The Experts below are selected from a list of 14580 Experts worldwide ranked by ideXlab platform
Hiroyuki Mizuguchi - One of the best experts on this subject based on the ideXlab platform.
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development of a novel oncolytic adenovirus expressing a Short Hairpin RNA against cullin 4a
Anticancer Research, 2020Co-Authors: Keisaku Wakabayashi, Fuminori Sakurai, Toshiyoshi Fujiwara, Ryosuke Ono, Hiroyuki MizuguchiAbstract:Background Arming of an oncolytic adenovirus (OAd) by inserting expression cassettes of therapeutic transgenes into the OAd genome is a promising approach to enhance the therapeutic effects of an OAd. Ideally, this approach would simultaneously promote the replication of an OAd in tumor cells and transgene product-mediated antitumor effects by expressing therapeutic transgenes. We previously demonstrated that knockdown of cullin 4A (CUL4A), which is an E3 ubiquitin ligase, significantly promoted adenovirus replication by increasing the c-JUN protein level. In addition, previous studies reported that CUL4A was highly expressed in various types of tumor, and was involved in tumor growth and metastasis. Materials and methods In this study, we developed a novel OAd expressing a Short-Hairpin RNA (shRNA) against CUL4A (OAd-shCUL4A). Results OAd-shCUL4 mediated higher levels of cytotoxic effects on various types of human tumor cell than a conventional OAd. Higher levels of OAd genome copy numbers were found in the tumor cells for OAd-shCUL4A, compared with a conventional OAd. Conclusion OAd-shCUL4A showed efficient antitumor effects by both enhancing OAd replication and inhibiting tumor cell growth.
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type i interferons impede Short Hairpin RNA mediated RNAi via inhibition of dicer mediated processing to small interfering RNA
Molecular therapy. Nucleic acids, 2017Co-Authors: Keisaku Wakabayashi, Mitsuhiro Machitani, Masashi Tachibana, Fuminori Sakurai, Kosuke Takayama, Hiroyuki MizuguchiAbstract:RNAi by Short Hairpin RNA (shRNA) is a powerful tool not only for studying gene functions in various organisms, including mammals, but also for the treatment of severe disorders. However, shRNA-expressing vectors can induce type I interferon (IFN) expression by activation of innate immune responses, leading to off-target effects and unexpected side effects. Several strategies have been developed to prevent type I IFN induction. On the other hand, it has remained unclear whether type I IFNs have effects on shRNA-mediated RNAi. Here, we show that the type I IFNs significantly inhibit shRNA-mediated RNAi. Treatment with recombinant human IFN-α significantly inhibited shRNA-mediated knockdown of target genes, while it did not inhibit small interfering RNA (siRNA)-mediated knockdown. Following treatment with IFN-α, increased and decreased copy numbers of shRNA and its processed form, respectively, were found in the cells transfected with shRNA-expressing plasmids. Dicer protein levels were not altered by IFN-α. These results indicate that type I IFNs inhibit shRNA-mediated RNAi via inhibition of dicer-mediated processing of shRNA to siRNA. Our findings should provide important clues for efficient RNAi-mediated knockdown of target genes in both basic researches and clinical gene therapy.
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Enhanced Oncolytic Activities of the Telomerase-Specific Replication-Competent Adenovirus Expressing Short-Hairpin RNA against Dicer
Molecular cancer therapeutics, 2016Co-Authors: Mitsuhiro Machitani, Keisaku Wakabayashi, Masashi Tachibana, Fuminori Sakurai, Toshiyoshi Fujiwara, Hiroyuki MizuguchiAbstract:Oncolytic viruses have been receiving much attention as potential agents for cancer treatment. Among the various types of oncolytic viruses, the telomerase-specific replication-competent adenovirus (TRAD), which carries the tumor-specific promoter-driven E1 gene expression cassette, exhibits efficient antitumor effects. The development of a novel TRAD that shows higher replication efficiency and antitumor activity would be highly beneficial for safer and more efficient cancer therapy. We recently demonstrated that the endoribonuclease Dicer significantly inhibits the replication of wild-type adenovirus (Ad) via the processing of viral-associated (VA)-RNAs, which are Ad-encoded small noncoding RNAs, and that the knockdown of Dicer leads to enhanced VA-RNA expression and Ad replication after infection with wild-type Ad. Based on these findings, we herein developed a novel TRAD expressing Short-Hairpin RNA against Dicer (shDicer; TRAD-shDicer). After infection, TRAD-shDicer efficiently induced the knockdown of Dicer. TRAD-shDicer showed significantly higher replication efficiency and tumor cell lysis activity compared with the conventional TRAD in tumor cells. The Dicer expression levels and viabilities of normal cells were not altered by infection with TRAD-shDicer. These results indicate that TRAD-shDicer is a potent antitumor reagent by virtue of its enhanced oncolytic activity. Mol Cancer Ther; 16(1); 251–9. ©2016 AACR.
