Lipid nanoparticle mRNA systems containing high levels of sphingomyelin engender higher protein expression in hepatic and extra-hepatic tissues

Abstract

Lipid nanoparticles (LNPs) for delivery of mRNA usually contain ionizable lipid/helper lipid/cholesterol/PEG-lipid in molar ratios of 50:10:38.5:1.5, respectively. These LNPs are rapidly cleared from the circulation following intravenous (i.v.) administration, limiting uptake into other tissues. Here, we investigate the properties of LNP mRNA systems prepared with high levels of "helper" lipids such as 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) or N-(hexadecanoyl)-sphing-4-enine-1-phosphocholine (egg sphingomyelin [ESM]). We show that LNP mRNAs containing 40 mol % DSPC or ESM have a unique morphology with a small interior "solid" core situated in an aqueous compartment that is bounded by a lipid bilayer. The encapsulated mRNA exhibits enhanced stability in the presence of serum. LNP mRNA systems containing 40 mol % DSPC or ESM exhibit significantly improved transfection properties in vitro compared with systems containing 10 mol % DSPC or ESM. When injected i.v., LNP mRNAs containing 40 mol % ESM exhibit extended circulation lifetimes compared with LNP mRNA systems containing 10 mol % DSPC, resulting in improved accumulation in extrahepatic tissues. Systems containing 40 mol % ESM result in significantly improved gene expression in spleen and bone marrow as well as liver post i.v. injection compared with 10 mol % DSPC LNP mRNAs. We conclude that LNP mRNAs containing high levels of helper lipid provide a new approach for transfecting hepatic and extrahepatic tissues.

Keywords: bone marrow; helper lipids; hepatocytes; lipid nanoparticles; mRNA; splenocytes.

Graphical abstract

Figure 1

LNP luc mRNA morphology progress from a solid core structure to a bilayer structure surrounding an aqueous compartment containing a small solid core as the ESM helper lipid content is increased LNP luc mRNA systems were formulated at an N/P ratio of 6 as described in methods to contain increasing amounts of the helper lipid egg sphingomyelin (ESM). (A) LNP composition ESM/MC3/Chol/PEG-lipid 10:50:38.5:1.5 mol/mol, (B) LNP composition ESM/MC3/Chol/PEG-lipid 20:44.25:34.25:1.5 mol/mol, (C) LNP composition ESM/MC3/Chol/PEG-lipid 30:38.5:30:1.5 mol/mol, (D) LNP composition ESM/MC3/Chol/PEG-lipid 40:33:25.5:1.5 mol/mol, (E) LNP composition ESM/MC3/Chol/PEG-lipid 50:27.25:21.25:1.5 mol/mol, (F) LNP composition ESM/MC3/Chol/PEG-lipid 55:24.5:19:1.5 mol/mol. For other details see Table S1.

Figure 2

LNP mRNA system containing high helper lipid contents exhibit dramatically improved transfection potencies compared with Onpattro-type formulations in vitro HuH7 cells were incubated with LNP luc mRNA systems containing DSPC or ESM at 10 or 40 mol % levels over a dose range of 0.03–3 μg mRNA/mL Luciferase expression (luminescence) was quantified after a 24 h incubation. (A) Luminescence observed for each formulation and dose. Data represent the arithmetic mean ± SD of three replicates. (B) Bar graphs depicting the mean fluorescence intensity (MFI) of HuH7 cells indicating the uptake of DiI-labeled LNPs. Data represent the arithmetic mean ± SD of three replicates and are normalized for the higher lipid content of 40 mol % systems as described in methods. ∗p < 0.05, ∗∗∗p < 0.001.

