Background Thalassemia is a prevalent inherited hematologic disorder imposing substantial health burdens, with unmet clinical needs for effective anemia-relieving therapies. Despite the rapid advancement of targeted drug development, systematic characterization of the global clinical trial landscape remains insufficient. Objective To examine the characteristics and publication status of clinical trials involving anemia-relieving medications for thalassemia registered on Clinicaltrials.gov. Methods Data of interventional clinical trials of anemia-relieving medications for thalassemia, registered on Clinicaltrials.gov, were collected. Key aspects such as the general characteristics, study design features, treatment strategies, outcome measures, and publication status were analyzed. Results Fifty-three clinical trials were included. Of these, 45.28% were completed. However, only 24.53% reported results. Most trials targeted adults/older adults, with 50.94% of the studies conducted in Asia. Among the trials analyzed, 47.17% were Phase 2 studies, and 37.74% employed randomization. Additionally, 47.17% used a parallel assignment intervention model, while 75.47% had no blinding. The most commonly studied drug was fetal hemoglobin inducers. Recently, research into novel therapeutic agents has risen considerably. While primary outcome measures varied across studies, hemoglobin level changes were the most commonly utilized measure. Conclusions Most interventional trials on anemia-relieving medications for thalassemia were non-randomized, non-blinded, and employed parallel assignment, primarily involving adult and older adult patients. While a significant portion of the research focused on fetal hemoglobin inducers, there has been a rising interest in erythroid maturation agents and pyruvate kinase-R agonists.

Allogeneic stem cell transplantation (allo-HSCT) has recently been approved as standard therapy for transfusion-dependent thalassemia (TDT) but remains limited to the use of HLA-matched sibling donors (MSDs), due to a lack of large-scale prospective studies evaluating the use of grafts from alternative donors. Here, we report the results of a non-randomised, interventional, phase 4 clinical trial evaluating allo-HSCT from alternative donors for the treatment of TDT. A total of 823 patients with TDT were transplanted with grafts from MSDs (n = 331) or alternative donors, including matched unrelated donors (MUDs; n = 352) and haploidentical related donors (Haplos; n = 140). Conditioning was with busulfan, cyclophosphamide, fludarabine and anti-thymocyte globulin. Graft-versus-host disease (GvHD) prophylaxis was cyclosporine, methotrexate (MTX) and mycophenolate mofetil (MMF) for recipients of MSDs and tacrolimus, MTX, MMF for others. The primary endpoints were 2-year overall survival (OS) and event-free survival (EFS). Two-year OS for MSDs, MUDs, Haplos was 97.2% (95% CI, 95.4-99.0), 93.1% (90.5-95.9) and 95.4% (91.9-99.1); EFS was 97.2% (95.4-99.0), 92.9% (90.1-95.7) and 94.7% (90.9-98.6); GvHD-free, relapse-free survival (GRFS) was 91.4% (88.4-94.6), 77.0% (72.6-82.7) and 75.6% (68.5-83.4) respectively. Two-year OS and EFS for MUDs were lower than those for MSDs (both P < 0.05) and were not significantly different with those for Haplos (both P > 0.05). Transplant-related mortality was 4.4% (3.0-5.9) and graft failure rate was 0.5%. The incidence of grades 2-4 acute GvHD and moderate-severe chronic GvHD from alternative donors were higher than those from MSDs (28.9% [24.7-33.2] vs 7.5% [4.9-10.7], P < 0.001; 12.3% [9.2-15.7] vs 5.0% [2.9-7.9], P < 0.01). In summary, these findings may help expand the donor pool for patients with TDT lacking MSDs (ClinicalTrials.gov: NCT04009525).

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.