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rapid construction of small interfering RNA expressing adenoviral vectors on the basis of direct cloning of Short Hairpin RNA coding dnas
Human Gene Therapy, 2006Co-Authors: Hiroyuki Mizuguchi, Fuminori Sakurai, Naoko Funakoshi, Tetsuji Hosono, Kenji Kawabata, Teruhide Yamaguchi, Takao HayakawaAbstract:In the conventional method for constructing an adenoviral (Ad) vector expressing small interfering RNA (siRNA), Short Hairpin RNA (shRNA)-coding oligonucleotides are introduced downstream of a polymerase III (or polymerase II)-based promoter cloned into a shuttle plasmid. An siRNA expression cassette, which is cloned into the shuttle plasmid, is then introduced into the E1 deletion region of the Ad vector plasmid by in vitro ligation or homologous recombination in Escherichia coli, and the linearized plasmid is transfected into 293 cells, generating an Ad vector expressing siRNA. Therefore, two-step plasmid manipulation is required. In this study, we developed a method by which shRNA-coding oligonucleotides can be introduced directly into the Ad vector plasmid. To do this, we constructed a new vector plasmid into which the human U6 promoter sequence was cloned in advance. Unique restriction enzyme sites were introduced at the transcription start site of the U6 promoter sequence in the vector plasmid. Luci...
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RNA interfering approach for clarifying the pparγ pathway using lentiviral vector expressing Short Hairpin RNA
FEBS Letters, 2004Co-Authors: Kazufumi Katayama, Masashi Tachibana, Hiroyuki Mizuguchi, Koichiro Wada, Hiroyuki Miyoshi, Kozo Ohashi, Rie Furuki, Takao Hayakawa, Atsushi Nakajima, Takashi KadowakiAbstract:Peroxisome proliferator-activated receptor γ (PPARγ) plays a central role in adipocyte differentiation and insulin sensitivity. Although PPARγ also appears to regulate diverse cellular processes in other cell types such as lymphocytes, the detailed mechanisms remain unclear. In this study, we established a lentivirus-mediated Short Hairpin RNA expression system and identified a potent Short Hairpin RNA which suppresses PPARγ expression, resulting in marked inhibition of preadipocyte-to-adipocyte differentiation in 3T3-L1 cells. Our PPARγ-knockdown method will serve to clarify the PPARγ pathway in various cell types in vivo and in vitro, and will facilitate the development of therapeutic applications for a variety of diseases.
Chaeok Yun - One of the best experts on this subject based on the ideXlab platform.
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vegf specific Short Hairpin RNA expressing oncolytic adenovirus elicits potent inhibition of angiogenesis and tumor growth
Molecular Therapy, 2007Co-Authors: Ji Young Yoo, Joo Hang Kim, Young Guen Kwon, Eok Cheon Kim, Nam Kyu Kim, Hye Jin Choi, Chaeok YunAbstract:RNA interference is being developed to treat cancer. Although highly target specific, its use has been limited by its Short duration of expression. To overcome this Shortcoming, we constructed an oncolytic adenovirus (Ad)-based Short Hairpin RNA (shRNA) expression system (Ad-ΔB7-shVEGF) against vascular endothelial growth factor (VEGF), a key mediator in angiogenesis. To demonstrate the VEGF-specific nature of this Ad-based shRNA, replication-incompetent Ad expressing VEGF-specific shRNA (Ad-ΔE1-shVEGF) was also generated. Ad-ΔE1-shVEGF was highly effective in reducing VEGF expression, and elicited an antiangiogenic effect in vitro and in vivo. Similarly, Ad-ΔB7-shVEGF exhibited potent antiangiogenic effects in the matrigel plug assay. Moreover, Ad-ΔB7-shVEGF demonstrated a greater antitumor effect and enhanced survival compared to the cognate control oncolytic Ad, Ad-ΔB7. Ad-ΔB7-shVEGF induced significant reduction in tumor vasculature, verifying the antiangiogenic mechanism. Furthermore, both the duration and magnitude of gene silencing by Ad-ΔB7-shVEGF was greater than Ad-ΔE1-shVEGF. These results suggest that the combined effects of oncolytic viral therapy and cancer cell-specific expression of VEGF-targeted shRNA elicits greater antitumor effect than an oncolytic Ad alone.
