Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells

Jing Zeng; My Anh Nguyen; Pengpeng Liu; Lucas Ferreira da Silva; Sébastien Levesque; Linda Y Lin; David G Justus; Karl Petri; Kendell Clement; Shaina N Porter; Archana Verma; Nola R Neri; Tolulope Rosanwo; Marioara-Felicia Ciuculescu; Daniela Abriss; Esther Mintzer; Stacy A Maitland; Selami Demirci; Hye Ji Cha; Stuart H Orkin; John F Tisdale; David A Williams; Lihua Julie Zhu; Shondra M Pruett-Miller; Luca Pinello; J Keith Joung; Vikram Pattanayak; John P Manis; Myriam Armant; Danilo Pellin; Christian Brendel; Scot A Wolfe; Daniel E Bauer

doi:10.1016/j.stem.2024.11.001

Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells

Cell Stem Cell. 2024 Dec 11:S1934-5909(24)00400-4. doi: 10.1016/j.stem.2024.11.001. Online ahead of print.

Authors

Jing Zeng¹, My Anh Nguyen¹, Pengpeng Liu², Lucas Ferreira da Silva³, Sébastien Levesque¹, Linda Y Lin¹, David G Justus⁴, Karl Petri⁵, Kendell Clement⁵, Shaina N Porter⁶, Archana Verma¹, Nola R Neri¹, Tolulope Rosanwo¹, Marioara-Felicia Ciuculescu⁷, Daniela Abriss⁷, Esther Mintzer², Stacy A Maitland², Selami Demirci⁸, Hye Ji Cha¹, Stuart H Orkin¹, John F Tisdale⁸, David A Williams¹, Lihua Julie Zhu⁹, Shondra M Pruett-Miller⁶, Luca Pinello⁵, J Keith Joung⁵, Vikram Pattanayak⁵, John P Manis¹, Myriam Armant⁷, Danilo Pellin¹, Christian Brendel¹, Scot A Wolfe², Daniel E Bauer¹⁰

Affiliations

¹ Division 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.
² Department of Molecular, Cell and Cancer Biology, Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
³ Division 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; Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Boston, MA 02129, USA; Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.
⁴ Department of Pathology, Harvard Medical School, Boston, MA 02115, USA; Program in Transfusion Medicine, Boston Children's Hospital, Boston, MA 02115, USA.
⁵ Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Boston, MA 02129, USA; Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.
⁶ Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
⁷ TransLab, Boston Children's Hospital, Boston, MA 02115, USA.
⁸ Cellular and Molecular Therapeutics Branch, National Heart Lung and Blood Institutes (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA.
⁹ Department of Molecular, Cell and Cancer Biology, Department of Molecular Medicine, Program in Bioinformatics and Integrative Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
¹⁰ Division 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. Electronic address: [email protected].

PMID: 39672163
DOI: 10.1016/j.stem.2024.11.001

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.

Keywords: BCL11A; enhancers; fetal hemoglobin; genotoxicity; hematopoietic stem cells; sickle cell disease; therapeutic gene editing.