Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases
Open accessAbstract
Chronic hepatitis B virus (HBV) infection remains an important global health problem. Stability of the episomal covalently closed circular HBV DNA (cccDNA) is largely responsible for the modest curative efficacy of available therapy. Since licensed anti-HBV drugs have a post-transcriptional mechanism of action, disabling cccDNA is potentially of therapeutic benefit. To develop this approach, we engineered mutagenic transcription activator-like effector nucleases (TALENs) that target four HBV-specific sites within the viral genome. TALENs with cognate sequences in the S or C open-reading frames (ORFs) efficiently disrupted sequences at the intended sites and suppressed markers of viral replication. Following triple transfection of cultured HepG2.2.15 cells under mildly hypothermic conditions, the S TALEN caused targeted mutation in ~35% of cccDNA molecules. Markers of viral replication were also inhibited in vivo in a murine hydrodynamic injection model of HBV replication. HBV target sites within S and C ORFs of the injected HBV DNA were mutated without evidence of toxicity. These findings are the first to demonstrate a targeted nuclease-mediated disruption of HBV cccDNA. Efficacy in vivo also indicates that these engineered nucleases have potential for use in treatment of chronic HBV infection.
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ZFNs and TALENs can cleave host genomes in a site-specific fashion and have been under investigation for specific degradation of HBV genomes in vitro and in vivo.
“Two genome editing technologies, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), can cleave the host genomes in a site-specific fashion and have been under investigation for specific degradation of the HBV genomes in vitro and in vivo.8,9,10”
From The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo (2014) · cites this paper as [9]
The CRISPR/Cas9 system was as effective as TALENs in mediating gene disruption in vitro, although its mutagenesis rate is lower (~5% by T7E1 and ~27% by clonal sequencing) in vivo.
“Our study demonstrated that the CRISPR/Cas9 system was as effective as TALENs in mediating gene disruption in vitro, although its mutagenesis rate is lower (~5% by T7E1 and ~27% by clonal sequencing) in vivo.”
From The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo (2014) · cites this paper as [9]
Previous studies reported that HBV-specific TALENs have a mutagenesis rate ranging between 30 and 70%.
“Previous studies reported that HBV-specific TALENs have a mutagenesis rate ranging between 30 and 70%.9,10”
From The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo (2014) · cites this paper as [9]
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Cites
Multiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339 · see citing works
Abràmoff, M. D., Magalhães, P. J., & Ram, S. J. (2004) · Utrecht University Repository (Utrecht University) · Cited by 11,816 · see citing works
Epidemiology of Viral Hepatitis and Hepatocellular Carcinoma
El‐Serag, H. B. (2012) · Gastroenterology · doi:10.1053/j.gastro.2011.12.061 · Cited by 3,384 · see citing works
High-Frequency off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells
Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D. (2013) · Nature Biotechnology · doi:10.1038/nbt.2623 · Cited by 3,292 · see citing works
Efficient genome editing in zebrafish using a CRISPR-Cas system
Hwang, W. Y., Fu, Y., Reyon, D., Maeder, M. L., Tsai, S. Q., Sander, J. D., Peterson, R. T., Yeh, J.-R. J., & Joung, J. K. (2013) · Nature Biotechnology · doi:10.1038/nbt.2501 · Cited by 2,937 · see citing works
Perz, J. F., Armstrong, G. L., Farrington, L. A., Hutin, Y. J. F., & Bell, B. P. (2006) · Journal of Hepatology · doi:10.1016/j.jhep.2006.05.013 · Cited by 2,572 · see citing works
Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.
