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\begin{document}

\title{Lipid nanoparticle-mediated delivery of mRNA for IL-21 achieves
clearance of hepatitis B virus in mouse models}



\author[1]{Zhongliang Shen}%
\author[1]{Shenyan Zhang}%
\author[2]{Nannan Liu}%
\author[1]{Qirong Jiang}%
\author[2]{Zixiang Gao}%
\author[3]{Weiju Hao}%
\author[2]{Qiang Deng}%
\author[2]{Jing Liu}%
\author[1]{Jiming Zhang}%
\author[1]{You-hua Xie}%
\affil[1]{Huashan Hospital Fudan University Department of Infectious Diseases}%
\affil[2]{Fudan University School of Basic Medical Sciences}%
\affil[3]{University of Shanghai for Science and Technology}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
\let\center\flushleft
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\endgroup





\selectlanguage{english}
\begin{abstract}
Chronic hepatitis B virus (HBV) infection causes hepatitis, liver
cirrhosis and hepatocellular carcinoma. Covalently closed circular DNA
(cccDNA) is the transcription template for HBV RNAs and not affected by
current treatment options. Effective therapeutics with ability to remove
cccDNA need to be developed. Previously, we established an HBV
persistence mouse model via hydrodynamic injection of a clinical isolate
(BPS) and identified IL-21 as a potent inducer of viral clearance. Here,
we aimed to explore the anti-HBV effects of IL-21 messenger RNA (mRNA)
delivered by lipid nanoparticle (LNP-IL-21) system. First, LNP-IL-21 was
prepared and analyzed for its safety, expression, biodistribution and
stability \emph{in vitro} and \emph{in vivo}. Next, LNP-IL-21 was
injected into two HBV persistence mouse models based on BPS and
recombinant cccDNA (rcccDNA) respectively. LNP-IL-21 administration
successfully cleared HBV serum markers, and more importantly, BPS
replicons and rcccDNA in livers, which was associated with activation of
viral specific immune responses. Notably, transfer of peripheral blood
mononuclear cells from BPS persistence mice stimulated \emph{ex vivo}
with LNP-IL-21 and viral antigen could induce HBV clearance in recipient
mice. These findings suggested that both LNP-IL-21-based gene and
cellular therapies provided novel therapeutic strategies against chronic
HBV infection.%
\end{abstract}%



\sloppy


\emph{Title} \textbf{}

Lipid nanoparticle-mediated delivery of mRNA for IL-21 achieves
clearance of hepatitis B virus in mouse models

Authors

Zhongliang Shen\textsuperscript{1,2\#,*}, Shenyan
Zhang\textsuperscript{1\#}, Nannan Liu\textsuperscript{2\#}, Qirong
Jiang\textsuperscript{1}, Zixiang Gao\textsuperscript{2}, Weiju
Hao\textsuperscript{3}, Qiang Deng\textsuperscript{2}, Jing
Liu\textsuperscript{2*}, Jiming Zhang\textsuperscript{1,2*}and Youhua
Xie\textsuperscript{1,2*}

\textsuperscript{1}Department of Infectious Diseases, Shanghai Institute
of Infectious Diseases and Biosecurity, Shanghai Key Laboratory of
Infectious Diseases and Biosafety Emergency Response, National Medical
Center for Infectious Diseases, Huashan Hospital, Fudan University,
Shanghai, China

\textsuperscript{2}Key Laboratory of Medical Molecular Virology
(Ministry of Education/National Health Commission/Chinese Academy of
Medical Sciences), Department of Microbiology and Parasitology, School
of Basic Medical Sciences, Shanghai Medical College, Fudan University,
Shanghai 200032, China

\textsuperscript{3} School of Materials and Chemistry, University of
Shanghai for Science and Technology, Shanghai 200093, China

\textbf{* Correspondence authors:}

\textbf{Zhongliang Shen and Jiming Zhang}

Address: Department of Infectious Diseases, Huashan Hospital, 12
Mid-Wulumuqi Road, Shanghai 200040, China.

Tel: +86 021-5288-8125; Fax: +86 021-6248-9191.

Email: shenzhongliangnjnu@163.com (Z.S.); jmzhang@fudan.edu.cn (J.Z.).

And

\textbf{Jing Liu and Youhua Xie}

Address: Shanghai Medical College, 131 Dong'an Road, Shanghai 200032,
China.

Tel: +86 021-5423-7972; Fax: +86 021-5423-7973.

Email: liujing212@fudan.edu.cn (J.L.); yhxie@fudan.edu.cn (Y.X.).

