A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells
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.
Authors
- Bernhard Gentner (ORCID: https://orcid.org/0000-0001-6024-4718)
- Edoardo Carsana
- Chiara Brandas (ORCID: https://orcid.org/0000-0003-1548-6720)
- Raffaella Di Micco (ORCID: https://orcid.org/0000-0002-0639-8238)
- Teresa Tavella
- Samuele Ferrari (ORCID: https://orcid.org/0000-0003-1122-1231)
- Ivan Merelli (ORCID: https://orcid.org/0000-0003-3587-3680)
- Kety Giannetti (ORCID: https://orcid.org/0000-0001-7989-2520)
- Matteo Maria Naldini (ORCID: https://orcid.org/0000-0002-9542-2884)
- Angelica Varesi (ORCID: https://orcid.org/0000-0002-1246-2320)
- Matteo Barcella (ORCID: https://orcid.org/0000-0002-2353-3999)
- Anastasia Conti (ORCID: https://orcid.org/0000-0002-8670-2649)
- Antonella Santoro (ORCID: https://orcid.org/0000-0002-1012-888X)
- Federico Midena
- Elena Lo Furno (ORCID: https://orcid.org/0000-0003-1414-0154)
- Simona Porcellini (ORCID: https://orcid.org/0009-0002-6179-6287)
- Luigi Naldini (ORCID: https://orcid.org/0000-0002-7835-527X)
- Lucrezia della Volpe (ORCID: https://orcid.org/0000-0002-9087-1293)
- Anna Villa (ORCID: https://orcid.org/0000-0003-4428-9013)
- Stefano Beretta (ORCID: https://orcid.org/0000-0003-4375-004X)
- Diego Gilioli (ORCID: https://orcid.org/0000-0003-0021-1759)
- Laura Alessandrini
- Roberta Vacca
- Giacomo Farina
Institutions
- The San Raffaele Telethon Institute for Gene Therapy (IT)
- Istituti di Ricovero e Cura a Carattere Scientifico (IT)
- Institute of Biomedical Technologies (IT)
- National Research Council (IT)
- Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele (IT)
Publication Details
- Journal
- Cell Reports Medicine
- Published
- 2024-11-01
- DOI
- https://doi.org/10.1016/j.xcrm.2024.101823
- Citations
- 17
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 3.97
Funders
- Fondazione Telethon
- Associazione Italiana per la Ricerca sul Cancro
- HORIZON EUROPE Framework Programme