2. Academic Validation
  3. A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells

A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells

  • Cell Rep Med. 2024 Nov 19;5(11):101823. doi: 10.1016/j.xcrm.2024.101823.
Lucrezia Della Volpe 1,  Federico Midena 1,  Roberta Vacca 1,  Teresa Tavella 2,  Laura Alessandrini 1,  Giacomo Farina 3,  Chiara Brandas 2,  Elena Lo Furno 2,  Kety Giannetti 2,  Edoardo Carsana 2,  Matteo M Naldini 1,  Matteo Barcella 2,  Samuele Ferrari 2,  Stefano Beretta 2,  Antonella Santoro 2,  Simona Porcellini 2,  Angelica Varesi 2,  Diego Gilioli 1,  Anastasia Conti 2,  Ivan Merelli 4,  Bernhard Gentner 5,  Anna Villa 6,  Luigi Naldini 1,  Raffaella Di Micco 7
Affiliations

Affiliations

  • 1 San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy; Vita-Salute San Raffaele University, 20132 Milan, Italy.
  • 2 San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.
  • 3 San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy; University of Milan-Bicocca, 20126 Milan, Italy.
  • 4 National Research Council, Institute for Biomedical Technologies, 20054 Segrate, Italy.
  • 5 San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy; Ludwig Institute for Cancer Research and Department of Oncology, University of Lausanne (UNIL) and Lausanne University Hospital (CHUV), 1066 Lausanne, Switzerland.
  • 6 San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy; National Research Council, Institute for Biomedical Technologies, 20054 Segrate, Italy.
  • 7 San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy; University School of Advanced Studies IUSS, 27100 Pavia, Italy. Electronic address: dimicco.raffaella@hsr.it.
PMID: 39536752 DOI: 10.1016/j.xcrm.2024.101823
Abstract

Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here, we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks, enhancing the reconstitution capacity of edited HSPCs. However, this results in lower HDR efficiency, rendering ex vivo culture necessary yet detrimental. Mechanistically, ex vivo activation triggers a multi-step process initiated by p38 mitogen-activated protein kinase (MAPK) phosphorylation, which generates mitogenic Reactive Oxygen Species (ROS), promoting fast cell-cycle progression and subsequent proliferation-induced DNA damage. Thus, p38 inhibition before gene editing delays G1/S transition and expands transcriptionally defined HSCs, ultimately endowing edited cells with superior multi-lineage differentiation, persistence throughout serial transplantation, enhanced polyclonal repertoire, and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications.

Keywords

CRISPR-Cas9; DNA damage; DNA damage response; cell cycle; clonal output; differentiation; gene editing; hematopoietic stem cells; p38 MAPK-ROS; proliferative stress; single-cell analyses.

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