\textbf{\# These authors contributed equally to this work.}

\textbf{Running title:} LNP-IL-21 induced HBV clearance \emph{in vivo}

Abstract

Chronic hepatitis B virus (HBV) infection causes hepatitis, liver
cirrhosis and hepatocellular carcinoma. Covalently closed circular DNA
(cccDNA) is the transcription template for HBV RNAs and not affected by
current treatment options. Effective therapeutics with ability to remove
cccDNA need to be developed. Previously, we established an HBV
persistence mouse model via hydrodynamic injection of a clinical isolate
(BPS) and identified IL-21 as a potent inducer of viral clearance. Here,
we aimed to explore the anti-HBV effects of IL-21 messenger RNA (mRNA)
delivered by lipid nanoparticle (LNP-IL-21) system. First, LNP-IL-21 was
prepared and analyzed for its safety, expression, biodistribution and
stability\emph{in vitro} and \emph{in vivo} . Next, LNP-IL-21 was
injected into two HBV persistence mouse models based on BPS and
recombinant cccDNA (rcccDNA) respectively. LNP-IL-21 administration
successfully cleared HBV serum markers, and more importantly, BPS
replicons and rcccDNA in livers, which was associated with activation of
viral specific immune responses. Notably, transfer of peripheral blood
mononuclear cells from BPS persistence mice stimulated \emph{ex vivo}
with LNP-IL-21 and viral antigen could induce HBV clearance in recipient
mice. These findings suggested that both LNP-IL-21-based gene and
cellular therapies provided novel therapeutic strategies against chronic
HBV infection.

Key words

LNP, IL-21, HBV, mouse models, antiviral therapy

Introduction

Currently, \textasciitilde{}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 \textasciitilde{}0.68 million people die of HBV-related liver
diseases annually {[}1{]}.

HBV is a prototypical member of \emph{hepadnaviridae} family and is
exclusively hepatotropic \emph{in vivo} {[}3{]}. After HBV's entry into
hepatocytes through receptor NTCP, its \textasciitilde{}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 \selectlanguage{greek}α \selectlanguage{english}(IFN-\selectlanguage{greek}α\selectlanguage{english}) 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
\emph{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 \emph{in vitro} and \emph{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-na\selectlanguage{ngerman}ïve 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

\textbf{Plasmid and recombinant viruses}

BPS plasmid containing 1.3 fold over-length HBV genome (GenBank
Accession Number: AF100309.1) has been described previously {[}7{]}.
Both recombinant human adenovirus serotype 5 harboring precursor of
recombinant cccDNA (Adv/prcccDNA) and serotype 8 adeno-associated virus
(AAV) have been also described previously {[}8, 14{]}.

\textbf{mRNA synthesis}

The murine IL-21 gene (GenBank Accession Number: NM\_021782.2) was
inserted into plasmid vector pCZ (Jirui Biotechnology, Shenzhen, China)
which incorporates 5' and 3' untranslated regions (UTRs) and includes a
polyA tail with 120 nucleotides in length. The plasmid was linearized
with BspQI and the mRNA was synthesized \emph{in vitro} using T7
polymerase. The UTP was substituted with 1-methylpseudo UTP and a cap
structure was added to increase the translation efficiency of IL-21
mRNA. The capped mRNA was purified using lithium chloride, and then
Nanodrop (Thermo Fisher Scientific) was used to determine its
concentration. The purified mRNA was subjected to 1\% agarose
electrophoresis for size check (\textbf{Figure S1} ).

\textbf{Preparation of LNP-IL-21 particles}

Lipid nanoparticle formulations were prepared using the microfluidic
mixing step as previously described {[}15{]}. Briefly, lipids were
dissolved in ethanol containing cationic lipids (ALC-0315),
lipid-anchored PEG-2000, cholesterol and disaturated phosphatidyl
choline (DSPC) with molar ratios of 46.3: 1.6: 42.7: 9.4. The IL-21 mRNA
was dissolved in 10 mM citrate buffer (pH = 4) and combined with above
lipid mixture. The ethanol phase and aqueous phase were then mixed using
a microfluidic mixer INanoL (Micro \& Nano Technology Co. Ltd.,
Shanghai, China) at a flow rate ratio of 1 to 3 with the total flow rate
of 12 ml/min. The LNP formulations were then diafiltrated using
ultrafiltration centrifugal tube (Millipore, 15ml/10kD). The samples
were then collected, diluted with 1 X PBS and stored at 4\selectlanguage{english} for further
analysis.

\textbf{Cells and transfection}

Primary mouse hepatocytes were isolated from BALB/c mice by following a
two-step perfusion protocol as previously described {[}16, 17{]}, and
cultured in Hepato-Stim Hepatocyte Defined medium (BD Biosciences).
Primary human hepatocytes were cultured as previously described {[}18,
19{]}. IL-21 mRNA transfection was performed using TurboFect
Transfection Reagent (Thermo Fisher Scientific) according to the
manufacturer's instructions. For LNP transfection, LNP particles were
diluted in serum free medium at a ratio of 1:5 and kept for 20 minutes
at room temperature, and then added into the cultured cells. Medium was
changed at 6-8 hours post transfection.

\textbf{Mice work}

Male BALB/c mice aged 6-8 weeks were purchased from SLAC, Shanghai and
housed in Experimental Animal Centre, Fudan University. Male C57BL/6
mice aged 6-8 weeks transgenic for Cre recombinase (Cre Tg C57BL/6) has
been described previously {[}14{]}. To construct BPS persistence mice,
BALB/c mice were injected with 10 \selectlanguage{greek}μ\selectlanguage{english}g of BPS plasmids through HDI method
via tail vein and selecting mice positive for serum HBV antigens for 4
weeks. To construct rcccDNA persistence mice, Cre Tg C57BL/6 mice were
injected with 1.5 \selectlanguage{ngerman}× 10\textsuperscript{9} plaque-forming units (PFU) of
Adv/prcccDNA in 200 \selectlanguage{greek}μ\selectlanguage{english}l PBS via tail vein and selecting mice positive for
serum HBV antigens for 4 weeks. For evaluating the therapeutic effects
of IL-21, HBV persistence mice were injected with 10 \selectlanguage{greek}μ\selectlanguage{english}g of LNP-IL-21 or
control LNP in 200 \selectlanguage{greek}μ\selectlanguage{english}l PBS via tail vein, and again two weeks later.
Blood samples, liver and spleen tissues were collected at indicated time
points for further analysis.

