Modified self-amplifying RNA mediates robust and prolonged gene expression in the mouse and ex vivo human 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®) 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 N1mΨ 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.