AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication

• Published in: PloS one 2014-05, Vol.9 (5), p.e97579-e97579
• Main Authors: Weber, Nicholas D, Stone, Daniel, Sedlak, Ruth Hall, De Silva Feelixge, Harshana S, Roychoudhury, Pavitra, Schiffer, Joshua T, Aubert, Martine, Jerome, Keith R
• Format: Article
• Language: English
• Subjects: Activation; Antiviral Agents - chemistry; Antiviral Agents - metabolism; Base Sequence; Biology and life sciences; Cancer; Cell Line, Tumor; Circular DNA; CRISPR; Cytotoxicity; Deoxyribonucleic acid; Dependovirus - genetics; DNA; DNA binding proteins; DNA biosynthesis; DNA Breaks, Double-Stranded; DNA damage; DNA End-Joining Repair; DNA replication; DNA, Viral - genetics; Double-strand break repair; Endonucleases - chemistry; Endonucleases - genetics; Endonucleases - metabolism; Enzymes; Gene Products, pol - antagonists & inhibitors; Gene Products, pol - chemistry; Gene Products, pol - genetics; Gene sequencing; Gene Targeting; Gene therapy; Genes; Genetic Vectors; Genomes; Genomics; Genotype & phenotype; Health aspects; HEK293 Cells; Hemophilia; Hepatitis; Hepatitis B; Hepatitis B virus; Hepatitis B virus - chemistry; Hepatitis B virus - genetics; Hepatocytes; Hepatocytes - virology; Homology; Humans; Infection; Infections; Infectious diseases; Laboratories; Medical research; Medicine; Molecular Sequence Data; Mutation; Next-generation sequencing; Non-homologous end joining; Nuclease; Nucleases; Patients; Protein Engineering; Public health; Replication; Rodents; Therapy; Toxicity; Trans-Activators - antagonists & inhibitors; Trans-Activators - chemistry; Trans-Activators - genetics; Vaccines; Viral Core Proteins - antagonists & inhibitors; Viral Core Proteins - chemistry; Viral Core Proteins - genetics; Virions; Virus Replication - genetics; Viruses; Zinc; Zinc finger proteins; Zinc Fingers - genetics
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0097579

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.