For \emph{ex vivo} LNP-IL-21 stimulation, PBMC isolated from BPS
persistence mice were cultured in RPMI containing 2 mM L-glutamine, 50
U/ml penicillin, and 10\% fetal bovine serum (all from Invitrogen,
China) and transfected with 1 \selectlanguage{greek}μ\selectlanguage{english}g of LNP-IL-21 or LNP. At 6 hours post
transfection, recombinant HBsAg (rHBsAg) was added in culture media (5
\selectlanguage{greek}μ\selectlanguage{english}g/mL). rHBsAg was kindly provided by Prof. Di Qu, Fudan University. At
3 days post rHBsAg stimulation, 1 \selectlanguage{ngerman}× 10\textsuperscript{6} cells were
injected into each treatment-naïve BPS persistence mice via tail vein.
Mouse handling procedures were approved by the Animal Ethics Committee
of School of Basic Medical Sciences, Fudan University. The approval
number was JS-028.

\textbf{Flowcytometry analysis}

Recombinant HBsAg (rHBsAg, serotype adr) was purchased from Bioforce
(China). Purified rHBsAg and hen egg lysozyme (HEL) were conjugated with
AlexaFlour 647 (Thermo Fisher Scientific) and iFlour488 (Abcam, China)
to generate HBsAg-AF647 and HEL-iFlour488, respectively. HEL-iFlour488
was used for monitoring nonspecific binding. Murine peripheral blood
cells were co-stained with Fixable LIVE/DEAD Dye (Life Technologies,
Thermo Fisher Scientific), anti-mouse B220 (clone RA3-6B2, eBioscience)
Super Bright 436, HBsAg-AF647 and HEL-iFlour488 in FcR-blocking reagent
(Biolegend) at 4°C for 30 minutes. All data were acquired with Attune\selectlanguage{english}
NxT Flow Cytometer (Invitrogen), and then analyzed with FlowJo
(TreeStar, Ashland, OR, USA). At least 5 x 10\textsuperscript{5} events
were acquired per run.

\textbf{Serum analyses}

HBsAg, HBsAb and HBeAg were analyzed with ELISA kits (Kehua, China). HBV
DNA, alanine aminotransferase (ALT), total bilirubin and creatine kinase
were analyzed using commercial quantitative assays (Adicon, China). Both
serum and intracellular IL-21 levels were measured using ELISA (Boster,
China).

\textbf{Cell viability assay}

1 x 10\textsuperscript{4} primary mouse or human hepatocytes were
cultured in 96-well plate and transfected with 0.025-0.1 \selectlanguage{greek}μ\selectlanguage{english}g of LNP-IL-21
or LNP, or left untreated. Cell viability was assessed at day 1-4 post
transfection with using a Cell Counting Kit-8 kit (Dojindo, Japan)
according to the manufacturer's instructions.

\textbf{RNA sequencing analysis}

BALB/c mouse was injected with LNP or 2 \selectlanguage{ngerman}× 10\textsuperscript{11} genome
equivalents (geq) of AAV in 200 \selectlanguage{greek}μ\selectlanguage{english}l PBS via tail vein, or left untreated.
At 3 days post injection, RNA extracted form liver tissues was subjected
to library construction and deep sequencing on Illumina NovaSeq 6000 by
Novogene Biotechnology Co. Ltd. (Shanghai, China). FPKM, fragments per
kilobase of transcript per million mapped reads, was used to estimate
gene expression levels. Differential expression analysis of two groups
was performed using the DESeq2 R package (1.20.0). Differentially
expressed genes (DEGs) with a fold change [?] 2 and a false discovery rate
(FDR) \textless{} 0.05 were identified and presented in \textbf{Table
S1} and \textbf{S2} .

\textbf{Southern blot, PCR and Western blot}

HBV core-associated DNA, nuclear DNA and core capsids were extracted
from liver tissues and subjected to Southern blot, PCR and Western blot
as previously described {[}7, 18{]}. The primer sequences were listed
in\textbf{Table S3} . The antibody against capsid was purchased from
DAKO (1:10000, Copenhagen, Denmark).

\textbf{T cell immune responses analysis}

Mouse splenocytes cultured in RPMI 1640 (Invitrogen) containing 2 mM
L-glutamine, 50 U/mL penicillin, and 10\% fetal bovine serum
(Invitrogen) were stimulated with 15 \selectlanguage{greek}μ\selectlanguage{english}g/mL rHBsAg or 10 \selectlanguage{greek}μ\selectlanguage{english}g/mL
recombinant HBcAg (Catalog \#hbv-232-a, ProSpec, China) for 72 h.
Interferon gamma (IFN-\selectlanguage{greek}γ\selectlanguage{english}) in culture medium was measured using ELISA kits
(Catalog \#D721025, Sango, China).

