Genetic Code Expansion Enables Oriented, Site-Specific Conjugation of DARPins to Lipid Nanoparticles for Selective mRNA Delivery to CD8+ T Cells.

Lipid nanoparticles (LNPs) are an effective platform for delivering therapeutic payloads, but their use for targeting specific cell populations in vivo is limited by a lack of specificity. To address this issue, we developed an innovative approach for creating targeted LNPs based on the post-insertion of protein-lipid conjugates. By expanding the genetic code, a single p-azido-L-phenylalanine residue was specifically incorporated into a DARPin against murine CD8, enabling copper-free click-chemistry with dibenzocyclooctyne (DBCO) to functionalize the particle surface with DARPin conjugates. DARPin-LNP characterization demonstrated preservation of particle size and acquisition of a characteristic negative charge after DARPin insertion, confirming successful post-insertion. In vitro experiments using primary murine splenic T-cells showed that targeted LNPs selectively transfected CD8+ T-lymphocytes. These data indicate that the developed platform enables selective in vitro delivery of nucleic acids to cytotoxic lymphocytes, highlighting its potential relevance for designing safer immunotherapeutic strategies.