Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes

Abstract

In recent years, in vitro transcribed messenger RNA (mRNA) has emerged as a potential therapeutic platform. To fulfill its promise, effective delivery of mRNA to specific cell types and tissues needs to be achieved. Lipid nanoparticles (LNPs) are efficient carriers for short-interfering RNAs and have entered clinical trials. However, little is known about the potential of LNPs to deliver mRNA. Here, we generated mRNA-LNPs by incorporating HPLC purified, 1-methylpseudouridine-containing mRNA comprising codon-optimized firefly luciferase into stable LNPs. Mice were injected with 0.005-0.250mg/kg doses of mRNA-LNPs by 6 different routes and high levels of protein translation could be measured using in vivo imaging. Subcutaneous, intramuscular and intradermal injection of the LNP-encapsulated mRNA translated locally at the site of injection for up to 10days. For several days, high levels of protein production could be achieved in the lung from the intratracheal administration of mRNA. Intravenous and intraperitoneal and to a lesser extent intramuscular and intratracheal deliveries led to trafficking of mRNA-LNPs systemically resulting in active translation of the mRNA in the liver for 1-4 days. Our results demonstrate that LNPs are appropriate carriers for mRNA in vivo and have the potential to become valuable tools for delivering mRNA encoding therapeutic proteins.

Keywords: Luciferase; Nanoparticle; Non-viral gene delivery; Pseudouridine; mRNA.

Fig. 1

Efficiency of complexing reagents for in vitro luciferase mRNA transfection. Firefly luciferase expression following transfection of (A) HEK293T cells and human monocyte derived dendritic cells with Lipofectin (1 μg mRNA), TransIT (0.1 μg mRNA) or LNP-encapsulated luciferase mRNA (0.1 μg mRNA). ApoE recombinant protein was incubated with mRNA-LNPs where indicated prior to addition to the cells. (B) Firefly luciferase expression in HEK293T cells at 6, 12, and 24 hours following transfection of luciferase-encoding mRNA complexed with either Lipofectin, TransIT or encapsulated in LNPs. Error bars are standard error of the mean (SEM). p-value determined by paired student’s t-test.

Fig. 2

Duration and translational pattern of mRNA-LNPs in mice injected by various routes. Representative IVIS images of groups of 3 BALB/c mice injected with 5.0 μg mRNA-LNP by the intradermal (i.d.), intramuscular (i.m.), subcutaneous (s.c.), intravenous (i.v.), intraperitoneal (i.p.) and intratracheal (i.t.) routes. Relative luminescence plot is shown and the scale of luminescence is indicated.

Fig. 3

Translational kinetics of mRNA-LNP delivered by different routes in vivo. Quantification of the bioluminescent signal measured in BALB/c mice injected with (A) 0.1 μg, (B) 1.0 μg or (C) 5.0 μg mRNA-LNPs by intradermal (i.d.), intramuscular (i.m.), subcutaneous (s.c.), intravenous (i.v.), intraperitoneal (i.p.) and intratracheal (i.t.) routes. Error bars are standard error of the mean (SEM).

Fig. 4

Total amount of protein produced for each amount of mRNA administered at each delivery site. Area under the curve was calculated as described in the Materials and Methods. The reduction beyond the expected linear dose response for 0.1 μg of mRNA delivered by the i.v. route was likely due to the observation that a similar amount of mRNA-LNPs remained in the eye socket for each dose delivered and this amount was a substantial fraction delivered for the 0.1 μg dose.