Pregnancy-inspired DNA nanocarriers reproduce a cGAS-driven pro-inflammatory monocyte phenotype

Introduction: Placental cell-free DNA (cfDNA) activates maternal monocytes to a proinflammatory phenotype through cGAS-STING signaling during pregnancy, thereby stimulating T cells. We sought to engineer DNA nanocarriers that reproduce this mechanism in human monocytes using clinically translatable materials as a strategy for tumor-associated macrophage (TAM) reprogramming. Methods: A synthetic mimic of fetal cfDNA (scfDNA) was designed, commercially synthesized, and complexed with polyethylenimine (PEI) to form scfDNA-PEI nanoparticles (scfDNA-PEI-NPs) for cytosolic delivery. THP-1 Dual reporter monocytes expressing interferon regulatory factor (IRF)-inducible luciferase were treated with scfDNA-PEI-NPs or human cytotrophoblast derived cfDNA (CTB-DNA) to quantify cGAS-STING-IRF activation. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release to define a non-toxic therapeutic window. Results: scfDNA-PEI-NPs triggered robust cGAS-STING-IRF activation in THP-1 monocytes, exceeding CTB-DNA responses. This response was abolished in cGAS-knockout cells and rescued by a cGAS-independent stimulator of the IRF pathway, confirming cGAS specificity of scfDNA-PEI-NPs. Dose-response analysis showed a > 10-fold separation between cGAS-activating and cytotoxic concentrations, establishing a therapeutic window for myeloid-selective cGAS activation. Conclusions: These findings provide proof-of-concept that pregnancy-inspired DNA nanocarriers can reproduce fetal cfDNA-driven activation of a proinflammatory monocyte phenotype in vitro. This platform lays the groundwork for future work on macrophage-targeted cGAS activation and proinflammatory reprogramming for tumor immunotherapy development.