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

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: bauer@bloodgroup.tch.harvard.edu.

PMID: 39672163
PMCID: PMC11805672 (available on 2026-02-06)
DOI: 10.1016/j.stem.2024.11.001

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

Jing Zeng et al. Cell Stem Cell. 2025.

. 2025 Feb 6;32(2):191-208.e11.

doi: 10.1016/j.stem.2024.11.001. Epub 2024 Dec 12.

Authors

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: bauer@bloodgroup.tch.harvard.edu.

PMID: 39672163
PMCID: PMC11805672 (available on 2026-02-06)
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.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests J.Z., S.H.O., and D.E.B. are inventors of patents related to BCL11A enhancer therapeutic gene editing. S.A.W. is a consultant for Editas Medicine. L.P. has financial interests in Edilytics, Excelsior Genomics, and SeQure Dx. K.P. has a financial interest in SeQure. J.K.J. and V.P. are co-founders of and have a financial interest in SeQure, Dx, Inc., a company developing technologies for gene editing target profiling. J.K.J. also has, or had during the course of this research, financial interests in several companies developing gene editing technology: Beam Therapeutics, Blink Therapeutics, Chroma Medicine, Editas Medicine, EpiLogic Therapeutics, Excelsior Genomics, Hera Biolabs, Monitor Biotechnologies, Nvelop Therapeutics (f/k/a ETx, Inc.), Pairwise Plants, Poseida Therapeutics, and Verve Therapeutics. K.P., L.P., J.K.J., and V.P.’s interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies. J.K.J. is a co-inventor on various patents and patent applications that describe gene editing and epigenetic editing technologies.

Update of

Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells.
Zeng J, Nguyen MA, Liu P, Ferreira da Silva L, Lin LY, Justus DG, Petri K, Clement K, Porter SN, Verma A, Neri NR, Rosanwo T, Ciuculescu MF, Abriss D, Mintzer E, Maitland SA, Demirci S, Tisdale JF, Williams DA, Zhu LJ, Pruett-Miller SM, Pinello L, Joung JK, Pattanayak V, Manis JP, Armant M, Pellin D, Brendel C, Wolfe SA, Bauer DE. Zeng J, et al. bioRxiv [Preprint]. 2023 May 27:2023.05.27.542323. doi: 10.1101/2023.05.27.542323. bioRxiv. 2023. Update in: Cell Stem Cell. 2025 Feb 06;32(2):191-208.e11. doi: 10.1016/j.stem.2024.11.001. PMID: 37292647 Free PMC article. Updated. Preprint.

References

1. Piel FB, Steinberg MH, and Rees DC (2017). Sickle Cell Disease. N. Engl. J. Med 377, 305. - PubMed
1. Motta I, Ghiaccio V, Cosentino A, and Breda L (2019). Curing Hemoglobinopathies: Challenges and Advances of Conventional and New Gene Therapy Approaches. Mediterr. J. Hematol. Infect. Dis 11, e2019067. - PMC - PubMed
1. Lettre G, and Bauer DE (2016). Fetal haemoglobin in sickle-cell disease: from genetic epidemiology to new therapeutic strategies. Lancet 387, 2554–2564. - PubMed
1. Belisário AR, Sales RR, Silva CM, Velloso-Rodrigues C, and Viana MB (2016). The Natural History of Hb S/Hereditary Persistence of Fetal Hemoglobin in 13 Children from the State of Minas Gerais, Brazil. Hemoglobin 40, 215–219. - PubMed
1. Talbot JF, Bird AC, and Serjeant GR (1983). Retinal changes in sickle cell/hereditary persistence of fetal haemoglobin syndrome. Br. J. Ophthalmol 67, 777–778. - PMC - PubMed

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

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

Affiliations

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

Authors

Affiliations

Abstract

Conflict of interest statement

Update of

References

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Medical