\textbf{Immunohistochemistry}

Liver tissues sections were subjected to H\&E staining plus
immunostaining with anti- anti-CD8 (Servicebio, China).

\textbf{Statistical analysis}

Means and standard errors (SEMs) from different treatment groups were
subjected to unpaired two-tailed \emph{t} -test. Group positivity
percentages are presented as Kaplan-Meier plot and subjected to log-rank
(Mantel-Cox) test. GraphPad 6 was used for plotting and statistical
tests.

Results

\textbf{Characterization of lipid nanoparticle carrying IL-21 mRNA
(LNP-IL-21)}

In order to prepare LNP-IL21, IL-21 mRNA was firstly produced
using\emph{in vitro} transcription system and added with a cap structure
at 5' terminal to improve its translational efficiency (\textbf{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 (\textbf{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 (\textbf{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 (\textbf{Figure
1B} ). Transmission electron microscopy presented a well-formed
spherical structure (\textbf{Figure 1C} ).

Secondly, to investigate the expression efficiency and cytotoxicity of
LNP-IL-21 \emph{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 (\textbf{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 (\textbf{Figure 1E,
Figure S2B} ).

Thirdly, the biodistribution of IL-21 expression patterns \emph{in
vivo}was 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 (\textbf{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 \textbf{Figure S3} , a total of 945 differently
expressed genes (DEGs) (442 up-regulated and 503 down-regulated
genes,\textbf{Table S1} ) was observed post LNP injection, while up to
1695 DEGs (939 up-regulated and 756 down-regulated genes, \textbf{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 \emph{in vitro} and \emph{in
vivo}without 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 \textbf{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 (\textbf{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. (\textbf{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 \emph{in vitro}
and\emph{in vivo} .

\textbf{LNP-IL-21 induced HBV clearance in BPS-based persistence mouse
model}

To investigate whether LNP-IL-21 had any therapeutic effects against HBV
persistence, BALB/c mice displaying persistently positive serum HBV
antigens up to 4 weeks post injection of BPS were subjected to two-dose
LNP-IL-21 injections, two weeks apart, or control LNP via tail vein.
Serum HBV markers and biochemical indexes were regularly measured every
2 weeks up to 8 weeks post injection of LNP (w.p.i.). As shown
in\textbf{Figure 2 A} and \textbf{C} , LNP-IL-21 administration caused
HBsAg and HBeAg levels to decrease much more quickly compared to mice
(\emph{n} = 6) receiving LNP injection, and become undetectable in all
LNP-IL-21-injected mice (\emph{n} = 6) by 6 w.p.i., while both viral
markers persisted in 5 out of 6 LNP-injected mice. Meanwhile, all
LNP-IL-21-injected mice which had cleared serum HBsAg displayed HBsAb
seroconversion by 6 w.p.i. (\textbf{Figure 2B} ), indicating anti-HBsAg
humoral responses was involved in HBsAg clearance. Accordingly, HBV DNA
in pooled serum started to decrease following LNP-IL-21 injection and
dropped to detection limit level by 8 w.p.i., while it remained largely
unchanged in LNP-injected group (\textbf{Figure 2D} ).
LNP-IL-21-injected mice manifested ALT and IL-21 elevations at 3 w.p.i.,
which reverted to normal levels by 6 and 8 w.p.i. respectively
(\textbf{Figure 2E-F} ). In contrast, no obvious changes in ALT or
IL-levels were detected in LNP-injected mice (\textbf{Figure 2E-F} ).
Total bilirubin and creatine kinase levels were generally normal in both
two groups (\textbf{Figure S4} ).

In addition to serum HBV markers analyses, both intrahepatic HBV DNA and
capsids were assayed using Southern and Western blots to confirm true
clearance at 8 w.p.i. (\textbf{Figure 3A)} . As shown in \textbf{Figure
3B} , HBV replication intermediates, BPS replicons DNA or capsids were
undetectable in all LNP-IL-21-injected mice (\emph{n} = 6) that had
cleared serum HBsAg, but persisted in 5 out of 6 LNP-injected mice.
Collectively, LNP-IL-21 efficiently and safely induced HBV clearance in
BPS-based persistence mouse model.

\textbf{LNP-IL-21 induced HBV clearance in rcccDNA-based persistence
mouse model}

Considering the facts that cccDNA is a key factor for HBV persistence
and mouse hepatocytes do not support cccDNA formation for unknown
reasons {[}22, 23{]}, we previously constructed a recombinant cccDNA
(rcccDNA) persistence mouse model based on Cre recombinase/Loxp system,
in which rcccDNA is produced from a precursor rccccDNA (prcccDNA) under
the action of Cre {[}14{]}. rcccDNA is highly similar to wild type
cccDNA in size except a small extraneous intron that is removed via
selective splicing upon transcription {[}14{]}.

