BACKGROUND:Graft failure and transplant-related mortality remain major barriers to haploidentical donor hematopoietic stem cell transplantation (HID-HSCT) in children and adolescents aged ≥7 years with transfusion-dependent thalassemia (TDT). Thus, a novel low-toxicity myeloablative conditioning regimen was developed to improve transplantation outcomes. METHODS:In this prospective, single-center study (clinical trial registration ChiCTR2300071890), TDT patients aged ≥7 years were enrolled to evaluate the safety and efficacy of a conditioning regimen comprising fludarabine, busulfan, thiotepa, and melphalan (F-BMT). For HID recipients, graft-versus-host disease (GVHD) prophylaxis included half-dose PTCY combined with the in vivo T-cell depletion (porcine anti-human lymphocyte immunoglobulin or rabbit anti-human thymocyte globulin), along with other immunosuppressive agents. The primary endpoint was the 2-year thalassemia-free survival (TFS) rate. RESULTS:From June 2023 to August 2024, 29 TDT patients aged ≥7 years undergoing HID-HSCT were enrolled (14 males, 15 females). The 2-year TFS and overall survival rates were both 93.1%. Neutrophil and platelet engraftment were achieved in all patients, with no reported graft failure. The observed incidence of Grade II-IV acute GVHD was 17.2%. Chronic GVHD occurred in 6.9% of the patients. Regimen-related toxicities were manageable, with only one case of sinusoidal obstruction syndrome and three cases of hemorrhagic cystitis, all of which resolved with supportive treatment. No mixed chimerism was observed. CONCLUSIONS:The F-BMT regimen provides effective myeloablation with reduced toxicity, supporting its feasibility for HID-HSCT in TDT patients aged ≥7 years.

Abstract α-Thalassemia is a severe inherited hemoglobin (Hb) disorder with limited curative options for patients with transfusion dependency, lacking suitable donors. We report, to our knowledge, the first-in-human application of lentiviral vector–mediated α-globin gene therapy in a girl aged 14 years with transfusion-dependent HbH 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 after busulfan conditioning. The patient achieved engraftment within 3 weeks and started to be free of transfusions from the fourth week onward. In the last year of follow-up (months 12-24), the average Hb level remained at ∼90 g/L without transfusion support, with HbA accounting for >85% of total Hb. Vector copy numbers were stable, and no evidence of clonal dominance, insertional mutagenesis, or replication-competent lentivirus was detected. Adverse events (AEs) were limited to expected busulfan-related toxicities, including grade 2 menstrual irregularity and ovarian failure (serious AE). 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.

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.

β-Thalassemia is associated with a range of complications, with pulmonary hypertension in affected individuals having a particularly high mortality rate. However, data on the use of pulmonary vasodilators in β-thalassemia are scarce. Here, we present the case of an adult male diagnosed with β-thalassemia and pulmonary hypertension. There was no improvement in symptoms or test results with low-dose Riociguat. After taking Luspatercept, the patient's hemoglobin level increased significantly. Notably, his PAH symptoms also improved, as reflected by better 6-minute walk test results and reduced tricuspid regurgitation velocity on echocardiography. These findings suggest that Luspatercept may be useful in reducing pulmonary vascular resistance and improving clinical outcomes in patients with β-thalassemia and pulmonary hypertension.">β-Thalassemia is associated with a range of complications, with pulmonary hypertension in affected individuals having a particularly high mortality rate. However, data on the use of pulmonary vasodilators in β-thalassemia are scarce. Here, we present the case of an adult male diagnosed with β-thalassemia and pulmonary hypertension. There was no improvement in symptoms or test results with low-dose Riociguat. After taking Luspatercept, the patient's hemoglobin level increased significantly. Notably, his PAH symptoms also improved, as reflected by better 6-minute walk test results and reduced tricuspid regurgitation velocity on echocardiography. These findings suggest that Luspatercept may be useful in reducing pulmonary vascular resistance and improving clinical outcomes in patients with β-thalassemia and pulmonary hypertension.

Congenital dyserythropoietic anemia (CDA) represents a heterogeneous group of rare hereditary disorders characterized by ineffective erythropoiesis and often presents with clinical features that overlap with thalassemia. Hematopoietic stem cell transplantation (HSCT) remains the only definitive curative intervention for CDA; however, the application of haploidentical HSCT in this context is limited and presents considerable challenges. Herein, we report two pediatric cases of CDA coexisting with thalassemia who underwent haploidentical related donor HSCT utilizing a novel conditioning regimen comprising three alkylating agents. Graft-versus-host disease (GVHD) prophylaxis was achieved using. posttransplant cyclophosphamide and anti-thymocyte globulin. Both patients attained sustained engraftment, transfusion independence, and remained free from severe transplant-related complications. These cases illustrate the feasibility and therapeutic potential of haploidentical HSCT for CDA, even in. the presence of concomitant thalassemia.