Background: Hemoglobin H Constant Spring (HbH-CS) (–/αCSα) is the most common non-deletion α-thalassemia that can lead to severe health problems. The clinical manifestations of HbH-CS patients are widely variable and a considerable proportion of patients with HbH-CS need regular or irregular transfusions to support normal growth, development and life. Currently, there is no specific therapy for transfusion dependent α-thalassemia, and allogeneic hematopoietic stem cell transplantation is the only available curative option. Because patients with HbH-CS has 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 repairing of the mutation by base editing may provide a potential cure for this type of disease. Aims: To repair the CS mutation of HBA2 gene and increase the expression of 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 of edited patient cells (RM-004) in transfusion-dependent HbH-CS. Here, we present the initial results from the first patient treated with RM-004. Methods: Patients (12-35 y of age) with HbH-CS receiving packed red blood cell (pRBC) transfusions of ≥100 mL/kg/y or ≥10 units/y in the previous 2 y 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, patient received myeloablative conditioning with busulfan form day-7 to day-3. Patient were monitored for stem cell engraftment/hematopoietic recovery, adverse events (AEs), Hb production, HbA/HbH/Hb-CS expression and packed red blood cell (pRBC) transfusion requirements. Results: The first subject was a 14-y female patient with HbH-CS, who received an annualized 29 units/y pRBC transfusions before enrollment. She received a single dose of RM-004 cells, achieved neutrophil and platelet engraftment on Day14 and Day20 post-RM-004 infusion, respectively. She received the last pRBC transfusion on Day13 after RM-004 treatment, and has been transfusion-free over 3 months. The total Hb stably over 9 g/dL since Day26 and maintained between 10~11 g/dL currently. The proportion of HbH and Hb-CS were 3.8% and 1.1% respectively before enrollment (with regular transfusions), both dropped to 0.3% at 3-month after RM-004 infusion; the proportion of HbA1 has increased to 97.2%. The safety profile was generally consistent with busulfan myeloablation and autologous hematopoietic stem cell transplantation. No serious adverse event reported. Conclusion: The first patient treated with RM-004 demonstrated successful engraftment and clinical meaningful increase in Hb level. Although the follow-up time is relatively short, available data have shown that the patient has entered a transfusion-independence state. This is the first-ever clinical report of patients with α-thalassemia treated successfully with gene editing and indicates that RM-004 is a promising approach for the treatment of HbH-CS. Data will be updated for the presentation.

Abstract Background: RM-001 is a novel non-viral cell therapy for β-hemoglobinopathies that reactivates fetal hemoglobin (HbF) through ex vivo CRISPR-Cas9 editing of the BCL11A binding site in the γ-globin gene (HBG1/2) promoters of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs). Methods: Both an investigator-initiated trial (IIT; ChiCTR2100053406 and ChiCTR2100052858, n=7) and a Phase 1 trial (ChiCTR2300069244, n=12) were conducted to evaluate the safety and efficacy of RM-001 in patients with transfusion-dependent β-thalassemia (TDT). Eligible participants were aged 6 to 35 years, with a history of packed red blood cell (pRBC) transfusions ≥100 mL/kg/year or ≥10 units/year during the 2 years preceding screening. 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. TI12 assessment began 60 days after the last transfusion. Patients completing the 24-month trial will transition to a long-term follow-up study. Results: As of July 15, 2025, 19 patients (mean age, 15.9 years; 5 patients aged ≥18 years, 7 aged ≥12 to <18 years, and 7 aged ≥6 to <12 years) had received RM-001, with a median follow-up of 26.3 months (range, 18.9 to 44.5). Before enrollment, patients received a mean of 56.9 units/year (range, 35.3 to 106.3) of pRBC transfusions. Genotypes included β⁰/β⁰ in 13 patients (68.4%), β⁰/β⁺ in 5 patients (26.3%), and β⁺/β⁺ in 1 patient (5.3%). All patients achieved neutrophil engraftment (median, 15 days) and platelet engraftment (median, 21 days). All 19 patients (100%) discontinued transfusions and remained transfusion-free for ≥15 months (range, 16.2 to 42.0). Patients stopped transfusions at a median of 22 days (range, 10 to 94) after RM-001 infusion, with stable hemoglobin levels ≥9 g/dL at a median of 31 days (range, 14 to 127). All patients had ≥18 months of follow-up after RM-001 infusion, with all (100%) achieving TI12, with mean total hemoglobin levels of 10.8 g/dL at month 3, 11.6 g/dL at month 6, 11.8 g/dL at month 12, and 12.0 g/dL at month 18, and corresponding fetal hemoglobin (HbF) levels of 9.2, 11.4, 11.6, and 12.0 g/dL. HBG1/2 allele editing proportions remained stable in patient´s bone marrow over time. The first 13 patients have finished 24-month follow-up and enrolled in a long-term study. The first 5 patients have been followed over 3 years and their Hb levels maintained stably above 11 g/dL. No treatment-related serious adverse events were reported. All adverse events resolved completely, with no deaths, treatment discontinuations, or malignancies observed. Conclusion: The data from 19 TDT patients infused with RM-001 demonstrated clinically meaningful and sustained increases in total Hb and HbF, leading to transfusion independence in all subjects. The safety profile of RM-001 was excellent and no product-related serious adverse events were reported during the study. These results indicate that RM-001 has potential to cure TDT with one-time treatment.

