Modified self-amplifying RNAs mediate robust and prolonged gene expression in the mammalian brain

In self-amplifying RNA (saRNA), substitution of cytidine with 5-hydroxymethylcytidine (hm5C) reduces innate immune responses and prolongs protein expression. When formulated as a vaccine and administered intramuscularly, lipid nanoparticles (LNPs) loaded with modified saRNA (saRNA-LNPs) afford robust and long-term protein expression. Here we report the protein expression and cell type tropism of modified saRNA-LNPs, encoding fluorescent proteins, when injected in the mammalian brain. saRNA encapsulated in an LNP formulation comprising ALC-0315 (present in Comirnaty) efficiently mediates robust and long-lasting protein expression in brain cells beyond five weeks, with detectable expression in some neurons at three months. hm5C saRNA substantially outperforms N1mΨ mRNA. Intriguingly, in addition to transfecting astrocytes and neurons at the injection site, saRNA-LNPs labels neurons retrogradely. Thus, saRNA-LNPs are an exciting nonviral gene transduction method that effectively transduces brain cells with excellent potency and mediates prolonged gene expression.