Progress With Developing Use of Gene Editing To Cure Chronic Infection With Hepatitis B Virus
Authors: Abdullah Ely, Buhle Moyo, Patrick Arbuthnot
Journal: Molecular Therapy (2016)

Chronic infection with hepatitis B virus (HBV) occurs in approximately 6% of the world's population. Carriers of the virus are at risk for life-threatening complications, and developing curative treatment remains a priority. The main shortcoming of licensed therapies is that they do not affect viral covalently closed circular DNA (cccDNA), a stable intermediate of replication. Harnessing gene editing to mutate cccDNA provides the means to inactivate HBV gene expression permanently. Reports have described use of engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) nucleases. Although inhibition of viral replication has been demonstrated, reliably detecting mutations in cccDNA has been difficult. Also, the dearth of murine models that mimic cccDNA formation has hampered analysis in vivo. To reach a stage of clinical use, efficient delivery of the editors to HBV-infected hepatocytes and limiting unintended off-target effects will be important. Investigating therapeutic efficacy in combination with other treatment strategies, such as immunotherapies, may be useful to augment antiviral effects. Advancing gene editing as a mode of treating HBV infection is now at an interesting stage and significant progress is likely to be made in the immediate future. Chronic infection with hepatitis B virus (HBV) occurs in approximately 6% of the world's population. Carriers of the virus are at risk for life-threatening complications, and developing curative treatment remains a priority. The main shortcoming of licensed therapies is that they do not affect viral covalently closed circular DNA (cccDNA), a stable intermediate of replication. Harnessing gene editing to mutate cccDNA provides the means to inactivate HBV gene expression permanently. Reports have described use of engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) nucleases. Although inhibition of viral replication has been demonstrated, reliably detecting mutations in cccDNA has been difficult. Also, the dearth of murine models that mimic cccDNA formation has hampered analysis in vivo. To reach a stage of clinical use, efficient delivery of the editors to HBV-infected hepatocytes and limiting unintended off-target effects will be important. Investigating therapeutic efficacy in combination with other treatment strategies, such as immunotherapies, may be useful to augment antiviral effects. Advancing gene editing as a mode of treating HBV infection is now at an interesting stage and significant progress is likely to be made in the immediate future. The year 2015 marked 50 years since the Nobel prize-winning discovery of the Australia antigen by Baruch Blumberg and his colleagues.1Blumberg BS. Hepatitis B: The Hunt for a Killer Virus. Princeton University Press, Princeton, NJ, USA2002Crossref Google Scholar,2Blumberg BS Alter HJ Visnich S A "NEW" antigen in leukemia sera.JAMA. 1965; 191: 541-546Crossref PubMed Scopus (908) Google Scholar This protein, which was the first recognized marker of infection with hepatitis B virus (HBV), is now known as a surface antigen of HBV (HBsAg). Since this seminal finding, the considerable medical significance of HBV became apparent and research on the virus progressed at an impressive pace. It is estimated that 2 billion people have been infected with HBV and about 240 million of these individuals are currently chronic carriers of the virus.3WHO 2013 Hepatitis B Virus Fact Sheet No. 204 (Updated July 2013)Google Scholar It is these persistently infected individuals who are at high risk for the life-threatening complications of cirrhosis and hepatocellular carcinoma (HCC). The mechanism of HBV-induced hepatocarcinogenesis is incompletely understood. However integration of viral DNA into the host genome is thought to contribute to the process of malignant transformation.4Sung WK Zheng H Li S Chen R Liu X Li Y et al.Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma.Nat Genet. 2012; 44: 765-769Crossref PubMed Scopus (638) Google Scholar Spread of HBV occurs by parenteral transmission and when infection with the virus occurs at a young age, the risk for chronicity is high.5Trépo C Chan HL Lok A Hepatitis B virus infection.Lancet. 2014; 384: 2053-2063Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar,6Botha JF Ritchie MJ Dusheiko GM Mouton HW Kew MC Hepatitis B virus carrier state in black children in Ovamboland: role of perinatal and horizontal infection.Lancet. 