Precondensed Plasmid DNA Enhances CAR‑T Cell Generation via Lipid Nanoparticles

The advent of chimeric antigen receptor (CAR) T-cell therapy has introduced a novel and personalized approach to cancer treatment. Despite its promise, the challenge of developing a system that bypasses the need for viral vectors remains significant, particularly in terms of achieving a clinical efficacy and sustained durability. To address these challenges, lipid nanoparticles (LNPs) produced through advanced microfluidic technology have been recently utilized to encapsulate plasmid DNA (pDNA) encoding CAR receptors. However, the intrinsic challenges associated with pDNA encapsulation, along with the critical requirement for efficient expression, remain substantial obstacles. Here, we show that incorporating DNA-condensing agents into the microfluidic manufacturing of LNPs effectively overcomes these limitations. Briefly, we conducted a preliminary investigation to characterize LNPs with and without the commercial condensing agent P3000-Reagent (PR), focusing on their physicochemical properties and scrutinizing the biological outcomes primarily in the HEK-293 cell line. Our results demonstrated that precondensation of the pDNA with PR differentially increased the transfection efficiency of the tested formulations, whereas confocal microscopy indicated reduced lysosomal colocalization and major nuclear localization. Finally, PR was found to enhance LNP efficiency upon multiple administrations to the immortalized T-lymphocyte Jurkat cell line, enabling the delivery of both a luciferase reporter gene and a functional CAR-encoding plasmid. Overall, these findings underscore the great potential of introducing DNA-condensing agents into the LNP preparation process, especially for systems designed for challenging delivery applications, such as multiadministration transfection protocols.