the molecular basis, pathophysiology, clinical manifestations, and current management strategies of Hb CS. The condition arises from a point mutation in the termination codon of the HBA2 gene (HBA2:c.427T > C; p.Ter143Gln), leading to unstable mRNA, defective hemoglobin synthesis, and red blood cell membrane abnormalities. The clinical presentation of Hb H/Constant Spring disease and related genotypes ranges from mild anemia to severe hemolysis. In cases of homozygous Hb Constant Spring, affected fetuses may present with hydrops fetalis and severe intrauterine anemia; however, clinical improvement is typically observed after birth, with anemia often stabilizing to a milder form during infancy or early childhood. Management strategies vary from observation in mild cases to regular blood transfusions in more severe cases. Emerging gene editing techniques, including prime editing, represent promising therapeutic modalities under preclinical investigation. CONCLUSION: Hb CS is characterized by complex molecular mechanisms and a broad clinical spectrum. This review aims to provide a comprehensive overview to support improved clinical management of affected individuals and to explore future directions in therapeutic development.

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2025-11-03 | Curing Hemoglobin H-Constant Spring through Base Editing of Autologous CD34+ Hematopoietic Stem and Progenitor Cells

Abstract Background: Hemoglobin H Constant Spring (HbH-CS) (–/αCSα) is the most common non-deletional α-thalassemia that can lead to severe health problems. The clinical severity of HbH-CS is widely variable and a considerable proportion of patients with HbH-CS need regular or irregular transfusions to support a normal growth, development, and quality of life. Currently, there is no cure for this type of disease worldwide. Because patients with HbH-CS have only a single base mutation in the Hemoglobin Subunit Alpha 2 (HBA2: c.427T>C) compared to that of asymptomatic carriers (–/αα), we speculate that correction of the mutation by base editing may provide a potential cure for this type of disease. Aims: To correct the CS mutation of HBA2 gene and increase the expression of the normal α-globin chain in erythrocytes, we used ex vivo Cytosine Base Editor (CBE)–based gene editing to modify HBA2 gene in hematopoietic stem and progenitor cells (HSPCs), producing RM-004. NCT06107400 is an investigator initiated, first-in-human study evaluating edited autologous patient cells (RM-004) in transfusion-dependent HbH-CS patients. Here, we present the initial results from the first patient treated with RM-004. Methods: Patients (12–35 years of age) with HbH-CS receiving packed red blood cells (pRBC) transfusions of ≥100 mL/kg/year or ≥10 units/year in the previous 2 years were eligible. Peripheral CD34+ HSPCs were collected by apheresis after mobilization with G-CSF and plerixafor. CD34+ cells were edited with CBEs using a guide RNA specific for the CS mutation. Prior to RM-004 infusion, patients received myeloablative conditioning with busulfan from day-7 to day-3. Patients were monitored for stem cell engraftment/hematopoietic recovery, adverse events (AEs), Hb production, expression of HbA/HbH/Hb-CS, proportion of CS negative red blood cells and requirements for pRBC transfusions. The primary efficacy endpoint was transfusion independence (TI12), defined as the proportion of patients maintaining a weighted average hemoglobin level ≥9 g/dL without pRBC transfusion for ≥12 consecutive months. Results: As of July 15, 2025, a 14-year female patient with HbH-CS had received RM-004 and has been followed for over 15 months. This patient received an annualized 29 units/year pRBC transfusions before enrollment. After a single dose infusion of RM-004 cells, the patient achieved neutrophils engraftment and platelets engraftment on Day14 and Day20, respectively. The last pRBC transfusion occurred on Day13 after RM-004 treatment, and the patient has remained transfusion-free for 14.7 months. Additionally, the total Hb increased to over 9 g/dL on Day26 and remained between 9 and 11 g/dL during follow-up, with a value of 10.1 g/dL at the latest follow-up (month 15). Therefore, the patient has achieved transfusion independence (TI12). Following the infusion of RM-004, the proportions of HbH and Hb-CS decreased from 3.8% and 1.1% to 0 and 0.3%, respectively. Concurrently, the proportion of HbA1 increased from 93% to 97.8%.The proportion of CS-positive RBCs significantly decreased from 100% to approximately 10%, indicating that 90% of peripheral blood erythrocytes were derived from successfully edited HSPCs. The safety profile was generally consistent with busulfan myeloablation and autologous hematopoietic stem cell transplantation. No serious adverse event were reported. Conclusion: These results present the first successful application of gene editing in treating a patient with transfusion-dependent α-thalassemia. The patient treated with RM-004 demonstrated successful engraftment and a clinically meaningful increase in hemoglobin levels, leading to transfusion independence. This proof-of-concept study indicates that RM-004 is a promising therapy for the treatment of HbH-CS.

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2025-10-31 | Clinical spectrum and genotypes of children with alpha-thalassemia in northeastern, Thailand

Background: Alpha-thalassemia is a common inherited hemoglobin disorder. Hemoglobin H (Hb H) disease, a form of alpha-thalassemia caused by the inactivation of three α-globin genes through deletional or non-deletional mutations, presents with a broad clinical spectrum, ranging from non-transfusion-dependent thalassemia (NTDT) to transfusion-dependent thalassemia (TDT). Objective: To characterize the clinical manifestations and genetic diversity of alpha-thalassemia in pediatric patients.

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2025-09-19 | Lentiviral vectors for hematopoietic stem cell gene therapy restore α-globin expression in α-thalassemia red blood cells.