1984; 1: 1210-1212Abstract PubMed Scopus (158) Google Scholar,7Stevens C Szmuness W Vertical transmission of hepatitis B and neonatal hepatitis B.Virus and the liver. 1980; (MTP Press Lancaster): 285-291Google Scholar Available therapies to treat HBV infection, which include reverse transcriptase inhibitors and interferon-α (IFN-α) derivatives, rarely completely clear the virus from carriers.5Trépo C Chan HL Lok A Hepatitis B virus infection.Lancet. 2014; 384: 2053-2063Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar,8Chevaliez S Hézode C Bahrami S Grare M Pawlotsky JM Long-term hepatitis B surface antigen (HBsAg) kinetics during nucleoside/nucleotide analogue therapy: finite treatment duration unlikely.J Hepatol. 2013; 58: 676-683Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar Vaccines, comprising eukaryotic recombinant HBsAg, may be used to prevent HBV infection. However, incomplete global coverage and the inadequacy of current therapy indicate that complications arising from chronic HBV will remain a significant global health problem for some time. Research using gene editing to inactivate the virus permanently has shown that advancing this approach may lead to a cure for chronic HBV infection. The virion of HBV has an envelope, with embedded large, middle and small HBsAgs that surround the viral nucleocapsid. The restricted host range of HBV and limited availability of cultured cells that are infectable with the virus have impeded research on the early stages of HBV replication. An initiating step of hepatocyte infection is the glycosaminoglycan-mediated clustering of viral particles on the surface of hepatocytes (Figure 1).9Sureau C Salisse J A conformational heparan sulfate binding site essential to infectivity overlaps with the conserved hepatitis B virus a-determinant.Hepatology. 2013; 57: 985-994Crossref PubMed Scopus (134) Google Scholar Thereafter virions enter hepatocytes following interaction of the myristolated large HBsAg with the sodium-dependent taurocholate cotransporting polypeptide (NTCP).10Yan H Zhong G Xu G He W Jing Z Gao Z et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Elife. 2012; 1: e00049Crossref Scopus (1386) Google Scholar,11Ni Y Lempp FA Mehrle S Nkongolo S Kaufman C Fälth M et al.Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.Gastroenterology. 2014; 146: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar Other host factors are also likely to be involved in uptake of virions. The viral genome, comprising partly double-stranded relaxed circular DNA (rcDNA), is released in the nucleus where it is "repaired" to form covalently closed circular DNA (cccDNA).12Nassal M HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.Gut. 2015; 64: 1972-1984Crossref PubMed Scopus (540) Google Scholar This replication intermediate serves as the template for transcription of viral pregenomic RNA (pgRNA) and protein-coding mRNAs. Viral gene expression is maintained by the X protein of HBV, which exerts its effects by modulating DNA methylation and modifying cccDNA-associated histones.13Ducroux A Benhenda S Rivière L Semmes OJ Benkirane M Neuveut C The Tudor domain protein Spindlin1 is involved in intrinsic antiviral defense against incoming hepatitis B Virus and herpes simplex virus type 1.PLoS Pathog. 2014; 10: e1004343Crossref PubMed Scopus (38) Google Scholar,14Levrero M Pollicino T Petersen J Belloni L Raimondo G Dandri M Control of cccDNA function in hepatitis B virus infection.J Hepatol. 2009; 51: 581-592Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar,15Pollicino T Belloni L Raffa G Pediconi N Squadrito G Raimondo G et al.Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.Gastroenterology. 2006; 130: 823-837Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar Arrangement of the open reading frames (ORFs) and regulatory elements within the cccDNA is remarkably compact (Figure 2). The four ORFs, surface (S), polymerase (P), core (C) and X, overlap with each other. Moreover, promoters, enhancers, and other regulatory elements are embedded in the ORFs. This multifunctional nature of the cccDNA limits its sequence plasticity. Consequently, emergence of escape mutants is fairly unusual, and HBV is susceptible to inactivation by engineered mutation.Figure 2Arrangement of viral open reading frames and cis regulatory elements within the cccDNA of HBV. The cccDNA of HBV comprises approximately 3.2 kb. Nucleotide co-ordinates are typically calculated from the unique EcoRI site of the HBV DNA (top center). Approximate location of promoters, enhancers, and cis regulatory elements are indicated as circular and rectangular symbols on the cccDNA, which is shown as a double