Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs

Wenyan Han¹, Hou-Yuan Qiu², Shangwu Sun³, Zhi-Can Fu⁴, Guo-Quan Wang², Xiaowen Qian⁵, Lijie Wang⁶, Xiaowen Zhai⁵, Jia Wei⁷, Yichuan Wang⁶, Yi-Lin Guo⁷, Guo-Hua Cao², Rui-Jin Ji², Yi-Zhou Zhang², Hongxia Ma⁶, Hongsheng Wang⁵, Mingli Zhao¹, Jing Wu¹, Lili Bi², Qiu-Bing Chen², Zifeng Li⁵, Ling Yu⁵, Xiaodun Mou⁶, Hao Yin⁸, Li Yang⁹, Jia Chen¹⁰, Bei Yang¹¹, Ying Zhang¹²

Affiliations

¹ Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
² Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China.
³ Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China.
⁴ Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China; Center for Molecular Medicine, Children's Hospital of Fudan University and Shanghai Key Laboratory of Medical Epigenetics, International Laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China.
⁵ Department of Hematology and Oncology, Children's Hospital of Fudan University, Shanghai 201102, China.
⁶ CorrectSequence Therapeutics, Shanghai 201210, China.
⁷ Center for Molecular Medicine, Children's Hospital of Fudan University and Shanghai Key Laboratory of Medical Epigenetics, International Laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China.
⁸ Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China; Department of Pathology and Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China; TaiKang Centre for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan, China; State Key Laboratory of Virology, Wuhan University, Wuhan, China.
⁹ Center for Molecular Medicine, Children's Hospital of Fudan University and Shanghai Key Laboratory of Medical Epigenetics, International Laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China. Electronic address: liyang_fudan@fudan.edu.cn.
¹⁰ Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; Shanghai Clinical Research and Trial Center, Shanghai 201210, China; Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine, ShanghaiTech University, Shanghai 200031, China. Electronic address: chenjia@shanghaitech.edu.cn.
¹¹ Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; Shanghai Clinical Research and Trial Center, Shanghai 201210, China; Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine, ShanghaiTech University, Shanghai 200031, China. Electronic address: yangbei@shanghaitech.edu.cn.
¹² Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China; State Key Laboratory of Virology, Wuhan University, Wuhan, China. Electronic address: ying.zhang84@whu.edu.cn.

PMID: 37989316
DOI: 10.1016/j.stem.2023.10.007

Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs

Wenyan Han et al. Cell Stem Cell. 2023.

. 2023 Dec 7;30(12):1624-1639.e8.

doi: 10.1016/j.stem.2023.10.007. Epub 2023 Nov 20.

Authors

Affiliations

¹ Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
² Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China.
³ Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China.
⁴ Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China; Center for Molecular Medicine, Children's Hospital of Fudan University and Shanghai Key Laboratory of Medical Epigenetics, International Laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China.
⁵ Department of Hematology and Oncology, Children's Hospital of Fudan University, Shanghai 201102, China.
⁶ CorrectSequence Therapeutics, Shanghai 201210, China.
⁷ Center for Molecular Medicine, Children's Hospital of Fudan University and Shanghai Key Laboratory of Medical Epigenetics, International Laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China.
⁸ Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China; Department of Pathology and Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China; TaiKang Centre for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan, China; State Key Laboratory of Virology, Wuhan University, Wuhan, China.
⁹ Center for Molecular Medicine, Children's Hospital of Fudan University and Shanghai Key Laboratory of Medical Epigenetics, International Laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China. Electronic address: liyang_fudan@fudan.edu.cn.
¹⁰ Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; Shanghai Clinical Research and Trial Center, Shanghai 201210, China; Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine, ShanghaiTech University, Shanghai 200031, China. Electronic address: chenjia@shanghaitech.edu.cn.
¹¹ Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; Shanghai Clinical Research and Trial Center, Shanghai 201210, China; Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine, ShanghaiTech University, Shanghai 200031, China. Electronic address: yangbei@shanghaitech.edu.cn.
¹² Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China; State Key Laboratory of Virology, Wuhan University, Wuhan, China. Electronic address: ying.zhang84@whu.edu.cn.

PMID: 37989316
DOI: 10.1016/j.stem.2023.10.007

Abstract

Reactivating silenced γ-globin expression through the disruption of repressive regulatory domains offers a therapeutic strategy for treating β-hemoglobinopathies. Here, we used transformer base editor (tBE), a recently developed cytosine base editor with no detectable off-target mutations, to disrupt transcription-factor-binding motifs in hematopoietic stem cells. By performing functional screening of six motifs with tBE, we found that directly disrupting the BCL11A-binding motif in HBG1/2 promoters triggered the highest γ-globin expression. Via a side-by-side comparison with other clinical and preclinical strategies using Cas9 nuclease or conventional BEs (ABE8e and hA3A-BE3), we found that tBE-mediated disruption of the BCL11A-binding motif at the HBG1/2 promoters triggered the highest fetal hemoglobin in healthy and β-thalassemia patient hematopoietic stem/progenitor cells while exhibiting no detectable DNA or RNA off-target mutations. Durable therapeutic editing by tBE persisted in repopulating hematopoietic stem cells, demonstrating that tBE-mediated editing in HBG1/2 promoters is a safe and effective strategy for treating β-hemoglobinopathies.

Keywords: BCL11A binding site; HBG 1/2 promoter; hematopoietic stem cell; off-target mutation; transformer base editor; β-hemoglobinopathies; γ-globin reactivation.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests J.C., L. Yang, B.Y., and H.Y. are the scientific cofounders of CorrectSequence Therapeutics, a company that uses gene-editing technologies. X.M., L.W., Y.W., and H.M. are the employees of CorrectSequence Therapeutics. J.C., B.Y., L. Yang, W.H., S.S., and Y.Z. filed a provisional patent application related to this work.

Publication types

Actions

MeSH terms

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

Substances

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

Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs

Affiliations

Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs

Authors

Affiliations

Abstract

Conflict of interest statement

Publication types

MeSH terms

Substances