Active Nuclear Shuttling Enables Efficient Virus-Free CAR Gene Integration Using Ready-to-Use Lipid Nanoparticles

Non-viral engineering of chimeric antigen receptor T (CAR -T) cells is highly desirable to overcome the cost, safety, and scalability limitations associated with viral vectors and electroporation. However, efficient nuclear delivery and stable genomic integration of DNA in primary human T cells remain major challenges. Here, we established a virus-free CAR-T manufacturing platform using lipid nanoparticles (LNPs) combined with a nuclear localization signal (NLS) shuttle strategy. We developed proprietary ready-to-use LNPs that enable on-demand encapsulation of nucleic acids. To overcome nuclear transport barriers, NLS-fused transposase or genome-editing nuclease was used to bind donor DNA in the cytoplasm and promote active nuclear import. This approach enabled highly efficient and low-toxicity delivery of mRNA and plasmid DNA into primary human T cells. NLS-assisted transposase delivery markedly enhanced genomic integration of the CAR gene, resulting in high expression levels and improved cell viability compared with electroporation-based methods. In addition, TRAC locus-specific targeted integration was achieved more efficiently through end-joining-based repair pathways than through homology-directed repair following LNP delivery. The resulting engineered CAR-T cells exhibited potent and antigen-specific cytotoxic activity. Together, these results demonstrate that NLS-assisted LNP delivery overcomes a key bottleneck in non-viral gene integration and provides a robust strategy for the generation of functional CAR-T cells.