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Updated: Jun 5, 2025
Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells
Published on: March 10, 2023
Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells
Jing Zeng1, My Anh Nguyen1, Pengpeng Liu2
1Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Harvard Stem Cell Institute, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
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.