circle. The core (C), polymerase (P), surface (S), and X overlapping open reading frames are shown as arrows immediately surrounding the genome. Initiation of translation from the preS1 start codon generates the large surface antigen and the preS2 AUG begins synthesis of the middle HBsAg. The preC codon initiates formation of the secreted HBV e antigen (HBeAg).View Large Image Figure ViewerDownload (PPT) cccDNA is very stable, and exists as a minichromosome in infected hepatocytes.12Nassal M HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.Gut. 2015; 64: 1972-1984Crossref PubMed Scopus (540) Google Scholar Difficulties with eradicating cccDNA have been the most important obstacle to developing an effective cure for HBV infection. Currently licensed treatments are capable of suppressing replication of HBV, but relapse following treatment withdrawal occurs frequently.16Halegoua-De Marzio D Hann H-W Then and now: The progress in hepatitis B treatment over the past 20 years.World J Gastroenterol. 2014; 20: 401-413Crossref PubMed Scopus (38) Google Scholar Ability to disable cccDNA permanently using gene-editing technology has therefore generated interest. A variety of gene-editing approaches is being developed to treat HBV infection and these are discussed below in more detail. Technologies include use of engineered clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) systems, zinc finger nucleases (ZFNs), and transcription-activator-like effector (TALE) nucleases (TALENs). Homing endonucleases (HEs) have been investigated for antiviral therapeutic use, but complicated requirements for engineering these nucleases have restricted their widespread use. The rationale for employing designer nucleases to effect mutation is that repeated digestion of the target DNA, followed by error-prone repair mediated by the nonhomologous end joining pathway, eventually leads to mutation of the cccDNA. Epigenome editing, based on recruitment of transcriptional silencing elements to the cccDNA, is another approach that is being investigated and is discussed below. Dependence on use of an RNA guide to direct the Cas protein to cleave targets at specific sites is an advantage of the CRISPR/Cas system. These RNA-guided nucleases are simple to engineer and this has been the main reason for the popularity of CRISPR/Cas systems. Generating derivatives of zinc finger proteins (ZFPs) is complicated by the unpredictable context-dependent efficiency of the DNA-binding finger modules. Producing artificial gene editors containing sequences of TALEs is easier as the monomers interacting with each nucleotide are not influenced by neighboring sequences. Advancing gene editing as a means of therapeutic inactivation of cccDNA has been hindered by the paucity of models that simulate natural HBV infection and difficulties with proving specificity of mutagenic effects on cccDNA. The problem of assessing therapeutic efficacy in vivo is further exacerbated by the inability of murine cells to generate cccDNA.17Raney AK Eggers CM Kline EF Guidotti LG Pontoglio M Yaniv M et al.Nuclear covalently closed circular viral genomic DNA in the liver of hepatocyte nuclear factor 1 alpha-null hepatitis B virus transgenic mice.J Virol. 2001; 75: 2900-2911Crossref PubMed Scopus (94) Google Scholar Probing viral sequences in rcDNA, integrated and free linear viral DNA, needs to be distinguished from analysis of cccDNA. Recent development of NTCP-expressing cells that are susceptible to infection by HBV18Watashi K Sluder A Daito T Matsunaga S Ryo A Nagamori S et al.Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (NTCP).Hepatology. 2014; 59: 1726-1737Crossref PubMed Scopus (191) Google Scholar goes some way to enabling discrimination of an effect on cccDNA. Many convenient and commonly used models of HBV replication do not depend on formation of cccDNA. For example, transient transfection of cultured liver-derived cells or murine hepatocytes in vivo supports production of HBV particles from replication-competent plasmids. Thus, although gene editors may cause inhibition of HBV replication in these models, the effect is a result of action on plasmid DNA and not cccDNA. cccDNA is present at low concentrations in HBV-infected cells, and naturally the number of copies per cell is estimated to be less than 1.5.19Laras A Koskinas J Dimou E Kostamena A Hadziyannis SJ Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients.Hepatology. 