Alpha thalassemia major (ATM) is an inherited blood disorder caused by the absence of all four α-globin genes (HBA2/1), resulting in severe anemia and lifelong transfusion dependence. While allogeneic hematopoietic stem cell transplantation (HSCT) offers a potential cure, donor availability remains limited. We present a gene therapy approach for autologous HSCT using lentiviral vectors (LVs) to deliver HBA2 under the regulation of optimized β-globin locus control region (LCR) enhancers, restoring α-globin expression in red blood cells. The best-performing LVs, erythroid vector-alpha (EV-α) and EV-α-UV, achieved up to 100% transduction efficiency in human hematopoietic stem and progenitor cells (HSPCs), optimal vector copy numbers, and safe integration profiles. ATM-derived HSPCs from three donors treated with these LVs yielded α/β-globin mRNA and chain ratios within the therapeutic range (∼0.5+), and restored hemoglobin levels by 50%-100%. These findings establish the safety and clinical potential of EV-α and EV-α-UV as a promising autologous stem cell gene therapy for ATM.

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small molecules

2026-07-19 | Evaluating mitapivat for the treatment of alpha or beta thalassemia.

Thalassemia is a group of diverse genetic disorders with worldwide distribution that affects hemoglobin synthesis. Until recently, the therapeutic approach to thalassemia was symptomatic, relying on red blood cell transfusions, treatment of comorbidities and of disease-related complications. However, novel therapeutic agents have recently been developed and are gradually being integrated into routine clinical practice. One of the most promising agents is mitapivat (AG348), an oral pyruvate kinase activator that enhances the erythrocytic adenosine triphosphate (ATP) production. After series of preclinical and clinical studies, mitapivat has been suggested to be a safe and effective disease modifier for thalassemia. Large double blind randomized clinical trials have indicated that mitapivat may increase baseline hemoglobin levels, reduce transfusion burden, control ineffective erythropoiesis and hemolysis and improve quality of life. Mitapivat presents as a potential game-changer in the management of patients with both α- and β-thalassemia, regardless of transfusion dependency. However, further post-marketing evidence is required in order to evaluate mitapivat profile under real‑world conditions and routine clinical practice.

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2026-06-08 | Unveiling molecular mechanisms and therapeutic targets in HbH-CS disease: a focus on oxidative stress and mitochondrial dysfunction.

This study aimed to investigate the molecular mechanisms and therapeutic targets related to oxidative stress and mitochondrial dysfunction in Hemoglobin H-Constant Spring (HbH-CS) disease. HbH-CS differentially expressed genes (DEGs) were selected from a microarray dataset. Oxidative stress-related genes (OSGs) and mitochondrial function-related genes (MiRGs) were retrieved from public databases. Oxidative stress and mitochondrial function-related genes (OMRGs) were defined as the intersection of HbH-CS DEGs, OSGs, and MiRGs. Programmed cell death (PCD) mechanisms and functional enrichment analyses were subsequently investigated. A protein-protein interaction (PPI) network was constructed to identify hub genes, and potential regulatory mechanisms and expression levels of candidate genes were further explored. A total of 98 OMRGs were identified, which were associated with cellular respiration, oxidative stress response, mitochondrial membrane, and apoptotic signaling. Apoptosis was determined to be the primary PCD mechanism. Three hub genes were identified: AKT serine/threonine kinase 1 (AKT1), B cell lymphoma/leukemia-2 (BCL2), and cytochrome c, somatic (CYCS). Additionally, RNA-binding motif protein 15B (RBM15B) was recognized as a shared N6-methyladenosine (m6A) regulator. The mRNA expression levels of these four genes were significantly downregulated in HbH-CS patients. These findings provide novel insights into the molecular mechanisms and identify potential therapeutic targets for clinical intervention.

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2026-03-04 | Transforming the treatment of Alpha-Thalassemia: a single-center retrospective study on hematopoietic stem cell transplantation in transfusion-dependent pediatric patients.

Hematopoietic stem cell transplantation (HSCT) is the only definitive cure for transfusion-dependent α-thalassemia, though comprehensive studies on its effectiveness are limited. In this retrospective study, we analyzed the clinical characteristics of 21 pediatric patients with transfusion-dependent α-thalassemia who underwent HSCT, all of whom received a standardized conditioning regimen consisting of busulfan, cyclophosphamide, fludarabine, and anti-thymocyte globulin. After a median follow-up of 25 months (range: 7–92 months), the two-year overall survival (OS) and event-free survival (EFS) rates were both 90.2% (95% CI: 66.2–97.4%), and the two-year graft-versus-host disease-free, relapse-free survival (GRFS) rate was 82.3% (95% CI: 52.6–94.3%). The transplant-related mortality rate at two years was 5.0% (95% CI: 0.7–30.0%), with no cases of graft failure observed. Among the 19 surviving patients, hemoglobin levels significantly increased compared to pre-transplant levels (p < 0.05), and all became transfusion-independent. Hematopoietic stem cell transplantation is a curative treatment for α-thalassemia. For patients with transfusion-dependent α-thalassemia, HSCT should be performed as early as possible at an experienced transplant center when a suitable donor is available.

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2026-02-27 | Systematic Review of Non-Coding Genomic Variants in Globin and Non-Globin Clusters and Their Impact on Phenotypic Severity in Thalassemia and Sickle Cell Disease.

