Liver humanized NSG-PiZ mice support the study of chronic hepatitis B virus infection and antiviral therapies

Hepatitis B virus (HBV) is a pathogen of major public health importance that is largely incurable once a chronic hepatitis B (CHB) infection is established. Only humans and great apes are fully permissive to HBV replication, and this species restriction has impacted HBV research by limiting the utility of small animal models of HBV. To combat the species restriction of HBV and enable more HBV studies in vivo , liver-humanized mouse models have been developed that harbor primary human hepatocytes (PHH) and are fully permissive to HBV infection and replication. Unfortunately, these models can be difficult to establish and are expensive commercially, which has limited their academic use. As an alternative mouse model to study HBV, we evaluated liver-humanized NSG-PiZ mice and showed that they are fully permissive to HBV and can develop CHB. Mice were infected with a precore mutant clinical isolate that has now been serially passaged through 3 generations of mice without loss of fitness. HBV selectively replicates in hCK18+ human hepatocytes within chimeric livers, and HBV+ mice secrete infectious virions and HBsAg into blood, while also harboring covalently closed circular DNA (cccDNA). HBV+ mice remain viremic for at least 169 days, which should enable the study of new curative therapies targeting CHB and respond to antiviral entecavir therapy. The extended duration of viremia is sufficient to enable the study of established and new therapeutic approaches targeting CHB. Furthermore, HBV+ PHH in NSG-PiZ mice can be transduced by the hepatotropic AAV3b and AAV.LK03 vector capsids, which should enable the study of curative gene therapies that target CHB. In summary, our data demonstrates that liver humanized NSG-PiZ mice can be used as a robust and cost-effective alternative to existing CHB models and may enable more academic research labs to study HBV disease pathogenesis and antiviral therapy in a setting that is fully permissive to ongoing replication. ### Competing Interest Statement KRJ is a paid advisor and holds equity in Excision Biosciences; he also has a sponsored research agreement with Emendo Biotherapeutics. * AAV : adeno-associated virus cccDNA : covalently closed circular DNA CHB : chronic hepatitis B CPX : ciclopirox ddPCR : droplet digital PCR ETV : entecavir GFP : green fluorescent protein HBcAg : hepatitis B core antigen HBeAg : hepatitis B e antigen HBsAg : hepatitis B surface antigen HBV : hepatitis B virus HCC : hepatocellular carcinoma hRPP30 : human RPP30 hCK18 : human cytokeratin 18 HEK : human embryonic kidney HHV : human herpesvirus HPV : human papilloma virus HSC : hematopoietic stem cells HSV : herpes simplex virus huAlb : human albumin IHC : immunohistochemistry MCT : monocrotaline mRPP30 : mouse RPP30 NSG : NOD-scid-gamma NTCP : sodium taurocholate co-transporting polypeptide PBS : phosphate buffered saline PHH : primary human hepatocytes qPCR : quantitative PCR rcDNA : relaxed circular DNA RTi : reverse transcriptase inhibitor uPA : urokinase plasminogen activator