Abstract
Base editing offers a powerful platform for precise, scarless genome modification without introducing DNA double-strand breaks. Here, we develop and apply a multiplex base editing strategy in human hematopoietic stem and progenitor cells (HSPCs) to enhance erythroid output and fetal hemoglobin (HbF) expression for the treatment of sickle cell disease (SCD). Building on insights from benign erythrocytosis, we first developed a cytosine base editor (CBE)-mediated strategy to introduce a naturally occurring EPOR truncation (tEPOR) found in a Finnish Olympic cross-country skier, which confers erythropoietin hypersensitivity. This single-nucleotide edit significantly increased erythroid proliferation without impairing viability, lineage potential, or terminal differentiation of primary human HSPCs. When introduced into wild-type HSPCs, the tEPOR allele drove a 4.6-fold increase (P<0.0001) in red blood cell production over the course of in vitro erythroid differentiation, demonstrating its potential utility for both therapeutic and ex vivo manufacturing applications.
We next evaluated whether tEPOR could be combined with therapeutic HbF-inducing edits, including CBE editing of the BCL11A erythroid enhancer and the HBG1/2 promoter. After systematic optimization of editing conditions, we achieved efficient multiplex base editing (ranging from 54-87% edited alleles) at all three target loci in SCD unmobilized peripheral blood-derived HSPCs, with no detectable loss of viability or impairment in erythroid differentiation. Notably, the triple-edited cells expressed significantly more HbF (84.6% HbF of total hemoglobins) compared to unedited cells (7.9% HbF) as well as those edited using a CBE at BCL11A (40.6% HbF) or HBG alone (59.5% HbF) and exceeded HbF levels seen in cells edited with the FDA-approved CRISPR-based therapy Casgevy (34.5% HbF). Moreover, we identified multiple combinations of edits that outperformed both Casgevy and other clinical-stage editing strategies in vitro.
These findings establish a modular framework for multiplex base editing in primary HSPCs and demonstrate that introduction of a benign erythrocytosis allele can enhance therapeutic genome editing outcomes in SCD. Our results suggest that augmenting erythropoiesis genetically may lower the threshold of edited cell engraftment required for clinical benefit, potentially reducing or eliminating the need for toxic conditioning in ex vivo therapies. Importantly, RNA-seq analyses of edited cells revealed no transcriptomic signs of genotoxicity or off-target stress beyond the biological effects of each individual edit, supporting the safety and modularity of this strategy.
Together, this work defines a new class of combinatorial genome editing strategies that pair disease-corrective and cell output-enhancing variants to improve therapeutic potency and broaden the applicability of genome editing. Future applications may include incorporating immune evasion, epitope shielding, or antigen deletion into a single multiplex editing payload to generate functionally enhanced, transplantation-ready cell therapies.