Background: Haemoglobinopathies such as beta-thalassemia (β-thal), alpha-thalassemia (α-thal) and sickle cell disease (SCD) are characterised by pathogenic gene variations (mutations) in the globin genes. Patients with haemoglobinopathies have the same disease-causing coding variations with very different disease phenotypes, from requiring blood transfusions to being non-symptomatic. The gap between the expected clinical outcomes based on primary coding mutations (the genotype) and the actual observed symptoms (the phenotype) often remains unexplained. We refer to the contribution of secondary genetic modifiers-specifically, non-coding variants of the genome that alter globin gene expression and pathophysiology-as the "missing heritability" of the clinical presentation [Primary Mutation + Missing Heritability (Non-Coding Variants) = Actual Clinical Phenotype]. Objectives: This systematic review aims to find evidence connecting genetic differences outside of the protein-coding region, as in promoters, enhancers or untranslated regions (UTRs), to the clinical severity (phenotype) of beta-thalassemia, alpha-thalassaemia and SCD. We summarise the molecular basis of phenotypic variation among haemoglobinopathy patients with identical variations to reveal their missing heritability and to enhance our understanding of prognostic strategies. Methods: This systematic review was performed in accordance with the PRISMA 2020 guidelines. We used search terms related to haemoglobinopathies, non-coding variation, SNP, promoters, enhancers and clinical severity to search major databases (PubMed and Google Scholar) as of October 2025. A total of 527 (out of 572) abstracts were fit for initial screening to identify the eligible reports. Due to heterogeneity in study designs and reported outcomes, findings were synthesised descriptively and grouped by variant mechanism (cis-acting and trans-acting). The final analysis included 89 articles that demonstrated a direct association between a non-coding genomic variant and a quantitative measure of clinical severity. Results: Two main groups of non-coding variants (NCVs) that modulate foetal haemoglobin (HbF) induction were identified. The first major group comprises cis-acting variants within globin gene clusters (HBG2 promoter XmnI polymorphism, HBB promoter mutations and α-globin enhancer variants), while the second major group comprises trans-acting quantitative trait loci (QTLs) (BCL11A and HBS1L-MYB loci). Non-globin NCVs in the UGT1A1 promoter were also found to influence the severity measures in β-thal and SCD. NCVs primarily alter the binding of transcription factors and the looping dynamics of chromatin, modulating the α/β chain balance ratio and γ-globin repression. The XmnI polymorphism is the most prominent cis-acting modifier associated with β-thal intermedia. The promoter polymorphisms in TNF-α and VCAM1 are associated with vascular complications in SCD. Conclusions: NCVs are fundamental when determining the clinical measures of haemoglobinopathies, in addition to coding variants. NCV screening should be integrated for clinical prognosis for the accurate prediction of haemoglobinopathy severity and associated high-risk complications. NCVs may represent promising targets for next-generation gene editing and therapeutic intervention strategies aimed at modifying the severity of β-thal, α-thal and SCD.

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2025-11-03 | Transfusion independence in thalassemia patients after failure of gene therapy or with hemoglobin h constant spring and hb e using luspatercept: A case series

Abstract Background: Individuals with thalassemia who depend on blood transfusions to maintain adequate hemoglobin levels are at high risk of sequelae resulting from frequent blood products. As such, there is increased interest in decreasing transfusion frequency for such patients and limiting side effects. Luspatercept is a recombinant fusion protein that enhances erythropoiesis by binding to select TGF-b ligands and can increase hemoglobin for patients with thalassemia. Luspatercept use was associated with significantly greater reductions in transfusion burden than for the placebo group for transfusion-dependent b-thalassemia (TDT) patients in the BELIEVE trial. Luspatercept was also associated with a significant increase in hemoglobin level among non-transfusion-dependent beta thalassemia (NTDT) in the BEYOND trial. However, currently, there is very limited evidence for the utility luspatercept in thalassemia patients after a failed gene therapy or among those with alpha thalassemia. Here we describe two cases with significant clinical response to luspatercept achieving transfusion independence in distinct thalassemia populations that have not yet been reported in the literature. Case 1: This patient underwent LentiGlobin gene therapy for b-thalassemia (β0/β0) at the age of 34 years in 2015. Prior to gene therapy, she required regular packed red blood cell (pRBCs) transfusions every 4 weeks. After admission for gene therapy per protocol HGB-204, her transfusion needs initially decreased to every 6 to 9 months. However, she was not able to fully wean off transfusions which slowly increased in frequency to every 3 months. She began treatment with luspatercept at the age of 42, and has maintained hemoglobin levels above 10 g/dL without need for a blood transfusion for almost two years since then. Case 2: This patient was diagnosed with hemoglobin H Constant Spring and homozygous HbE by hemoglobin electrophoresis and hemoglobinopathy genetic testing (c.427T&gt;C , Hb Constant Spring; c79G&gt;A, HbE). Up to her third decade of life, she was requiring regular pRBCs transfusions every 3-4 weeks with a pre-transfusion hemoglobin goal of 9-10 g/dL. She also had evidence of significant iron overload due to her transfusion frequency and suboptimal adherence to iron chelation (maximum liver iron content (LIC) of 16.4 mg/g dry weight) with a normal cardiac T2* (42.7 +/- 2.6 msec). She started treatment with luspatercept at the age of 30 years and her transfusion needs soon decreased and she achieved transfusion independence for more than 2 years with occasional transfusion only every 18 months. Concurrently, she was weaned off iron chelation around two years following initiation of luspatercept with an LIC of &lt;3.5 and a ferritin of less than 500 ng/mL. She continues to be on luspatercept with no tolerability issues. These two patient cases demonstrate that luspatercept can also reduce transfusion needs or achieve independence following gene therapy or in alpha thalassemia. Other case reports have previously explored the potential clinical benefits of luspatercept in other settings. Our cases add to the existing experiences by elucidating possible novel applications of luspatercept to additional thalassemia populations. Our findings suggest that luspatercept can successfully increase and stabilize baseline hemoglobin for these patients, and thus could represent a safe and efficacious long-term disease-modifying therapy option for transfusion-dependent alpha thalassemias and patients who failed or had a suboptimal or incomplete response to gene therapy. Further research, including prospective studies and/or randomized trials, will be necessary to evaluate these indications for luspatercept on a larger study population in the future.

