Formulation of RNA-Loaded Lipid Nanoparticles

RNA-loaded LNP formulations were formed using the ethanol dilution method^{28 (link)}. The liver-targeted mRNA formulation (mDLNP) was developed and reported in our previous paper^{27 (link)}, and the base formulations were prepared as previously described^{35 (link), 11} . Unless otherwise stated, all lipids with specified molar ratios were dissolved in ethanol and RNA was dissolved in 10 mM citrate buffer (pH 4.0). The two solutions were rapidly mixed at an aqueous to ethanol ratio of 3:1 by volume (3:1, aq.:ethanol, vol:vol) to satisfy a final weight ratio of 40:1 (total lipids:mRNA), then incubated for 10 min at room temperature. To prepare SORT LNP formulations containing anionic SORT lipids (such as 18PA, 14PA and 18BMP), the anionic lipids were dissolved in tetrahydrofuran (THF) first then mixed with other lipid components in ethanol, finally yielding formulations with mRNA buffer (10 mM, pH 3.0) as described above. All formulations were named based on the additional lipids. Taking DOTAP mDLNP as an example, the internal molar ratio of mDLNP was fixed as reported in our published paper with 5A2-SC8/DOPE/Cholesterol/DMG-PEG of 15/15/30/3^{27 (link)}. DOTAP, as the additional lipid, was dissolved into the above ethanol lipid mixture with specified amount, making the molar ratio of 5A2-SC8/DOPE/Cholesterol/DMG-PEG/DOTAP equal to 15/15/30/3/X, then rapidly mixed with aq. mRNA solutions following the above standard protocol, finally producing SORT LNPs named Y% DOTAP, where Y means the molar percent of DOTAP in total lipids. Formulations with other additional lipids were formed similarly with the above methods (Supplementary Fig. 1 and Table 1). As a representative example, liver targeted SORT LNPs (20% DODAP) could be prepared as follows. A solution of lipids in ethanol was prepared consisting of 7.59 mM 5A2-SC8, 7.59 mM DOPE, 15.18 mM Cholesterol, 1.52 mM DMG-PEG2000, and 7.97 mM DODAP to make the final molar ratio of 19.05/19.05/38.1/3.81/20. To reach a final 40/1 (wt/wt) of total lipids to total RNAs ratio, 1.16 μL lipid solution could be used per μg RNA. For example, to make a final 5 μg RNA formulation, 5.8 μL lipid mixture and 9.2 μL ethanol were mixed first (total 15 μL), then 45 μL mRNA solution was prepared consisting of 5 μg RNA in citrate buffer (10 mM, pH 4.0). The 45 μL mRNA solution was rapidly combined into 15 μL of the ethanol lipid solution to form 20% DODAP SORT LNPs. For Cas9/sgRNA ribonucleoprotein (RNP) encapsulation, 1X PBS was used for formulation and the molar ratio of Cas9 and sgRNA was fixed at 1:3. After SORT LNP formation, the fresh LNP formulations were diluted with 1X PBS to 0.5 ng/μL mRNA (with final ethanol concentration < 5%) for in vitro assays and size detection by Dynamic Light Scattering (DLS, Malvern MicroV model; He-Ne laser, λ = 632 nm). For in vivo experiments, the formulations were dialyzed (Pur-A-Lyzer Midi Dialysis Kits, WMCO 3.5kDa, Sigma-Aldrich) against 1X PBS for 2h, and diluted with PBS to 15 μL/g for intravenous (IV) injections.