Long-term inhibition of Hepatitis B virus gene expression by a primary microrna expressing ancestral adeno-associated viral vector

Abstract

Current treatments for chronic infection with the hepatitis B virus (HBV) rarely cure carriers from the disease. Previously reported use of serotype 8 adeno-associated viral (AAV8) vectors to deliver expression cassettes encoding anti-HBV artificial primary microRNAs (apri-miRs) has shown promise in preclinical studies. A recently designed synthetic ancestral AAV (Anc80L65) with high liver transduction efficiency is a promising new addition to the anti-HBV vector toolbox. This study engineered Anc80L65 to express HBx-targeting apri-miRs. Single dose administration of the vectors to cultured cells and HBV transgenic mice effected reductions of secreted HBV surface antigen (HBsAg). Circulating HBV particles and HBV core antigen (HBcAg) were also significantly diminished in mice receiving the anti-HBV apri-miR-expressing ancestral AAVs. Downregulation of HBV biomarkers occurred over a period of 12 months. Absence of inflammatory responses or liver toxicity indicated that the vectors had a good safety profile. These data suggest that a single dose of apri-miR-expressing Anc80L65 is safe and capable of mediating durable suppression of HBV gene expression. Targeting HBx, which is required for transcriptional activity of covalently closed circular DNA of HBV, makes this Anc80L65-derived vector a promising candidate for functional cure from chronic HBV infection.

Keywords: AAV; Ancestral AAV; Hepatitis B virus; Microrna.

Fig. 1

Study design. (a) The scAAV vector genome was engineered to carry mono- or polycistronic artificial primary microRNA (apri-miR) sequences under control of a mouse transthyretin receptor (mTTR) promoter. ITR (inverted terminal repeats), int (intron) and TTS (transcription termination signal) are indicated schematically. (b) Engineered scAAV genomes were packaged into AAV2, AAV8 or Anc80L65 capsids. c-f. Upon administration, vectors mediate expression of pri-miRs (c) which are processed in several steps to generate pre-microRNA (pre-miR, d), and miR guides 5, 8 and 9 (e). Produced miRs then target all four HBV transcripts (green, purple and orange arrows shows target sites (f)) (created with Biorender)

Fig. 2

Assessment of scAAV activity in culture. a. Assessment of HBV gene expression knockdown by scAAV vectors following transfection of Huh7 cells with psiCHECK-HBx. Data are represented as a mean ratio of Renilla to Firefly luciferase reporter activity. b. Transfection with pCH 9/3901 then transduction with scAAVs was performed before measurement of HBsAg. c-f. Evaluation of HBsAg (c), secreted HBV viral particle equivalents (d, VPEs), intracellular HBV RNA (e) and HBcAg levels in HepG2-hNTCP cells infected with HBV and then transduced with scAAVs. Data shown are relative to control vectors encoding anti-HCV apri-miRs. The means (± SEM) were derived from n = 3–4 samples. The Student’s two-tailed paired t-test or Tukey’s multiple comparison test was used to ascertain statistically significant differences relative to controls. pvalues < 0.05 (*), < 0.001 (**), < 0.0001 (***) were regarded as statistically significant

Fig. 3

Expression and processing of artificial primary microRNA from scAnc80. Northern blot hybridization was performed on total RNA extracted from murine liver samples at 1 or 12 months post infection with scAAVs using probes complementary to sequence for guides (a) 5, (b) 8 or (c) 9. The figure shows data from a representative of 3 mice per group. After stripping miR probes from the blots, rehybridization with a probe complementary to U6 small nuclear RNA probe was carried out to confirm equal loading. MW, molecular weight

Fig. 4

HBV gene silencing in transgenic mice. Eight HBV transgenic mice per group were injected intravenously with saline or 1 × 10¹¹ scAAV genome copies. a. Serum HBsAg and (b) HBV particles were measured over a 12-month period using ELISA or qPCR respectively. Immunohistochemical staining was performed on mouse liver sections at (c) 1 or (d) 12 months post scAAV infection. Liver sections from a representative of 3 mice per group taken at 20 × magnification are shown. Graphs on the right show quantification of HBcAg-positive cells (brown) from 10 randomly selected fields per group. All values were normalized and presented relative to anti-HCV vectors. e. HBV transcript levels relative to GAPDH transcripts determined using RT-qPCR. RNA was extracted from livers harvested from 4 mice per group at 1 and 12 month post AAV injection. The means (± SEM) were derived from groups comprising 4 to 6 mice. One-way ANOVA was used to ascertain statistically significant differences relative to anti-HCV vector controls. pvalues < 0.05 (*), < 0.001 (**), < 0.0001 (***) were regarded as statistically significant

Fig. 5

Measurement of pro-inflammatory response in scAnc80 infected mice. Cytokine quantification was performed 6 h after injection with scAAVs, saline or poly (I: C). Cytometric bead array assay quantification of serum cytokines from 6 mice per group: (a) IL-12p70, (b) IFN- γ, (c) IL-10, (d) IL-6, (e) MCP-1 and (f) TNF-α. Concentrations of (g) IFIT-1, (h) IFN-β, (i) and OAS-1 mRNAs in liver samples from 4 mice per group determined using RT-qPCR. The means (± SEM) were derived from six or four mice per group. The Student’s two-tailed paired t-test was used to ascertain statistically significant differences relative to the saline control. pvalues < 0.05 (*), < 0.001 (**), < 0.0001 (***) were regarded as statistically significant and pvalues ≥ 0.05 were regarded as statistically insignificant (NS)

Fig. 6

Assessment of possible scAnc80-induced liver damage. Histochemical staining was performed using (a) H&E and (b) Sirius red on liver tissues obtained from 3 mice per group injected with saline or scAAVs. Arrows indicate areas of immune cell tissue infiltration. Mice were sacrificed at 1 or 12 months after scAAV administration and livers harvested. Representative low power fields are shown with scale bars of 200 μm. The visualization of tissue was carried out at 40 × magnification. c. Alanine aminotransferase (ALT) activity assay from murine serum samples. Serum was collected at weeks 1, 2, 4, and 8 after injection of groups of 3 mice with saline or scAAVs. The dashed line indicates the upper limit of normal ALT activity (100 IU/L). The means (± SEM) were derived from 3 mice per group. d. Evaluation of weight changes in HBV transgenic mice. The means (± SEM) were derived from 6 mice per group