Language：English   Publishing type：Research paper (scientific journal)  

SOX2 is a transcription factor essential for early mammalian development and for the maintenance of stem cells. Recently, SOX2 was identified as a lineage specific oncogene, recurrently amplified and activated in lung and esophageal squamous cell carcinoma (SCC). In this study, we have developed a zinc finger-based artificial transcription factor (ATF) to selectively suppress SOX2 expression in cancer cells and termed the system ATF/SOX2. We engineered the ATF using six zinc finger arrays designed to target a 19 bp site in the SOX2 distal promoter and a KOX transcriptional repressor domain. A recombinant adenoviral vector Ad-ATF/SOX2 that expresses ATF/SOX2 suppressed SOX2 at the mRNA and protein levels in lung and esophageal SCC cells expressing SOX2. In these kinds of cells, Ad-ATF/SOX2 decreased cell proliferation and colony formation more effectively than the recombinant adenoviral vector Ad-shSOX2, which expresses SOX2 short hairpin RNA (shSOX2). Ad-ATF/SOX2 induced the cell cycle inhibitor CDKN1A more strongly than Ad-shSOX2. Importantly, the ATF did not suppress the cell viability of normal human cells. Moreover, Ad-ATF/SOX2 effectively inhibited tumor growth in a lung SCC xenograft mouse model. These results indicate that ATF/SOX2 would lead to the development of an effective molecular-targeted therapy for lung and esophageal SCC.

DOI： 10.18632/oncotarget.21523

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Language：English   Publishing type：Research paper (scientific journal)  

Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms.

DOI： 10.1016/j.bbrc.2016.09.142

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Language：English   Publishing type：Research paper (scientific journal)  

Previously, we reported that an artificial zinc-finger protein (AZP)-staphylococcal nuclease (SNase) hybrid (designated AZP-SNase) inhibited DNA replication of human papillomavirus type 18 (HPV-18) in mammalian cells by binding to and cleaving a specific HPV-18 ori plasmid. Although the AZP-SNase did not show any side effects under the experimental conditions, the SNase is potentially able to cleave RNA as well as DNA. In the present study, to make AZP hybrid nucleases that cleave only viral DNA, we switched the SNase moiety in the AZP-SNase to the single-chain FokI dimer (scFokI) that we had developed previously. We demonstrated that transfection with a plasmid expressing the resulting hybrid nuclease (designated AZP-scFokI) inhibited HPV-18 DNA replication in transient replication assays using mammalian cells more efficiently than AZP-SNase. Then, by linker-mediated PCR analysis, we confirmed that AZP-scFokI cleaved an HPV-18 ori plasmid around its binding site in mammalian cells. Finally, a modified MTT assay revealed that AZP-scFokI did not show any significant cytotoxicity. Thus, the newly developed AZP-scFokI hybrid is expected to serve as a novel antiviral reagent for the neutralization of human DNA viruses with less fewer potential side effects.

DOI： 10.1007/s12033-014-9751-3

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Language：English   Publishing type：Research paper (scientific journal)  

We previously reported that our sandwiched zinc-finger nucleases (ZFNs), in which a DNA cleavage domain is inserted between two artificial zinc-finger proteins, cleave their target DNA much more efficiently than conventional ZFNs in vitro. In the present study, we compared DNA cleaving efficiencies of a sandwiched ZFN with those of its corresponding conventional ZFN in mammalian cells. Using a plasmid-based single-strand annealing reporter assay in HEK293 cells, we confirmed that the sandwiched ZFN induced homologous recombination more efficiently than the conventional ZFN; reporter activation by the sandwiched ZFN was more than eight times that of the conventional one. Western blot analysis showed that the sandwiched ZFN was expressed less frequently than the conventional ZFN, indicating that the greater DNA-cleaving activity of the sandwiched ZFN was not due to higher expression of the sandwiched ZFN. Furthermore, an MTT assay demonstrated that the sandwiched ZFN did not have any significant cytotoxicity under the DNA-cleavage conditions. Thus, because our sandwiched ZFN cleaved more efficiently than its corresponding conventional ZFN in HEK293 cells as well as in vitro, sandwiched ZFNs are expected to serve as an effective molecular tool for genome editing in living cells.