2006; 44: 694-702Crossref PubMed Scopus (158) Google Scholar Assays to measure cccDNA and evaluate induced mutation at intended target sites are difficult and sometimes unreliable.12Nassal M HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.Gut. 2015; 64: 1972-1984Crossref PubMed Scopus (540) Google Scholar Use of primers that flank the gap of rcDNA was initially thought to provide the means for specific amplification of cccDNA. However, this polymerase chain reaction (PCR)-based assay is indeed not specific for cccDNA. Presence of the duplicated sequence of the "flap" at the 5' end of the minus strand of rcDNA results in synthesis of sequences with overlapping complementary 3' ends during PCR.12Nassal M HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.Gut. 2015; 64: 1972-1984Crossref PubMed Scopus (540) Google Scholar The benchmark for identifying cccDNA is Southern blot hybridization, and radioactive probes are generally required to detect the viral replication intermediate with sufficient sensitivity. Southern blot hybridization is however not amenable to identifying and quantifying mutagenesis at target sites. PCR coupled to treatment with endonucleases, such as T7E1, and next-generation sequencing are better to evaluate target mutation. To separate cccDNA from other viral DNA, such as rcDNA, linear DNA, and viral sequences that have been integrated into the host's genome, Hirt extraction coupled to treatment with ATP-dependent DNase (plasmid-safe) has been employed.20Bloom K Ely A Arbuthnot P A T7 endonuclease I assay to detect TALEN-mediated targeted mutation of HBV cccDNA.Methods Mol Biol. 2016; (in press)Google Scholar,21Bloom K Ely A Mussolino C Cathomen T Arbuthnot P Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases.Mol Ther. 2013; 21: 1889-1897Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar With this approach, non-cccDNA should be degraded to leave cccDNA for amplification. Of course appropriate controls and standards need to be included to verify reliability of individual assays. Digestion with plasmid-safe DNase has a disadvantage in that nicked cccDNA may also be removed during the extraction procedure. As a result, existing cccDNA may be diminished and any real effect of gene editors on cccDNA may be underestimated. Despite difficulties with reliable assay of mutation in cccDNA, progress has been made with using gene editing to disrupt cccDNA by gene editors, and this is described below and in Table 1.Table 1Summary of selected studies using gene editing to disable HBV replicationType of gene editorMost effective viral target sitesModels used to evaluate efficacyEfficacyEvidence for efficacy against cccDNAReferenceCultured cellsIn vivoZFNC, PZFN-encoding sequences delivered with AAVs to HepAD38 cells in cultureSustained complete inhibition of HBV replication. Little if any off-target effectsNot assessedThe question of cccDNA mutation was addressed, but reproducible evidence was difficult to obtainWeber et al.24Weber ND Stone D Sedlak RH De Silva Feelixge HS Roychoudhury P Schiffer JT et al.AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.PLoS One. 2014; 9: e97579Crossref PubMed Scopus (88) Google ScholarTALENS, CCell culture. Murine hydrodynamic injectionSignificant decrease of markers of viral replication in stable HBV-producing cellsHBsAg decreased by 90% and circulating VPEs by 90%Mutagenesis of Hirt and plasmid-safe-treated DNA extracted from HepG2.2.15 cellsBloom et al.21Bloom K Ely A Mussolino C Cathomen T Arbuthnot P Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases.Mol Ther. 2013; 21: 1889-1897Abstract Full Text Full Text PDF PubMed Scopus (162) Google ScholarTALENC, PCell culture. Murine hydrodynamic injectionSignificant decrease of markers of viral replication in transiently transfected Huh7 cellsSignificant decrease of serum HBeAg, HBsAg, VPEs and hepatic pgRNAMutagenesis of Hirt and plasmid-safe treated DNA extracted from Huh7 cells. (Target HBV DNA transfected as linear duplex)Chen et al.27Chen J Zhang W Lin J Wang F Wu M Chen C et al.An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases.Mol Ther. 2014; 22: 303-311Abstract Full Text Full Text PDF PubMed Scopus (124) Google ScholarCRISPR/CasX, PTransient transfection of cultured cells. Murine hydrodynamic injectionSignificant decrease of markers of viral replication in transfected cells. Efficacy good against different viral genotypes. Synergistic effect of two sgRNAs used in combinationSignificant decrease of markers of viral replication. Decrease in intrahepatic plasmid DNA-containing HBV sequencesNone providedLin et at.31Lin SR Yang HC Kuo YT Liu CJ Yang TY Sung KC et al.The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo.Mol Ther Nucleic Acids. 