Narrow regions screening across HBV whole-genome for multifunctional, highly-conserved siRNA triggers

We sequenced the HBV genome using the Hepatitis Virus Database (HVDB) and motif-based sequence analysis tools (MEME) (Supplementary Fig. 1). Twenty-two conserved motifs were identified (Supplementary Table 1) and 20 siRNA sequences targeting the conserved motifs distributed across the entire HBV genome were synthesized for further examination (Fig. 1a). These siRNAs were subsequently categorized into three groups based on their theoretical target mRNAs, as shown in Supplementary Table 2 and Fig. 1b (class I: targets pgRNA, PreS/S RNAs; class II: targets pgRNA, PreS/S, X RNAs; class III: targets only pgRNA).

Fig. 1

Genome-wide scanning of conservative and efficient anti-HBV siRNA triggers. a The structure of HBV RNAs and the location of siRNA candidates. The siRNA candidates are shown as short lines. The locations of four HBV ORFs (HBs, HBc, HBx and Pol) and the relative transcripts (3.5 kb, 2.4 kb, 2.1 kb, and 0.7 kb) are indicated. The distribution of 22 conserved motifs in HBV genome are also indicated. b Twenty siRNA candidates were transfected by RNAiMAX transfection reagents and screened at the dose of 50 nM in HepG2-HBVEGFP cells (HBsAg derived from recombination cccDNA. The reporting cell line was constructed in-house) and PLC/PRF/5 cells (HBsAg derived from HBV integrates. The cell line carrying natural HBV integrates was derived from clinical patents). The supernatants were collected at 72 h post-transfection, and the HBsAg levels in the culture medium were determined by commercial ELISA kits and was normalized to that of cells treated with a negative control siRNA (siNC). HepG2-NTCP (NTCP receptor-overexpressing HepG2 cell line that supports HBV infection) cells were infected with HBV at a multiplicity of infection (MOI) of 200 for 3 days, then transfected with siRNA candidates at the dose of 50 nM using transfection reagents (RNAiMAX). HBeAg expression in the culture medium was analyzed at 72 h post-transfection, and the intracellular pgRNA and total RNA levels were detected with the corresponding primers. c The inhibitory efficacy of siRNAs on EGFP expression levels in HepG2-HBVEGFP cells were visualized by high content imaging (HCI) and screening (HCS) assay using Operetta CLS (PerkinElmer). Scale bar indicated 200 μm. d HepG2-NTCP cells were infected with HBV at the MOI of 200, then treated with serial dilutions of siRNA (0, 6.25, 12.5, 25, 50, and 100 nM) using RNAiMAX transfection reagent. HBeAg expression levels in the supernatants were determined by ELISA kits, and the IC50 values were calculated. The si74 and si77 (two siRNA triggers from ARC-520) were used as positive controls. Data were shown as means ± SDs (n = 3 ~ 4)

The interference efficiency of the siRNA candidates was assessed using the following cell models: PLC/PRF/5 is a clinical hepatocarcinoma-derived cell line that carries natural HBV integration with varying breakpoints and secrets HBsAg from intHBV.41,42 The HepG2-HBVEGFP cell line, which is established in-house, contains recombinant cccDNA with an EGFP reporter gene and secrets HBsAg from cccDNA.37,38 As shown in Fig. 1b, most siRNA candidates (class I and class II) significantly reduced cccDNA-derived HBsAg levels in HepG2-HBVEGFP cells, except for class III siRNAs (si-2303-si-2831). Conversely, class II siRNAs (si-1606~si-1912) lost their ability to target intHBV-derived HBsAg transcripts in PLC/PRF/5 cells. Similarly, all candidate siRNAs efficiently reduced the secretion of HBeAg and intracellular pgRNA levels in HepG2-NTCP (HBV receptor transduced cell line) infection model compared to the negative control siRNA (si-NC), whereas class III siRNAs failed to reduce HBV total RNA levels. These results suggested that only narrow regions across the HBV genome were reasonable for designing multifunctional siRNA reagents that target all cccDNA- and intHBV-derived mRNAs with a single siRNA.