ABSTRACT Background Hemoglobin H (HbH) disease is a relatively common genetic disorder worldwide. However, the lack of long‐term follow‐up studies involving large patient cohorts has limited our understanding of its clinical features, disease progression, and outcomes. Methods We conducted a retrospective study of 1421 patients diagnosed with HbH disease who were admitted to six hospitals in the Guangxi Zhuang Autonomous Region, China, between 2010 and 2022. We analyzed hemoglobin levels and their association with different genotypes and ages. Key hematological parameters, serum ferritin levels, and visit frequency were compared between patients with HbH‐constant spring (HbH‐CS) and those with deletional HbH. Additionally, we assessed the survival status of 117 patients over 30 years of age and examined complications and causes of death in four patients who died during the study period. Results In the cohort, 73.0% of patients had HbH‐CS, while 24.2% had deletional HbH. Patients with HbH‐CS exhibited a significantly younger average age, lower mean and minimum hemoglobin (Hb) levels, and higher visit frequencies and serum ferritin levels compared with those with deletional HbH. Iron overload was common in HbH‐CS patients, with a mean serum ferritin level of 1284 ± 1231 ng/mL (measured in 671 patients). Among the four deceased patients (aged 44.8 to 68.5 years), at least two deaths were attributed to severe anemia and related complications. Conclusion This study provides valuable insights into the natural history, clinical progression, and long‐term outcomes of HbH disease in a large patient cohort over the past decade. It highlights key differences between HbH‐CS and deletional HbH, particularly in terms of disease severity, iron overload, and survival outcomes. Clinical Trail Registration : The authors have confirmed clinical trial registration is not needed for this submission.

