Engineered extracellular vesicles for targeted TREX1 delivery attenuate neuroinflammation after cerebral ischemia

Ischemic stroke stands as a principal driver of global mortality and permanent functional deficits. Notably, the clinical efficacy of current interventions is severely restricted by post-ischemic neuroinflammation. Cerebral ischemic injury prompts an inflammatory surge mediated by the cGAS-STING signaling cascade, a process initiated by the recognition of aberrantly localized cytosolic DNA. Three-prime repair exonuclease 1 (TREX1), a cytosolic DNA exonuclease, negatively regulates STING signaling; however, efficient delivery of TREX1 to the ischemic brain remains a major challenge. Here, we engineered mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) as carriers for TREX1 delivery. This was accomplished by using a palmitoylation signal-fused construct (PALM-TREX1), which enables efficient loading of the protein into extracellular vesicles. The C1C2 domains of lactadherin and the RGD-4C peptide were combined into a single recombinant fusion protein, enabling EV surface functionalization for enhanced ischemic targeting via phosphatidylserine interaction. In a mouse model of middle cerebral artery occlusion (MCAO), RGD-modified TREX1-loaded EVs (RGD-EV-TREX1) preferentially accumulated in ischemic regions, suppressed STING pathway activation, and reduced microglial activation and pro-inflammatory cytokine expression. The reduction in neuronal DNA damage and apoptosis ultimately facilitated improved neurological functional recovery, positioning RGD-EV-TREX1 as a promising cell-free therapeutic strategy for ischemic stroke.