2014; 3: e186Abstract Full Text Full Text PDF PubMed Scopus (310) Google ScholarCRISPR/CasX, CStable and transiently transfected HBV-producing cells. Murine hydrodynamic injection to produce rcccDNAGood inhibition of markers of viral replication and effective against different viral genotypesSignificant decrease of markers of viral replication: serum HBsAg, HBeAg and hepatic viral core proteinReduction of rcccDNA and targeted mutation of 34-44%Dong et at.38Dong C Qu L Wang H Wei L Dong Y Xiong S Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication.Antiviral Res. 2015; 118: 110-117Crossref PubMed Scopus (186) Google ScholarCRISPR/CasP, X, CLentiviral vectors used to transduce cultured HBV- producing cells with CRISPR/Cas. Murine hydrodynamic injectionGood inhibition of secretion of HBsAg and decreased intracellular viral pgRNASignificant decrease of markers of serum HBsAg and circulating VPEsReduction of cccDNA demonstrated using Southern blot hybridizationRamanan et al.39Ramanan V Shlomai A Cox DB Schwartz RE Michailidis E Bhatta A et al.CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus.Sci Rep. 2015; 5: 10833Crossref PubMed Scopus (219) Google ScholarAAV, adeno-associated virus; Cas, CRISPR-associated; cccDNA, covalently closed circular DNA; CRISPR, clustered regularly interspaced short palindromic repeats; HBsAg, hepatitis B virus surface antigen; HBeAg, hepatitis B virus e antigen; pgRNA, pregenomic RNA; rcccDNA, recombinant cccDNA; TAlEn, transcription activator-like effector nuclease; VPEs, viral particle equivalents; ZFN, zinc finger nuclease; C, HBV core DNA; P, HBV polymerase DNA; S, HBV surface DNA; X, HBV X DNA. Open table in a new tab AAV, adeno-associated virus; Cas, CRISPR-associated; cccDNA, covalently closed circular DNA; CRISPR, clustered regularly interspaced short palindromic repeats; HBsAg, hepatitis B virus surface antigen; HBeAg, hepatitis B virus e antigen; pgRNA, pregenomic RNA; rcccDNA, recombinant cccDNA; TAlEn, transcription activator-like effector nuclease; VPEs, viral particle equivalents; ZFN, zinc finger nuclease; C, HBV core DNA; P, HBV polymerase DNA; S, HBV surface DNA; X, HBV X DNA. In one of the first studies to describe anti-HBV utility of engineered DNA-binding proteins, Zimmerman et al.22Zimmerman KA Fischer KP Joyce MA Tyrrell DL Zinc finger proteins designed to specifically target duck hepatitis B virus covalently closed circular DNA inhibit viral transcription in tissue culture.J Virol. 2008; 82: 8013-8021Crossref PubMed Scopus (77) Google Scholar reported on the efficacy of ZFPs targeted to the enhancer region of duck HBV (DHBV). Significant nontoxic reductions in the core antigen, pgRNA, total viral RNA, and surface antigen were induced by the ZFPs following transfection of cells in culture. As the ZFPs did not possess nuclease domains, their mechanism of action is likely to have resulted from obstructing access of transcription factors required for DHBV gene expression. A later study employed target-cleaving ZFNs to inhibit HBV replication.23Cradick TJ Keck K Bradshaw S Jamieson AC McCaffrey AP Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs.Mol Ther. 2010; 18: 947-954Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar Although there was evidence for digestion of target sequences, proof of mutation of cccDNA was not provided. Subsequently, another group generated ZFNs that were delivered using self-complementary adeno-associated viruses in a cell culture model of HBV replication.24Weber ND Stone D Sedlak RH De Silva Feelixge HS Roychoudhury P Schiffer JT et al.AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.PLoS One. 2014; 9: e97579Crossref PubMed Scopus (88) Google Scholar The three ZFN pairs targeted P/X, P/C, and P sequences. All three ZFNs showed targeted disruption in cultured cells but one of the gene editors caused significant cytotoxicity. Inhibitory effects of the lead ZFNs were sustained and high-throughput sequencing indicated that the ZFNs acted specifically on HBV sequences. The HepAD38 cells that were used in this study replicate HBV in a controllable manner: the virus proliferates on withdrawal of doxycycline from the culture medium, subsequently establishing a pool of cccDNA molecules in the liver-derived cells.25Ladner SK Otto MJ Barker CS Zaifert K Wang GH Guo JT et al.Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication.Antimicrob Agents Chemother. 