Back to Search
Start Over
AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.
- Source :
-
PloS one [PLoS One] 2014 May 14; Vol. 9 (5), pp. e97579. Date of Electronic Publication: 2014 May 14 (Print Publication: 2014). - Publication Year :
- 2014
-
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.
- Subjects :
- Antiviral Agents chemistry
Base Sequence
Cell Line, Tumor
DNA Breaks, Double-Stranded
DNA End-Joining Repair
DNA, Viral genetics
Dependovirus genetics
Endonucleases chemistry
Endonucleases metabolism
Gene Products, pol antagonists & inhibitors
Gene Products, pol chemistry
Genetic Vectors
HEK293 Cells
Hepatitis B virus chemistry
Hepatocytes virology
Humans
Molecular Sequence Data
Protein Engineering
Trans-Activators antagonists & inhibitors
Trans-Activators chemistry
Viral Core Proteins antagonists & inhibitors
Viral Core Proteins chemistry
Viral Regulatory and Accessory Proteins
Virus Replication genetics
Zinc Fingers genetics
Antiviral Agents metabolism
Endonucleases genetics
Gene Products, pol genetics
Gene Targeting
Hepatitis B virus genetics
Trans-Activators genetics
Viral Core Proteins genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 9
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 24827459
- Full Text :
- https://doi.org/10.1371/journal.pone.0097579