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cell therapies

2026-06-23 | Chronic Red Cell Exchange for the Management of Alpha Thalassemia Major Complicated by Iron Overload.

Alpha thalassemia major is a severe hemoglobinopathy characterized by absent or markedly reduced alpha-globin production, necessitating lifelong blood transfusions. Chronic simple transfusions can lead to significant iron overload, often requiring iron chelation therapy. However, some patients are unable to tolerate chelation or chelation is insufficient, highlighting the need for alternative strategies for managing iron overload. This study is one of the first to evaluate the feasibility, safety, and efficacy of red blood cell exchange (RBCX) as a therapeutic option for managing iron overload while improving hemoglobin function in an alpha thalassemia major patient with iron overload despite chelation therapy. RBCX therapy was performed approximately every 3 weeks over 1 year, with frequent adjustment of exchange parameters to meet pre-transfusion target levels of functional hemoglobin. Serum ferritin levels, hemoglobin electrophoresis, and functional hemoglobin levels were tracked with each exchange transfusion. For each RBCX treatment, variant hemoglobins decreased by 3.8-fold (from 19.9% to 8.9%) and functional hemoglobin levels increased approximately 3.0 g/dL. RBCX was well tolerated and associated with a marked reduction in systemic iron burden, with serum ferritin declining 7.4-fold (86.4%) over 1 year of therapy, followed by a rise after cessation of RBCX. Cardiac iron remained within normal limits throughout the study period. In contrast, liver iron demonstrated a transient increase shortly after initiation of RBCX, but ultimately declined to levels below baseline after 1 year, even during a period in the absence of iron chelation. These findings suggest that RBCX exerts a meaningful effect on iron homeostasis and raises the possibility of a synergistic benefit when combined with iron chelation. This case highlights that RBCX is a viable therapeutic strategy to treat iron overload in patients with alpha thalassemia major.

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2026-06-18 | Delayed Recognition and Significant Morbidity in Adults with Non-Transfusion-Dependent Thalassemia.

Non-transfusion-dependent thalassemia (NTDT) is characterized by variable degrees of anemia, ineffective erythropoiesis, and iron overload, with a heightened risk of age-related complications. However, the clinical profiles of patients who first present to thalassemia care in adulthood, as well as the gaps in management, are poorly described. In a multi-institutional study, we identified 82 patients with NTDT aged ≥18 years who were referred to three U.S. thalassemia centers between 2013 and 2023. Data were collected by manual chart review and included clinical history, laboratory tests, and imaging. The median age at initial visit was 36.8 years (range 18-74), and 37 (45%) of the patients had α-thalassemia, while 45 (55%) had β-thalassemia. Complications assessed included symptomatic anemia, prior splenectomy, extramedullary hematopoiesis, iron overload, pulmonary hypertension, cardiac arrhythmia, endocrine complications, and thrombosis. Iron overload was common, with 71% of available liver MRIs showing hepatic iron >5 mg Fe/g dry weight. Among patients with LIC >5 mg Fe/g, only 24.4% were on chelation at referral. Strikingly, 49% of patients were recommended to start regular transfusions following the consultation, predominantly for symptomatic anemia or complications of ineffective erythropoiesis. Patients with NTDT referred for initial comprehensive thalassemia care in adulthood had high rates of morbidities and undertreated iron overload. The criteria for initiating regular transfusions must be widely implemented to mitigate long-term complications and improve the quality of life of adults with NTDT.

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2026-06-08 | Derivation and Pluripotency Validation of Six iPSC Lines From Amniotic Fluid Carrying Intermediate α-Thalassemia Genotypes (--3.7/αSEA and --4.2/αSEA).

Thalassemia intermedia (TI), or hemoglobin H (Hb H) disease, presents significant clinical heterogeneity, and its pathogenesis remains unclear. This study aimed to establish induced pluripotent stem cells (iPSCs) from amniotic fluid (AF) cells of fetuses diagnosed with intermediate α-thalassemia genotypes (-α3.7/--SEA and -α4.2/--SEA) to provide a robust in vitro model for investigating disease mechanisms and exploring therapeutic strategies. AF cells from six TI fetuses were reprogrammed using nonintegrating episomal vectors via electro-transfection. The resulting iPSC lines underwent comprehensive characterization, including alkaline phosphatase (AP) staining, immunofluorescence (IF) for pluripotency markers (e.g., OCT-4, SOX-2, and TRA-1-60), quantitative reverse transcription real-time PCR (RT-qPCR) for the expression of pluripotency genes, G-band karyotyping, and genetic analysis for thalassemia. Differentiation potential was assessed in vivo via teratoma formation in immunodeficient mice and in vitro through directed hematopoietic differentiation. Six stable iPSC lines were successfully established. All lines retained the progenitor's intermediate α-thalassemia genotype and exhibited normal karyotypes. They displayed typical embryonic stem cell (ESC)-like morphology, were positive for AP, and expressed key pluripotency markers. In vivo, all lines robustly formed teratomas containing tissues derived from all three germ layers (endoderm, mesoderm, and ectoderm). Furthermore, in vitro hematopoietic differentiation protocols successfully induced the iPSCs to generate CD34+/CD43+ hematopoietic progenitors, which were functionally validated by their ability to form hematopoietic colonies in colony-forming unit (CFU) assays. We have successfully derived and comprehensively characterized six patient-specific iPSC lines from intermediate α-thalassemia fetuses. These cell lines demonstrate robust pluripotency and maintain the capacity for hematopoietic differentiation. They represent a valuable resource for investigating TI pathophysiology, screening novel drugs, and developing future cell-based therapeutic strategies.

