Characterization of lipid nanoparticle carrying IL-21 mRNA
(LNP-IL-21)
In order to prepare LNP-IL21, IL-21 mRNA was firstly produced usingin vitro transcription system and added with a cap structure at
5’ terminal to improve its translational efficiency (Figure
S1A-B ). Primary mouse hepatocytes transfected with IL-21 mRNA indeed
secreted great amounts of IL-21 into culture media compared to untreated
cells (Figure S1C ). Next, cationic lipids were combined with
set ratios of lipid-anchored PEG, cholesterol, disaturated phosphatidyl
choline (DSPC) mixed through microfluidic devices with IL-21 mRNA to
generate LNP-IL-21 (Figure 1A ). The diameters of resulting
LNP-IL-21, described as the z-average measurement, ranged from 125.9 to
129.9 nm with polymer dispersity index (PDI) below
0.2 (Figure 1B ).
Transmission electron microscopy presented a well-formed spherical
structure (Figure 1C ).
Secondly, to investigate the expression efficiency and cytotoxicity of
LNP-IL-21 in vitro , both primary mouse hepatocytes and primary
human hepatocytes were transfected with LNP-IL-21, control LNP or left
untreated. ELISA assay revealed that LNP-IL-21-transfected cells, but
not LNP-transfected or untreated cells, could secrete great amounts of
IL-21 into culture media (Figure 1D, Figure S2A ). And, CCK8
assay revealed that transfection with LNP-IL-21 in a dose-dependent
manner did not obviously affect cell viability (Figure 1E,
Figure S2B ).
Thirdly, the biodistribution of IL-21 expression patterns in vivowas evaluated via injection of LNP-IL-21 or LNP particles into BALB/c
mice via tail vein. At 3 days post injection, mice were sacrificed to
collect major organs, including heart, liver, spleen, lung and kidney
tissues. In addition to prominent IL-21 expression in liver tissues from
LNP-IL-21-injected mice, IL-21 was also observed in spleen and, to a
lesser extent, in kidney tissues (Figure 1F ), indicating LNP
preferred to target liver tissues.
Among commonly used AAV types for gene therapy, serotype 8 is generally
accepted to harbor higher liver-tropism [20]. In order to compare
intrahepatic gene expression profiles based on transduction by different
gene therapy vectors, liver tissues collected from BALB/c mice injected
with LNP or serotype 8 AAV vector were subjected to RNA transcriptome
sequencing. As shown in Figure S3 , a total of 945 differently
expressed genes (DEGs) (442 up-regulated and 503 down-regulated genes,Table S1 ) was observed post LNP injection, while up to 1695
DEGs (939 up-regulated and 756 down-regulated genes, Table S2 )
observed post AAV injection, indicating LNP-based delivery system might
be much safer than AAV system. Collectively, these results showed that
LNP-IL-21 potently expressed IL-21 in vitro and in vivowithout provoking obvious adverse effects.
Given that the storage conditions are vital factors for the
effectiveness of LNP particles[21], LNP-IL-21 were kept at 4℃, a
commonly recommended temperature for LNP storage, for 0, 2, 4 and to 6
weeks separately prior to be analyzed for stability and expression
efficiency. As shown in Figure 1G-H , particle sizes were
similarly comparable at week 0 and 2, but afterwards significantly
increased from week 4 to 6 accompanied by decreases of mRNA
concentration. To analyze the expression efficiency, primary mouse
hepatocytes or BALB/c mice were transfected or injected with LNP-IL-21
stored for different weeks. IL-21 levels in culture medium or serum
displayed highly positive association with mRNA concentration of
LNP-IL-21 (Figure 1H-J ). Notably, serum IL-21 levels peaked at
1 week post injection of LNP-IL-21 (w.p.i.) and dropped to detection
limit by nearly 3-4 w.p.i. (Figure 1J ). These data suggested
that storing LNP-IL-21 at 4℃ for up to 2 weeks did not apparently impair
its stability and IL-21 expression efficiency in vitro andin vivo .