LAUSR

Lipid nanoparticle (LNP) formulation was the first approved delivery strategy for liver-targeted siRNA delivery and currently represent the most advanced platform for delivery of therapeutic mRNA in clinical use, after the approval of mRNA-based vaccines against the SARS-CoV-2 coronavirus. Still, poor efficiency of LNPs to promote cytosolic delivery of both mRNA and siRNA after internalization by target cells limits their potency and thus clinical application as RNA delivery vehicles – especially in extrahepatic tissues and tumors. It is still unclear how endosomal escape of LNP-delivered RNA cargo occurs, and why only a minute proportion of all therapeutic molecules reach the cytoplasm. Here, we explored the intracellular sorting, integrity and endosomal escape of both mRNA and siRNA loaded LNPs to characterize the nature of RNA release from endosomal compartments and pinpoint current bottlenecks. Using live-cell imaging and super-resolution microscopy of LNPs with fluorescently labeled RNA payload, we identify multiple distinct steps of substantial inefficiency in the cytosolic delivery of nucleic acid cargoes. We demonstrate that membrane damages marked by recruitment of galectins are conducive to cytosolic RNA delivery, while membrane perturbations recruiting the ESCRT machinery do not permit endosomal escape. By quantitative single-vesicle analysis, we show that only a small fraction of the nucleic acid cargo contained in the endosome is released to the cytosol upon LNP-triggered membrane damage detected by galectins. Unexpectedly, we also observe that only a subset of damaged endosomes contain RNA payload. Through FRET and super-resolution microscopy, using LNPs formulated with both fluorescently labeled ionizable lipid (MC3-BODIPY) and RNA, we find that RNA cargo and ionizable lipid segregate during endosomal sorting – both within single endosomes and to different endosomal compartments. Finally, we visualize localized MC3-BODIPY enrichment in endosomal membranes and membrane damage in direct proximity to siRNA-LNPs tethered to the luminal vesicle membrane. Taken together, this work has identified multiple mechanistically distinct barriers limiting intracellular RNA delivery by LNPs.