Formulation and delivery of lipid nanoparticles with mRNA

C12–200 (courtesy of Alnylam Pharmaceuticals) was prepared⁶⁵ and mixed together with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE; Avanti Polar Lipids), cholesterol (Sigma) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (PEG; Avanti) at a 50:20:20:10 weight ratio in ethanol. Then, an aqueous phase containing human ATG5 or GFP mRNA (TriLink Biotechnologies) in 10 mM citrate buffer was prepared. Syringe pumps were used to mix the ethanol and aqueous phases at a 1:3 ratio to generate C12–200 lipid nanoparticles (LNPs),¹⁹ which were then dialyzed against 1x PBS in a 20k MWCO cassette at 4 °C for 2 h. mRNA concentration was quantified using a modified Quant-iT RiboGreen RNA Assay (Invitrogen).⁶⁶ Cells (hESCs and hESC-derived neurons) were treated with 2 μg/mL C12–200 LNP containing human ATG5 or GFP mRNA for 4 days with replenishment on day 2, or as indicated. See the key resources table for further information of the LNPs used in this study.