C57BL/6 mice transgenic for Cre recombinase were injected with
adenovirus carrying prcccDNA (Adv/prcccDNA) via tail vein, and mice
positive for serum HBV antigens for up to 4 weeks were injected with
LNP-21 or LNP. As seen in \textbf{Figure 4A, C-D} , serum HBsAg, HBeAg
and HBV DNA were cleared in all LNP-IL-21-treated mice (\emph{n} = 6) by
8 w.p.i., but persisted at considerably highly levels in LNP-treated
group (\emph{n} = 6). LNP-IL-21-induced HBV clearance was associated
with HBsAb seroconversion, together with IL-21 and ALT transient
elevations which reverted to base line levels by 8 w.p.i.
(\textbf{Figure 4B, E-F} ). Serum biochemical indexes for safety
analysis, including total bilirubin and creatine kinase, maintained
largely normal between two groups (\textbf{Figure S5} ). At 8 w.p.i.,
expectedly, no intrahepatic HBV replication intermediates, rcccDNA or
capsids were detected in LNP-IL-21-injected mice (n = 6) that had
achieved HBsAg clearance (\textbf{Figure 5} ), indicating true HBV
clearance.

\textbf{LNP-IL-21 treatment successfully indued potent anti-HBV immune
responses}

LNP-IL-21 induced HBV clearance in both BPS and rcccDNA persistence
mouse models was associated with HBsAb seroconversion (\textbf{Figure 2B
and 4B} ), suggesting LNP-IL-21 treatment is probably able to activate
HBsAg-specific humoral immune responses. To prove it, at 8 w.p.i., PBMC
were collected from LNP-IL-21-cured BPS mice achieving HBsAb
seroconversion (\emph{n} = 6) and LNP-injected mice (5 out 6 mice
positive for HBsAg), and subjected to flow cytometry analysis for
HBsAg-specific B cells (\textbf{Figure 6 A, C-D} ). The percentages of
HBsAg-specific B cells in LNP-IL-21-cured mice were significantly higher
than those in LNP-injected mice (\textbf{Figure 6 E} ), and
representative images of two mice in each group were presented
in\textbf{Figure 6F} .

On the other hand, ALT transient elevations were observed in above two
mouse models post LNP-IL-21 injection (\textbf{Figure 2E and 4E} ),
indicating acute liver injury. In order to investigate whether LNP-IL-21
treatment participated in activation of cellular immune responses, which
is responsible for the removal of HBV-harboring hepatocytes to achieve
cure, mouse liver tissues were collected form LNP-IL-21-and LNP-injected
mice at 2 and 8 w.p.i., and then subjected to H\&E and anti-CD8 staining
(\textbf{Figure 6 A,} ). As shown in \textbf{Figure 6B} , marked
intrahepatic immune cell infiltration, including
CD8\textsuperscript{+}cell infiltration, was observed in
LNP-IL-21-injected mice at 2 w.p.i., but not at 8 w.p.i., which roughly
coincided with HBV clearance. No obvious immune infiltration was
observed in LNP-injected mice at 2 or 8 w.p.i.. What's more, at 8
w.p.i., splenocytes from LNP-IL-21-cured mice, but not LNP-injected
mice, secreted much more IFN-\selectlanguage{greek}γ \selectlanguage{english}post HBcAg or HBsAg stimulation
(\textbf{Figure 6G} ), indicating activated T cell immune responses. In
a summary, LNP-IL-21-based gene therapy successfully induced both potent
anti-viral humoral and cellular immune responses contributing to HBV
clearance.

\textbf{LNP-IL-21-engineered PBMC induced HBV clearance in BPS
persistence mice}

Previously, we described an IL-21-based cellular therapy, in which
splenocytes from BPS persistence mice were subjected to recombinant
IL-21 and rHBsAg co-stimulation before infusion into BPS persistence
mice, could induce HBV clearance in nearly half of recipient mice in a
cellular immune response dependent manner {[}8{]}. Given the ability of
LNP-IL-21 administration to induce potent anti-HBV humoral and cellular
immune responses \emph{in vivo} (\textbf{Figure 2-6} ) and the
possibility and operability of LNP-IL-21-based cellular therapy, PBMC,
but not splenocytes, were collected from BPS persistence mice and
co-stimulated with rHBsAg and LNP-IL-21or LNP before injection into
treatment-na\selectlanguage{ngerman}ïve BPS persistence mice (\textbf{Figure 7A} ). Compared to
mice receiving LNP/rHBsAg co-treated PBMC, those receiving
LNP-IL-21/rHBsAg co-treated PBMC displayed accelerated decreases of
serum HBsAg and HBeAg (\textbf{Figure 7B and D} ), accompanied by HBsAb
seroconversion (\textbf{Figure 7C} ) and transient ALT elevations
(\textbf{Figure 7E} ), and remained negative for serum HBV antigens by 4
w.p.i., indicating activation of humoral and cellular immune responses.
Accordingly, clearance of serum HBV antigens following injection of
LNP-IL-21/rHBsAg-co-stimulated PBMC correlated with disappearance of BPS
replicons DNA and capsids in mouse livers \textbf{(Figure 7G} ), proving
true clearance.

