The PXB-mouse: Humanized Liver Model of HBV infection for Therapeutic Development

Hepatitis B virus (HBV) is a global public health challenge that impacts millions of people worldwide. Chronic HBV infection can lead to cirrhosis and hepatocellular carcinoma, severe diseases that are difficult to treat. 254 million people are living with chronic HBV and, while an effective vaccine for HBV exists, a further ~1.2 million people acquire the infection annually, especially in areas where healthcare resources are limited. Effective treatment options also exist, but they are not curative. HBV belongs to the hepadnavirus family, and contains a 3.2 kilobase relaxed circular DNA (rcDNA) genome. A hindrance to understanding the biology of HBV and developing therapeutics to cure the disease is the fact that HBV is significantly host restricted, and infection is limited to human and chimpanzee hepatocytes¹. Thus, traditional preclinical models, such as mice and rats, that are routinely used in drug development are not suitable for HBV studies.

What makes PXB-mice a good model for HBV research?

The PXB-mouse®, a cDNA-uPA/SCID mouse transplanted with primary human hepatocytes, has high human hepatocyte engraftment, with up to 95% humanization. PXB-mice have physiologically normal livers with stable expression of human genes and proteins including human Phase I and Phase II metabolic pathways and excretion pathways^2-4. Importantly, PXB-mice express human sodium taurocholate co-transporting polypeptide (NTCP), making them susceptible to HBV infection⁵. PXB-mice can be infected with clinically relevant HBV variants (Figure 1) and support the entire HBV life cycle (Figures 2 and 3)^5,6. As such, rcDNA is converted to covalently closed circular DNA (cccDNA) in the liver of PXB-mice (Figure 3). This is in contrast to pseudo-infection models such as the AAV-HBV and HBV minicircle models, which have varying degrees of cccDNA formation or impaired processing of rcDNA^7-9. While those models can be useful for certain types of studies, such as those where an immunocompetent background is required, the PXB-mouse is an ideal choice for determining efficacy of a wide range of therapeutic modalities from small molecules to oligonucleotides and gene editors to antibodies^10-13. Humanized liver mice are also ideally suited to mechanistic studies aimed at better understanding host-pathogen interactions^14-16.

Figure 1: Viral kinetics of 9 patients on nucleos(t)ide analog (NA) treatment and when inoculated in the PXB-mouse model. (A) HBV DNA levels of the patients selected for this study following administration of the NAs tenofovir (TDF) in monotherapy or TDF and emtricitabine in combination therapy. Each line represents an individual patient. The dashed line represents the lower limit of quantification (LLOQ). (B) Mice (n = 2 per isolate) were inoculated with 0.1 mL of baseline serum samples from patients chronically infected with HBV genotype B (Patient 1, 2, 5, 7, 9) or C (Patient 3, 4, 6, 8). HBV DNA was measured by qRT-PCR and reported as copies/mL. The dashed line represents the LLOQ (4 x 10⁴copies/mL) and the solid line represents the lower limit of detection (LLOD) (8 x 10² copies/mL). (Adapted from Burdette et al., 2022⁶)

Figure 2: Multiphasic HBV kinetic patterns in PXB-mice from inoculation to steady state. A) Early HBV kinetic phases (1 to 5) in 9 individual mice (circles) during the first 8 days post inoculation. (B) Late HBV kinetic phases (6 and 7). Average serum HBV DNA ± SD shown based on inoculum size and donor. Abbreviations: Ph, phase; Grp, group. (C) Representative images of HBV core antigen (HBcAg) immunostaining in PXB-mouse livers at 2 or 10 weeks post-HBV inoculation. Corresponding Serum HBV-DNA levels are <10⁴ copies/mL (2 weeks) or 2.5 x 10⁹ copies/mL (10 weeks). Arrows indicate HBV positive cells. Bar, 50 µm. Abbreviation: P, portal vein. (Adapted from Ishida et al., 2018)⁵Figure 3: Levels of HBV biomarkers in PXB-mice at 2 - 13 weeks post inoculation with HBV Genotype C. (A) Serum HBV DNA (B) Intrahepatic HBV DNA (C) HBV RNA and (D) Intrahepatic cccDNA. *not detected; #not measured; dashed line indicates LLOQ. (Adapted from Ishida et al., 2018)⁵

Extensive Validation of the PXB-mouse Highlights its Translational Value

The PXB-mouse has been extensively validated as a model to test a variety of therapeutic modalities, including current standard of care options such as nucleos(t)ide inhibitors (Figures 4 & 5) and pegylated interferon (PegIFN) (Figures 4 & 5). Treatment with entecavir, a commonly used nucleo(t)ide inhibitor, which targets viral replication results in a dramatic decrease in serum HBV DNA levels in the PXB-mouse, but as expected, it does not decrease levels of HBV surface antigen (HBsAg) or cccDNA. On the other hand, PegIFN, which works via immune modulation to clear HBsAg, has a small impact on serum HBV DNA, but dramatically reduces levels of HBsAg, HBV e antigen (HBeAg) and HBV core-related antigen (HBcrAg) (Figures 4 & 5). Combination of the two therapeutics results in larger drops in all serum markers as well as substantial reductions in levels of HBV DNA, cccDNA, precore mRNA and HBs mRNA in the livers of HBV-infected PXB-mice (Figure 4 C & D). This aligns with clinical data showing that combined treatment with entecavir and PEG-IFN resulted in higher levels of HBsAg loss and HBsAg seroconversion than patients treated with entecavir alone¹⁷.

