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

In self-amplifying ribonucleic acid (saRNA), substitution of cytidine with 5-hydroxymethylcytidine (hm5C) reduces innate immune responses and prolongs protein expression. Administration routes to date for hm5C modified saRNA encapsulated within lipid nanoparticle (LNPs) include intramuscular, as a potent low dose vaccine, but expression levels, patterns, and cell tropism in other key organs are lacking but critical for advancing RNA treatments/technology. Here we report the protein expression and cell type tropism of modified saRNA-LNPs, encoding fluorescent proteins, when injected in the mouse brain or applied to human cortical brain slices. saRNA encapsulated in an LNP formulation comprising ALC-0315 (present in Comirnaty(R)) efficiently mediates robust and long-lasting protein expression in mouse brain cells beyond five weeks, with detectable expression in some neurons at three months. hm5C saRNA substantially outperforms N1m{Psi} mRNA. In addition to transfecting astrocytes and neurons at the injection site, saRNA-LNPs labels neurons retrogradely. Excitingly, the saRNA-LNPs afford robust protein expression in human cortical brain slices, obtained during standard surgical procedures for epilepsy treatment, with expression emerging within twenty-four hours and lasting beyond six days. Thus, saRNA-LNPs are an exciting nonviral gene delivery method that effectively transfects brain cells and will catalyze new opportunities for mechanistic neuroscience research and therapeutic development.