Discussion

In practice, successful functional cure, termed as loss of serum HBsAg
with or without HBsAg appearance, could be achieved in a small fraction
of CHB patients after antiviral treatment. However, hepatitis B may be
reactivated due to cccDNA persistence in hepatocytes {[}2, 6{]}. In an
effort to achieve CHB pure cure that is removal of cccDNA from HBV
infected hepatocytes, new therapeutic methods including drug candidates
and delivery systems are urgently required {[}1, 6{]}. We previously
reported that injection of IL-21-expressing AAV (AAV-IL-21) into both
BPS-based and rcccDNA-based HBV persistence mice could induce clearance
of serum HBV markers, and more importantly BPS replicons DNA and rcccDNA
in liver tissues, via activation of HBV specific cytotoxic
CD8\textsuperscript{+} T lymphocytes mediating removal of HBV-infected
cells {[}7, 8{]}. Although AAV vectors have been approved for gene
therapy in cancers and other diseases, an increasing body of evidence
suggests AAV delivery system causes some adverse symptoms, such as
abnormal~immune~response and DNA integration {[}10{]}. In the past few
years, with technology advances in mRNA synthesis \emph{in vitro} and
delivery systems, LNP-based mRNA delivery has been applied broadly to
clinical and basic research due to its acceptable safety profile {[}21,
24{]}. In this work, we compared gene expression profile of liver
tissues between LNP-injected and AAV-injected mice via transcriptome
sequencing. Although fewer DEGs were observed in LNP-injected mouse
compared to AAV-injected mouse (\textbf{Figure S3} ), the biofunctions
of these DEGs are not fully understood and probably contribute to
clarification of difference between these two systems. And, lipid and
cholesterol, essential for maintenance of cell membrane structure, are
the main components of LNP, suggesting LNP delivery system might be much
safer than AAV. Next, we established LNP-IL-21particle and evaluated its
therapeutic effects in animal models. \emph{In vitro} study revealed
that LNP-IL-21 displayed a well-formed spherical structure with diameter
of 128.3 nm, and induced efficient IL-21 expression without obvious
cytotoxicity in primary mouse and human hepatocytes (\textbf{Figure 1,
Figure S2} ). \emph{In vivo} study indicated that livers in mice were
the major organs targeted by LNP-IL-21, and were the main source of
secreted IL-21 (\textbf{Figure 1} ). Therefore, overall serum IL-21
levels were probably determined by half-time of IL-21 mRNA in
hepatocytes and metabolic rate of serum IL-21. Meantime, storing
LNP-IL-21 at 4\selectlanguage{english} for 2 weeks did not affect its structure integrity and
expression efficiency. In the following study, in order to further
improve its tolerance to storage temperature and time, more works are
needed to optimize LNP and IL-21 mRNA structure, such as modification of
the proportion of each component in LNP and codon optimization of IL-21
open reading frame.

LNP-IL-21 treatment also achieved HBV pure clearance in BPS-based and
rcccDNA-based HBV persistence mice (\textbf{Figure 2-5} ) without
provoking obvious changes in total bilirubin and creatine kinase
(\textbf{Figure S4-5} ), which is similarly observed in those
AAV-IL-21-cured ones {[}8{]}. LNP-IL-21-incued clearance was associated
with both cellular and anti-HBsAg humoral responses (\textbf{Figure 6} )
while only cellular response was detected in AAV-IL-21-cured mice
{[}8{]}. Previous studies revealed that IL-21 was involved in helping B
cells to develop antibody-producing cells and in CD8\textsuperscript{+}T
cell activation {[}25, 26{]}. LNP-IL-21 treatment indeed improved the
frequencies of HBsAg-specific B cells and viral specific T cell
activities, contributing to HBV clearance (\textbf{Figure 6} ). Given
the facts that AAV itself is easy to establish persistent infection in
humans {[}27{]} and that AAV-mediated HBV transduction in adult mice
leads to HBV persistence {[}28{]}, we speculated that AAV vector likely
prohibited IL-21 from priming B cell activation during the process of
AAV-IL-21-induced HBV clearance. IL-21 functions through binding to its
receptor IL21R, broadly distributed on surface of different cells, to
activate intracellular signaling pathways {[}25{]}. We previously
observed that IL-21 could activate JAK-STAT pathway in
CD8\textsuperscript{+} T cells in mice {[}8{]}. Future work delineating
cellular and molecular mechanism for IL-21-mediated B and T cells
activation in LNP-IL-21 treated mice would be carried out to identify
key molecules and pathways through single-cell sequencing technology.

In order to avoid potential dangers caused by systemic administration of
IL-21 with LNP-IL-21, we developed LNP-IL-21-based cellular therapy, in
which PBMC were co-stimulated with LNP-IL-21 and rHBsAg before infusion
into HBV persistence mice, and found it is much more sufficient in HBV
clearance than IL-21-based cellular therapy {[}8{]}, in which
splenocytes were co-stimulated with recombinant IL-21 protein and
rHBsAg. At the meantime, LNP-IL-21-based cellular therapy, but not
IL-21-based cellular therapy, was able to activate both humoral and
cellular responses. The possible reason is that IL-21 derived from the
prokaryotic expression system in IL-21-based cellular therapy could not
exert complete biological functions compared to IL-21 expressed from
LNP-transfected cells in LNP-IL-21-based cellular therapy. Secondly,
LNP-IL-21-based cellular therapy offers the possibility of autologous
therapy for CHB patients. To achieve the goal, whether PBMC from
patients could be switched from tolerant phase to activated state
through LNP-IL-21-based cellular therapy treatment needs to be
determined before infusion into patients. Lastly, in terms of mechanism
research, \emph{ex vivo} stimulated cells can be easily characterized,
sorted and quantitated with regard to HBV specific humoral and cellular
immune responses.

Conclusion

Both LNP-IL-21-mediated gene and cellular therapies efficiently and
safely induced HBV clearance in HBV persistence mouse models through
activation of HBV specific humoral and cellular immune responses.