Introduction : RM-001 is a novel non-viral cell therapy for β-hemoglobinopathies, which is designed to reactivate fetal hemoglobin (HbF) via ex vivo CRISPR-Cas9 gene-editing of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) at the binding site of BCL11A on the promoter of the γ-globin genes (HBG1/2). Here we report that in both investigator initiated trial (IIT) and phase I trial, all the patients (pts) with transfusion-dependent β-thalassemia (TDT) achieved transfusion-free after RM-001 treatment. Methods: Both IIT (ChiCTR2100053406 and ChiCTR2100052858, n =7) and phase I trial (ChiCTR2300069244, n =12) have been conducted to evaluate the safety and efficacy of RM-001 in treating TDT. Patients (6-35 y of age) with TDT and a history of ≥100 mL/kg/y or ≥10 units/y packed red blood cell (pRBC) transfusions in the 2 years before screening were eligible. Primary efficacy endpoint is transfusion independence defined as proportion of pts maintaining a weighted average hemoglobin (Hb) ≥9 g/dL without pRBC transfusion for ≥12 consecutive months (TI12). Key secondary endpoint is proportion of pts maintaining a weighted average Hb ≥ 9 g/dL without RBC transfusion for ≥ 6 consecutive months (TI6). Evaluation of TI12 and TI6 started 60 days after last pRBC transfusion. Pts completing the 24-month trial will be enrolled in a long-term follow-up study. Results: As of July 24, 2024, 19 pts (mean age 15.9, 5 pts aged ≥18, 7 pts aged ≥12 to <18y and 7 pts aged ≥6 to <12y) have received RM-001, with a median follow-up of 14.5 (7-32.6) months. Pts received a mean of 53.0 (35.3-106.3) units/y pRBC transfusions before enrollment; 13 pts (68.4%) have the most severe genotype (β0/β0), 5 pts (26.3%) have β0/β+ genotype and the remaining one (5.3%) has a β+/β+ genotype. Following infusion, all pts engrafted neutrophils and platelets (median 15 and 21 days, respectively). All of 19 (100%) pts stopped transfusions and maintained transfusion-free ≥ 6 (6-31.8) months. Pts stopped transfusions at a median of 22 (10-95) days post-RM-001 infusion and achieved stable Hb ≥9 g/dL at a median of 31 (14-127) days. Of the 9 pts had ≥15 months of follow-up after RM-001 infusion, 9 (100%) achieved TI12; of the 16 pts had ≥9 months of follow-up, all (100%) achieved TI6; the remaining 3 pts had ≥7 months of follow-up, all of them are transfusion-free ≥ 6 months. For all pts, the mean total Hb and HbF were 10.8 g/dL and 9.2 g/dL at Month 3, respectively. Of the 13 pts had ≥12 months of follow-up, the mean total Hb and HbF were 11.8 (10.1-13.3) g/dL and 11.7 (10.0-13.2) g/dL at Month 12, respectively. Proportion of edited HBG1/2 alleles was stable over time in bone marrow cells. The first 5 pts have finished 24-month follow-up and enrolled in a long-term study. No RM-001-related serious adverse event report. All of adverse events have been resolved. There were no deaths, discontinuations, or malignancies. Conclusion: The data from 19 TDT pts infused with RM-001 demonstrated clinically meaningful and sustained increases in total Hb and HbF, leading to transfusion-free in all of subjects. The safety profile of RM-001 is very well and no product-related serious adverse event was reported during the study. These results indicate that RM-001 has the potential to cure TDT with one-time treatment.

Erythroleukemia, a subtype of acute myeloid leukemia (AML), is a life-threatening malignancy that affects the blood and bone marrow. Despite the availability of clinical treatments, the complex pathogenesis of the disease and the severe side effects of chemotherapy continue to impede therapeutic progress in leukemia. In this study, we investigated the antitumor activity of L76, an acylphloroglucinol compound derived from Callistemon salignus DC., against erythroleukemia, along with its underlying mechanisms. MTT assays were performed to evaluate the inhibitory effects of L76 on cancer cell viability, while flow cytometry was used to analyze apoptosis and cell cycle arrest in HEL cells. The molecular mechanisms of L76 were further explored using Western blotting, microscopic analysis, and cellular thermal shift assays (CETSA). Our in vitro experiments demonstrated that L76 inhibits proliferation, induces G1/S cell cycle arrest, and promotes apoptosis in human leukemia cells. Mechanistically, L76 exerts its effects by targeting STAT3 and p38-MAPK, and by inhibiting the PI3K/AKT/mTOR signaling pathway. In conclusion, this study highlights the potential of L76 as an anti-erythroleukemia agent, demonstrating its ability to target STAT3 and p38-MAPK, and to inhibit the PI3K/AKT/mTOR signaling pathway. These findings suggest that L76 could be a promising candidate for the treatment of erythroleukemia.

