Creation of a transgenic pig model permissive to HBV infection - PROJECT SUMMARY The lack of a convenient animal model that is susceptible to hepatitis B virus (HBV) infection has prevented the development of curative therapies for a disease chronically infecting over 300 million people worldwide. To address the need for a universally applicable large animal model that can be used to advance understanding of HBV biology and test curative strategies for chronic infection, the long-term goal is to establish an immunocompetent pig model susceptible to HBV infection. Prior studies have shown that the introduction of the HBV entry receptor, human sodium taurocholate cotransporting polypeptide (hNTCP), into pig hepatocytes in vitro was sufficient to mediate HBV infection and covalently closed circular DNA (cccDNA) genome formation. The central hypothesis is that pigs expressing hNTCP will be capable of replicating HBV at high levels and subsequently will establish chronic HBV infection in the liver with similar features to human patients. To test this hypothesis, the efficiency of HBV infection and replication in pigs will be first evaluated in vitro and in vivo. The proposing team, led by the PI, has previously established a method of non-viral endoscopic hydrodynamic gene delivery into pig liver that greatly simplifies liver DNA delivery in vivo, supporting this first aim. Using the knowledge from these experiments, the second aim is to create the transgenic pig model expressing hNTCP that would be fully permissive to HBV infection. This will be accomplished by knocking hNTCP cDNA into a safe harbor locus in the pig genome using CRISPR in fibroblasts, followed by somatic nuclear transfer into embryos and implantation into female pigs. After gestation, transgenic pigs will be assessed for hNTCP expression and normal physiology and biliary metabolism, and then tested for acute vs. chronic HBV infection upon virus introduction, monitoring viral markers, immune responses to HBV proteins, and liver pathological changes. The innovation of the proposed research lies both in the (i) methods for interrogation of the most suitable DNA construct to accomplish the overall objective and (ii) the final deliverable – a transgenic animal that is susceptible to HBV infection, which is currently not available to the HBV field. The significance lies in providing a convenient, immunocompetent pig model capable of authentic HBV to test novel HBV therapeutic strategies. Thus, the proposed research benefits all, facilitating testing therapies stimulating HBV immunity toward the development of a cure for chronic HBV infection.