Conflicts of interests

Nothing to decla\textbf{r} e.

Data availability

Data that support the findings of this study are available from the
corresponding authors upon request.

Acknowledgments

This study was supported by the NSFC (32170150, 81701986, 81971931,
81971921, 3157010371), Shanghai Rising Star program (20QA1401800),
National Key Project for Infectious Diseases of China (2017ZX10202202
and 2018ZX10301208).

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Figure legends

\textbf{Figure 1. Preparation and characterization of LNP particles
formulated with IL-21 mRNA (LNP-IL-21).} (\textbf{A} ) Schematic
presentation of components used to generate LNP-IL-21 particles.
(\textbf{B} ) The size distribution of three samples of
LNP-IL-21measured using dynamic light scattering. (\textbf{C} )
Transmission electronic microscopy image of LNP-IL-21. (\textbf{D} )
Primary mouse hepatocytes cultured in 24-well plate were transfected
with 0.3 \selectlanguage{greek}μ\selectlanguage{english}g of LNP-IL-21 or control LNP, or left untreated. At 2 days
post transfection, IL-21 in culture medium was measured using ELISA.
(\textbf{E} ) Primary mouse hepatocytes cultured in 96-well plate were
transfected with 0.025-0.1 \selectlanguage{greek}μ\selectlanguage{english}g of LNP-IL-21, control LNP or left
untreated. Cell viability was detected at day 1-4 post transfection
using CCK-8 assays. (\textbf{F} ) Male BALB/c mice aged 6-8 weeks were
injected with 10 \selectlanguage{greek}μ\selectlanguage{english}g of LNP-IL-21 or LNP via tail vein. At 3 days post
injection, mice were sacrificed to collect major organs including heart,
liver, spleen, lung and kidney. Intracellular IL-21 levels were
determined using ELISA. LNP-IL-21 particles were stored at 4 for 0-6
weeks and used for further analysis. Measurement of LNP-IL-21 in
particle size (\textbf{G} ) and mRNA concentration (\textbf{H} ). Same
volumes of LNP-IL-21 particles stored for different weeks were
transfected into primary mouse hepatocytes (\textbf{I} ) or injected
into BALB/c mice (\textbf{J} , solid arrow) and IL-21 levels in culture
medium or serum measured using ELISA. The number of mice (\emph{n} ) in
each group was indicated. Group means and SEM within groups were plotted
and statistical significances calculated using unpaired two-tailed
\emph{t} -test. Dotted lines represented cut-off thresholds (\textbf{F}
and \textbf{J} ). **\emph{p} \textless{} 0.01; ***\emph{p} \textless{}
0.001. DSPC, disaturated phosphatidyl choline. PDI, polymer dispersity
index. w.p.i., weeks post injection of LNP.

\textbf{Figure 2. LNP-IL-21 administration induced HBV clearance in BPS
persistence mice.} BPS persistence mice remaining positive serum HBV
antigens for 4 weeks were injected (solid arrows) with two-dose
LNP-IL-21injections, two weeks apart, or control LNP. Sera collected at
indicated time points were analyzed for HBsAg (\textbf{A} ), HBsAb
(\textbf{B} ), HBeAg (\textbf{C} ), HBV DNA (\textbf{D} , pooled serum),
ALT (\textbf{E} ) and IL-21(\textbf{F} , pooled serum). Means and SEMs
within group were presented with the group size (\emph{n} ) indicated.
Group positivity percentage data were presented for HBsAg, HBsAb and
HBeAg (\textbf{A-C} , right panels). Dotted lines represented cut-off
thresholds (\textbf{A-C} , left panels, and \textbf{F} ), quantification
lower limit (\textbf{D} ) or pre-treatment baseline (\textbf{E} ).
Statistical significances were calculated using log-rank (Mantel-Cox)
test for group positivity percentages (\textbf{A-C} , right panels) or
unpaired two-tailed \emph{t} -test (\textbf{E} ). **\emph{p} \textless{}
0.01; ***\emph{p} \textless{} 0.001. w.p.i., weeks post injection of
LNP. geq, genome equivalents.

\textbf{Figure 3. LNP-IL-21 administration induced intrahepatic BPS
replicons DNA clearance in BPS persistence mice.} (\textbf{A} ) BPS
persistence mice remaining positive serum HBV antigens for 4 weeks were
injected with two-dose LNP-IL-21 injections (\emph{n} = 6), two weeks
apart, or control LNP (\emph{n} = 6). Sera collected at indicated time
points were analyzed for HBsAg. At 8 weeks post injection of LNP
(w.p.i.), mice were sacrificed to collect liver tissues. (\textbf{B} )
HBV core-associated DNA, BPS replicons DNA in nuclear DNA and capsid
proteins were extracted from liver tissues and subjected to Southern and
Western blots respectively. Serum HBsAg status of respective mouse was
indicated.