Introduction: Splenomegaly and hypersplenism are common complications of thalassemia patients due to the excessive clearance of defective red blood cells from the spleen. To date, splenectomy has been considered one of the most effective treatments for splenomegaly, reducing clinical severity among thalassemia patients. Thus, we aim to investigate the differences in splenectomy rates and hematological indices among thalassemia patients with different genotypes. Method: In this study, we analyzed the clinical data of thalassemia in 2,130 patients admitted to the 923rd Hospital of the People's Liberation Army from January 2006 to December 2020, and the statistical software SPSS 26.0 was applied to analyze the data. Result: Of the 2,130 patients with thalassemia, 265 patients underwent splenectomy. It was determined that significantly more patients with hemoglobin H (HbH) disease, a form of alpha-thalassemia, have undergone splenectomy than beta-thalassemia patients (20% vs. 7%). Further, HbH disease patients were diagnosed at a significantly older age than beta-thalassemia patients. Conclusion: The greater probability of HbH disease patients undergoing splenectomy is likely influenced by multiple factors, including their lower dependency on transfusion, leading to high spleen compensatory stress on the spleen, and the destruction of defective erythrocytes. In contrast, beta-thalassemia is clinically more severe and less tolerant of hemoglobin fluctuations. Based on these findings, clinicians are suggested to pay more attention to HbH disease patients as many of them are still under-transfused, which could lead to chronic hemolysis and more severe hepatosplenomegaly. These results might offer insight for improving the clinical management of patients with different types of thalassemia. (c) 2023 S. Karger AG, Basel

Background: Reactivating fetal globin (HbF) is a promising treatment for β-hemoglobinopathies. Using gene editing to mimic these mutations should reactivate γ-globin in patients with transfusion-dependent β-thalassemia (TDT) and ameliorate the symptoms of patients. RM-001 is a novel cell therapy that uses non-viral, ex vivo CRISPR-Cas9 gene editing in autologous hematopoietic stem and progenitor cells (HSPCs) at the promoter of the γ-globin genes (HBG1/2) to disrupt the binding site of BCL11. Aims: Here, we present available safety and efficacy results from all patients that received RM-001 infusion from two ongoing clinical trials (ChiCTR2100053406 & ChiCTR2100052858), including a recent treated TDT patient (β0/β0) who also carries two α-globin genes deletion (--/αα). Methods: Patients (6–35 y of age) with TDT receiving packed red blood cell (pRBC) transfusions of ≥100 mL/kg/y or ≥10 units/y in the previous 2 y were eligible. Peripheral CD34+ HSPCs were collected by apheresis after mobilization with G-CSF and plerixafor. CD34+ cells were edited with CRISPR-Cas9 using a guide RNA specific for the binding site of BCL11A on the HBG1/2 promoter. Prior to RM-001 product infusion (day 0), 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, HbF and F-cell expression, and pRBC transfusion requirements. Bone marrow cells were obtained at 3, 6, 12 and 24 months after RM-001 infusion to measure the on-target allelic editing frequency using next-generation sequencing. Results: Data presented here for all 6 TDT patients have been treated with RM-001. As of February 28, 2023, patients were followed up for 2 to 15 months and 5 of them have been followed up more than 12 months. Five patients have β0/β0 genotype (CD17/CD41-42, n=1; CD41-42/CD41-42, n=4) and the other has β0/β+ genotype (CD41-42/IVS-II-654). In addition to β-thalassemia (CD41-42/CD41-42), the sixth patient (25yo) also carries two α-globin genes deletion (--SEA/αα). Patients had received a mean of 56.2 units/y pRBC transfusions (range: 39-79.6 units/y). All patients received a single dose of RM-001 cells, and achieved both neutrophil and platelet engraftments 2 to 3 weeks after RM-001 infusion (neutrophil: day 13-19, platelet: day 10-22). All patients ceased pRBC transfusions within 1 month after RM-001 infusion and achieved transfusion-independent (TI, total Hb continued ≥ 9g/dL) within 2 months (Figure). At 4 month post-RM-001 infusion, HbF reached 10g/dL in the first 5 patients and continuously maintained over this level through the reported period. At 2 month post-RM-001 infusion, the sixth patient had a total Hb of 11.7g/dL with 78.0% of HbF. The safety profile was generally consistent with busulfan myeloablation and autologous hematopoietic stem cell transplantation. No RM-001 related SAE report. Summary/Conclusion: The updated data reported here from 6 patients with TDT infused with RM-001 demonstrated clinically meaningful increases in total hemoglobin (Hb) and HbF levels. All patients stopped receiving pRBC transfusions within 1 month after RM-001 infusion and remained transfusion-free through the time of this analysis. The safety profile of RM-001 is generally consistent with myeloablative conditioning and autologous hematopoietic stem cell transplantation. These results strongly support continued investigation of RM-001 as a potential cure for patients with TDT. Submitted on behalf of the RM-001 Investigators.Keywords: Gene therapy, Thalassemia, Autologous hematopoietic stem cell transplantation

