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