Kim, Y. G., Cha, J., & Chandrasegaran, S. (1996) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.93.3.1156 · Cited by 2,099 · see citing works
TALENs: A widely applicable technology for targeted genome editing
Joung, J. K., & Sander, J. D. (2013) · Nature Reviews Molecular Cell Biology · doi:10.1038/nrm3486 · Cited by 1,650 · see citing works
Tiollais, P., Pourcel, C., & Dejean, A. (1985) · Nature · doi:10.1038/317489a0 · Cited by 1,213 · see citing works
Sells, M. A., Chen, M. L., & Acs, G. (1987) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.84.4.1005 · Cited by 1,121 · see citing works
A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity
Mussolino, C., Morbitzer, R., Lütge, F., Dannemann, N., Lahaye, T., & Cathomen, T. (2011) · Nucleic Acids Research · doi:10.1093/nar/gkr597 · Cited by 714 · see citing works
Self-citationHigh-level hepatitis B virus replication in transgenic mice
Guidotti, L., Matzke, B., Schaller, H., & Chisari, F. (1995) · Journal of Virology · doi:10.1128/jvi.69.10.6158-6169.1995 · Cited by 656 · see citing works
TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction
Doyle, E., Booher, N. J., Standage, D., Voytas, D. F., Brendel, V., VanDyk, J. K., & Bogdanove, A. J. (2012) · Nucleic Acids Research · doi:10.1093/nar/gks608 · Cited by 587 · see citing works
Control of cccDNA function in hepatitis B virus infection
Levrero, M., Pollicino, T., Petersen, J., Belloni, L., Raimondo, G., & Dandri, M. (2009) · Journal of Hepatology · doi:10.1016/j.jhep.2009.05.022 · Cited by 552 · see citing works
Woodchuck hepatitis virus X protein is required for viral infection in vivo
Zoulim, F., Saputelli, J. R., & Seeger, C. (1994) · Journal of Virology · doi:10.1128/jvi.68.3.2026-2030.1994 · Cited by 456 · see citing works
Nassal, M. (1992) · Journal of Virology · doi:10.1128/jvi.66.7.4107-4116.1992 · Cited by 450 · see citing works
Hydrodynamic injection of viral DNA: A mouse model of acute hepatitis B virus infection
Yang, P. L., Althage, A., Chung, J., & Chisari, F. V. (2002) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.202398599 · Cited by 381 · see citing works
The woodchuck hepatitis virus X gene is important for establishment of virus infection in woodchucks
Chen, H. S., Kaneko, S., Gironés, R., Anderson, R. W., Hornbuckle, W. E., Tennant, B. C., Côté, P. J., Gerin, J. L., Purcell, R. H., & Miller, R. H. (1993) · Journal of Virology · doi:10.1128/jvi.67.3.1218-1226.1993 · Cited by 370 · see citing works
Newbold, J. E., Xin, H., Tencza, M. G., Sherman, G., Dean, J., Bowden, S., & LOCARNINI, S. A. (1995) · Journal of Virology · doi:10.1128/jvi.69.6.3350-3357.1995 · Cited by 313 · see citing works
Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions
Sells, M. A., Zelent, A., Shvartsman, M., et al. (1988) · Journal of Virology · doi:10.1128/jvi.62.8.2836-2844.1988 · Cited by 269 · see citing works
Mismatch cleavage by single-strand specific nucleases
Till, B. J. (2004) · Nucleic Acids Research · doi:10.1093/nar/gkh599 · Cited by 263 · see citing works
Ding, D., Lou, X., Hua, D., Yu, W., Li, L., Wang, J., Gao, F., Zhao, N., 任国平, Li, L., et al. (2012) · PLoS Genetics · doi:10.1371/journal.pgen.1003065 · Cited by 206 · see citing works
Arbuthnot, P. B., Capovilla, A., & Kew, M. C. (2000) · Journal of Gastroenterology and Hepatology · doi:10.1046/j.1440-1746.2000.02069.x · Cited by 198 · see citing works
Self-citationLaras, A., Koskinas, J. S., Dimou, E., Kostamena, A., & Hadziyannis, S. J. (2006) · Hepatology · doi:10.1002/hep.21299 · Cited by 194 · see citing works
Assembly of custom TALE-type DNA binding domains by modular cloning
Morbitzer, R., Elsäesser, J., Hausner, J., et al. (2011) · Nucleic Acids Research · doi:10.1093/nar/gkr151 · Cited by 186 · see citing works
Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs
Cradick, T. J., Keck, K., Bradshaw, S., Jamieson, A. C., & McCaffrey, A. P. (2010) · Molecular Therapy · doi:10.1038/mt.2010.20 · Cited by 184 · see citing works
Homing endonucleases: structure, function and evolution
Jurica, M. S., & Stoddard, B. L. (1999) · Cellular and Molecular Life Sciences · doi:10.1007/s000180050372 · Cited by 167 · see citing works
Artificial DNA cutters for DNA manipulation and genome engineering
Aiba, Y., Sumaoka, J., & Komiyama, M. (2011) · Chemical Society Reviews · doi:10.1039/c1cs15039a · Cited by 144 · see citing works
Transient cold shock enhances zinc-finger nuclease–mediated gene disruption