\textbf{Figure 4. LNP-IL-21 administration induced HBV clearance in
rcccDNA persistence mouse model.} rcccDNA persistence mice remaining
positive serum HBV antigens for 4 weeks were injected (solid arrows)
with two-dose LNP-IL-21 injections, two weeks apart, or control LNP.
Sera collected at indicated time points were analyzed for HBsAg
(\textbf{A} ), HBsAb (\textbf{B} ), HBeAg (\textbf{C} ), HBV DNA
(\textbf{D} , pooled serum), ALT (\textbf{E} ) and IL-21(\textbf{F} ,
pooled serum). Means and SEMs within group were presented with the group
size (\emph{n} ) indicated. Group positivity percentage data were
presented for HBsAg, HBsAb and HBeAg (\textbf{A-C} , right panels).
Dotted lines represented cut-off thresholds (\textbf{A-C} , left panels,
and \textbf{F} ), quantification lower limit (\textbf{D} ) or
pre-treatment baseline (\textbf{E} ). Statistical significances were
calculated using log-rank (Mantel-Cox) test for group positivity
percentages (\textbf{A-C} , right panels) or unpaired two-tailed\emph{t}
-test (\textbf{E} ). *\emph{p} \textless{} 0.05; **\emph{p}\textless{}
0.01; ***\emph{p} \textless{} 0.001. w.p.i., weeks post injection of
LNP. geq, genome equivalents.

\textbf{Figure 5. LNP-IL-21 administration induced intrahepatic rcccDNA
clearance in rcccDNA persistence mice.} (\textbf{A} ) rcccDNA
persistence mice remaining positive serum HBV antigens for 4 weeks were
injected with two-dose LNP-IL-21 injections (\emph{n} = 6), two weeks
apart, or control LNP (\emph{n} = 6). Sera collected at indicated time
points were analyzed for HBsAg. At 8 weeks post injection of LNP
(w.p.i.), mice were sacrificed to collect liver tissues. (\textbf{B} )
HBV core-associated DNA and capsid proteins extracted from liver tissues
were measured using Southern and Western blots respectively. rcccDA and
murine GAPDH (mGAPDH) were amplified from unclear DNA in liver tissues
using PCR and subjected to 1\% agarose electrophoresis for amplicon size
check. Serum HBsAg status of respective mouse was indicated.

\textbf{Figure 6. LNP-IL-21 treatment induced potent HBV-specific
humoral and cellular immune responses in BPS persistence
mice.}(\textbf{A} ) BPS persistence mice remaining positive serum HBV
for 4 weeks were injected with two-dose LNP-IL-21 injections, two weeks
apart, or control LNP. Blood samples were collected at indicated time
points. At 2 and 8 weeks post injection of LNP (w.p.i.), mice were
sacrificed to collect liver or/and spleen tissues. (\textbf{B} ) H\&E
and anti-CD8 staining were performed on liver tissues. Scale bar: 100
\selectlanguage{greek}μ\selectlanguage{english}m. At 8 w.p.i., serum HBsAg (\textbf{C} ) and HBsAb (\textbf{D} ) were
measured using ELISA, and HBsAg-specific B cells in PBMC from LNP-IL-21-
or LNP-injected mice were measured using flow cytometry analysis
(\textbf{E} ) with representative images of two mice in each group
presented (\textbf{F} ). At 8 w.p.i., spleen cells from above two groups
were stimulated with recombinant HBsAg or HBcAg proteins. After 3 days
post stimulation, IFN-\selectlanguage{greek}γ \selectlanguage{english}in culture medium was measured using ELISA.
Group means and SEMs were presented with group sizes (\emph{n} )
indicated. Statistical significances were calculated using unpaired
two-tailed \emph{t} -test. **, \emph{P} \textless{} 0.01; ***,
\emph{P}\textless{} 0.001.

\textbf{Figure 7. Transfer of PBMC co-stimulated with LNP-IL-21 and
rHBsAg induced clearance of HBV serum markers and intrahepatic BPS
repliocns DNA in BPS persistence mice.}(\textbf{A} ) PBMC from untreated
BPS persistence mice were cultured and treated with 1\selectlanguage{greek}μ\selectlanguage{english}g of LNP-IL-21 or
LNP in presence of 15 \selectlanguage{greek}μ\selectlanguage{english}g/ml rHBsAg. After 72 hours, 1 \selectlanguage{ngerman}×
10\textsuperscript{6} PBMC were injected into each untreated BPS
persistence mice via tail vein. Sera collected at indicated time points
were analyzed for HBsAg (\textbf{B} ), HBsAb (\textbf{C} ), HBeAg
(\textbf{D} ), ALT (\textbf{E} ) and IL-21 (\textbf{F} ). Means and SEMs
within group were presented with the group size (\emph{n} ) indicated.
Dotted lines represented cut-off thresholds (\textbf{B-D} , and
\textbf{F} ) or pre-treatment baseline (\textbf{E} ). (\textbf{G} ) At 4
weeks post injection of PBMC (w.p.i.), BPS replicons DNA and capsid
proteins extracted from liver tissues were measured using Southern and
Western blots respectively. Serum HBsAg status of respective mouse was
indicated. Statistical significance was unpaired two-tailed \emph{t}
-test. *\emph{p} \textless{} 0.05; **\emph{p}\textless{} 0.01.

Supplementary table legends

\textbf{Table S1. Profile of differentially expressed genes (DEGs) in
mouse liver post LNP injection}

\textbf{Table S2. Profile of differentially expressed genes (DEGs) in
mouse liver post AAV injection}

\textbf{Table S3. Primer sequences used for PCR analysis}\selectlanguage{english}
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