CRISPR-Cas12a gene editing of the HBG1/2 promoters leads to sustained normalization of total hemoglobin and increased fetal hemoglobin in patients with severe sickle cell disease: Updated Results from the RUBY trial

Abstract

Abstract Introduction: Elevated levels of fetal hemoglobin (HbF, α2γ2) can attenuate or eliminate symptoms in sickle cell disease (SCD), including painful vaso-occlusive events (VOEs). Renizgamglogene autogedtemcel (reni-cel) is an investigational gene-edited autologous hematopoietic stem cell therapy designed to induce sustained production of HbF for the treatment of SCD by reactivating y-globin gene (HBG1/2) expression. Reni-cel is generated by editing cluster of differentiation 34+ (CD34+) cells at the BCL11A transcriptional repressor binding site in the HBG1/2 promoters using a highly specific gene-editing nuclease, Acidaminococcus sp. CRISPR-associated protein 12a (AsCas12a). This genomic modification replicates naturally occurring mutations associated with hereditary persistence of HbF that cause lifelong elevated HbF and are known to alleviate symptoms of β-hemoglobinopathies. In preclinical studies, editing this genomic region in CD34+ cells from patients with SCD led to ≥80% editing, robust HbF production, and a significant reduction in sickling of erythroid progeny. The RUBY trial (NCT04853576, now closed) was a Phase I/II, multicenter, open-label, single-arm study evaluating the safety, tolerability, and efficacy of reni-cel in patients with severe SCD. Here, we report results with extended follow-up and greater patient accrual; the final and full cohort analysis will be presented. Methods: Eligible patients were 12–50 years old and diagnosed with severe SCD (defined as ≥2 severe VOEs/year in the 2 years prior to informed consent). After plerixafor mobilization, autologous CD34+ hematopoietic stem and progenitor cells were collected from each patient by apheresis. Reni-cel was generated from each patient's pooled cells by gene editing the HBG1/2 promoters using an engineered variant of AsCas12a. Patients then received myeloablative conditioning with pharmacokinetically adjusted busulfan before a single infusion of reni-cel (≥3×106 CD34+ cells/kg). Patients were monitored for engraftment, on-target allelic editing levels, VOEs, total hemoglobin (Hb), HbF production, percentage of F-cells, mean HbF concentration/F-cell (MCH-F/F-cell), markers of hemolysis, and adverse events (AEs) for 24 months. Results: As of February 24, 2025, 32 patients received reni-cel. Median (range) age was 25.5 (12–41) years, 56.3% were female, 96.9% had the βS/βS genotype, and 96.9% were Black or African American. After reni-cel infusion, median (range) follow-up time was 13.2 (0.9–26.9) months, with 18 patients having >12 months of follow-up. Neutrophil engraftment was achieved after a median (range) of 21.0 (14–29) days, and platelet engraftment after a median (range) of 25.0 (16–51) days (n=29). Patients achieved rapid and durable normalization of total Hb; mean (standard deviation [SD]) total Hb was 13.8 (1.8) g/dL at Month 6 (n=23) and was maintained throughout the follow-up period. Mean (SD) percentage of HbF was 47.6% (3.5%) by Month 6 (n=24) and remained at >40% through last follow-up. Percentage of F-cells and MCH-F/F-cell also increased early. Mean (SD) percentage of F-cells increased to 99.4% (0.8%) at Month 6 and was maintained above 98% through last follow-up. Mean MCH-F/F-cell was sustained above the anti-sickling threshold of 10 pg/F-cell through last follow-up. Key markers of hemolysis, including absolute reticulocyte count, indirect bilirubin, lactate dehydrogenase, and haptoglobin, improved or normalized by Month 6 and were largely maintained through last follow-up. As of the data cutoff date, all but one patient (96.9%) were VOE-free after reni-cel infusion, compared with a mean (SD) of 4.9 (2.9) severe VOEs/year in the 2 years prior to enrollment. Levels of on-target allelic editing were high and remained stable in both peripheral blood nucleated cells and bone marrow–derived CD34+ cells, with mean (SD) editing levels of 75.8% (9.2%) and 87.8% (3.4%) observed at Month 12, respectively. The safety profile of reni-cel was consistent with busulfan. Two serious AEs related to reni-cel were reported.Conclusions: Data from the RUBY trial continue to demonstrate early and sustained normalization of total Hb and durable increases in HbF following reni-cel infusion, with 31 of 32 patients VOE-free at the time of this data cut. These updated findings, derived from a larger patient cohort and extended follow-up period, validate this novel gene-editing approach as a potential one-time treatment for patients with severe SCD.