担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Public Library of Science (PLoS)  

DOI： 10.1371/journal.pone.0056633

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

PubMed

researchmap

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/bi801800k

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The vascular endothelial growth factor A (VEGF-A) gene is an attractive therapeutic target because both activation and repression of the gene are useful for treatment or cure of many diseases related to abnormal angiogenesis. To modulate the endogenous gene expression artificially, we previously designed a six-finger AZP to recognize a 19-bp DNA in the VEGF-A gene, and fused the AZP with a nuclear localization signal and a repressor domain to generate an artificial transcription factor (ATF). Using the ATF, we demonstrated efficient modulation of the VEGF-A expression. In the present study, we evaluate the ability of the ATF to modulate the gene expression in more detail. First we examined the ability of the ATF under hypoxia condition. ELISA of the VEGF-A protein in the culture medium revealed that the gene-delivered ATF repressed the expression rate of the VEGF-A gene under hypoxia condition.

DOI： 10.1093/nass/nrm173

Scopus

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Recently, we designed artificial zinc-finger proteins (AZPs) that prevent a viral replication protein, E2, of human papillomavirus type 18 (HPV-18) from binding to its replication origin and demonstrated that the gene-delivered AZPs inhibited HPV-18 DNA replication in mammalian cells. In the present study, we examined another approach to delivery of AZPs. We constructed cell-permeable AZPs by fusing an AZP previously generated for inhibition of HPV-18 DNA replication with a cell-penetrating peptide (CPP), and 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 reduced HPV-18 DNA replication to 3% of that of a control experiment. Western blot analysis detected 7% of the CPP-AZP added to the culture medium in the cell lysates, and demonstrated that greater internalization of CPP-AZP into mammalian cells causes greater inhibition of viral DNA replication. Furthermore, CPP-AZP did not show any significant cytotoxixity in MTT assays. Thus, our results demonstrate that cell-permeable AZPs could serve as potent protein-based antiviral drugs.

DOI： 10.1093/nass/nrm214

Scopus

PubMed

researchmap

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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.

PubMed

researchmap

担当区分：筆頭著者,　最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

ABSTRACT

Prevention of virus infections is a major objective in agriculture and human health. One attractive approach to the prevention is inhibition of virus replication. To demonstrate this concept in vivo, an artificial zinc finger protein (AZP) targeting the replication origin of theBeet severe curly top virus(BSCTV), a model DNA virus, was created. In vitro DNA binding assays indicated that the AZP efficiently blocked binding of the viral replication protein (Rep), which initiates virus replication, to the replication origin. All of the transgenicArabidopsisplants expressing the AZP showed phenotypes strongly resistant to virus infection, and 84% of the transgenic plants showed no symptom. Southern blot analysis demonstrated that BSCTV replication was completely suppressed in the transgenic plants. Since the mechanism of viral DNA replication is well conserved among plants and mammals, this approach could be applied not only to agricultural crop protection but also to the prevention of virus infections in humans.

DOI： 10.1128/jvi.79.4.2614-2619.2005

researchmap

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Proceedings of the National Academy of Sciences  

We describe a facile method to activate or repress transcription of endogenous genes in a quantitative and specific manner by treatment with designed regulatory proteins (DRPs), in which artificial transcription factors (ATFs) are fused to cell-penetrating peptides (CPPs). Penetration of DRPs into cells is mediated by an N-terminal CPP fused to a nuclear localization signal; a DNA-binding domain and a transactivation domain follow. The DNA-binding domain was targeted to the vascular endothelial growth factor (VEGF)-A gene. An agonist DRP was rapidly taken up by cells and transported to the nucleus; soon after, the cells began transcribing the gene and secreting VEGF-A protein in a dose-dependent manner. Multiple copies of a short oligopeptide derived from a minimal transactivation domain of human β-catenin was stronger than VP-16. The SRDX domain from the plant transcription factor, SUPERMAN, changed the DRP to a hypoxia-induced antagonist of VEGF-A. DRPs combine many of the potential benefits of transgenes with those of recombinant proteins.

DOI： 10.1073/pnas.0406473101

researchmap