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2026-04-15 | In Utero Hematopoietic Cell Transplantation in Fetuses with Alpha Thalassemia Major: A Phase 1 Clinical Trial.

In utero hematopoietic cell transplantation (IUHCT) has the potential to treat patients who have hemoglobinopathies by harnessing the unique period of fetal tolerance to maternal cells, thereby enabling semi-allogeneic transplantation without conditioning or immunosuppression. We conducted a phase 1 clinical trial of IUHCT in fetuses with Alpha Thalassemia Major (ATM), a diagnosis that requires serial in utero transfusions (IUT) for survival. We also analyzed outcomes in fetuses with ATM treated with IUT alone in the same time period. Six fetuses underwent transplantation with maternal CD34+ cells (1.33 x 108 ± 2.67x107 cells/kg +1% T cells) at 23.1 ± 1.1 weeks' gestation. All received serial IUT and were delivered at or near term. Perinatal outcomes were broadly similar to those undergoing IUT alone. Two mothers received prophylactic antibiotics because of late positive cultures of harvested cells (without fetal adverse events). Low-level maternal microchimerism was detected in all recipients; T-cell hyporeactivity to maternal antigens was detected in 2/6 offspring but persisted in only one child. Cord blood from ATM-affected pregnancies demonstrated marked expansion of host hematopoietic stem and progenitor cells compared with healthy controls, suggesting a competitive disadvantage for donor cells in this disease context. Neurodevelopmental assessments were largely reassuring, with high quality of life scores. Overall, this protocol was safe and feasible but demonstrates the limitations of IUHCT for ATM without concurrent bone marrow conditioning. The favorable perinatal and neurologic outcomes in most offspring underscore the benefits of prenatal transfusions and the unmet medical need for developing definitive therapies for ATM. NCT02986698.

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2025-11-11 | Tackling the challenge of in utero blood exchange transfusion for fetal anemia at 15 weeks' gestation.

Severe fetal anemia due to parvovirus infection and alpha-thalassemia major can occur during the first trimester of pregnancy. In utero transfusion before 16 weeks of gestation is beneficial but challenging because the umbilical vein is too narrow to be precisely punctured. In addition, the fetal circulating volume is small, and the heart of an affected fetus is compromised. To avoid vascular overload, exchange transfusion may be considered. The technical difficulties make in utero simple transfusions seldom performed before 16 weeks of gestation, and exchange transfusion before 16 weeks of gestation has not been reported. Hence, we reported our technique of successful in utero exchange transfusion in a fetus with alpha-thalassemia major at 15 weeks of gestation. The keys of success were as follows: Fetal blood was not withdrawn for diagnostic investigation before the first transfusion for the following pragmatic reasons.

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proteins

2026-04-20 | Hemoglobin alpha-like subunit mu is expressed during ontogeny and is elevated in alpha thalassemia.

The alpha globin gene cluster on chromosome 16 encodes alpha-like globins whose expression changes during development. HBM located in this cluster was considered a pseudogene until its transcript was discovered in 2005, but without a detectable protein product. Here, we provide the first detailed description of HBM-encoded globin at the protein level (hemoglobin mu chain [HBM]) across different age groups using liquid chromatography coupled with tandem mass spectrometry. Using targeted analysis, we demonstrate that HBM protein is readily detectable in circulation with its levels falling with increasing gestational age. We also report HBM levels in relation to other hemoglobin chains in neonatal, pediatric, and adult blood. Furthermore, analysis of alpha thalassemia patient samples revealed that HBM levels were elevated 3- to 17-fold, indicating a compensatory role, which could be explored for its therapeutic potential. This study enhances our understanding of erythropoiesis and globin chain production through ontogeny.

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2024-12-05 | Sickle Trait and Alpha Thalassemia Increase NOS-Dependent Vasodilation of Human Arteries Through Disruption of Endothelial Hemoglobin-eNOS Interactions.

