Cell-specific mRNA delivery via nanobody-functionalized lipid nanoparticles

mRNA-based therapeutics formulated in lipid nanoparticles (mRNA-LNPs) have emerged as a groundbreaking platform technology for vaccination, immunotherapy, protein replacement therapy, and gene editing. However, a major bottleneck in their application is the lack of cell-specific delivery methods, which limits their efficacy and safety. To overcome this challenge, we developed a novel mRNA-LNP platform with targeted delivery capabilities. To this end, lipid nanoparticles were functionalized with nanobodies (VHH) specific to Aminopeptidase N (APN), a cell surface protein on gut epithelial cells. These nanobodies were produced in genetically engineered E. coli, incorporating the non-canonical amino acid azido-phenylalanine into the VHH sequence to enable their precise conjugation onto lipid nanoparticles containing DSPE-PEG₂₀₀₀-TCO via a two-step click chemistry (SPAAC and IEDDA) reaction. Our findings demonstrate that APN-targeted, mRNA-loaded LNPs selectively target APN-expressing cells, enhancing LNP uptake and mRNA delivery to these cells. Furthermore, we show that directing the nanobody-functionalized mRNA-LNPs toward APN promotes their transcytosis across the gut epithelial barrier in porcine apical-out intestinal organoids and in vivo. Together, these findings highlight the potential of this programmable platform for the cell-specific delivery of mRNA-based vaccines and therapeutics. While this study focuses on porcine APN, the approach is adaptable across species, providing a versatile and customizable solution for the precise delivery of mRNA payloads to specific cells.

Aminopeptidase N; Apical-out intestinal organoids; Cell-specific delivery; Nanobodies; mRNA-lipid nanoparticles.