DOI： 10.1016/j.bmcl.2013.12.096

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Language：English   Publishing type：Research paper (scientific journal)  

Previously we demonstrated that inhibition of replication-associated protein (Rep) binding to its replication origin by artificial zinc-finger proteins (AZPs) is a powerful method to prevent plant virus infection in vivo. In the present study, we applied the AZP technology to Tomato yellow leaf curl virus (TYLCV), which is a limiting factor in tomato cultivation worldwide. First, we determined 5'-ATCGGTGT ATCGGTGT-3' in the 195-bp intergenic region of the TYLCV-Israel strain, a strain reported first among TYLCV strains, as the Rep-binding site by gel shift assays. We then constructed a 6-finger AZP that bound to a 19-bp DNA including the Rep-binding site. We demonstrated that the binding affinity of the AZP was >1,000-fold greater than that of Rep and that the AZP inhibited Rep binding completely in vitro. Because the binding capability of the AZP was same as that of the AZP previously designed for geminivirus-resistant Arabidopsis thaliana, we predict that the present AZP will prevent TYLCV infection in vivo.

DOI： 10.1007/s12033-012-9552-5

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Language：English   Publishing type：Research paper (scientific journal)  

Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP-SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP-SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP-SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP-SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.

DOI： 10.1371/journal.pone.0056633

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Language：English   Publishing type：Research paper (scientific journal)  

We previously developed a novel type of zinc finger nucleases (ZFNs), sandwiched ZFNs that can discriminate DNA substrates from cleavage products and thus cleave DNA much more efficiently than conventional ZFNs as well as perform with multiple turnovers like restriction endonucleases. In the present study, we used the sandwiched ZFN to unidirectionally clone exogenous genes into target vectors by cleaving heterogeneous sites that contained heterogeneous spacer DNAs between two zinc-finger protein binding sites with a single sandwiched ZFN. We demonstrated that the sandwiched ZFN cleaved a 40-fold excess of both insert and vector plasmids within 1h and confirmed by sequencing that the resulting recombinants harbored the inserted DNA fragment in the desired orientation. Because sandwiched ZFNs can recognize and cleave a variety of long (≥ 26-bp) target DNAs, they may not only expand the utility of ZFNs for construction of recombinant plasmids, but also serve as useful meganucleases for synthesis of artificial genomes.

DOI： 10.1016/j.bbrc.2011.09.145

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Language：English   Publishing type：Research paper (scientific journal)  

Repression of vascular endothelial growth factor A (VEGF-A) is an attractive approach to cancer therapy. Although zinc-finger-based artificial transcription factors (ATFs) were constructed for human VEGF-A and constitutive expressions of ATFs were previously shown to downregulate the endogenous VEGF-A gene expression, repression of VEGF-A specifically in hypoxic tumors is desirable for therapeutic application of ATF technology. Here, we describe hypoxia-driven expression of the ATF for hypoxia-specific repression of human VEGF-A gene. We constructed a hypoxia-driven promoter for the ATF expression and placed it upstream of the ATF-encoding region. The resulting hypoxia-driven expression plasmids induced the expression of ATFs specifically in hypoxia, and the hypoxia-specific expression of ATFs effectively downregulated the VEGF-A expression in hypoxia, but not in normoxia. Thus, the engineered expression system of ATFs may enable repression of VEGF-A expression specifically in hypoxic tumors without affecting normal, healthy tissues.

