Beyond the Endosomal Bottleneck: Understanding the Efficiency of mRNA/LNP Delivery

Abstract The development of lipid nanoparticles (LNP) holds great promise for vaccine and therapeutic applications. However, ineffective transport of nucleic acid cargo from endosomes to the cytosol, termed “endosomal escape,” has remained a bottleneck to achieving greater LNP efficacy. Poor understanding of the mechanisms of endosomal escape and a lack of suitable techniques to assess the efficiency of escape have also impeded progress in this field. To address this gap, a modified SNAP Switch /SNAP‐tag assay is developed to compare the cytosolic delivery capabilities of various LNP formulations, comparing ionizable lipids used in major commercial LNP products. The results show that all the LNP formulations tested exhibited low (<10%) endosomal escape efficiency. Interestingly, a limited correlation is found between the amount of mRNA delivery to the cytosol and protein expression. While endosomal escape of mRNA LNP cargo is necessary for protein expression, subcellular processes after delivery to the cytosol play a critical role in maximising protein expression. These findings challenge existing paradigms and reveal critical parameters for optimizing nucleic acid delivery and therapeutic effectiveness of next‐generation LNP.