Ex Vivo\nDelivery of mRNA to Immune Cells via a Nonendosomal\nRoute Obviates the Need for Nucleoside Modification

Base modification and the use of lipid nanoparticles\nare thought\nto be essential for efficient in vivo delivery and expression of mRNA.\nHowever, for ex vivo immune cell engineering, the need for either\nof the two is unclear. Previous reports have suggested that nucleic\nacids may be efficiently delivered to immune cells ex vivo, through\na nonendosomal delivery route, but the need for base modification\nhas not been determined. Herein, we demonstrate that when a nonendosomal\ndelivery method is used, unmodified mRNA performs equally well to\nthe commonly used base-modified mRNA, including the <i>N</i><sup>1</sup> methyl pseudouridine modification, in terms of protein\nexpression and inflammatory response in cells. However, if an endosomal\ndelivery route is used, then <i>N</i><sup>1</sup> methyl\npseudouridine modification is necessary for high expression and low\ninflammatory response, as demonstrated by others as well. Overall,\nwe show that nonendosomal mRNA delivery renders nucleoside modifications\nnonessential and that unmodified mRNA combined with nonendosomal delivery\nroute may be used for efficient ex vivo mRNA-based engineering of\nimmune cells.