mRNA was synthesized in vitro by T7 RNA polymerase–mediated transcription, using either uridine 5′-triphosphate or 100% substituted with 1mΨTP, from a linearized DNA template, which incorporates 5′ and 3′ untranslated regions and a polyadenosine tail. For process A, NTPs were included at equimolar concentrations, and the resulting mRNA was purified by 2′-deoxy-T20 oligo affinity chromatography. For process B, NTPs were included at custom molar ratios and purified by ion pair RP-HPLC. After purification, mRNA was buffer-exchanged into 2 mM sodium citrate (pH 6.5), passed through a 0.22-μm filter, and stored at −20°C until use.
LNP formulations were prepared as previously described (48 (link)). Briefly, lipids dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 of ionizable:helper:structural:polyethylene glycol were mixed with acidified mRNA at a ratio of 3:1 mRNA:lipid. Formulations were dialyzed against PBS (pH 7.4) for at least 18 hours, concentrated using Amicon Ultra centrifugal filters (EMD Millipore Corp., Merck KGaA, Darmstadt, Germany), passed through a 0.22-μm filter, and stored at 4°C until use. Particle size was measured by dynamic light scattering and found to be <100 nm; encapsulation was >90%, as measured by the Quant-iT RiboGreen RNA quantitation kit (Thermo Fisher Scientific), and endotoxin was <10 endotoxin units (EU)/ml. mRNA was formulated with DOTAP (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s recommended protocol.
LNP formulations were prepared as previously described (48 (link)). Briefly, lipids dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 of ionizable:helper:structural:polyethylene glycol were mixed with acidified mRNA at a ratio of 3:1 mRNA:lipid. Formulations were dialyzed against PBS (pH 7.4) for at least 18 hours, concentrated using Amicon Ultra centrifugal filters (EMD Millipore Corp., Merck KGaA, Darmstadt, Germany), passed through a 0.22-μm filter, and stored at 4°C until use. Particle size was measured by dynamic light scattering and found to be <100 nm; encapsulation was >90%, as measured by the Quant-iT RiboGreen RNA quantitation kit (Thermo Fisher Scientific), and endotoxin was <10 endotoxin units (EU)/ml. mRNA was formulated with DOTAP (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s recommended protocol.
