Inside out: optimization of lipid nanoparticle formulations for exterior complexation and in vivo delivery of saRNA

Abstract

Self-amplifying RNA (saRNA) is a promising biotherapeutic tool that has been used as a vaccine against both infectious diseases and cancer. saRNA has been shown to induce protein expression for up to 60 days and elicit immune responses with lower dosing than messenger RNA (mRNA). Because saRNA is a large (~9500 nt), negatively charged molecule, it requires a delivery vehicle for efficient cellular uptake and degradation protection. Lipid nanoparticles (LNPs) have been widely used for RNA formulations, where the prevailing paradigm is to encapsulate RNA within the particle, including the first FDA-approved small-interfering siRNA therapy. Here, we compared LNP formulations with cationic and ionizable lipids with saRNA either on the interior or exterior of the particle. We show that LNPs formulated with cationic lipids protect saRNA from RNAse degradation, even when it is adsorbed to the surface. Furthermore, cationic LNPs deliver saRNA equivalently to particles formulated with saRNA encapsulated in an ionizable lipid particle, both in vitro and in vivo. Finally, we show that cationic and ionizable LNP formulations induce equivalent antibodies against HIV-1 Env gp140 as a model antigen. These studies establish formulating saRNA on the surface of cationic LNPs as an alternative to the paradigm of encapsulating RNA.

Fig. 1

Characterization of saRNA lipid nanoparticle formulations. a Schematic of saRNA formulated on the interior or exterior of the lipid nanoparticles, with ionizable (C12-200) or cationic (DDA, DOTAP) complexing lipids. b Particle diameter (in nm) as determined by Nanoparticle tracking analysis (NTA) (bar graph) and their related polydispersity index (empty circles). c Surface charge of the LNPs as determined by zeta potential analysis measured on by the Zetasizer instrument. Bars represent means ± standard deviations for n = 3 for particle size and surface charge data

Fig. 2

Transmission electron micrographs of LNP formulations. LNPs with saRNA on the interior (a–c) or exterior (d–f) of the particle, with C12-200 (a, d), DDA (b, e), and DOTAP (c, f) as the complexing lipid. Scale bar = 100 nm

Fig. 3

Transfection efficiency of fLuc-encoding saRNA delivered on the interior or exterior of LNPs. saRNA was transfected under standard conditions without FCS ((−) FCS), in the presence of FCS ((+) 50% FCS), or in the presence of exogenous RNAse ((+) RNAse). Luciferase expression was evaluated 24 h after transfection and is expressed as relative light units (RLU). Bars represent means ± standard deviations for n = 3

Fig. 4

Effect of interior or exterior complexation on the protection of saRNA from RNAse degradation. a Formulations were subjected to RNAse, then purified by phenol–chloroform extraction, and analyzed by gel electrophoresis. Bars represent means ± standard deviations of the intensity of the nondegraded RNA band normalized to an equivalent sample that was not subjected to RNAse treatment for n = 2 samples. b Representative image of denaturing gel electrophoresis (Int: interior, Ext: exterior)

Fig. 5

In vivo luciferase expression of saRNA complexed to the interior or exterior of LNPs. a In vivo imaging (IVIS) visualization of mice injected intramuscularly with 5 μg of fLuc saRNA per leg and imaged 7 days after injection. b Quantification of IVIS luciferase expression with a line at the mean ± standard deviation for n = 5 mice (n = 10 legs) per group. Units are expressed as relative light units (RLU). Asterisk indicates significance applying an unpaired t-test with *p < 0.05 (p-values indicated); n.s., nonsignificant

Fig. 6

Antibody titers after immunization with HIV-1 Env gp140 saRNA complexed to the interior or exterior of LNPs. Bars represent means HIV-1 Env gp140-specific serum IgG antibody titer ± SEM, as determined by ELISA for n = 5 mice at each time point