Insect-Cell-Produced AAV2 Vectors Activate Myeloid Innate Immune Response Featuring NETosis

Recombinant adeno-associated virus (rAAV) is a leading platform for in vivo gene therapy. However, host immune responses remain a major barrier to its safe and effective clinical application. As insect cell systems have emerged as a valuable production platform with unique advantages, we examined the immunogenicity of insect-cell-derived rAAV2. Temporal transcriptomic profiling in mice revealed a rapid and sustained innate immune response dominated by myeloid cell activation. Notably, we observed prominent enrichment of the neutrophil extracellular trap (NETosis) pathway. Functional assays in isolated neutrophils confirmed that rAAV2 directly induces a pro-NETotic response marked by reactive oxygen species (ROS) production and NET formation. Degradation of extracellular DNA via DNase I significantly attenuated hepatic inflammatory signaling and enhanced vector transduction, indicating that NET formation functions as a biological barrier to rAAV2-mediated gene transfer. Our findings identify NETosis as a previously underappreciated immune mechanism triggered by insect-cell-produced AAV vectors, offering new insights into vector immunogenicity and revealing potential targets to improve the safety and efficacy of AAV-based gene therapies.