The conservativity of the siRNA candidates was calculated (Supplementary Table 3). The efficacy of siRNA candidates was confirmed using 1.3× overlength HBV plasmids of different genotypes (A/B/C/D) and HBV-infected primary human hepatocytes (PHHs) (Supplementary Fig. 2). The interference efficiency was also confirmed using the EGFP reporter protein (Fig. 1c), and compared with two siRNA triggers from ARC-520 (si74 and si77) (Fig. 1d). siRNAs with high interference potency across different genotypes were selected for further analysis.

Combined siRNA triggers possessed pan-genotypic and multifunctional anti-HBV efficacy

To obtain pan-genotypic and multifunctional siRNA triggers against all HBV-derived transcripts, different siRNA combinations were evaluated for their conservation (Supplementary Table 4) and anti-HBV activities. As shown in Fig. 2a, the combination of si-1260 and si-1848 (terms “siHBV” in the following text) provided means of pan-genotypic functionality (19-mer coverage of 98.55%) against all forms of HBV transcripts, thus was selected for further analysis. In addition, siHBV-encapsulating LNPs effectively reduced intracellular HBc and HBx protein levels in a dose-dependent manner (Fig. 2b). Potent and equipotent inhibition of viral antigens against different HBV genotypes was also observed, confirming pan-genotypic anti-HBV activity (Fig. 2c).

Fig. 2

Screening of pan-genotypic and multifunctional siRNA combinations and chemical modifications. a HepG2 cells transfected with pHBV1.3 plasmids (Genotype D), HepAD38 (HepG2 cells that stably integrated with a 1.1× overlength HBV genome to support HBV replication) cells, PLC/PRF/5 and HepG2-NTCP cells that infected with HBV at the MOI of 200 were treated with siRNA at the doses of 50 nM or 25 nM, or siRNA combinations at the dose of 25 nM each, using RNAiMAX transfection reagents. The HBsAg and HBeAg levels in the supernatants were determined via ELISA kits at 72 h post-transfection, the intracellular HBV pre-genomic RNA (pgRNA) and HBV total RNA were determined via real-time quantitative PCR (RT-qPCR). b HepAD38 cells were treated with varying doses of LNP-formulated siRNA (0, 6.25, 12.5, 25, 50, and 100 nM) and then the expression levels of intracellular HBx and HBc were determined by western blot analysis. c HepG2 cells were transfected with pHBV1.3 plasmids of genotypes A, B, C, and D. One day following pHBV1.3 transfection, the cells were transfected with different doses of LNP-formulated siRNA (0, 6.25, 12.5, 25, 50, and 100 nM), then the supernatants were collected and applied for HBsAg and HBeAg detection at 72 h post-transfection. d HepG2-HBVEGFP cells were transduced with Ad-Cre and transfected with serial dilutions of si-1260 or si-1848 (0, 6.25, 12.5, 25, 50, and 100 nM) with varying chemical modifications as shown in Supplementary Table 5. HBsAg expression in the supernatant was determined 3 days after transfection. Cell viability was detected by cell counting kit-8 (CCK-8) analysis at 2 days post-transfection. Data were shown as means ± SDs (n = 3). e The unmodified and modified si-1260 and (f) si-1848 were incubated with 10% fetal bovine serum (FBS) in PBS for indicated periods, and the stability of siRNA was evaluated using gel-red electrophoresis and photographed with imaging system

Co-injection of unmodified dissociative siHBV with the prcccDNA/pCMV-Cre plasmid (4 μg each) system by high volume hydrodynamic injection (HDI) significantly reduced HBV antigen levels (1.4–2.3 log10 IU/mL reduction of HBsAg, vs Mock), whereas the antigen levels recovered significantly at 4 days post-injection, indicating the short half-lives of un-stabilized and unformulated siRNAs (Supplementary Fig. 3a–c). Naked siRNAs are subject to degradation (especially by the innate immune response triggered in vivo or degradation by nucleases). Chemical modification may dramatically increase in vivo stability and abrogate the undesirable immunostimulatory properties of siRNA.43 Thus, the siHBV was chemically stabilized at desired sites with 2′-O-methyl group (2′-OMe), 2′-Fluro (2′-F), and the sulfate bond modification in the backbone, the incorporation of which could reduce immunogenicity, improve stability and extend half-life of siRNAs (Supplementary Table 5). These siRNAs were screened for biological activity, cytotoxicity, and serum stability (Fig. 2d–f and Supplementary Fig. 3d, e). Both siRNA triggers with partial or full 2′-OMe and 2′-F modifications efficiently preserved the knockdown bioactivity, and significantly improved serum stability after incubation with 10% FBS at 37 °C for indicated durations, with limited cellular toxicity. Since si-1260 was sensitive but si-1848 was well-tolerated to chemical modifications, the siRNAs with satisfactory bioactivity, minimal chemical modifications, and superior stability (si1260-2m and si1848-2m) were selected for further evaluation.

