Trivalent ions kinetic-gating for producing high-concentration and shelf-stable plasmid DNA/PEI particles

Abstract Scalable, cost-effective manufacturing remains a major barrier to the clinical translation of viral vector–mediated gene therapies. The widely used transfection method for producing adeno-associated virus (AAVs) and lentivirus uses plasmid DNA (pDNA)/polyethyleneimine (PEI) particles loaded with multiple plasmids; however, these particles must be prepared at low concentrations and used immediately, limiting scalability and reproducibility. Here we show a kinetic-gating strategy in which transient binding of trivalent citrate ions slows complexation, modulating charge-neutralization kinetics and delaying particle nucleation, enabling the formation of stable, highly concentrated pDNA/PEI particles. By incorporating citrate, we prevent aggregation and achieve uniform assembly at high concentrations, enabling a ten-fold increase in DNA concentration (to 0.2 mg/mL) and reduced dosing volumes. The method is robust across mixing conditions, compatible with standard manufacturing workflows, and maintains AAV production efficiency across scales. These results establish a simple and generalizable approach to control polyelectrolyte assembly kinetics, improving the scalability and reliability of viral vector production.