DOI： 10.1007/s12033-010-9288-z

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Language：English   Publishing type：Research paper (scientific journal)  

Zinc-finger-based artificial transcription factors (ATFs) have been used to regulate expression of target genes both in vitro and in vivo. However, if we develop ATF expression further, target gene regulation by ATFs may be more effective. Here, we report a new transcriptional regulation system in which an ATF that is designed to upregulate a target gene also activates itself. To construct the system, we inserted tandem copies of the ATF-binding sites upstream of a promoter for ATF expression. Using the endogenous human VEGF-A gene, we demonstrated that the new expression system amplified ATF expression in a manner dependent on the number of copies of the ATF-binding site, and that the 'self-propagating ATF' upregulated VEGF-A gene expression more efficiently than a control promoter with no ATF-binding site.

DOI： 10.1016/j.bmcl.2010.05.002

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Activation of vascular endothelial growth factor A (VEGF-A) is an attractive approach to treatment of ischemic diseases. Although zinc-finger-based artificial transcription factors (ATFs) were constructed for human VEGF-A and constitutive expression of ATFs upregulated the endogenous VEGF-A gene expression, activation of VEGF-A specifically in ischemic tissues is desirable for therapeutic application of ATF technology. Here, we describe hypoxia-specific activation of human VEGF-A gene by hypoxia-driven expression of the ATF. We constructed a hypoxia-driven promoter for the ATF expression and placed it upstream of the ATF-encoding regions. The resulting hypoxia-driven expression plasmid induced the ATF expression in hypoxia but not in normoxia, and the hypoxia-specific expression of the ATF activated the VEGF-A expression specifically in hypoxia. Thus, the engineered expression system of ATFs may enable activation of VEGF-A expression specifically in ischemic tissues without affecting normal, healthy tissues in vivo.

DOI： 10.1016/j.bbrc.2009.10.060

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Language：English   Publishing type：Research paper (scientific journal)  

Zinc-finger nucleases (ZFNs) are a powerful tool for manipulation of genomic DNA. Recently, we reported a new ZFN composed of one artificial zinc-finger protein (AZP) and a single-chain FokI dimer (scFokI) that refines ZFN technology. While AZP-scFokI cleaved DNA specifically around the AZP-target site, several nucleotide positions were cleaved due to the mobility of the scFokI domain. In the present study, we aimed to improve the DNA-cleavage specificity at the nucleotide level. To this end, we sandwiched a scFokI domain between two AZPs to reduce the mobility of the scFokI moiety when bound to DNA. We demonstrated that the AZP-sandwiched scFokI cleaved DNA at a single nucleotide position of a target plasmid, in which two AZP-binding sites were connected with a 6-bp spacer, with multiple turnovers. Further improvement of AZP-sandwiched scFokI will lead to development of ideal artificial meganucleases.

DOI： 10.1016/j.bbrc.2009.10.030

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

DOI： 10.1093/nass/nrp145

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

DOI： 10.1093/nass/nrn094

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

DOI： 10.1093/nass/nrn095

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Language：English   Publishing type：Research paper (scientific journal)  

Many studies show that a tegument protein, VP22, of herpes simplex virus possesses an unusual capacity for intercellular trafficking, while several studies have reported that the intercellular trafficking was observed only in cells after fixation. Therefore, the trafficking ability in living cells remains controversial. To settle the question, we first examined secretion of VP22 in living cells. In this report, we fused VP22 with beta-galactosidase (betaGal) and investigated the secretion of VP22-betaGal in living cells by monitoring betaGal activity in the culture medium. Under our conditions, a significant amount of VP22-betaGal was detected in the culture medium, and it increased with time. Particularly, 6 days after transfection, 72% of all VP22-betaGal expressed was detected in the culture medium. Lactate dehydrogenase assays revealed that leakage of VP22-betaGal from damaged cells was not the main cause of the high level of secretion. We thus conclude that VP22 possesses a remarkable ability to be secreted from living cells.