Optimal LNP formulation enables efficient delivery of siHBV

LNPs were selected as the nanocarriers for efficient and tissue-specific delivery of siHBV in vivo. Considering the antigenicity of methoxyl PEG against pre-existing anti-PEG antibodies (data not shown here), HO-PEG2000-DMG (with a hydroxyl terminus) lipids at varying molar ratios were explored to replace mPEG2000-DMG (with a methoxyl terminus) which is commonly used in approved LNP formulations. The diameters of the nanoparticles ranged from 60 to 100 nm, and 3% molar ratio of PEGylated lipids (both HO-PEG and mPEG) resulted in a smaller mean value of ~75 nm. For all formulations, the encapsulation efficiency (EE%) of siRNA was > 97% (Fig. 3a, b). Many studies reported that the unwanted uptake of LNP by the immune system is a major cause of LNP toxicity and limits its efficacy.23,44 Thus, the in vivo performance of LNPs/siRNA was studied in mice using Cy5-labeled siRNA (Cy5-siRNA) as a model siRNA. Biodistribution of LNPs/Cy5-siRNA in different organs of mice was determined at 4 h after injection (at a dose of 0.5 mg/kg), showing reduced lung, spleen and kidney distribution of 3% PEG2000-DMG-based LNPs, comparing to that of 1.5% PEG2000-DMG-based LNPs, whereas the biodistribution of LNPs in the liver was comparable (Supplementary Fig. 4a–c). The 3% PEG2000-DMG-based LNPs also showed better LNP uptake when co-cultured with Huh7 cells for 4 h in vitro (Supplementary Fig. 4d). Hepatocytes and liver-associated lymphocytes were collected 4 h after injection, and the results indicated that 3% PEG2000-DMG-based LNPs significantly reduced immune cell uptake with comparable hepatocyte uptake of Cy5-siRNA (Fig. 3c and Supplementary Fig. 5a–d). Meanwhile, the 3% OH-PEG2000-DMG-based LNP displayed higher hepatocyte uptake than that of 3% mPEG2000-DMG-based LNP at the dose of 0.05 mg/kg (Supplementary Fig. 5e). The splenocytes were collected, showing reduced off-target immune cell uptake as well (at the dose of 0.5 mg/kg) (Supplementary Fig. 6).

