AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication
Authors: Ruth Hall Sedlak
Daniel Stone
Pavitra Roychoudhury
Harshana S De Silva Feelixge
Keith R Jerome
Nicholas D Weber
Martine Aubert
Joshua T Schiffer
Journal: PLoS ONE (2014)
Discipline: Multidisciplinary
Keywords: Hepatitis B virus; HBV; Zinc finger nucleases; ZFNs; Adeno-associated virus
Affiliations: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America; Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America., Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America., Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America., Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America; Division of Allergy and Infectious Disease, Department of Medicine, University of Washington, Seattle, Washington, United States of America; Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America., Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America; Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America; Department of Microbiology, University of Washington, Seattle, Washington, United States of America.

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. 

Retracted: No

PubPeer comments:
 [Gene-therapy will finally fail-after causing immense harm.] [Because after the DSB, one cant control what happens - sometimes the vector (the viral bit, the nuclease, etc) integrates, and almost always there are translocations. ] [And this, when we are mostly looking at a tree - completely unaware of what is happening in the forest due to off-targets] And it is not only in eukaryotes, even in viruses, the plasmid integrates. SRR1206324.15967.1 15967 length=935 GCGGCGATTGAGACCTTCGTCTGCGAGGCGAGGGAGTTTTACTTCTAGGGAGCCTGCTCCGTCGTCTAACAGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTCAAGGCCCAGCTGACCAGGCTGAAACCGCAAAACCAACTGCAATGGCGCCGTGCTGAGCGTGGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAGCCGGCACCTGAACACTGGGAGGAGGTGCGGGCGCAAGTTCAACAACGGCGAGATCAACTTCTGAGCGGCCGCTGGGGATCCAGACATGATAAGACATACATTGATGAGTTTGTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAAATAAACAAGTTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTTCTAGAGCATGGCTACGTAGATAAGTAGGCATGGCGGGTTAATCATTAACTACACAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGAACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAACAACAGTAGTTTCCGGAAGTGTTGATAGGATAGGGGCATTTGGTGGTCTATAAGCTGGAGGAGTGCGAATCCACACTCCGAAAGGACACCAAATACTCTATAACGGTTTCTCTTCCAAAAGTGAGACAAGAAATTTGAAACCACAAGAGTTGCCTGAACTTTAGGCCCATATTAGTGGTTGACATAACTGACTACTAGGTCTCTAGATGCTGG ![file] This long read shows the integration ZFN and the AAV vector in the Hepatitis B virus exactly where the ZFN gRNA2 is designed to cut (743 CACTTCCGGAAACTACTGTTGTT 721) List of gRNAS 1 CGCTGAATCCTGCGGACGACCCTTCTCGGGGTCGCTTGGGAC 2 CACTTCCGGAAACTACTGTTGTTAGACGACGAGGCAGGT 3 GTTGTCCTCTCCCGCAAATATACATCGTATCCATGGCTGCT ~ ~ ,