Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells

RNA-based therapeutics hold great promise for treating diseases and lipid nanoparticles (LNPs) represent the most advanced platform for RNA delivery. However, the fate of the LNP-mRNA after endosome-engulfing and escape from the autophagy-lysosomal pathway remains unclear. To investigate this, mRNA (encoding human erythropoietin) was delivered to cells using LNPs, which shows, for the first time, a link between LNP-mRNA endocytosis and its packaging into extracellular vesicles (endo-EVs: secreted after the endocytosis of LNP-mRNA). Endosomal escape of LNP-mRNA is dependent on the molar ratio between ionizable lipids and mRNA nucleotides. Our results show that fractions of ionizable lipids and mRNA (1:1 molar ratio of hEPO mRNA nucleotides:ionizable lipids) of endocytosed LNPs were detected in endo-EVs. Importantly, these EVs can protect the exogenous mRNA during in vivo delivery to produce human protein in mice, detected in plasma and organs. Compared to LNPs, endo-EVs cause lower expression of inflammatory cytokines. Lipid nanoparticles (LNPs) are potential platforms for RNA-based therapeutics, but the fate of LNP-RNAs upon internalization into the cell is unclear. Here, the authors show that LNP-mRNAs and ionizable lipids escape the endosomes and are re-released via extracellular vesicles which could deliver the functional mRNA to other cells.