Key words
LNP, IL-21, HBV, mouse models, antiviral therapy
Introduction
Currently, ~250 million people are chronically infected
with hepatitis B virus (HBV), which seriously threatens human life and
health [1]. Chronic hepatitis B (CHB) infection is recognized a
major factor for the development of liver fibrosis, liver cirrhosis and
hepatocellular carcinoma [2]. World Health Organization estimated
that ~0.68 million people die of HBV-related liver
diseases annually [1].
HBV is a prototypical member of hepadnaviridae family and is
exclusively hepatotropic in vivo [3]. After HBV’s entry into
hepatocytes through receptor NTCP, its ~3.2 kb partially
double-stranded relaxed circular DNA (rcDNA) genome is delivered into
the nucleus and converted into covalently closed circular DNA (cccDNA),
which then serves as the sole transcription template for all HBV mRNAs
[2, 3]. 3.5 kb pregenomic RNA (pgRNA) functions as the template for
translation of polymerase (Pol) and hepatitis core protein (HBc) while
3.5 kb preC RNA encodes hepatitis e antigen (HBeAg) [3]. The
subsequent binding of pgRNA to Pol initiates synthesis of progeny viral
genome inside the newly formed nucleocapsid via reverse transcription.
The mature nucleocapsids with rcDNA are either secreted from infected
cells after enveloped by large/middle/small hepatitis B antigens
(L/M/SHBsAg), translated from 2.4 and 2.1 kb RNAs, or recycled back to
nucleus to replenish cccDNA pool [3, 4]. Indeed, it is generally
accepted that cccDNA is vital for HBV persistence [4]. Currently,
clinical antiviral drugs, such as interferon α (IFN-α) and nucleot(s)ide
analogous, fail to eradicate cccDNA pool even if in patients after
functional cure [5, 6]. It is urgent to develop new antiviral
therapies targeting cccDNA.
Previously, we established an HBV persistence mouse model based on a
single hydrodynamic injection (HDI) of an HBV replicon plasmid (termed
BPS) via tail vein [7]. Mechanism study of persistence revealed that
IL-21 is a potent inducer of HBV clearance and injection of
IL-21-expressing plasmids or adeno-associated virus (AAV-IL-21) into
mice with pre-established BPS persistence facilitated HBV clearance
[7, 8]. Although gene therapy based on AAV vector has been approved
for clinical treatment, such viral delivery system is restricted, to
some extent, by the cost, viral immunogenicity and exogenous DNA
integration [9, 10]. Recently,
lipid nanoparticle (LNP) delivery
system carrying messenger RNA (mRNA) of interested gene(s) has been
believed to overcome these challenges and emerged as an effective
platform for the treatment of infectious diseases and cancers
[11-13].
In this work, we aimed to develop
LNP formulated with IL-21 mRNA (LNP-IL-21) and determine its therapeutic
effects against HBV. Firstly, in order to compare the differences caused
by LNP and AAV vectors in vivo , transcriptome sequencing was
performed on liver tissues of mice post LNP or AAV injection. Secondly,
we characterized the stability, safety, expression efficiency and
biodistribution of LNP-IL-21 in vitro and in vivo .
Thirdly, IL-21-induced antiviral effects and underlying mechanisms were
explored in two different mouse models of HBV persistence. Lastly, and
more importantly, we also evaluated the antiviral effects of
LNP-IL-21-based cellular therapy, in which peripheral blood mononuclear
cells (PBMC) from BPS persistence mice were stimulated with LNP-IL-21
plus recombinant HBsAg (rHBsAg) before infusion into treatment-naïve BPS
persistence mice. Results presented here demonstrated that both
LNP-IL-21-based gene and cellular therapies displayed potent antiviral
effects via activation of HBV specific humoral and cellular immune
responses.
Materials and methods