Predictors of Hepatitis B Cure Using Gene Therapy to Deliver DNA Cleavage Enzymes: A Mathematical Modeling Approach

Abstract

Most chronic viral infections are managed with small molecule therapies that inhibit replication but are not curative because non-replicating viral forms can persist despite decades of suppressive treatment. There are therefore numerous strategies in development to eradicate all non-replicating viruses from the body. We are currently engineering DNA cleavage enzymes that specifically target hepatitis B virus covalently closed circular DNA (HBV cccDNA), the episomal form of the virus that persists despite potent antiviral therapies. DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR-associated system 9 (Cas9) proteins, can disrupt specific regions of viral DNA. Because DNA repair is error prone, the virus can be neutralized after repeated cleavage events when a target sequence becomes mutated. DNA cleavage enzymes will be delivered as genes within viral vectors that enter hepatocytes. Here we develop mathematical models that describe the delivery and intracellular activity of DNA cleavage enzymes. Model simulations predict that high vector to target cell ratio, limited removal of delivery vectors by humoral immunity, and avid binding between enzyme and its DNA target will promote the highest level of cccDNA disruption. Development of de novo resistance to cleavage enzymes may occur if DNA cleavage and error prone repair does not render the viral episome replication incompetent: our model predicts that concurrent delivery of multiple enzymes which target different vital cccDNA regions, or sequential delivery of different enzymes, are both potentially useful strategies for avoiding multi-enzyme resistance. The underlying dynamics of cccDNA persistence are unlikely to impact the probability of cure provided that antiviral therapy is given concurrently during eradication trials. We conclude by describing experiments that can be used to validate the model, which will in turn provide vital information for dose selection for potential curative trials in animals and ultimately humans.

Author Summary

Innovative new approaches are being developed to eradicate viral infections that until recently were considered incurable. We are interested in engineering DNA cleavage enzymes that can cut and incapacitate persistent viruses. One hurdle is that these enzymes must be delivered to infected cells as genes within viral vectors that are not harmful to humans. In this paper, we developed a series of equations that describe the delivery of these enzymes to their intended targets, as well the activity of DNA cutting within the cell. While our mathematical model is catered towards hepatitis B virus infection, it is widely applicable to other infections such as HIV, as well as oncologic and metabolic diseases characterized by aberrant gene expression. Certain enzymes may bind DNA more avidly than others, while different enzymes may also bind cooperatively if targeted to different regions of viral DNA. We predict that such enzymes, if delivered efficiently to a high proportion of infected cells, will be critical to increase the probability of cure. We also demonstrate that our equations will serve as a useful tool for identifying the most important features of a curative regimen, and ultimately for guiding clinical trial dosing schedules to ensure hepatitis B eradication with the smallest number of possible doses.