A new humanized mouse model to study HBV gene editing

Project Summary Chronic Hepatitis B virus (HBV) infection (CHB) affects hundreds of millions of people across the world. Despite effective vaccines, and antiviral therapies that can suppress viral replication, these infections are largely incurable, and new therapies for CHB are desperately needed. We have investigated gene editing using targeted nucleases to cleave covalently closed circular DNA (cccDNA), the template for HBV replication, which results in its degradation or inactivation in infected hepatocytes. Our work and the work of others has been hindered due to the fact that existing small animal models for HBV have substantial limitations, or are complex and expensive to establish. To this end, we established and validated a robust and low-cost liver humanized NSG-PiZ mouse as a model for the study of HBV. Here we propose to use these mice for the evaluation of novel curative gene editing therapies that target CHB. Specific Aim 1. Evaluate the anti-HBV efficacy of HBV gene therapy in liver-humanized NSG-PiZ mice. We will first evaluate the efficiency of HBV gene editing in NSG-PiZ mice, using AAV-SaCas9 vectors we previously validated in HBV-infected humanized FRG mice. We will then compare lipid nanoparticle (LNP) delivery of nuclease mRNA with AAV vectors for the transduction of PHH in our model. We will compare the antiviral efficacy of different classes of gene editing nuclease in our NSG-PiZ mouse model of CHB, and evaluate chromatin modifying agents for their ability to make cccDNA accessible to gene editing nucleases in vitro and in vivo. Finally, we will assess combined gene-editing, reverse transcriptase inhibitor (RTi), and entry inhibitor antiviral therapy in our model of CHB. Specific Aim 2. Evaluate the tolerability and host genomic consequences of AAV- and LNP-delivered nuclease therapies targeting HBV in liver-humanized NSG-PiZ mice. We will first determine the maximum tolerated dose (MTD) and minimal effective dose for AAV-nuclease and LNP-nuclease therapies in our CHB model. We will then monitor the effects of different nuclease classes on treatment-related off-target genotoxicity in vivo and determine whether use of liver-specific promoters can mitigate therapy-associated genotoxicity. We will also determine whether therapy-associated genotoxicity is reduced when nuclease expression is transiently provided by LNPs. Finally, we will evaluate therapy-specific immune responses and therapy- associated hepatotoxicity and neurotoxicity after transient or persistent nuclease expression in immunocompetent mice.