A versatile gemini amphiphile-based platform with STING-activating properties for efficient gene delivery into dendritic cells

[Display omitted] •A gemini amphiphile (GA)-based material platform to identify high-performing non-viral vectors for pDNA and mRNA transfection.•GA delivered antigen-encoded mRNA to the DCs and enabled strong antigen expression and presentation, while also promoting DC maturation through the intracellular STING pathway.•GA vectors demonstrated potent co-delivery capability for the STING agonist, cGAMP, thereby inducing more pronounced DC maturation.•GA can be co-assembled with other lipid components into LNP-like quinary complexes system for mRNA delivery. Encouraging results from clinical trials have underscored the potential of gene-pulsed dendritic cell (DC) vaccines as a viable approach for immunotherapy. However, insufficient gene delivery and functional deficiencies in antigen presentation, migratory capacity and cytokine release of DC vaccines limited its broader clinical application. Here, a combinatorial design of cationic gemini amphiphile (GA) molecular library based on the four-component Ugi reaction (Ugi-4CR) has been developed to identify versatile non-viral vectors for gene-engineered DC vaccines. We demonstrated that the leading GA enabled robust transfection of plasmid DNA or mRNA into the DCs through the formation of minimalist binary complexes, which exhibited great advantages of low carrier/gene ratios, high gene loading efficiency and well biocompatibility. The identified GA stimulated strong type I interferon (IFN) expression via the intracellular stimulator of interferon genes (STING) pathway and enhanced DC maturation and activation, which could potentially facilitate strong immunogenicity of DC vaccines. Importantly, DC maturation and activation could be further strengthened by co-delivering maturation stimuli with mRNA/GA complexes, and adoptive transfer of gene-engineered DC vaccines elicited strong T cell responses and thus inhibited tumor growth in vivo. The further studies indicated GAs can be formulated into lipid nanoparticle (LNP)-like quinary complexes that presented 6.9-fold higher mRNA delivery efficiency on the DCs than the commercial Lipofectamine 2000. These results suggest that GA-based molecular library is a powerful platform to develop versatile gene delivery vectors for immune cell engineering.