Efficient hepatic delivery and protein expression enabled by optimized mRNA and ionizable lipid nanoparticle

Abstract

mRNA is a novel class of therapeutic modality that holds great promise in vaccination, protein replacement therapy, cancer immunotherapy, immune cell engineering etc. However, optimization of mRNA molecules and efficient in vivo delivery are quite important but challenging for its broad application. Here we present an ionizable lipid nanoparticle (iLNP) based on iBL0713 lipid for in vitro and in vivo expression of desired proteins using codon-optimized mRNAs. mRNAs encoding luciferase or erythropoietin (EPO) were prepared by in vitro transcription and formulated with proposed iLNP, to form iLP171/mRNA formulations. It was revealed that both luciferase and EPO proteins were successfully expressed by human hepatocellular carcinoma cells and hepatocytes. The maximum amount of protein expression was found at 6 h post-administration. The expression efficiency of EPO with codon-optimized mRNA was significantly higher than that of unoptimized mRNA. Moreover, no toxicity or immunogenicity was observed for these mRNA formulations. Therefore, our study provides a useful and promising platform for mRNA therapeutic development.

Keywords: Codon optimization; Erythropoietin; Lipid nanoparticle; mRNA delivery; mRNA therapy.

Graphical abstract

Fig. 1

Characterization of the physicochemical properties of iLP171/mRNA formulation. (a) Schematic diagram and (b) physicochemical parameters of iLP171/mRNA. PDI, polydispersity index. (c) TEM image of iLP171/mRNA nanoparticle. (d) Determination of pKa value of iLP171 by in situ TNS fluorescence titration. The pKa value was calculated with GraphPad Prism 8.0 software. Data are presented as mean ± SD. n = 6.

Fig. 2

Cellular uptake and intracellular distribution of iLP171/mRNA in Huh7 cells. (a) Confocal images of Huh7 cells after transfected with Lipofectamine2000/siRNA, Lipofectamine2000/mRNA, iLP171/siRNA or iLP171/mRNA for 4 h. (b and c) The cellular uptake of nucleic acid recorded by FACS. (c) Quantitative analysis of (b). Data were shown as the mean ± SD. ns: no significant difference. Scale bar: 20 μm.

Fig. 3

In vitro protein expression mediated by iLP171/mRNA. (a) Cumulative luciferase expression in Huh7 cells after being transfected with iLP171/mLuc. (b) EPO protein expression in Huh7 receiving the transfection of iLP171/mEPO (mRNA without codon optimization) or iLP171/omEPO (mRNA with codon optimization). (c) The EPO protein expression of iLP171/omEPO in Huh7 cells was recorded at various time points after cells were transfected at three different concentrations.

Fig. 4

In vivo biodistribution of iLP171/mRNA. The animals were treated with PBS, or iLP171/siRNA, or iLP171/mRNA by intravenous injection. At indicated time points, whole-bodies of the mice (a) or the main organs (b) were fluorescently examined using an in vivo imaging system. Red circles indicate the liver positions in living mice. The mean fluorescence intensities of the liver in living mice (c) and the main organs from sacrificed animals (d–f) were quantitatively analyzed. SmG, submandibular gland; T, thymus; H, heart; L, lung; P, pancreas; Sp, spleen.

Fig. 5

In vivo protein expression of iLP171/mRNA in mice. (a) Bioluminescence imaging of the mice receiving intravenous injection of PBS or iL171/mLuc (2 mg/kg). n = 2 or 3. (b) Luciferase expression recorded with the homogenates of 50 μg of tissues collected at 1.5 h, 6 h and 24 h after administration. (c) Quantitative analysis of the livers in (a). (d) iLP171/omEPO was injected into mice at a dose of 1 mg/kg, and EPO protein level in circulation was determined at 6 h and 24 h post injection with ELISA Kit.

Fig. 6

In vivo safety evaluation of iLP171/mRNA. The PBS, iLP171/mLuc or iLP171/omEPO were intravenously injected into the mice at the dose of 2 mg/kg (for iLP171/mLuc) or 1 mg/kg (for iLP171/omEPO). (a) H&E staining of the major organ sections were performed 48 h after injection. (b and c) Cytokine stimulation of these formulations recorded at 6 h (b) and 24 h (c) post injection. Scale bar: 200 μm.