citations

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

Authors:

Della Volpe L, Midena F, Vacca R, Tavella T, Alessandrini L, Farina G, Brandas C, Lo Furno E, Giannetti K, Carsana E, Naldini MM, Barcella M, Ferrari S, Beretta S, Santoro A, Porcellini S, Varesi A, Gilioli D, Conti A, Merelli I, Gentner B, Villa A, Naldini L, Di Micco R.

In:

Source: Cell Rep Med
Publication Date: (2024)
Issue: 5(11): 101823

Research Area:

Cancer Research/Cell Biology

Immunotherapy / Hematology

Gene Expression

Basic Research

Molecular Biology

Regenerative medicine

Drug Discovery

Cells used in publication:

CD34+ cell, human: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® X-Unit

Experiment

Primary cell cultures
CD34+ HSPCs were either freshly purified from human CB after obtaining informed consent and upon approval by the San Raffaele Hospital ethical committee (TIGET09) or purchased frozen from Lonza. After thawing, HSPCs were seeded at the concentration of 5 x 10^5 cells/mL in serum-free StemSpan SFEM medium (StemCell Technologies) supplemented with 10 µM 16,16-Dimethyl Prostaglandin E2 (added only at thawing) (Cayman), L-glutamine (2mM) and Penicillin-Streptomycin (100 IU/mL penicillin, 100 mg/mL streptomycin), 1µM SR-1 (Biovision), 50nM UM171 (STEMCell Technologies), and human early-acting cytokines (SCF 100 ng/mL, Flt3-L 100 ng/mL, TPO 20 ng/mL, and IL-6 20 ng/mL; Peprotech).

Gene editing
HSPCs were edited according to a previously optimized protocol 60 after an overnight. or three days of ex vivo culture, cells were nucleofected with 2.5–1.25 µM of RNPs and electroporation enhancer (Integrated DNA Technologies) according to manufacturer’s instructions using P3 Primary Cell 4D-Nucleofector X Kit and program EO-100 (Lonza). Ribonucleoproteins (RNPs) were assembled by incubating for 10 min at r.t at 1:1.5 M ratio S.p. Cas9 protein (Aldevron) with synthetic gRNAs (Integrated DNA Technologies) targeting AAVS1 (high specificity) or IL2RG locus (low specificity).12 Transduction with AAV6 was performed at a dose of 1x10^4 vg/cell 15 min after electroporation.

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.

Open in PubMed