Background Conditioning regimens are crucial in allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially for older patients (≥7 years) with transfusion-dependent thalassemia (TDT) recieving haplo indentical transplantation (HID). The development of low-toxicity conditioning yet myeloablative regimens is essential to address the dual challenges of high graft rejection rates and transplant-related mortality. Objective This study aimed to evaluate the efficacy and safety of the F-BMT conditionig regimen in improving allo-HSCT outcomes for TDT patients, especially for older patients with HID transplantation . Methods This prospective, multicenter clinical trial was conducted at 923rd Hospital of the Joint Logistics Support Force, the First Affiliated Hospital of Guilin Medical University, and the Eighth Affiliated Hospital of Southern Medical University.Patients with TDT aged 3–25 years were enrolled. The F-BMT regimen included fludarabine 30 mg/m² (days –7 to –2), busulfan 130 mg/m² (day –7), thiotepa 250 mg/m² (day –5), melphalan 100 mg/m² (day –3). For graft versus host disease (GVHD) prentation, porcine anti-human lymphocyte immunoglobulin (p-ALG) (25 mg/kg) or antithymocyte globulin (r-ATG) (2.5 mg/kg ) on days –1, cyclophosphamide 25 mg/kg (days +3 and +4)，methotrexate 5 mg/m² (day +5), cyclosporine (initiated on day +5, continued for 12 months). For HID donors, additional agents were administered: p-ALG (25 mg/kg ) or r-ATG (2 mg/kg) on day day +5, ruxolitinib (days +5 to +28). The primary endpoint was 1-year thalassemia-free survival (TFS). (ChiCTR2300077054) Results From June 2023 to May 2025, 91 patients were enrolled. The median age was 10.0 years (range: 4-19 years), among them 76 patients aged ≥7y, and 63 patients with HID donors. With a median follow-up duration of 12 months (range: 3-25 months). 1-year TFS was 94.24%, 1-year overall survival (OS) was 96.44%.Two patients (2.2%) died from severe pneumonia or sepsis. The remaining 89 patients achieved both neutrophil and platelet engraftment within 28 days post-transplantation, with full donor chimerism. One patient experienced secondary graft failure due to infection, and one patient had primary graft failure. Both of these patients underwent secondary transplantation with successful engraftment.The engraftment rate by day +28 post-transplantation was 97.78%. The mean time to neutrophil engraftment was 13.7 ± 1.5 days, and the mean time to platelet engraftment was 13.1 ± 3.9 days. By day +100 post-transplantation, the cumulative incidence of grade II-IV aGVHD was 7.1%, with only 4.4% (n=4) of patients developing grade III-IV aGVHD. Grade 1-2 hemorrhagic cystitis (HC) occurred in 3.3% (n=3) of patients. Viral reactivation was observed in 26% (n=24) of patients, including Cytomegalovirus (CMV) reactivation in 18 patients and Epstein-Barr virus (EBV) reactivation in 8 patients; 2 of these patients had concurrent CMV and EBV reactivation.Infections occurred in 12% (n=11) of patients within the first 3 months post-transplantation, including bacteremia in 11% (n=10). One patient developed posterior reversible encephalopathy syndrome (PRES), one had an invasive fungal infection, and two patients developed veno-occlusive disease (VOD). Post-transplant lymphoproliferative disorder (PTLD) was diagnosed in one patient and was cured after Rituximab injection.. Chronic graft-versus-host disease (cGVHD) occurred in 8.9% (6 cases) of evaluable patients, involving the pulmonary and hepatic systems, with all cases rated as moderate; all cases were cured with appropriate therapy. No cases of thrombotic microangiopathy (TMA) were observed. Conclusion This study demonstrates that the F-BMT conditioning regimen followed by HSCT represents a low-toxicity protocol with significant efficacy and safety in TDT patients, even in older patients with HID donors.

Objective: This study analyzed anti-mullerian hormone (AMH) levels in patients with non-transfusion-dependent thalassemia (NTDT) to evaluate their ovarian function.