Doyon, Y., Choi, V. M., Xia, D. F., Vo, T. D., Gregory, P. D., & Holmes, M. C. (2010) · Nature Methods · doi:10.1038/nmeth.1456 · Cited by 137 · see citing works
Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients
Wong, D. K.-H., Yuen, M., Yuan, H., Sum, S. S., Hui, C., Hall, J. G., & Lai, C. (2004) · Hepatology · doi:10.1002/hep.20353 · Cited by 132 · see citing works
Review article: current antiviral therapy of chronic hepatitis B
Ayoub, W. S., & Keeffe, E. B. (2008) · Alimentary Pharmacology & Therapeutics · doi:10.1111/j.1365-2036.2008.03731.x · Cited by 131 · see citing works
Targeted DNA Mutagenesis for the Cure of Chronic Viral Infections
Schiffer, J. T., Aubert, M., Weber, N. D., Mintzer, E., Stone, D., & Jerome, K. R. (2012) · Journal of Virology · doi:10.1128/jvi.00052-12 · Cited by 111 · see citing works
Rabe, B., Glebe, D., & Kann, M. (2006) · Journal of Virology · doi:10.1128/jvi.02303-05 · Cited by 105 · see citing works
TALENs and ZFNs are associated with different mutation signatures
Kim, Y., Kweon, J., & Kim, J. (2013) · Nature Methods · doi:10.1038/nmeth.2364 · Cited by 97 · see citing works
Zimmerman, K. A., Fischer, K. P., Joyce, M., & Tyrrell, D. L. (2008) · Journal of Virology · doi:10.1128/jvi.00366-08 · Cited by 90 · see citing works
Yang, W., Mason, W. S., & Summers, J. W. (1996) · Journal of Virology · doi:10.1128/jvi.70.7.4567-4575.1996 · Cited by 88 · see citing works
Review article: current antiviral therapy of chronic hepatitis B
Ayoub, W. S., & Keeffe, E. B. (2011) · Alimentary Pharmacology & Therapeutics · doi:10.1111/j.1365-2036.2011.04869.x · Cited by 81 · see citing works
Immune Suppression Uncovers Endogenous Cytopathic Effects of the Hepatitis B Virus
Meuleman, P., Libbrecht, L. J., Wieland, S. F., et al. (2006) · Journal of Virology · doi:10.1128/jvi.80.6.2797-2807.2006 · Cited by 67 · see citing works
Hendrie, P. C., Hirata, R. K., & Russell, D. W. (2003) · Journal of Virology · doi:10.1128/jvi.77.24.13136-13145.2003 · Cited by 33 · see citing works
Chudy, M., Hanschmann, K. ‐., Kress, J., et al. (2012) · Journal of Clinical Virology · doi:10.1016/j.jcv.2012.08.013 · Cited by 26 · see citing works
Chattopadhyay, S., Ely, A., Bloom, K. M., et al. (2009) · Biochemical and Biophysical Research Communications · doi:10.1016/j.bbrc.2009.09.004 · Cited by 18 · see citing works
Self-citationTremblay, J. P., Xiao, X., Aartsma-Rus, A. M., Barbas, C. F., Blau, H. M., Bogdanove, A. J., Boycott, K. M., Braun, S., Breakefield, X. O., Bueren, J. A., et al. (2013) · Molecular Therapy · doi:10.1038/mt.2013.4 · Cited by 12 · see citing works
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The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo
Lin, S.-R., Yang, H.-C., Kuo, Y.-T., Liu, C.-J., Yang, T.-Y., Sung, K.-C., Lin, Y.-Y., Wang, H.-Y., Wang, C.-C., Shen, Y.-C., et al. (2014) · Molecular Therapy - Nucleic Acids · doi:10.1038/mtna.2014.38 · Cited by 386 · see citing works
Targeting Hepatitis B Virus With CRISPR/Cas9
Seeger, C., & Sohn, J. A. (2014) · Molecular Therapy - Nucleic Acids · doi:10.1038/mtna.2014.68 · Cited by 294 · see citing works
- Authors
- Bloom, K., Ely, A., Mussolino, C., Cathomen, T., & Arbuthnot, P.
- Affiliations
- University of the Witwatersrand (ZA)
- University Medical Center Freiburg (DE)
- Corresponding author
- Arbuthnot, Patrick
- Venue
- Molecular Therapy
- Publisher
- Elsevier BV
- Published
- 2013-10
- Volume
- 21
- Issue
- 10
- Pages
- 1889-1897
- ISSN
- 1525-0016 (ISSN-L)
- PMC
- PMC3808145
- OpenAlex
- W2084686098
- Cited by
- 217
- References
- 42
- Citations by year
- 2026: 4 · 2025: 6 · 2024: 4 · 2023: 18 · 2022: 11 · 2021: 10 · 2020: 16 · 2019: 17 · 2018: 12 · 2017: 28 · 2016: 33 · 2015: 37 · 2014: 19 · 2013: 1
- FWCI
- 21.86
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular Therapy (publishedVersion, cc-by-nc-nd)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Topics
- Health Sciences › Medicine › Epidemiology › Hepatitis B Virus Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- hepatitis B virus, covalently closed circular DNA, TALEN, chronic hepatitis B, viral replication inhibition, targeted genome editing, hydrodynamic injection, mouse model, HepG2.2.15 cells
- MeSH terms
- Animals, Base Sequence, Cell Line, Deoxyribonucleases, Disease Models, Animal, DNA Replication, DNA, Circular, DNA, Viral, Genetic Vectors, Hepatitis B, Hepatitis B virus, Humans, Molecular Sequence Data, Transfection, Virus Replication, Protein Engineering, Genetic Therapy, Mutagenesis, Mice, Hep G2 Cells
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