Nanoparticle stereochemistry-dependent endocytosis improves in vivo mRNA delivery

We report the application of single-molecule-based sequencing technology for high-throughput profiling of lipid nanoparticles (LNPs) carrying mRNA in murine liver non parenchymal cells. We report that LNPs containing stereopure 20α-hydroxycholesterol (20α) deliver mRNA to these cells up to three-fold more efficiently than LNPs containing both 20α- and 20ß- hydroxycholesterols (20mix). We show from the sequencing data that 20mix LNPs were sorted into phagocytic pathways, resulting in different functional delivery between stereopure and non-stereopure LNPs. We performed scRNA-seq using the 10X Chromium System, loading ~2,000 cells per condition, and processed the resulting data using Cell Ranger, resulting in an average read depth of ~100,000 reads per cell. These data suggest that stereochemistry-dependent interactions between LNPs and target cells can be exploited to improve mRNA delivery. Examination of FACS isolated liver non parenchymal cells three hours after systemically injecting mice with PBS, 20α LNPs carrying 0.3 mg/kg Cre mRNA, or 20mix LNPs carrying 0.3 mg/kg Cre mRNA