Cre/LoxP-HBV plasmids generating recombinant covalently closed circular DNA genome upon transfection

Abstract

New therapies against hepatitis B virus (HBV) require the elimination of covalently closed circular DNA (cccDNA), the episomal HBV genome. HBV plasmids containing an overlength 1.3-mer genome and bacterial backbone (pHBV1.3) are used in many different models, but do not replicate the unique features of cccDNA. Since the stable cccDNA pool is a barrier to HBV eradication in patients, we developed a recombinant circular HBV genome (rcccDNA) to mimic the cccDNA using Cre/LoxP technology. We validated four LoxP insertion sites into the HBV genome using hydrodynamic tail vein injection into murine liver, demonstrating high levels of HBV surface antigen (HBsAg) and HBV DNA expression with rcccDNA formation. HBsAg expression from rcccDNA was >30,000 ng/mL over 78 days, while HBsAg-expression from pHBV1.3 plasmid DNA declined from 2753 ng/mL to 131 ng/mL over that time in immunodeficient mice (P < 0.001), reflective of plasmid DNA silencing. We then cloned Cre-recombinase in cis on the LoxP-HBV plasmids, achieving plasmid stability in bacteria with intron insertion into Cre and demonstrating rcccDNA formation after transfection in vitro and in vivo. These cis-Cre/LoxP-HBV plasmids were then used to create HBx-mutant and GFP reporter plasmids to further probe cccDNA biology and antiviral strategies against cccDNA. Overall, we believe these auto-generating rcccDNA plasmids will be of great value to model cccDNA for testing new therapies against HBV infection.

Keywords: Cre/LoxP; Drug discovery; HBV; cccDNA.

Figure 1.. Recombinant cccDNA can be formed with LoxP insertion points resulting in similar production of viral biomarkers and is stable in mice long-term compared to HBV plasmids.

(A) Four different LoxP insertion sites were identified in the HBV genome and predicted to not interfere with important viral functions. 15 μg of pLoxP-HBV plasmid (HBx, Core, PreS2, PreS1) were injected with 5 μg pCMV.NLS-Cre or Control plasmid into female NSG mice. HBsAg levels (B) and serum DNA levels (C) were measured at one week post-injection, showing increased expression with presence of Cre (n=4). (D) Immunofluorescence staining for Core expression in the same mice revealed Core staining only with the presence of Cre recombinase. (E,F) Long-term expression of rcccDNA generated from the LoxP-HBx construct was assessed in male NSG mice. 15 μg LoxP-HBx (n=4) or pHBV1.3 (n=3) were hydrodynamically injected with 5 μg pCMV-NLS-Cre or control plasmid. Serum HBsAg levels (E) and serum DNA levels (F) were monitored with elevated HBV expression seen with rcccDNA. Data represent mean ± standard error of mean (SEM); statistical significance (P<0.05). (** P<0.01; *** P<0.001; **** P<0.0001)

Figure 2.. A plasmid with Cre recombinase in cisis stable and forms recombinant cccDNA molecules.

(A) A CMV.NLS-Cre cassette, with inserted internal intron to inhibit bacterial expression, was cloned into the LoxP-HBV plasmids, such that the Cre transcript would utilize the HBV polyadenylation site. Upon introduction into mammalian cells, expressed Cre will excise the HBV genome forming to separate circular DNA molecules inside the cell. (B) The stability of the Cre/LoxP-HBV plasmids in E. coli were assessed by HindIII restriction digest, showing that their total size is the 7.9 kB of the original plasmid. (C) Upon transfection into 293T cells, recombination can be confirmed using PCR across the recombination junction showing predicted band sizes are present. (D) Livers of female NSG mice were transfected with 20 μg Cre/LoxP-HBV plasmids using hydrodynamic tail vein injection and HBsAg levels in the serum assessed week 1 post-injection (n=4). Data represent mean ± standard error of mean (SEM).

Figure 3.. HBx protein regulates HBV core expression in rcccDNA.

HBx function was probed by injecting 15 μg pHBV1.2 or pHBV1.2*7 with 5 μg pSI-X expressing HBx or control plasmid (pCMV-Gaussia) into mice. Similarly, 15 μg pCre/LoxP-PreS2 or pCre/LoxP-PreS2*7 with 5 μg pSI-X or control plasmid was injected into mice. At one week post-injection, mice were harvested and tissue analyzed. (A) Immunofluorescent staining for core protein across the six conditions was undertaken, depicting dependence on HBx for rcccDNA expression. Representative photos are shown. (B) Immunofluorescent staining for HBV core was quantified in four fields at low magnification (>300 hepatocytes per field) and percentage positive reported (*** P<0.001; **** P<0.0001, n.s. = nonsignificant). Data represent mean ± standard error of mean (SEM); statistical significance (P<0.05).

Figure 4.. A fluorescent rcccDNA reporter exhibits knockdown after CRISPR targeting.

Green fluorescent protein (GFP) was cloned into the HBsAg ORF abolishing the overlapping polymerase reading frame. The final molecule after rcccDNA formation is depicted. (B,C) 293T cells were transfected with equal amounts of pCre/LoxP-HBV-GFP(S) and pCas9-gRNA21 or control plasmid. At 48 hours post-transfection, expression of GFP could be detected in 293T cells with control plasmid (B), whereas transfection with pCas9-gRNA21 revealed greatly reduced GFP expression (C).