Blood transfusion is a principal treatment for thalassemia at present, but there is still an absence of effective examination to evaluate the efficacy of blood transfusion and judge the timing of blood transfusion. Carbon monoxide (CO) breath test is a non-invasive and rapid examination way that could dynamically monitor red blood cell destruction in patients with thalassemia. A total of 61 patients with transfusion-dependent thalassemia and 21 healthy controls were recruited in the study.

Nucleoporin 98 (NUP98) fusion oncoproteins are associated with various hematologic malignancies. Acute myeloid leukemia (AML) with NUP98-NSD1 typically co-occurs with FLT3-ITD mutations, exhibiting poor initial responses to traditional chemotherapy. This case report describes a relapsed and refractory AML case co-expressing NUP98/NSD1 and FLT3/ITD after matched sibling haplo-identical allogeneic hematopoietic stem cell transplantation, achieving molecular remission with a salvage therapy combining selinexor, venetoclax, and azacitidine. To our knowledge, this is the first report demonstrating the effectiveness of this combination therapy for relapsed/refractory NUP98-NSD1+/FLT3-ITD + AML. This report highlights the potential synergy between selinexor and established AML therapies, suggesting a promising approach to improve outcomes for refractory AML patients.

Abstract Background: Haematopoietic stem cell transplantation (HSCT) is a crucial treatment for severe aplastic anemia (SAA). However, there is no standardized preconditioning regimen for patients without matched sibling or unrelated donors. Achieving successful engraftment remains a significant challenge in haploidentical HSCT, controlling transplantation-related complications and reducing transplantation-related mortality are urgent issues that need to be addressed. New conditioning regimens are still worth exploring. Objective: We conducted a prospective, multi-center clinical study to explore the efficacy and safety of a novel preconditioning regimen consisting of fludarabine, cyclophosphamide, and melphalan (FCM) in haploidentical HSCT for the treatment of SAA. Methods: Patients who were diagnosed with SAA according to NCCN guidelines and required haplo-identical HSCT were recruited. Patients with active infections or bleeding, severe liver or kidney dysfunction, ECOG > 2, <3 or >65 years old, psychiatric disorders, or pregnancy were excluded. The preconditioning regimen included: 30mg/m² fludarabine for 6 days, 50mg/kg cyclophosphamide on day -7 and day -5, 100mg/m² melphalan on day -2, and 2mg/kg ATG on day -1. The prevention of acute graft-versus-host disease (aGVHD) consisted of post-transplantation cyclophosphamide (PTCy) and short-term methotrexate ± other drugs, detailed aGVHD prophylaxis was determined according to each center’s decision. Chimerism was assessed on days +28, +60, +90 post-transplantation and then once half year. The primary endpoints was graft failure rate, the second endpoint were the incidence of transplantation-related complications, 2-year overall survival (OS) and disease-free survival (DFS). This study was approved by the Ethic Committee of 920th Hospital of Joint Logistics Support Force and was registered at www.clinicaltrial.gov as NCT06378060. Results: From April 2024 to May 2025, 25 SAA patients from 7 transplantation centers who underwent haploidentical HSCT were enrolled. The male-to-female ratio was 11:14, with a median age of 26 years (IQR 15-38). All patients achieved successful engraftment, with median times to neutrophil and platelet engraftment of 13 days (IQR 11-14) and 12days (IQR 11-14), respectively. One patient experienced poor graft function and developed aGVHD after donor stem cell transfusion, resulting in death on day +110. No seizures, severe mucositis, cardiac events, or bleeding events occurred during transplantation. The incidences of grade II-IV and grade III-IV aGVHD were 20% and 4%, respectively. Hemorrhagic cystitis occurred in 8% of patients. Epstein-Barr Virus (EBV) and cytomegalovirus (CMV) reactivation rates were 16% and 20%, respectively. Three patients developed pulmonary fungal infections post-transplantation, they were resolved after antifungal treatment. One patient developed post-transplant lymphoproliferative disorder (PTLD), she was cured by the treatment of rituximab. Till August 2025, the median follow-up time was 13.1 months, with 24 patients surviving. The transplantation-related mortality (TRM) rate was 4%. Conclusion: The FCM conditioning regimen in haplo-identical HSCT for severe aplastic anemia achieves a high engraftment rate with good safety and low infection rates.

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.

