Abstract
Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.
Highlights
Hepatitis B virus (HBV) remains a significant cause of morbidity and mortality worldwide [1]
Several double strand breaks (DSB)-inducing enzymes, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), homing endonucleases (HEs), and the CRISPR/Cas system could be used in such an approach
HBV-specific ZFNs In order to verify that HBV-specific ZFN pairs could have broad activity against multiple HBV genotypes, we analyzed sequence conservation and the relative evolutionary rates of each ZFN target site across 3847 HBV sequences found in Genbank (Figure 1B)
Summary
Hepatitis B virus (HBV) remains a significant cause of morbidity and mortality worldwide [1]. One potential approach involves eliminating or modifying viral sequences enough to sufficiently disrupt HBV gene functions This could be achieved by introducing double strand breaks (DSBs) into viral DNA using targeted endonucleases. Episomal DNA that is linearized from DSBs may be susceptible to direct degradation by cellular DNases, which could result in reduced viral replication or virus elimination. This strategy of targeted gene disruption has been proposed as a novel anti-viral therapy [6,7,8]. Several DSB-inducing enzymes, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), homing endonucleases (HEs), and the CRISPR/Cas system could be used in such an approach
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