Fig. 3

Optimization of low antigenic and hepatocyte-targeted LNP-based siRNA delivery system. a Schematics of the preparation procedures of LNPs. b Different formulations of LNPs (abbreviated as 1.5% OH, 3% OH, 1.5% OMe and 3% OMe) were prepared, and the sizes, polydispersity index (PDI), ζ potential and encapsulation efficiency (EE%) were analyzed. c C57BL/6 mice were intravenously injected with different formulations of LNPs/Cy5-siRNA at the dose of 0.5 mg/kg, 4 h post-injection, the hepatocytes and liver-associated lymphocytes were isolated, and the percentage of Cy5-positive hepatocytes, T cells, B cells and Kupffer cells (KCs) were determined by flow cytometry analysis. Data were shown as means ± SDs (n = 4). d C57BL/6 mice were intravenously injected with different formulations of LNPs/siApoB at the doses of 0.5 mg/kg and 0.05 mg/kg, mice were sacrificed at 72 h post-injection. Relative ApoB mRNA expression levels in the liver of mice (compared with β-actin mRNA levels) were determined at 72 h post-injection, and (e) the sera total cholesterol (T-CHO) levels were detected via commercial kits. Data were analyzed using two-way ANOVA with Sidak multiple comparison correction, and shown as means ± SDs (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Apolipoprotein B (ApoB) is the major apolipoprotein of low-density lipoproteins (LDL) that specifically expressed in hepatocytes, and siRNA targeting ApoB (siApoB) was utilized as another model siRNA to evaluate hepatocyte-targeted delivery and knockdown efficiency of siRNA-encapsulating nanoparticles. Briefly, siApoB encapsulating LNPs were intravenously injected into mice at doses of 0.05 and 0.5 mg/kg, respectively. The liver was dissected 72 h after injection and analyzed for intrahepatic ApoB mRNA levels. In addition, intrahepatic and seral total cholesterol (T-CHO) and total triglycerides (TG) levels were determined. The results showed that mice treated with LNPs/siApoB exhibited significantly reduced ApoB mRNA expression and circulating T-CHO levels (Fig. 3d, e). Specifically, 3% HO-PEG2000-DMG-based LNP showed higher knockdown efficiency than that of 3% mPEG2000-DMG-based LNP at the dose of 0.05 mg/kg. Subsequently, the 3% HO-PEG2000-DMG SM-102-based LNP (terms “tLNP”) was selected for further evaluation of liver targeted efficacy and knockdown activity in primary mouse hepatocytes (Supplementary Fig. 7a) and in mice. The tLNP/siApoB formulation was intravenously injected into mice at doses of 1, 0.5, 0.1,0.05, 0.01, 0.005 and 0.001 mg/kg. Blood and liver samples were harvested 72 h after injection. The results showed that the formulation potently reduced intrahepatic ApoB mRNA levels in a dose-dependent manner, with an ED50 value of 0.02303 mg/kg (Supplementary Fig. 7b), the effects of which were better than that of 3% mPEG2000-DMG-based LNP (with an ED50 value of 0.06127 mg/kg) (Supplementary Fig. 7g). Additionally, intrahepatic TG and T-CHO levels elevated dose-dependently, while seral T-CHO levels reduced accordingly (Supplementary Fig. 7c–f).

tLNP/siHBV demonstrates satisfactory biosafety

The tLNP was explored for the delivery of chemically modified siHBV to evaluate their antiviral efficacy and biosafety in vitro and in vivo. The tLNP was prepared (see Supplementary Methods), and the stability of siRNA encapsulating tLNP was evaluated at −20 °C, 4 °C, or 25 °C for a storage period ranging from 1 week to 4 weeks. The results presented in Supplementary Fig. 8a, b suggested that tLNP/siRNA was stable under the tested conditions. The in vitro analysis revealed that tLNP-formulated siHBV (tLNP/siHBV) suppressed HBsAg and EGFP in a dose-dependent manner in HepG2-HBVEGFP cells, with an IC50 value of 1.517 nM (Fig. 4a–c). The dose-dependent anti-HBV effects of tLNP/siHBV were also validated in stable HBV-inducing cell lines, HepAD38 and HepDE19 cells, as well as HBV integrative cell line, PLC/PRF/5 cells (Supplementary Fig. 9).

Fig. 4

The efficacy and safety of tLNP/siHBV formulations. a HepG2-HBVEGFP cells were transduced with Ad-Cre at the MOI of 8 and then transfected for 72 h with varying concentrations of modified siHBV, and HBsAg expression was determined via commercial kits. Data were shown as means ± SDs (n = 3). b The IC50 was calculated by GraphPad Prism software (Version 9.00, San Diego, California, USA). c The EGFP expression levels of the treated HepG2-HBVEGFP cells were visualized with high content imaging (HCI) system. Scale bar indicated 200 μm. d Global transcriptional profiling of HepG2 cells following treatment with tLNP/siNC and tLNP/siHBV at the dose of 50 nM for 16 h. The volcano plots depicting gene expression changes between tLNP/siHBV and tLNP/siNC treated cells (n = 3). e Transcriptional dysregulation of mice liver after intravenous injection of tLNP/siHBV (1 mg/kg) or tLNP/siNC (1 mg/kg) for 48 h (n = 3). The volcano plots depicting gene expression changes between tLNP/siHBV and tLNP/siNC were shown. f Treatment and sampling schedule of toxicity study in mice. 1 mg/kg and 2.5 mg/kg doses of tLNP/siHBV formulations were intravenously injected into C57BL/6 mice. Mice that treated with PBS and 1 mg/kg of tLNP/siNC (i.v.) were utilized as negative controls, and the lipopolysaccharides (LPS) (5 mg/kg, s.c.) and polyI:C treated mice (10 mg/kg, s.c.) were included as positive controls in the assay. g Cytokine concentrations in the serum including TNF-α, (h) IFN-γ, and (i) IL-6 were determined via commercial ELISA kits at indicated time points. Data were shown as means ± SDs (n = 5)

