Ex Vivo Delivery of mRNA to Immune Cells via a Nonendosomal Route Obviates the Need for Nucleoside Modification.

Base modification and the use of lipid nanoparticles are thought to be essential for efficient in vivo delivery and expression of mRNA. However, for ex vivo immune cell engineering, the need for either of the two is unclear. Previous reports have suggested that nucleic acids may be efficiently delivered to immune cells ex vivo, through a nonendosomal delivery route, but the need for base modification has not been determined. Herein, we demonstrate that when a nonendosomal delivery method is used, unmodified mRNA performs equally well to the commonly used base-modified mRNA, including the methyl pseudouridine modification, in terms of protein expression and inflammatory response in cells. However, if an endosomal delivery route is used, then methyl pseudouridine modification is necessary for high expression and low inflammatory response, as demonstrated by others as well. Overall, we show that nonendosomal mRNA delivery renders nucleoside modifications nonessential and that unmodified mRNA combined with nonendosomal delivery route may be used for efficient ex vivo mRNA-based engineering of immune cells.