Severe malaria is associated with impaired nitric oxide (NO) synthase (NOS)-dependent vasodilation, and reversal of this deficit improves survival in murine models. Malaria might have selected for genetic polymorphisms that increase endothelial NO signaling and now contribute to heterogeneity in vascular function among humans. One protein potentially selected for is alpha globin, which, in mouse models, interacts with endothelial NOS (eNOS) to negatively regulate NO signaling. We sought to evaluate the impact of alpha globin gene deletions on NO signaling and unexpectedly found human arteries use not only alpha but also beta globin to regulate eNOS. The eNOS-hemoglobin complex was characterized by multiphoton imaging, gene expression analysis, and coimmunoprecipitation studies of human resistance arteries. Novel contacts between eNOS and hemoglobin were mapped using molecular modeling and simulation. Pharmacological or genetic disruption of the eNOS-hemoglobin complex was evaluated using pressure myography. The association between alpha globin gene deletion and blood pressure was assessed in a population study. Alpha and beta globin transcripts were detected in the endothelial layer of the artery wall. Imaging colocalized alpha and beta globin proteins with eNOS at myoendothelial junctions. Immunoprecipitation demonstrated that alpha globin and beta globin form a complex with eNOS and cytochrome b5 reductase. Modeling predicted negatively charged glutamic acids at positions 6 and 7 of beta globin to interact with positively charged arginines at positions 97 and 98 of eNOS. Arteries from donors with a glutamic acid-to-valine substitution at beta globin position 6 (sickle trait) exhibited increased NOS-dependent vasodilation. Alpha globin gene deletions were associated with decreased arterial alpha globin expression, increased NOS-dependent vasodilation, and lower blood pressure. Mimetic peptides that targeted the interactions between hemoglobin and eNOS recapitulated the effects of these genetic variants on human arterial vasoreactivity. Alpha and beta globin subunits of hemoglobin interact with eNOS to restrict NO signaling in human resistance arteries. Malaria-protective genetic variants that alter the expression of alpha globin or the structure of beta globin are associated with increased NOS-dependent vasodilation. Targeting the hemoglobin-eNOS interface could potentially improve NO signaling in diseases of endothelial dysfunction such as severe malaria or chronic cardiovascular conditions.

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2023-08-08 | Concomitant Presence of Hb Agrinio and - -Med Deletion in a Greek Male Patient with Hemoglobinopathy H: More Severe Phenotype and Literature Review

Hemoglobin (Hb) Agrinio is a rare non-deletional a-globin mutation observed almost exclusively in Greek, Spanish or other Mediterranean families. The clinical manifestations of a carrier of a single Hb Agrinio mutation (single heterozygosity) depend on the concomitant presence or absence of other mutations or variants in the beta, alpha or other modifying genes. We present a Greek patient harboring a Hb Agrinio variant plus the - -Med alpha deletional allele, having an infrequent severe form of alpha thalassemia, in contrast to the typical alpha thalassemic patient and requiring regular red blood cell (RBC) transfusions and chelation treatment. We also provide a concise literature review regarding alpha thalassemic hemoglobin variants and their molecular and clinical combinations. A phase 2, double-blind, randomized, placebo-controlled, multicenter clinical trial to determine the efficacy and safety of luspatercept (BMS-986346/ACE-536) for the treatment of anemia in adults with alpha thalassemia with the participation of our center is currently recruiting patients (NCT05664737).

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2023-03-24 | Evidence of in vivo exogen protein uptake by red blood cells: a putative therapeutic concept.

For some molecular players in red blood cells (RBCs), the functional indications and molecular evidence are discrepant. One such protein is transient receptor potential channel of canonical subfamily, member 6 (TRPC6). Transcriptome analysis of reticulocytes revealed the presence of TRPC6 in mouse RBCs and its absence in human RBCs. We transfused TRPC6 knockout RBCs into wild-type mice and performed functional tests. We observed the "rescue" of TRPC6 within 10 days; however, the "rescue" was slower in splenectomized mice. The latter finding led us to mimic the mechanical challenge with the cantilever of an atomic force microscope and simultaneously carry out imaging by confocal (3D) microscopy. We observed the strong interaction of RBCs with the opposed surface at around 200 pN and the formation of tethers. The results of both the transfusion experiments and the atomic force spectroscopy suggest mechanically stimulated protein transfer to RBCs as a protein source in the absence of the translational machinery. This protein transfer mechanism has the potential to be utilized in therapeutic contexts, especially for hereditary diseases involving RBCs, such as hereditary xerocytosis or Gárdos channelopathy.

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2021-07-20 | PTD-mediated delivery of α-globin chain into Κ-562 erythroleukemia cells and α-thalassemic (HBH) patients’ RBCs ex vivo in the frame of Protein Replacement Therapy

Abstract Background α-Thalassemia, a congenital hemoglobinopathy, is characterized by deficiency and/or reduced levels of α-globin chains in serious forms of α-thalassemia (HbH disease/Hb Bart’s). This research work deals with a Protein Replacement Therapy approach in order to manage α-thalassemia manifestations, caused by the excess of β-globin chain into HbH RBCs. The main goal was to produce the recombinant human α-globin chain in fusion with TAT, a Protein Transduction Domain, to ex vivo deliver it into HbH patients RBCs, to replace the endogenous missing α-globin chain. Results Cloning of the α-globin coding sequence, fused to the nucleotide sequence of TAT peptide was conducted and the human recombinant fusion proteins, 10xHis-Xa SITE -α-globin-HA and 10xHis-Xa SITE -TAT-α-globin-HA were produced. The ability of human recombinant 10xHis-Xa SITE -α-globin-HA to interact in vitro with the previously produced 10xHis-Xa SITE -TAT-β-globin-HA and form α-/β-globin heterodimers, was assessed and confirmed by size exclusion chromatography. The recombinant 10xHis-Xa SITE -TAT-α-globin-HA was successfully delivered into human proerythroid K-562 cells, during the preliminary transduction evaluation experiments. Finally, the recombinant, TAT-fused α-globin was successfully transduced into RBCs, derived from HbH patients and reduced the formation of HbH-Inclusion Bodies, known to contain harmful β 4 -globin chain tetramers. Conclusions Our data confirm the successful ex vivo transduction of recombinant α-globin chains in HbH RBCs to replace the missing a-globin chain and reduce the HbH-inclusion bodies, seen in α-thalassemias. These findings broaden the possibility of applying a Protein Replacement Therapy approach to module sever forms of α-thalassemia, using recombinant α-globin chains, through PTD technology.

