A NOVEL BASE-EDITING STRATEGY FOR THERAPEUTIC FETAL HEMOGLOBIN REACTIVATION IN SICKLE CELL DISEASE

Sickle cell disease (SCD) is a common monogenic disorder caused by a point mutation in the HBB gene, leading to the formation of sickle hemoglobin (HbS). Under low oxygen, HbS polymerizes, distorting red blood cells and triggering hemolysis, inflammation, and vaso-occlusion. Although allogeneic hematopoietic stem cell transplantation offers a cure, its use is limited by donor compatibility and transplant risks. Gene editing strategies that reactivate fetal hemoglobin (HbF) have emerged as promising alternatives. Among them, adenine base editors (ABEs) enable precise A-to-G conversions without inducing double-stranded breaks, lowering genotoxic risks. This project focuses on developing an ex vivo ABE-based approach to upregulate HbF in human hematopoietic stem and progenitor cells (HSPCs), mimicking naturally occurring non-deletional hereditary persistence of fetal hemoglobin (HPFH). Target sites include the HBG1/2 promoter region (−113 to −198) and the +55 and +58 erythroid enhancers of BCL11A. We have identified HBG-123 and HBG-175 as top candidates, based on editing efficiency and HbF induction (via NGS and F-cell analysis) in CD34⁺ HSPCs. Editing efficiencies averaged 66.8% and 49.4%, resulting in HbF levels of 23.3% and 41.2%, respectively, with maintained erythroid differentiation and minimal indel formation (<1%). Further optimizations—adjusting gRNA:ABE ratios, electroporation timing, and using engineered Cas9/TadA with custom UTRs—boosted editing efficiency and HbF expression without impairing HSPC clonogenicity. Functional evaluation in single- cell colony assays with stromal co-culture, which enhances erythroid output and hemoglobinization compared to methylcellulose, enabled parallel assessment of editing and HbF expression by HPLC. Results showed strong correlation between A-to-G conversion and HbF for HBG-175 (r = 0.9), while HBG-123 clones, though highly edited (∼90%), showed more variability and lower HbF. Together, these findings establish a scalable and efficient base editing platform for HbF induction in HSPCs. We are currently performing in vivo validation and already started the development of a GMP-compliant production process. Implementing this technology in Brazil, where SCD is highly prevalent, will support local capacity-building and broaden access to advanced gene-editing therapies.