Cholesterol in mRNA‐Lipid Nanoparticles can be Replaced with the Synthetic Mycobacterial Monomycoloyl Glycerol Analogue MMG‐1

Abstract Messenger RNA (mRNA) vaccines based on lipid nanoparticles (LNPs) are stabilized with cholesterol, which is thought to be a critical LNP component because it is essential for membrane integrity and endosomal escape. Here, it is shown that cholesterol in LNPs can be replaced with an immunopotentiating lipid, i.e., a synthetic analogue of the C‐type lectin receptor agonist monomycoloyl glycerol (MMG‐1), without compromising physicochemical properties, in vivo transfection efficiency, and immunogenicity of the mRNA‐loaded LNPs (mRNA‐LNPs). Replacement of cholesterol with MMG‐1 results in LNPs that mediate intracellular delivery of mRNA, which is translated into high levels of protein in vivo. Replacement of cholesterol with MMG‐1 in LNPs improves the transfection efficiency in T cells, B cells, and macrophages in the spleen and lymph nodes of mice. In mice, MMG‐1‐based LNPs loaded with mRNA encoding the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) induce S‐specific CD8 + T‐cell responses and virus‐neutralizing antibody responses, which are on par with the responses induced by cholesterol‐based LNPs. Upon vaccination and a subsequent SARS‐CoV‐2 challenge of Syrian golden hamsters, replacement of cholesterol with MMG‐1 in mRNA‐LNPs enhances S‐specific immunoglobulin G titers and reduces the SARS‐CoV‐2 load in the nasal cavity. These findings provide insights for improved design of LNPs for mRNA vaccine delivery.