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2024-08-01 | METTL16 participates in haemoglobin H disease through m6A modification.

Haemoglobin H (HbH) disease is caused by a disorder of α-globin synthesis, and it results in a wide range of clinical symptoms. M6A methylation modification may be one of the mechanisms of heterogeneity. Therefore, this article explored the role of methyltransferase like 16 (METTL16) in HbH disease. The results of epigenetic transcriptome microarray were analysed and verified through bioinformatic methods and qRT-PCR, respectively. The overexpression or knock down of METTL16 in K562 cells was examined to determine its role in reactive oxygen species (ROS), cell cycle processes or iron overload. YTH domain family protein 3 (YTHDF3) was knocked down in K562 cells and K562 cells overexpressing METTL16 via siRNA to investigate its function. In addition, haemoglobin expression was detected through benzidine staining. qRT-PCR, WB, methylated RNA Immunoprecipitation (MeRIP) and (RNA Immunoprecipitation) RIP experiments were conducted to explore the mechanism of intermolecular interaction. METTL16, YTHDF3 and solute carrier family 5 member 3 (SLC5A3) mRNA and the methylation level of SLC5A3 mRNA were downregulated in HbH patients. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) mRNA expression was negatively correlated with HGB content among patients with HbH-CS disease. Overexpression of METTL16 increased ROS and intracellular iron contents in K562 cells, changed the K562 cell cycle, reduced hemin-induced haemoglobin synthesis, increased the expressions of SLC5A3 and HBG and increased SLC5A3 mRNA methylation levels. Knockdown of METTL16 reduced ROS and intracellular iron contents in K562 cells. Hemin treatment of K562 cells for more than 14 days reduced the protein expressions of METTL16 and SLC5A3 and SLC5A3 mRNA methylation levels. Knockdown of YTHDF3 rescued the intracellular iron content changes induced by the overexpression of METTL16. The RIP experiment revealed that SLC5A3 mRNA can be enriched by METTL16 antibody. METTL16 may affect the expression of SLC5A3 by changing its m6A modification level and regulating ROS synthesis, intracellular iron and cycle of red blood cells. Moreover, METTL16 possibly affects the expression of haemoglobin through IGF2BP3, which regulates the clinical phenotype of HbH disease.

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2024-04-16 | miR-214 aggravates oxidative stress in thalassemic erythroid cells by targeting ATF4

Oxidative damage to erythroid cells plays a key role in the pathogenesis of thalassemia. The oxidative stress in thalassemia is potentiated by heme, nonheme iron, and free iron produced by the Fenton reaction, due to degradation of the unstable hemoglobin and iron overload. In addition, the levels of antioxidant enzymes and molecules are significantly decreased in erythrocytes in α- and β-thalassemia. The control of oxidative stress in red blood cells (RBCs) is known to be mediated by microRNAs (miRNAs). In erythroid cells, microR-214 (miR-214) has been reported to respond to external oxidative stress. However, the molecular mechanisms underlying this phenomenon remain unclear, especially during thalassemic erythropoiesis. In the present study, to further understand how miR-214 aggravates oxidative stress in thalassemia erythroid cells, we investigated the molecular mechanism of miR-214 and its regulation of the oxidative status in thalassemia erythrocytes. We have reported a biphasic expression of miR-214 in β- and α-thalassemia. In the present study the effect of miR-214 expression was investigated by using miR -inhibitor and -mimic transfection in erythroid cell lines induced by hemin. Our study showed a biphasic expression of miR-214 in β- and α-thalassemia. Subsequently, we examined the effect of miR-214 on erythroid differentiation in thalassemia. Our study reveals the loss-of-function of miR-214 during translational activation of activating transcription factor 4 mRNA, leading to decreased reactive oxygen species levels and increased glutathione levels in thalassemia erythroid cell. Our results suggest that the expression of activating transcription factor 4 regulated by miR-214 is important for oxidative stress modulation in thalassemic erythroid cells. Our findings can help to better understand the molecular mechanism of miRNA and transcription factors in regulation of oxidative status in erythroid cells, particularly in thalassemia, and could be useful for managing and relieving severe anemia symptoms in patients in the future.

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2022-01-21 | [Regulation effect of siRNA on β-globin in erythrocytes of hemoglobin H disease].

