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 20a-hydroxycholesterol (20a) deliver mRNA to these cells up to three-fold more efficiently than LNPs containing both 20a- 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. Overall design: Examination of FACS isolated liver non parenchymal cells three hours after systemically injecting mice with PBS, 20a LNPs carrying 0.3 mg/kg Cre mRNA, or 20mix LNPs carrying 0.3 mg/kg Cre mRNA