Abstract
Precise genome editing in hematopoietic stem cells (HSCs) holds immense potential for the treatment of genetic blood disorders, such as severe combined immunodeficiencies (SCID) and hemoglobinopathies such as sickle cell disease (SSD). Among genome editing strategies, homology-directed repair (HDR)-based knock-in (KI) is particularly attractive for enabling site-specific gene correction or insertion. However, achieving high KI efficiency in HSCs has been a long-standing challenge due to their quiescent nature, their inherent preference for error-prone non-homologous end joining (NHEJ), and their susceptibility to DNA damage-induced apoptosis. These barriers have greatly limited the clinical application of HDR-based editing in long-term HSCs.
In this study, we report that transient inhibition of ataxia-telangiectasia mutated (ATM) kinase markedly improves KI efficiency in both murine and human HSCs. Using a CRISPR/Cas9 RNP and AAV6 donor system, we edited primary mouse HSCs (CD150⁺CD201⁺c-Kit⁺Sca1⁺Lineage⁻) with or without an ATM inhibitor in ex vivo culture. ATM inhibition increased KI frequency from ~40% to ~70% in vitro. Upon transplantation into myeloablated recipient mice, the frequency of genome-edited HSCs (CD150⁺CD48⁻KSL) in the bone marrow of primary recipient mice rose from ~5% to ~30% with ATN inhibitor. More strikingly, secondary transplantation revealed a >100-fold enhancement of long-term engrafting, genome-edited HSCs, with frequencies increasing from ~0.3% in controls to ~40% in ATM-inhibited cells.
Mechanistically, phosphoproteomic analysis revealed that ATM inhibition suppressed activation of the ATM–p53–caspase 3 pathway, as well as phosphorylation of H2AX and 53BP1, all key regulators of the DNA damage response (DDR) and apoptosis. Capillary Western blotting confirmed that ATM inhibition mitigated Cas9/AAV-induced stress signaling, leading to improved survival and preservation of HDR-edted HSCs.
Furthermore, we applied this strategy to a murine model of X-linked severe combined immunodeficiency (X-SCID). ATM inhibition improved targeted integration and restored expression of the IL-2 receptor γ chain (CD132), a critical component for immune function, demonstrating therapeutic benefit. Importantly, this approach also enhanced KI efficiency in human cord blood-derived CD34⁺ cells, supporting the translational relevance of this strategy.
Collectively, these findings establish transient ATM inhibition as a powerful and clinically applicable method to enhance KI-mediated genome editing in HSCs, while preserving their long-term repopulating capacity. This strategy may serve as a foundation for future therapies aiming to durably correct genetic hematological diseases in human patients.