Objective: To investigate the regulatory effect of targeted siRNA on β-globin in erythroid cells cultured by targeted differentiation in vitro and provide new theoretical support for gene therapy for hemoglobin H (HbH) disease. Methods: Based on the β-globin gene expression results, the optimal siRNA sequence and its effective action dose were screened in erythroid cells, and the effect of the effective dose of the optimal siRNA on the regulation of β-globin expression and apoptosis in erythroid cells was examined. The effective dose of the optimal siRNA was applied to erythroid cells with HbH disease. The effects of transfected siRNAs on red line cells with HbH disease were comprehensively evaluated by measuring the expression of β-globin, reactive oxygen species (ROS) , and apoptosis rates. Results: Within 96 hours after transfection, siRNA2 significantly downregulated β-globin expression in in vitro cultured erythroid cells, but not α-globin. siRNA silencing effect and duration of effect were dose-dependent. siRNA2 downregulated β-globin expression, reduced intracellular ROS production, and decreased apoptosis rate in erythroid cells with HbH disease. Conclusion: Targeted siRNAs can downregulate β-globin expression, reduce intracellular ROS production, and downregulate apoptosis rate in erythroid cells with HbH disease. 目的： 研究靶向siRNA对体外定向分化培养的红系细胞β-珠蛋白的调控作用，为血红蛋白H（Hemoglobin H, HbH）病的基因治疗提供新的理论支持。 方法： ①根据β-珠蛋白基因表达结果，在红系细胞中筛选出最佳siRNA序列及其有效作用剂量，检测有效剂量的最佳siRNA对红系细胞β-珠蛋白表达的调控和细胞凋亡的影响。②将有效剂量的最佳siRNA作用于HbH病红系细胞，检测转染后细胞β-珠蛋白表达、活性氧（ROS）和细胞凋亡率，综合评估转染siRNA对HbH病红系细胞的影响。 结果： ①siRNA(2)可在转染后96 h内显著下调体外培养的红系细胞β-珠蛋白的表达，但对α-珠蛋白无明显调控作用。siRNA(2)的沉默效应和效应持续时间均与作用剂量有关。②siRNA(2)可下调HbH病红系细胞β-珠蛋白表达，减少细胞内ROS生成，并减少细胞凋亡率。 结论： 靶向性siRNA可下调HbH病红系细胞β-珠蛋白表达，减少细胞内ROS产生，下调细胞凋亡率。.

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2019-08-27 | miR-144 regulates oxidative stress tolerance of thalassemic erythroid cell via targeting NRF2.

Thalassemia has a high prevalence in Thailand. Oxidative damage to erythroid cells is known to be one of the major etiologies in thalassemia pathophysiology. Oxidative stress status of thalassemia is potentiated by the heme, nonheme iron, and free iron resulting from imbalanced globin synthesis. In addition, levels of antioxidant proteins are reduced in α-thalassemia and β-thalassemia erythrocytes. However, the primary molecular mechanism for this phenotype remains unknown. Our study showed a high expression of miR-144 in β- and α-thalassemia. An increased miR-144 expression leads to decreased expression of nuclear factor erythroid 2-related factor 2 (NRF2) target, especially in α-thalassemia. In α-thalassemia, miR-144 and NRF2 target are associated with glutathione level and anemia severity. To study the effect of miR-144 expression, the gain-loss of miR-144 expression was performed by miR inhibitor and mimic transfection in the erythroblastic cell line. This study reveals that miR-144 expression was upregulated, whereas NRF2 expression and glutathione levels were decreased in comparison with the untreated condition after miR mimic transfection, while the reduction of miR-144 expression contributed to the increased NRF2 expression and glutathione level compared with the untreated condition after miR inhibitor transfection. Moreover, miR-144 overexpression leads to significantly increased sensitivity to oxidative stress at indicated concentrations of hydrogen peroxide (H2O2) and rescued by miR-144 inhibitor. Taken together, our findings suggest that dysregulation of miR-144 may play a role in the reduced ability of erythrocyte to deal with oxidative stress and increased RBC hemolysis susceptibility especially in thalassemia.

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2001-11-01 | The studies of hemoglobinopathies and thalassemia in China—the experiences in Shanghai Institute of Medical Genetics

Background: In the past two decades, a large-scale survey of hemoglobinopathies and thalassemia was carried out in mainland China, involving nearly one million people in 28 provinces. The incidences of hemoglobin (Hb) variants, α-thalassemia and β-thalassemia were 0.33%, 2.64% and 0.66%, respectively. The chemical structural analysis identified 67 Hb variants. Among them, 20 are new variants. The analysis of the α-globin gene organization in 111 HbH patients showed 76 cases (68.5%) were of the deletion type, 8 had Hb Constant Spring and the other cases were of non-deletion type. The results of the molecular characterization of more than 200 β-thalassemia alleles showed that the most common types of β-thalassemia mutations in China are CD 41/42 (−4 bp), IVS-II-nt.654 C→T, CD 17 A→T, CD 71/72 (+A) and −28 A→G. Methods: To explore the simple method for molecular diagnosis of β-thalassemia, multiplex allele-specific amplification (MAS-PCR) was used that could simultaneously detect the above five common types of β-thalassemia mutations. Results: Based on the molecular analysis of β-thalassemia intermedia, β-thalassemia homozygotes or compound heterozygotes combined with α-thalassemia, as well as the conjunctive abnormalities of β-thalassemia heterozygote with triplicated haplotype of α-globin genes, were the most common cause of thalassemia intermedia in China. We also used the RT-PCR quantitation method to show that the most common β-thalassemia allele, IVS-II-nt.654 C→T, still produced a small amount (about 15%) of normally spliced β-globin mRNA, therefore, causing β+-thalassemia. In clinical trials of hydroxyurea (HU) treatment for β-thalassemia patients, we found that HU may enhance the expression of the β-globin gene in some patients, leading to an alleviation of clinical symptoms. In the studies of the reversal of aberrant splicing of IVS-II-nt.654 C→T allele by the antisense approach, we constructed a mammalian expression vector that can produce an antisense RNA targeting against the aberrant splice sites of IVS-II-nt.654 C→T pre-mRNA. Conclusions: The results indicated that the antisense RNA produced from the vector could efficiently suppress the aberrant splicing pattern and restore the correct splicing pathway in vitro and in vivo, leading to the improvement of globin chain biosynthesis in thalassemia cells.

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