DOI： 10.1007/s00705-008-0094-x

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Language：English   Publishing type：Research paper (scientific journal)  

Human papillomavirus (HPV) is one of the important pharmaceutical targets because infection of the high-risk types causes invasive cervical cancer. However, effective antiviral drugs for HPV have not been developed so far. In the present study, we constructed cell-permeable artificial zinc-finger proteins (AZPs) by fusing an AZP previously generated for inhibition of HPV-18 DNA replication with a cell-penetrating peptide (CPP) as candidates for new antiviral drugs against HPV. We confirmed that these CPP-AZP fusions reduced the replication rate in transient replication assays when added to the culture medium. In particular, 250 nM CPP-AZP (designated AZP-R9) containing a 9-mer of arginine as the CPP reduced HPV-18 DNA replication to 3% of that of a control, and the 50% effective concentration (EC50) was <31 nM. Furthermore, a cytotoxicity assay revealed that the 50% inhibitory concentration (IC50) of AZP-R9 was >10 microM. Therefore, the selectivity index, defined as IC50/EC50, was >300, which is better than that of the antiviral cidofovir for HPVs. Thus, our results demonstrate that cell-permeable AZPs could serve as potent protein-based antiviral drugs.

DOI： 10.1007/s00705-008-0125-7

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

DOI： 10.1093/nass/nrm214

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

DOI： 10.1093/nass/nrm173

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

DOI： 10.1093/nass/nrl156

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

DOI： 10.1093/nass/nrl145

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Language：English   Publishing type：Research paper (scientific journal)  

Recently, we demonstrated that plant DNA virus replication was inhibited in planta by using an artificial zinc finger protein (AZP) and created AZP-based transgenic plants resistant to DNA virus infection. Here we apply the AZP technology to the inhibition of replication of a mammalian DNA virus, human papillomavirus type 18 (HPV-18). Two AZPs, designated AZP(HPV)-1 and AZP(HPV)-2, were designed by using our nondegenerate recognition code table and were constructed to block binding of the HPV-18 E2 replication protein to the replication origin. Both of the newly designed AZPs had much higher affinities towards the replication origin than did the E2 protein, and they efficiently blocked E2 binding in vitro. In transient replication assays, both AZPs inhibited viral DNA replication, especially AZP(HPV)-2, which reduced the replication level to approximately 10%. We also demonstrated in transient replication assays, using plasmids with mutant replication origins, that AZP(HPV)-2 could precisely recognize the replication origin in mammalian cells. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses but also to mammalian DNA viruses.

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Language：English   Publishing type：Research paper (scientific journal)  

A novel protein molecular targeting system was created using a cytoplasmic face of a plasma membrane-targeting system in Saccharomyces cerevisiae. The technique involves a molecular display for the creation of a novel reaction site and interaction sites in the field of biotechnology. In a model system, a fluorescent protein was targeted as a reporter to the cytoplasmic face of the plasma membrane. The C-terminal transmembrane domain (CTM) of Ras2p and Snc2p was examined as the portions with anchoring ability to the cytoplasmic face of the plasma membrane. We found that the CTM of Snc2p targeted the enhanced cyan fluorescent protein (ECFP)-protein A fusion protein on the cytoplasmic face of the plasma membrane more strongly than that of Ras2p. To develop it for use as a detection system for protein-protein interactions, the Fc fragment of IgG (Fc) was genetically fused with the enhanced yellow fluorescent protein (EYFP) and expressed in the cytoplasm of the ECFP-protein A-anchored cell. A microscopic analysis showed that fluorescence resonance energy transfer (FRET) between ECFP-protein A and EYFP-Fc occurred, and the change in fluorescence was observed on the cytoplasmic face of the plasma membrane. The detection of protein-protein interactions at the cytoplasmic face of a plasma membrane using FRET combined with a cytoplasmic face-targeting system for proteins provides a novel method for examining the molecular interactions of cytoplasmic proteins, in addition to conventional methods, such as the two-hybrid method in the nuclei.

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