Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. Unintended on-target outcomes of double-strand break (DSB) repair in hematopoietic stem and progenitor cells (HSPCs), such as long deletions and centromere-distal chromosome fragment loss, are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing quiescent HSPCs bypasses long deletion and micronuclei formation and preserves efficient on-target editing and engraftment function.
Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells.
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作者:Zeng Jing, Nguyen My Anh, Liu Pengpeng, da Silva Lucas Ferreira, Levesque Sébastien, Lin Linda Y, Justus David G, Petri Karl, Clement Kendell, Porter Shaina N, Verma Archana, Neri Nola R, Rosanwo Tolulope, Ciuculescu Marioara-Felicia, Abriss Daniela, Mintzer Esther, Maitland Stacy A, Demirci Selami, Cha Hye Ji, Orkin Stuart H, Tisdale John F, Williams David A, Zhu Lihua Julie, Pruett-Miller Shondra M, Pinello Luca, Joung J Keith, Pattanayak Vikram, Manis John P, Armant Myriam, Pellin Danilo, Brendel Christian, Wolfe Scot A, Bauer Daniel E
期刊: | Cell Stem Cell | 影响因子: | 20.400 |
时间: | 2025 | 起止号: | 2025 Feb 6; 32(2):191-208 |
doi: | 10.1016/j.stem.2024.11.001 |
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