S271: an updated follow-up of brl-101, crispr-cas9-mediated gene editing of the bcl11a enhancer for transfusion-dependent beta-thalasse

Background: β-Thalassemia is an inherited hemolytic disease that is prevalent worldwide. Over 200 mutations in the HBB gene, which encodes adult hemoglobin (HbA), result in β-thalassemia. Hereditary persistence of fetal hemoglobin (HbF) can alleviate the symptoms of anemia. CRISPR-Cas9-mediated disruption of the BCL11A erythroid enhancer results in the reduction of BCL11A expression and the induction of fetal γ-globin, which is a practicable therapeutic strategy for treating transfusion-dependent β-thalassemia (TDT). Aims: We initiated a clinical trial to evaluate the safety and efficacy of BRL-101, a gene editing autologous HSPCs product, in the treatment of TDT, including the most severe β0/β0 patients lacking β-globin chain expression. Methods: We conducted a nonrandomized, single-dose, open-label clinical trial of BRL-101 in pediatric patients with TDT at Xiangya Hospital (NCT04211480) and 923rd Hospital (NCTO4205435). The inclusion criteria included age 5-15 years, clinically diagnosed as β-thalassemia major, phenotypes including β0/β0, β+/β0, βE/β0 genotype, and subject’s body condition eligible for autologous stem cell transplantation. CD34+ HSPCs were edited with CRISPR-Cas9 RNP at the +58 erythroid specific enhancer region of the BCL11A gene. Patients were monitored for routine blood test, editing frequency, total Hb, Hb fractions on HPLC, F-cell percentage (circulating erythrocytes with detectable levels of HbF) and AEs. Results: All 6 patients, including 4 β0/β0 phenotype patients, received BRL-101 treatment and successfully achieved transfusion independence. The neutrophil engraftment occurred within 1 month for all patients, the platelet engraftment and last pRBC transfusions occurred within 2 months for two-thirds of patients (table 1). The total Hb levels began to increase steadily until they reached healthy levels, and HbF levels were elevated sustainedly (figure 1). F-cell expression increased to more than 90% within 6 months (figure 2). The CRISPR-Cas9-edited HSPCs engrafted and differentiated into multiple lineages that retained the gene editing. Editing frequency of PBMCs from patients increased to more than 60% during the follow-up periods (figure 3). No drug-related AEs leaded to study withdrawal or death of patients during treatment. The majority of AEs were consistent with that of mobilization, apheresis, myeloablation, and autologous hematopoietic stem cell transplantation. All of these AEs have been resolved. Four SAEs were reported during treatment, including left cheek soft tissue infection, acute upper respiratory infection, infective fever and thrombocytopenia. Only thrombocytopenia was attributed to BRL-101 and was gradually relieved. Summary/Conclusion: All 6 patients with different genotypes achieved transfusion independence, including the patients with the most severe β0/β0 phenotype. After BRL-101 infusion, the levels of total Hb and HbF increased significantly, and gene editing is retained in multiple lineages of HSPCs. The safety profile of BRL-101 is generally consistent with mobilization, apheresis, myeloablation and autologous hematopoietic stem cell transplantation. This study demonstrate that BRL-101 is a safe, potential functional cure for the treatment of TDT.Keywords: Thalassemia, Hematopoietic stem and progenitor cells, Human CD34+ cell