Programmable ternary polyplexes redirect systemic mRNA delivery to the spleen for in vivo CAR engineering

In vivo engineering of chimeric antigen receptor (CAR) immune cells has emerged as a promising alternative to ex vivo cell manufacturing, but its clinical translation remains limited by the lack of delivery systems capable of selective programming immune cells within lymphoid organs. Here we report a rationally engineered ternary polyplex that redirects intravenously-administered CAR mRNA from the lungs to the spleen through programmable surface-charge modulation while maintaining nanoparticle stability. The polyplex consists of amphiphilic polyaspartamide/mRNA secondary complexes coated with a pH-responsive charge-conversion polymer, enabling efficient systemic delivery and endosomal release of mRNA. Systemic optimization of polymer architecture, hydrophobicity, and surface charge identifies design principles governing spleen-selective delivery, resulting in luminescence intensities that are over 250-fold higher in the spleen than in the liver and over 330-fold higher than in the lungs following intravenous administration. The optimized formulation preferentially transfects splenic dendritic cells, macrophages, and natural killer cells, enabling transient in vivo expression of anti-CD19 CAR. This immune cell programming promotes macrophage activation, enhances CAR-mediated phagocytic activity, and induces antigen-specific depletion of peripheral B cells for several days. Transcriptomic profiling further reveals coordinated activation of innate immune pathways associated with macrophage polarization, dendritic cell activation, and natural killer cell responses. These findings establish programmable ternary polyplexes as a platform for spleen-selective mRNA delivery and provide a nonviral strategy for in vivo CAR immune cell engineering.