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

Abstract

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.