Figure 3

mRNA encapsulated in LNP containing high levels of helper lipid exhibits improved stability properties when incubated with serum compared with free mRNA or mRNA encapsulated in Onpattro-type LNP formulations LNP GFP mRNA systems containing 10% DSPC (DSPC/MC3/Chol/PEG-lipid 10:50:38.5:1.5 mol/mol) or 40% ESM (ESM/MC3/Chol/PEG-lipid 40:33:25.5:1.5 mol/mol) were prepared as described in methods (N/P = 6) and were incubated in 50% FBS at 37°C for 48 h mRNA stability was assessed using an Agilent 2100 Bioanalyzer (see methods). Data are representative of two separate experiments.

Figure 4

LNP mRNA systems containing 40 mol % ESM exhibit longer circulation lifetimes following i.v. administration than Onpattro-type formulations DiD-labeled LNP GFP mRNA were administered at a dose of 1.5 mg mRNA/kg and blood was collected via the saphenous vein at 0, 1.5, 4, 8, and 24 h. The circulating LNP levels were determined by measuring the DiD fluorescent intensity from collected blood. Each data point represents the arithmetic mean ± SD of data collected from five mice. (A) LNP GFP mRNA systems with the Onpattro lipid composition (DSPC/MC3/Chol/PEG-lipid 10:50:38.5:1). (B) LNP GFP mRNA systems containing 40 mol % ESM (ESMMC3/Chol/PEG-lipid 40:33:25.5:1.5).

Figure 5

LNP GFP mRNA systems containing 40 mol % ESM exhibit enhanced accumulation in spleen and bone marrow 48 h post injection compared with LNP GFP mRNA systems with Onpattro (10 mol % helper lipid) lipid compositions DiD-labeled LNP GFP mRNA were administered i.v. at a dose of 1.5 mg mRNA/kg. LNP GFP mRNA systems with the Onpattro lipid composition contained DSPC/MC3/Chol/PEG-lipid 10:50:38.5:1. LNP GFP mRNA systems containing 40 mol % ESM had the lipid composition ESMMC3/Chol/PEG-lipid 40:33:25.5:1.5. Organs were isolated 48 h post administration and DiD fluorescence was determined in cell isolates using flow cytometry. The MFI was calculated from the FACS contour plots (see methods) and normalized to PBS-treated values. Bar graphs depict mean fold increase of DiD in indicated tissues. Each data point represents arithmetic mean ± SD of five mice. ∗p < 0.05.

Figure 6

LNP GFP mRNA systems containing 40 mol % ESM exhibit superior protein expression in liver (C57B16 mice) at 4 and 24 h post injection compared with systems containing 10 mol % ESM Flow cytometry results showing LNP uptake and GFP expression in hepatocytes. For original flow data see Figure S3A. (A) Bar graphs represent the arithmetic mean ± SD of the percentage of DiD+ cells and (B) the arithmetic mean ± SD of the percentage of cells co-expressing DiD and GFP. ∗p < 0.05. The data represent three measurements of duplicate mice in three different experiments.

Figure 7

LNP GFP mRNA systems containing 40 mol % ESM exhibit superior protein expression in spleen (C57B16 mice) at 24 h post injection compared with systems containing 10 mol % ESM Flow cytometry results showing LNP uptake and GFP expression in splenocytes. For original flow data see Figure S3B. (A) Bar graphs represent the arithmetic mean ± SD of the percentage of DiD+ cells and (B) the arithmetic mean ± SD of the percentage of cells co-expressing DiD and GFP. ∗p < 0.05. The data represent three measurements of duplicate mice in three different experiments.

Figure 8

LNP GFP mRNA systems containing 40 mol % ESM exhibit superior protein expression in bone marrow (C57B16 mice) at 4 and 24 h post injection compared with systems containing 10 mol % ESM Flow cytometry results showing LNP uptake and GFP expression in bone marrow cells. For original flow data see Figure S3C. (A) Bar graphs represent the arithmetic mean ± SD of the percentage of DiD+ cells and (B) the arithmetic mean ± SD of the percentage of cells co-expressing DiD and GFP. ∗p < 0.05. The data represent three measurements of duplicate mice in three different experiments.