Background: Reactivating fetal globin (HbF) is a promising treatment for β-hemoglobinopathies. Natural mutations in the promoter region of γ-globin genes ( HBG1/2) that disrupt the binding of the transcriptional repressors BCL11A could lead to a lifelong persistence of fetal γ-globin expression. Using gene editing to mimic these mutations should reactivate γ-globin in patients with transfusion-dependent β-thalassemia (TDT) and ameliorate the symptoms of patients. RM-001 is a novel cell therapy that uses non-viral, ex vivo CRISPR-Cas9 gene editing in autologous hematopoietic stem and progenitor cells (HSPCs) at the promoter of the γ-globin genes ( HBG1/2) to disrupt the binding site of BCL11A. Aims: ChiCTR2100053406 and ChiCTR2100052858 are ongoing multi-center, first-in-human studies of RM-001 for TDT. Here, we present available safety and efficacy results from 7 patients that have been dosed with RM-001. Methods: Patients (6-35 y of age) with TDT receiving packed red blood cell (pRBC) transfusions of ≥100 mL/kg/y or ≥10 units/y in the previous 2ys were eligible. Peripheral CD34+ HSPCs were collected by apheresis after mobilization with G-CSF and plerixafor. CD34+ cells were edited with CRISPR-Cas9 using a guide RNA specific for the binding site of BCL11A on the HBG1/2 promoter. Prior to RM-001 product infusion (day 0), 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, HbF and F-cell expression, and pRBC transfusion requirements. Bone marrow cells were obtained at 3, 6, 12 and 24 months after RM-001 infusion to measure the on-target allelic editing frequency using next-generation sequencing. Results: Data presented here for 7 TDT patients have been treated with RM-001. As of July 31, 2023, patients were followed up from 1 to 20 months and 5 of them have been followed up more than 15 months. Six patients have β 0/β 0 genotype (CD17/CD41-42, n=1; CD41-42/CD41-42, n=5) and the other has β 0/β + genotype (CD41-42/IVS-II-654). In addition to β-thalassemia (CD41-42/CD41-42), two patients also carry a Southeast Asian deletion of α-globin genes (– SEA/αα). Patients had received a mean of 55.8 units/y pRBC transfusions (range: 39-79.6 units/y). All patients received a single dose of RM-001 cells, and achieved both neutrophil and platelet engraftments 2 to 3 weeks after RM-001 infusion (neutrophil: day 11-19, platelet: day 10-22). All patients ceased pRBC transfusions within 1 month after RM-001 infusion and remained transfusion-free through the reported period (Figure). For the 6 patients that have been followed up more than 6 months, HbF reached 9g/dL at 4 month post-RM-001 infusion and continuously maintained over this level through the reported period. From 6 month post-RM-001 infusion, hemoglobin in all patients consists of HbF (97.6%-99.8%) and HbA2 only, including the fifth patient who has a β0/β+ genotype (99.5% HbF). Five participants have remained transfusion independent more than 15 months and the mean HbF in the first 4 patients was 11g/dL(10.9-11.3 g/dL) at 18 month post-RM-001 infusion. The safety profile was generally consistent with busulfan myeloablation and autologous hematopoietic stem cell transplantation. No RM-001 related SAE report. Summary/Conclusion: This updated data reported here from 7 patients with TDT infused with RM-001 demonstrated clinically meaningful increases in total hemoglobin (Hb) and HbF levels. All patients stopped receiving pRBC transfusions within 1 month after RM-001 infusion and remained transfusion-free through the time of this analysis. The safety profile of RM-001 is generally consistent with myeloablative conditioning and autologous hematopoietic stem cell transplantation. These results strongly support continued investigation of RM-001 as a potential cure for patients with TDT. Data will be updated for the presentation. Submitted on behalf of the RM-001 Investigators.

