Abstract
Chronic hepatitis B virus (HBV) infection is prevalent, deadly, and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients.
Highlights
Hepatitis B virus (HBV) chronically infects over 250 million people worldwide
Since integration of hepatitis B virus (HBV) DNA into the host human genome is not part of the canonical HBV life-cycle, we evaluated the effects of Cas[9] targeting in the context of de novo HBV infection, where episomal closed circular DNA (cccDNA) serves as the only template for viral gene expression and replication
Largely unexplored in mammalian systems, bacteria and archaea utilize sequence specific DNA nucleases to interfere with viral replication[17]
Summary
Hepatitis B virus (HBV) chronically infects over 250 million people worldwide. Chronically infected individuals are at an increased risk for deadly complications, including cirrhosis, end-stage liver disease and hepatocellular carcinoma, resulting in approximately 600,000 deaths per year[1]. Approved HBV therapies act post-transcriptionally to inhibit viral replication and fail to target or eliminate the cccDNA pool, which exhibits extraordinary stability and persistence[3]. These drugs must often be taken indefinitely to prevent viral rebound. Agents that act directly on viral DNA to deplete this reservoir may represent more desirable and possibly curative therapeutic alternatives[4]. To this end, targeted nucleases may provide an efficient and specific way to damage the HBV genome while sparing host genomic DNA5–7. We hypothesized that by directly targeting the HBV genome for cleavage using CRISPR/Cas[9], we could suppress HBV by mutagenizing critical genomic elements or decreasing the stability of cccDNA and other viral intermediates through repeated linearization of the circular genomes (Fig. 1a)
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