The RNAi-mediated, miRNA-like off-target effects of siRNAs are major concerns for their clinical application. Chemical modification-related toxicities arising from non-specific binding to cellular proteins and immune recognition of exogenous nucleic acids are major drivers of siRNA hepatotoxicity.45,46 We performed global transcriptome profiling of HepG2 cell and mouse livers following treatment with tLNP-formulated siNC (chemically modified) (tLNP/siNC) or tLNP/siHBV. As shown in Fig. 4d, e, tLNP/siHBV demonstrated limited sequence-specific off-target effects, interruption on liver transcriptome, and induction of immune responses.

To further facilitate the clinical utilization of siRNA therapeutics, the biosafety profile of tLNP/siHBV was analyzed in mice. A single dose of tLNP/siHBV was intravenously injected at doses of 1 mg/kg and 2.5 mg/kg, respectively. tLNP/siNC was administrated at a dose of 1 mg/kg. Lipopolysaccharide (LPS) and polyI:C were intraperitoneally injected at doses of 5 and 10 mg/kg, and used as positive controls. Blood was sampled at 3, 24, and 48 h. As shown in Fig. 4f–i, LPS induced a remarkable elevation of IL-6, IFN-γ and TNF-α as expected, whereas neither tLNP/siNC nor tLNP/siHBV triggered cytokine expression during 48 h observation period. Intrahepatic inflammatory response-related genes were also determined, confirming the low immunogenicity of tLNP/siHBV (Supplementary Fig. 10).

tLNP/siHBV exhibits potent anti-HBV activity in rAAV-HBV1.3 and rAAV-rcccDNA/rAAV-Cre mouse models

The antiviral efficacy of tLNP/siHBV was further assessed in the rAAV-HBV1.3 mouse model, in which HBV replication was based on an adenovirus-associated virus (AAV) containing a transgene encompassing a 1.3× overlength HBV genome of genotype D. Single doses of tLNP/siHBV at 0.1, 0.3, and 1 mg/kg were intravenously injected to characterize the dose-response and kinetics of the viral products. Negative controls included PBS and tLNP/siNC, while Entecavir (ETV), the first-line antiviral medication for CHB treatment, served as a positive control. Blood was sampled weekly as shown in Fig. 5a and the results suggested that tLNP/siHBV significantly reduced HBsAg (0.56–2.65log10 reduction, vs PBS), HBeAg (0.93–1.82log10 reduction) and circulating HBV DNA (1.28–2.29log10 reduction) in a dose-dependent manner (Fig. 5b–d). In particular, a single dose of tLNP/siHBV (1 mg/kg) achieved 2.65log10 IU/mL reduction of HBsAg relative to the PBS control. The reduction in viral products was durable, with resolution of the effects of single-dose treatment occurring between day 7 and 28 in a dose- and time-dependent manner. In contrast, although ETV reduced viral replication with high efficiency, it failed to decrease viral antigen levels throughout the treatment course. Taken together, tLNP/siHBV could not only reduce viral antigen burden, but also block the synthesis of viral DNA.

