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Peer-reviewedCC BY 4.0Proceedings of the National Academy of Sciences of the United States of America

Transient ATM inhibition enhances knock-in efficiency in hematopoietic stem cells by attenuating the DNA damage response

  • Munkh-Erdene Natsagdorj1
  • Hiromasa Hara1,2,3,4
  • Kohei Iida1
  • Yuji Kashiwakura5
  • Nemekhbayar Baatartsogt5
  • Enkhzaya Ganzorig1
  • Daniele Canarutto6,7
  • Sandra Pinho8
  • Luigi Naldini6,7
  • Tsukasa Ohmori5
  • Yutaka Hanazono1,2,9
  • Fumio Nakahara1
9 affiliations
  1. 1Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Shimotsuke 329-0498, Tochigi, Japan
  2. 2Laboratory of Regenerative and Cellular Medicine, Center for Development of Advanced Medical Technology, Jichi Medical University, Shimotsuke 329-0498, Tochigi, Japan
  3. 3Division of Animal Resource Development, Center for Development of Advanced Medical Technology, Jichi Medical University, Shimotsuke 329-0498, Tochigi, Japan
  4. 4Center for Experimental Medicine, Jichi Medical University, Shimotsuke 329-0498, Tochigi, Japan
  5. 5Department of Biochemistry, School of Medicine, Jichi Medical University, Shimotsuke 329-0498, Tochigi, Japan
  6. 6San Raffaele Telethon Institute for Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele Scientific Institute, Milan 20132, Italy
  7. 7Faculty of Medicine and Surgery, Faculty of Medicine and Surgery, Vita-Salute San Raffaele University, Milan 20132, Italy
  8. 8Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago, Chicago, IL 60612
  9. 9Translational Research Laboratory, Center for Development of Advanced Medical Technology, Jichi Medical University, Shimotsuke 329-0498, Tochigi, Japan
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Abstract

Precise genome editing in hematopoietic stem cells (HSCs) holds great potential for treating inherited blood disorders; however, low knock-in (KI) efficiency and loss of functional HSCs after genome editing remain major obstacles. Here, we demonstrate that transient inhibition of Ataxia-Telangiectasia Mutated (ATM) kinase markedly enhanced KI efficiency in mouse HSCs genome-edited with Cas9/ribonucleoprotein (RNP) and AAV donor DNA. Phosphoproteomic and capillary western blot analyses revealed that ATM inhibition suppressed aberrant Cas9-AAV-induced ATM activation and the subsequent DNA damage response, reduced p53-dependent apoptosis, and enhanced survival of KI HSCs. In serial transplantation assays, ATM inhibition preserved long-term engrafting KI HSCs, increasing their frequency from ~0.3 to 40% in secondary recipients, an over 100-fold improvement. Furthermore, in an X-linked severe combined immunodeficient mouse model, ATM inhibition increased KI efficiency and restored T- and B cell differentiation. Comprehensive safety analyses, including long-read nanopore sequencing, amplicon deep sequencing, whole genome sequencing and clonal tracking, did not detect increased genomic alterations, chromosomal abnormalities, or clonal expansion following transient ATM inhibition. Taken together, these findings establish transient ATM inhibition as an effective strategy to enhance KI-mediated genome editing while maintaining the long-term repopulating capacity of HSCs.

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Cite

BibTeXRIS
BibTeX
@article{natsagdorj2026transient,
  title = {{Transient ATM inhibition enhances knock-in efficiency in hematopoietic stem cells by attenuating the DNA damage response}},
  author = {Munkh-Erdene Natsagdorj and Hiromasa Hara and Kohei Iida and Yuji Kashiwakura and Nemekhbayar Baatartsogt and Enkhzaya Ganzorig and Daniele Canarutto and Sandra Pinho and Luigi Naldini and Tsukasa Ohmori and Yutaka Hanazono and Fumio Nakahara},
  journal = {Proceedings of the National Academy of Sciences of the United States of America},
  year = {2026},
  month = oct,
  doi = {10.1073/pnas.2531364123},
  url = {https://doi.org/10.1073/pnas.2531364123},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Transient ATM inhibition enhances knock-in efficiency in hematopoietic stem cells by attenuating the DNA damage response
AU  - Munkh-Erdene Natsagdorj
AU  - Hiromasa Hara
AU  - Kohei Iida
AU  - Yuji Kashiwakura
AU  - Nemekhbayar Baatartsogt
AU  - Enkhzaya Ganzorig
AU  - Daniele Canarutto
AU  - Sandra Pinho
AU  - Luigi Naldini
AU  - Tsukasa Ohmori
AU  - Yutaka Hanazono
AU  - Fumio Nakahara
PY  - 2026
DA  - 2026/10/20
JO  - Proceedings of the National Academy of Sciences of the United States of America
DO  - 10.1073/pnas.2531364123
UR  - https://doi.org/10.1073/pnas.2531364123
AB  - Precise genome editing in hematopoietic stem cells (HSCs) holds great potential for treating inherited blood disorders; however, low knock-in (KI) efficiency and loss of functional HSCs after genome editing remain major obstacles. Here, we demonstrate that transient inhibition of Ataxia-Telangiectasia Mutated (ATM) kinase markedly enhanced KI efficiency in mouse HSCs genome-edited with Cas9/ribonucleoprotein (RNP) and AAV donor DNA. Phosphoproteomic and capillary western blot analyses revealed that ATM inhibition suppressed aberrant Cas9-AAV-induced ATM activation and the subsequent DNA damage response, reduced p53-dependent apoptosis, and enhanced survival of KI HSCs. In serial transplantation assays, ATM inhibition preserved long-term engrafting KI HSCs, increasing their frequency from ~0.3 to 40% in secondary recipients, an over 100-fold improvement. Furthermore, in an X-linked severe combined immunodeficient mouse model, ATM inhibition increased KI efficiency and restored T- and B cell differentiation. Comprehensive safety analyses, including long-read nanopore sequencing, amplicon deep sequencing, whole genome sequencing and clonal tracking, did not detect increased genomic alterations, chromosomal abnormalities, or clonal expansion following transient ATM inhibition. Taken together, these findings establish transient ATM inhibition as an effective strategy to enhance KI-mediated genome editing while maintaining the long-term repopulating capacity of HSCs.
SN  - 0027-8424
SN  - 1091-6490
ER  -