Amplification of\nProtein Expression by Self-Amplifying\nmRNA Delivered in Lipid Nanoparticles Containing a β‑Aminoester\nIonizable Lipid Correlates with Reduced Innate Immune Activation

Self-amplifying mRNA (saRNA) is witnessing increased\ninterest as\na platform technology for protein replacement therapy, gene editing,\nimmunotherapy, and vaccination. saRNA can replicate itself inside\ncells, leading to a higher and more sustained production of the desired\nprotein at a lower dose. Controlling innate immune activation, however,\nis crucial to suppress unwanted inflammation upon delivery and self-replication\nof RNA <i>in vivo</i>. In this study, we report on a class\nof β-aminoester lipids (βAELs) synthesized through the\nMichael addition of an acrylate to diethanolamine, followed by esterification\nwith fatty acids. These lipids possessed one or two ionizable amines,\ndepending on the use of nonionic or amine-containing acrylates. We\nutilized βAELs for encapsulating saRNA in lipid nanoparticles\n(LNPs) and evaluated their transfection efficiency <i>in vitro</i> and <i>in vivo</i> in mice, while comparing them to LNPs\ncontaining ALC-0315 as an ionizable lipid reference. Among the tested\nlipids, OC7, which comprises two unsaturated oleoyl alkyl chains and\nan ionizable azepanyl motif, emerged as a βAEL with low cytotoxicity\nand immunogenicity relative to ALC-0315. Interestingly, saRNA delivered\nvia the OC7 LNP exhibited a distinct <i>in vivo</i> transfection\nprofile. Initially, intramuscular injection of OC7 LNP resulted in\nlow protein expression shortly after administration, followed by a\ngradual increase over a period of up to 7 days. This pattern is indicative\nof successful self-amplification of saRNA. In contrast, saRNA delivered\nvia ALC-0315 LNP demonstrated high protein translation initially,\nwhich gradually declined over time and lacked the amplification seen\nwith OC7 LNP. We observed that, in contrast to saRNA OC7 LNP, saRNA\nALC-0315 LNP induced potent innate immune activation by triggering\ncytoplasmic RIG-I-like receptors (RLRs), likely due to the highly\nefficient endosomal membrane rupturing properties of ALC-0315 LNP.\nConsequently, the massive production of type I interferons quickly\nhindered the amplification of the saRNA. Our findings highlight the\ncritical role of the choice of ionizable lipid for saRNA formulation\nin LNPs, particularly in shaping the qualitative profile of protein\nexpression. For applications where minimizing inflammation is desired,\nthe use of ionizable lipids, such as the βAEL reported in this\nstudy, that elicit a low type I interferon response in saRNA LNP is\ncrucial.