DNA methyltransferase 1 (DNMT1) is a major epigenetic regulator of the formation of large macromolecular complexes that repress human γ-globin expression by maintaining DNA methylation. However, very little is known about the association of DNMT1 variants with β-thalassemia phenotypes. We systematically investigated associations between variants in DNMT1 and phenotypes in 1142 β-thalassemia subjects and identified a novel missense mutation (c.2633G>A, S878F) in the DNMT1 bromo-adjacent homology-1 (BAH1) domain. We functionally characterized this mutation in CD34+ cells from patients and engineered HuDEP-2 mutant cells. Our results demonstrate that DNMT1 phosphorylation is abrogated by substituting serine with phenylalanine at position 878, resulting in lower stability and catalytic activity loss. S878F mutation also attenuated DNMT1 interactions with BCL11A, GATA1, and HDAC1/2, and reduced recruitment of DNMT1 to the γ-globin (HBG) promoters, leading to epigenetic derepression of γ-globin expression. By analyzing the F-cell pattern, we demonstrated that the effect of DNMT1 mutation on increased fetal hemoglobin (HbF) is heterocellular. Furthermore, introduction of S878F mutation into erythroid cells by clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) recapitulated γ-globin reactivation. Thus, the natural S878F DNMT1 mutation is a novel modulator of HbF synthesis and represents a potential new therapeutic target for β-hemoglobinopathies.

Objective: This study focused on the efficacy and safety of thalidomide for patients with thalassemia intermedia (TI) in a multicenter trial. Methods：Clinical and laboratory data of 62 patients subjected to thalidomide therapy in four centers were retrospectively analyzed. We evaluated the efficacy and safety of thalidomide in the short-term (three months) and long-term follow-up (12 and 24 months). Response to thalidomide was defined as follows: Main Responder (MaR) showing an increase in Hb level of >2.0 g/dl or removal from blood transfusion and Minor Responder (MiR) achieving elevated hemoglobin (Hb) level of 1.0-2.0 g/dl or ≥50% reduction in blood transfusion frequency. Results：The overall response rate (ORR) of 62 patients with TI was 93.5% (58/62), with MaR and MiR rates accounting for 62.9% (39/62) and 30.6% (19/62) in short-term follow-up and 66.1% (41/62) and 27.4% (17/62) in long-term follow-up, respectively. The clinical response during long-term follow-up was maintained and the Hb level remained stable during the observation period. The response was still observed in patients with dose reduction despite a slight decrease in Hb level. However, Hb decreased rapidly to the baseline level after drug discontinuation. No effect of thalidomide on spleen size in nonsplenectomized patients was evident. Minimal side-effects were documented throughout, except peripheral neurotoxicity in one patient. Nevertheless, the mean serum ferritin (SF) level was significantly increased after treatment. Conclusion: Thalidomide had significant therapeutic effects on patients with TI, and the response was sustained with acceptable short-term and long-term adverse reactions. While these preliminary results support the potential long-term efficacy and safety of thalidomide as a therapeutic agent for TI, several issues need to be addressed before its application in the clinic.

我国地中海贫血(简称地贫)患者中数量最多的是中间型α地贫,也称血红蛋白H病(hemoglo-bin H disease,Hb H病)[1],由于未得到足够重视,该类型患者的生存状况甚至比不上规范高量输血的重型β地贫(thalassaemia major,TM)[2-3].近年来,国际上根据临床严重程度和输血需求将地贫分为输血依赖型地贫(transfusion dependent thalassaemia,TDT)和非输血依赖型地贫(non transfusion dependent thalassaemia,NTDT)两类,并制定了相应的管理指南[2-4].部分Hb H病患者属于TDT,需规范高量输血和铁螯合剂治疗,这些患者中部分是发病时即为TDT,部分是后来出现并发症或贫血加重转为TDT的[2-3,5-6].本文基于笔者所在医疗单位收治的病例,略述Hb H病的治疗.