Fig. 5

Anti-HBV efficacy of tLNP/siHBV in rAAV-HBV1.3 mouse model. a Treatment and sampling schedule of the single-dose study. Mice were injected with rAAV-HBV1.3 (2.5 × 1010 viral genome (v.g.) per mice), 2 ~ 4 weeks later, the mice were grouped according to seral HBsAg levels, and treated with PBS, tLNP/siNC (1 mg/kg), tLNP/siHBV of different doses (0.1, 0.3, and 1 mg/kg), or given with Entecavir (ETV, 1 mg/kg) orally. b Mice were bled once weekly, and the seral levels of HBsAg, (c) HBeAg and (d) HBV DNA were monitored during the treatment course. Data were shown as means ± SDs (n = 8). e Treatment and sampling schedule of the multi-dose study. HBV-replicating mice model was obtained and grouped according to seral HBsAg levels. Mice were then treated with PBS, tLNP/siHBV at the doses of 1 mg/kg and 0.3 mg/kg, and at the frequency of once weekly (Q1W) or once bi-weekly (Q2W). f Mice were bled once weekly, and the seral levels of HBsAg, (g) HBeAg, and (h) HBV DNA during the treatment course were determined by commercial kits. i Body weights were monitored during the treatment course. Data were shown as means ± SDs (n = 8)

Repeated doses (two groups, respective 0.3 and 1 mg/kg) of tLNP/siHBV were intravenously injected into mice of HBV replication weekly (five doses in total) or bi-weekly (three doses in total). Blood was sampled weekly for serological testing and observation period continued for three weeks after treatment discontinuation (Fig. 5e). The results showed a significant reduction in HBsAg in a dose- and time-dependent manner (Fig. 5f). Over 2log10 IU/mL reduction of viral surface antigen was observed on day 7 after administration, and the inhibition efficiency maintained at 1.85-2.65log10 IU/mL (relative to the PBS group) when the dose was 1 mg/kg during the weekly dosing course. Similar effects were seen for HBeAg (1.63–2.59log10 reduction) and HBV DNA (1.43–1.72lgo10 reduction) levels (Fig. 5g, h). Three weeks after the final dose, 0.57log10 IU/mL repression efficiency of HBsAg was still observed. Whereas intrahepatic viral products except HBsAg recovered largely after three weeks of drug withdrawal, which might be due to the stable existence of cccDNA and AAV episome (Supplementary Fig. 11). Toxicity evaluation was performed. Neither body weight changes nor histological abnormalities were observed (Fig. 5i and Supplementary Fig. 12a). Seral anti-HBs antibody levels were determined, and the results showed that one out of eight (1/8, 12.5%) mice produced anti-HBs antibodies after receiving five weekly doses of tLNP/siHBV at doses of 0.3 and 1 mg/kg.

The efficacy of tLNP/siHBV was further evaluated in a rAAV-rcccDNA/rAAV-Cre mouse model, which was established independently to support the long-lasting maintenance of recombinant cccDNA and antigenemia.47 The results demonstrated that a single dose of tLNP/siHBV at doses of 0.1, 03, and 1 mg/kg led to a significant reduction of HBsAg levels (1.83-2.74log10 IU/mL, relative to the PBS group), and multi-doses of tLNP/siHBV at a dose of 1 mg/kg resulted in a durable reduction in HBsAg levels during treatment courses (Supplementary Fig. 13).

Combined siHBV and mIL-2 mRNA therapy leads to synergistic viral and immune control of hepatitis B

The desired treatment endpoint is to achieve seroclearance of HBsAg, with or without seroconversion, which is associated with a prolonged prognosis. The siHBV therapy only result in viral control in a limited number of subjects, and immunological evaluation showed that siHBV alone did not alter the phenotype or improve the function of CD8+ T cells in the treated mice (Supplementary Fig. 12b–d). We then investigated whether combining siHBV with immunomodulators such as IL-2 could improve therapeutic outcomes. Thus, an mIL-2 encoding mRNA was constructed (see Supplementary Methods), encapsulated within tLNP (tLNP/IL2), and confirmed for proper expression and induction of downstream STAT5 phosphorylation (Fig. 6b and Supplementary Figs. 14 and 15a, b). Intravenous injection of tLNP/IL2 into mice increased the percentage of CD69-positive and CD107a-positive immune cells in the liver, indicating proper expression and functionality (Supplementary Fig. 15c, d). The safety of IL-2 overexpression after tLNP/IL2 injection was also confirmed that then (Supplementary Fig. 15e–h).

