Mannich reaction-based combinatorial libraries identify antioxidant ionizable lipids for mRNA delivery with reduced immunogenicity.

The immunogenicity of lipid nanoparticles (LNPs) used for the delivery of nucleoside-modified messenger RNA limits the levels and durability of expression of the encoded protein. Here, by leveraging the Mannich reaction for ionizable lipid synthesis, and via the in vitro and in vivo screening of six combinatorial libraries of synthesized lipids, we report the identification of an antioxidant ionizable lipid, C-a16, exhibiting reduced immunogenicity. When incorporated into LNPs for mRNA delivery, C-a16 mitigated the generation of intracellular reactive oxygen species, thereby extending the duration of protein expression. In mice, and compared with commercial LNPs, LNPs incorporating C-a16 and co-delivering Cas9 mRNA and guide RNA for the editing of the transthyretin gene led to 2.8-fold higher editing efficiency; LNPs with C-a16 delivering fibroblast growth factor 21 mRNA increased the expression of the protein 3.6-fold; and when delivering mRNA encoding a tumour neoantigen or the spike protein of SARS-CoV-2, LNPs with C-a16 induced stronger antigen-specific immune responses. Our findings support the further testing of C-a16 as a promising ionizable lipid for mRNA delivery in therapeutic applications.