The management of patients with myelodysplastic syndrome (MDS) refractory to hypomethylating agents (HMAs) remains a challenge with few reliably effective treatments. Preclinical studies have shown that the inhibition of the nuclear export protein XPO1 causes nuclear accumulation of p53 and disruption of NF-κB signaling; both of which are relevant targets for MDS. Selinexor is an XPO1 inhibitor with demonstrated efficacy in MDS patients. Herein, we report three patients with MDS refractory to HMAs, however, when selinexor and venetoclax were added to the treatment regimen, the patients achieved a complete response and a significant reduction in spleen size. All patients successfully underwent hematopoietic stem cell transplantation. These cases demonstrate that the combination therapy can achieve CR and significant reductions in spleen size, offering a promising therapeutic option for patients with limited treatment choices. Combination therapy would also offer a potential way for patients to bridge to transplantation. Formal evaluations of this regimen in patients with MDS refractory to HMAs may be meaningful.

Relapse remains the main cause of treatment failure in patients with myeloid malignancies even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We observed a particularly low incidence of relapse in patients prepared with fludarabine, busulfan and melphalan in our previous study and this multicenter retrospective analysis aimed to confirm the feasibility of the regimen and to identify the potential prognostic factors. This study was performed using registry data from adults patients with myeloid malignancies who underwent their first allo-HSCT following fludarabine(≥100 mg/m2), busulfan (≥3.2 mg/kg) and melphalan (≥100 mg/m2) based conditioning at nine transplantation centers in China between Jan. 2020 and Mar. 2022. A total of 221 consecutive patients (AML n = 171, MDS-IB-1 or 2 n = 44, CMML n = 6) with median age of 46 were enrolled in this study. The median follow-up was 507 days for survivors. The 2-year NRM, CIR, OS and DFS were 10.6% ± 2.2%, 14.8% ± 3.3%, 79.4% ± 3.7% and 74.6% ± 3.7%, respectively. In multivariate analyses, high HCT-CI (≥3) was the only independent factor for higher NRM [hazard ratio (HR), 2.96; 95% confidence interval (CI), 1.11 to 7.90; p = 0.030] and ECOG score ≥2 was the only independent factor for inferior OS (HR, 2.43; 95%CI, 1.15 to 5.16; p = 0.020) and DFS (HR, 2.12; 95%CI, 1.13 to 4.02; p = 0.020). AML diagnosis and positive measurable residual disease (MRD) at transplantation were predictors for higher CIR (HR = 7.92, 95%CI 1.05-60.03, p = 0.045; HR = 3.64, 95%CI 1.40-9.44, p = 0.008; respectively), while post-transplantation cyclophosphamide based graft-versus-host disease prophylaxis was associated with lower CIR (HR = 0.24 95%CI 0.11-0.54, p = 0.001). The intensity of conditioning regimen did not impact CIR, NRM, DFS and OS. These results supported that double alkylating agents of busulfan and melphalan based conditioning regimens were associated with low relapse rate and acceptable NRM in adult patients with myeloid malignancies. The optimal dose remained to be confirmed by further prospective studies.

Background: Thalassemia comprises a diverse group of genetic disorders that affects the synthesis of globin chains, with a global distribution. The use of erythrocyte lifespan (ELS) to assess differences among patients with different types of thalassemia and the efficacy of splenectomy has not been implemented.

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.

Background:IVS-II-5 G>C (HBB: c.315+5 G>C) is a rare β-thalassemia mutation. However, there is no clear evidence regarding the effect of this defect or co-inheritance of other β-thalassemia mutations on phenotypes.Methods:The clinical phenotypes associated with compound heterozygosity for the IVS-II-5 G>C mutation and other β-thalassemia mutations, together with the genetic modifiers' potential effect of the genetic modifiers α-thalassemia, were studied in 13 patients. In addition, analyses of red cell indices, hemoglobin component, iron status, and α-globin genes were carried out in 19 heterozygotes.Results:Next-generation sequencing of 24 undiagnosed patients with transfusion-dependent thalassemia (TDT) or non-transfusion-dependent thalassemia (NTDT) identified 13 carriers of the IVS-II-5 G>C mutation. There was a wide spectrum of phenotypic severity in compound heterozygotes and 6 (46.2%) of 13 were transfusion dependent. Analysis of 19 heterozygotes indicated that most were hematologically normal without appreciable microcytosis or hypochromia, and approximately half had normal hemoglobin A2 levels at the same time.Conclusion:Compound heterozygotes for IVS-II-5 G>C and other severe β-thalassemia mutations are phenotypically severe enough to necessitate appropriate therapy and counselling. Co-inheritance of this nucleotide substitution with other β-thalassemia mutations may account for a considerable portion of the incidence of undiagnosed patients with NTDT and TDT in Guangxi. Therefore, the IVS-II-5 G>C mutation can pose serious difficulties in screening and counselling.