1997; 41: 1715-1720Crossref PubMed Google Scholar Reintroduction of doxycycline prevents replication from integrated sequences and consequently HBV proliferation should only be driven by the established pool of cccDNA. Weber et al.24Weber ND Stone D Sedlak RH De Silva Feelixge HS Roychoudhury P Schiffer JT et al.AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.PLoS One. 2014; 9: e97579Crossref PubMed Scopus (88) Google Scholar attempted to exploit this property of HepAD38 cells but were unfortunately unable to obtain reproducible evidence of ZFN-induced mutation of cccDNA. Zhao et al.,26Zhao X Zhao Z Guo J Huang P Zhu X Zhou X et al.Creation of a six-fingered artificial transcription factor that represses the hepatitis B virus HBx gene integrated into a human hepatocellular carcinoma cell line.J Biomol Screen. 2013; 18: 378-387Crossref Scopus (12) Google Scholar investigated the use of a ZFP-based artificial transcription factor (ATF) to inhibit function of the enhancer I region and diminish X expression. Two three-fingered ZFP motifs were linked to form a six-fingered DNA binding domain that recognized an 18 base pair sequence. To confer transcriptional repression characteristics, the ZFP was coupled to a Krüppel associated box (KRAB) domain. Evaluation showed that the ATF suppressed transcription specifically and caused cell cycle arrest specifically in cells containing integrated X. The efficacy of anti-HBV TALENs was first demonstrated by Bloom et al.21Bloom K Ely A Mussolino C Cathomen T Arbuthnot P Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases.Mol Ther. 2013; 21: 1889-1897Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar in 2013. Four dimeric TALENs were designed to target within the S (S TALEN), C (C TALEN), and P (P1 TALEN and P2 TALEN) ORFs. Bioinformatic analysis revealed that the TALENs had few if any off-target binding sites within human and murine genomes. The S and C TALENs were the most effective and caused intended mutation in the stable HBV-producing HepG2.2.15 cell line. Analysis using a T7E1 assay, carried out on amplicons derived from putative cccDNA, revealed mutagenesis of up to 35 and 12% by the S and C TALENs respectively. Hydrodynamic injection (HDI) of mice, using plasmids encoding the TALENs and the HBV genome, was used to assess efficacy in vivo. With this model, the S and C TALENs reduced HBsAg levels by 90% and circulating viral particle equivalents (VPEs) by 70%. Targeted mutagenesis of plasmid HBV sequences by the S and C TALENs was between 58 and 87% respectively, and no associated cytotoxic effects were detected. Sequencing demonstrated that deletions were the commonest mutations. An investigation by Chen et al.27Chen J Zhang W Lin J Wang F Wu M Chen C et al.An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases.Mol Ther. 2014; 22: 303-311Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar also provided evidence that TALENs are effective anti-HBV agents. In this study, three pairs of anti-HBV TALENs were designed against conserved regions within C (L1/R1 and L2/R2) and P (L3/R3) sequences of the HBV genome. As was previously reported,21Bloom K Ely A Mussolino C Cathomen T Arbuthnot P Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases.Mol Ther. 2013; 21: 1889-1897Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar the TALENs caused reductions in markers of viral replication in cell culture and following HDI of mice.27Chen J Zhang W Lin J Wang F Wu M Chen C et al.An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases.Mol Ther. 2014; 22: 303-311Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar A decrease in the concentration of detectable putative cccDNA molecules, ranging from 10–50%, was observed following transfection of Huh7 cells with linear HBV-encoding DNA and plasmids expressing C-targeting TALENs. HDI-mediated transfection with similar sequences showed diminished hepatic HBV pgRNA and reduced serum concentrations of HBV e antigen, HBsAg, and VPEs in mice receiving the TALEN-encoding sequences. The authors analyzed target sequences from various HBV subtypes, and showed that efficacy of the TALENs was good across different viral isolates. A synergistic effect when the TALENs were coadministered with IFN-α was also demonstrated. Since the first descriptions of how CRISPR/Cas9 may be employed for gene editing,28Antoniou MN Skipper KA Anakok O Optimizing retroviral gene expression for effective therapies.Hum Gene Ther. 2013; 24: 363-374Crossref PubMed Scopus (38) Google Scholar,29Cong L Ran FA Cox D Lin S Barretto R Habib N et al.Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9965) Google Scholar,30Aubert M Boyle NM Stone D Stensland L Huang ML Magaret AS et al.In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease.Mol Ther Nucleic Acids. 2014; 3: e146Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar numerous reports have shown the versatility of the system. The facile nature of CRISPR/Cas design has led to this t