Fig. 6

Additive antiviral efficacy of tLNP/siHBVIL2 in rAAV-HBV1.3 mouse model. a The morphology of tLNP/siHBV and tLNP/siHBVIL2 was observed by cryogenic electron microscopy (Cryo-EM). b mIL-2 mRNA encapsulating tLNP (tLNP/IL2) was intravenously injected into C57BL/6 mice at the doses of 0.1, 0.2, and 0.5 mg/kg, plasma was collected at indicated time points and the mIL-2 expression levels were determined via commercial ELISA kits. Data were shown as means ± SDs (n = 3). c C57BL/6 mice of HBV replicating models were divided into groups according to seral HBsAg levels, mice were then intravenously injected with PBS, tLNP/siHBV (1 mg/kg) and tLNP/siHBVIL2 (siHBV kept at the dose of 1 mg/kg, and mIL-2 mRNA was given at the doses of 0.1, 0.2, and 0.5 mg/kg). Mice were treated at weekly dose frequency for five doses, then the treatment stopped for another three weeks, the seral viral antigens and DNA were monitored at weekly frequency. The dot plots of anti-HBs, HBsAg and HBV DNA levels at the end of the experiment were displayed with median levels indicated (n = 7 ~ 8). d Heatmap displaying the percentage of mice of different groups. e Histological analysis of liver sections of HBV-replicating mice treated with or without tLNP/siHBV (1 mg/kg) and tLNP/siHBVIL2 (siHBV: 1 mg/kg, mIL-2 mRNA: 0.1–0.5 mg/kg). Immunohistochemical staining of HBc and CD8 levels in liver sections of multi-dose treated mice of HBV replication. Scale bar indicated 100 μm. f The HBc- positive cells per mm2 liver sections and (g) the infiltration of CD8- positive cells per mm2 liver sections were displayed. Data were analyzed using unpaired two-tailed Students’ t test analysis, and presented as means ± SDs (n = 4). h The statistical analysis of the proportion of HBsAg- (S109) and core- (C93) specific CD8+ and (i) CD4+ T cells in gated CD3+ T cells. Data were analyzed using unpaired two-tailed Students’ t test analysis, and presented as means ± SDs (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

The mIL-2 mRNA and siHBV were further encapsulated within a single tLNP (tLNP/siHBVIL2) at weight ratios of 1:10, 1:5, and 1:2, and showed satisfactory size, PDI and ζ potentials, with an encapsulation efficiency of ~97% (Fig. 6a and Supplementary Fig. 16a). The nanoparticles were administrated in five doses in HBV-replicating mouse models at a once weekly dose frequency and continued observation for three weeks after the final dose (Supplementary Fig. 16b). The results showed that tLNP/siHBVIL2 demonstrated potent viral antigen inhibitory efficiency comparable to that of tLNP/siHBV monotherapy, but exhibited higher inhibitory effects on HBV DNA (Supplementary Fig. 16c–f). Specifically, four out of eight (4/8, 50%) mice retained significant HBsAg control (<100 IU/mL) and five out of eight (5/8, 62.5%) retained strong HBV DNA reduction (>2log10 reduction) at the end of the observation, two of the mice (2/8, 25%) also produced anti-HBs antibodies (Fig. 6c, d). Furthermore, the proportion of HBc-positive cells in liver sections was significantly reduced (Fig. 6e, f). Other intrahepatic parameters were determined, revealing that tLNP/siHBVIL2 induced more persistent viral replication and transcription inhibition compared to tLNP/siHBV monotherapy, whereas intrahepatic cccDNA levels remained unchanged (Supplementary Fig. 17b–h). Mechanistically, tLNP/siHBVIL2 treatment induced a dramatic increase in the proportion of HBsAg-specific (S109 tetramer-stained) and core-specific (C93 tetramer-stained) CD8+ and CD4+ T cells in the liver, as observed by flow cytometry (Fig. 6h, i and Supplementary Fig. 17a). Histological studies further indicated that tLNP/siHBVIL2 induced significant infiltration of CD8+ T cells into the liver, enabling prolonged viral control without apparent liver injury (Fig. 6e, g and Supplementary Figs. 18 and 19).