Figure 4: Effect of antivirals on HBV-infected PXB-mice. Four HBV-infected mice received either no treatment, 2 mg/kg of entecavir (ETV) alone daily, 30 µg/kg of PEG-IFN alone twice weekly, or 2 mg/kg entecavir (daily) plus 30 µg/kg PEG-IFN (twice weekly) for 12 weeks. (A) Time course of serum HBV DNA titer in each mouse. (B) Serum HBsAg, HBeAg, and HBcrAg levels in each mouse at baseline and after 12 weeks of treatment. (C) Intrahepatic HBV DNA and HBV cccDNA levels in each mouse. An HBV-infected mouse without treatment (no treatment) was also analyzed. Data are presented as the mean ± standard deviation (SD) for three samples of liver tissue from each mouse. (D) Intrahepatic precore mRNA and HBs mRNA. *, P ≤ 0.05. LLOD, lower limit of detection. (Adapted from Uchida et al.,2017)¹⁰

Figure 5: Effect of ETV and PegIFNa alone or in combination on HBV viral markers in livers from HBV-infected PXB-mice. HBsAg (red) and cell nuclei (blue) (upper panel), and HBcAg (green), cell nuclei (blue) and human hepatocytes (red) (lower panel) were detected by immunohistochemistry in the mouse liver samples at the end of treatment (day 35). (Adapted from Mueller et al., 2018)¹¹

Translationally Relevant Data for a Variety of Therapeutic Modalities

The PXB-mouse can be used to test the anti-HBV efficacy of a wide range of therapeutic modalities. A recent study used a lipid nanoparticle (LNP)-delivered siRNA targeting human DOCK11 in HBV-infected PXB-mice¹³. DOCK11 had previously been identified as a host factor that is involved in the maintenance of cccDNA¹⁸. Depletion of human DOCK11 in the livers of HBV-infected PXB-mice resulted in a clear reduction in blood HBV DNA, serum HBsAg and HBeAg and liver HBV DNA levels (Figure 6A-F). Importantly a significant decrease in the copies of cccDNA in the siRNA-treated mice was also observed (Figure 6G).Figure 6: LNP-encapsulated chemically modified DOCK11-targeting siRNA reduces HBV replication and cccDNA in HBV-infected human hepatocyte chimeric mice (PXB-mice) (A) Experimental design (B–G) Relative expression levels of DOCK11 mRNA and protein in the liver (B), the copy number of blood HBV-DNA (C), serum HBsAg (D), and HBeAg (E) levels, and the copy numbers of HBV cccDNA (F) and HBV-DNA (G) in the liver of HBV-infected PXB-mice after administering LNP-siRNA DOCK11-I. (Adapted from Okada et al., 2024)¹³

Gene editing methodologies for modulating HBV have also been tested in the PXB-mouse. CRISPR/Cas9 editing was used to target cccDNA in HBV-infected PXB-mice¹². The editor was delivered using an AAV vector and resulted in a significant decrease in intrahepatic HBV DNA and cccDNA levels (Figure 7). More recently, base editing was also used successfully to result in a durable reduction in both serum HBV DNA and serum HBsAg levels in infected PXB-mice¹⁹. More details on this approach can be found in our recent blog post.

Figure 7: Effects of the AAV2/WJ11-Cas9 system in the persistently HBV genotype C-infected PXB-mice. HBV DNA (A) and HBV cccDNA (B) levels in the liver of negative control, AAV2-treated control, and AAV2/WJ11-Cas9-treated chimeric mice. (Adapted from Kayesh et al, 2020)¹².

Conclusion

The PXB-mouse is the gold standard for testing in vivo efficacy of anti-HBV therapeutics. Get the translational data you need to move your lead candidate forward by studying it in a model that not only supports the entire natural HBV lifecycle but also recapitulates human-specific drug metabolism.

For a limited time, PhoenixBio is offering the chance to carry out a study in HBV-infected PXB-mice for as low as $99,000. 

Using 12 animals, this study design provides the opportunity to test your compound against an approved nucleos(t)ide inhibitor. 

Figure 8: Study design in 12 HBV-infected PXB-mice. Three groups of 4 HBV-infected PXB-mice will be dosed with vehicle, entecavir and your compound of interest. Serum HBV DNA will be measured every two weeks until the end of the study on Day 28. Serum/Plasma and snap frozen and formalin-fixed paraffin embedded liver samples will be collected and shipped to you for further analyses. 

Reach out today to discuss your study!

References

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