Targeted KEAP1 disruption enhances antioxidant defense and mesenchymal stromal cell therapy for chronic limb-threatening ischemia

Chronic limb-threatening ischemia (CLTI) is a severe vascular disorder characterized by tissue hypoxia and oxidative stress that limit the efficacy of regenerative therapies. Mesenchymal stem/stromal cells (MSCs) hold promise for CLTI treatment through paracrine angiogenic and immunomodulatory signaling, yet their survival and function are compromised in the reactive oxygen species-rich ischemic microenvironment. Here, we utilized CRISPR-Cas9 to generate a targeted knockout of Kelch-like ECH-associated protein 1 (KEAP1), the negative regulator of the antioxidant transcription factor NRF2, in human bone marrow-derived MSCs. KEAP1 editing activated the NRF2 pathway, reduced intracellular oxidative stress, and reprogrammed redox and paracrine gene networks. Edited MSCs exhibited enhanced viability, sustained secretion of proangiogenic cytokines, and improved tissue perfusion and arteriogenesis in a murine model of CLTI. These findings establish KEAP1 gene editing as a permanent, integration-free strategy to augment MSC resistance and therapeutic efficacy in oxidative ischemic environments.