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vegf specific Short Hairpin RNA expressing oncolytic adenovirus elicits potent inhibition of angiogenesis and tumor growth
Molecular Therapy, 2007Co-Authors: Ji Young Yoo, Joo Hang Kim, Young Guen Kwon, Eok Cheon Kim, Nam Kyu Kim, Hye Jin Choi, Chaeok YunAbstract:RNA interference is being developed to treat cancer. Although highly target specific, its use has been limited by its Short duration of expression. To overcome this Shortcoming, we constructed an oncolytic adenovirus (Ad)-based Short Hairpin RNA (shRNA) expression system (Ad-DeltaB7-shVEGF) against vascular endothelial growth factor (VEGF), a key mediator in angiogenesis. To demonstrate the VEGF-specific nature of this Ad-based shRNA, replication-incompetent Ad expressing VEGF-specific shRNA (Ad-DeltaE1-shVEGF) was also generated. Ad-DeltaE1-shVEGF was highly effective in reducing VEGF expression, and elicited an antiangiogenic effect in vitro and in vivo. Similarly, Ad-DeltaB7-shVEGF exhibited potent antiangiogenic effects in the matrigel plug assay. Moreover, Ad-DeltaB7-shVEGF demonstrated a greater antitumor effect and enhanced survival compared to the cognate control oncolytic Ad, Ad-DeltaB7. Ad-DeltaB7-shVEGF induced significant reduction in tumor vasculature, verifying the antiangiogenic mechanism. Furthermore, both the duration and magnitude of gene silencing by Ad-DeltaB7-shVEGF was greater than Ad-DeltaE1-shVEGF. These results suggest that the combined effects of oncolytic viral therapy and cancer cell-specific expression of VEGF-targeted shRNA elicits greater antitumor effect than an oncolytic Ad alone.
Peter W Andrews - One of the best experts on this subject based on the ideXlab platform.
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specific knockdown of oct4 in human embryonic stem cells by inducible Short Hairpin RNA interference
Stem Cells, 2009Co-Authors: Gaetano Zafarana, Stuart Avery, Katie Avery, Harry Moore, Peter W AndrewsAbstract:Manipulation of gene function in embryonic stem cells by either over expression or downregulation is critical for understanding their subsequent cell fate. We have developed a tetracycline-inducible Short Hairpin RNA interference (shRNAi) for human embryonic stem cells (hESCs) and demonstrated doxycycline dose-dependent knockdown of the transcription factor OCT4 and the cell surface antigen β2-microglobulin. The induced knockdown of OCT4 resulted in rapid differentiation of hESCs with a significant increase in transcription of genes associated with trophoblast and endoderm lineages, the extent of which was controlled by the degree of induction. Transgene toxicity, which may occur in conditional over-expression strategies with hESCs, was not observed with wild-type Tet repressor protein. The system allows efficient, reversible, and long-term downregulation of target genes in hESCs and enables the generation of stable transfectants for the knockdown of genes essential for cell survival and self-renewal, not necessarily possible by nonconditional shRNAi methods.
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specific knockdown of oct4 in human embryonic stem cells by inducible Short Hairpin RNA interference
Stem Cells, 2009Co-Authors: Gaetano Zafarana, Stuart Avery, Katie Avery, Harry Moore, Peter W AndrewsAbstract:Manipulation of gene function in embryonic stem cells by either over expression or downregulation is critical for understanding their subsequent cell fate. We have developed a tetracycline-inducible Short Hairpin RNA interference (shRNAi) for human embryonic stem cells (hESCs) and demonstrated doxycycline dose-dependent knockdown of the transcription factor OCT4 and the cell surface antigen beta2-microglobulin. The induced knockdown of OCT4 resulted in rapid differentiation of hESCs with a significant increase in transcription of genes associated with trophoblast and endoderm lineages, the extent of which was controlled by the degree of induction. Transgene toxicity, which may occur in conditional over-expression strategies with hESCs, was not observed with wild-type Tet repressor protein. The system allows efficient, reversible, and long-term downregulation of target genes in hESCs and enables the generation of stable transfectants for the knockdown of genes essential for cell survival and self-renewal, not necessarily possible by nonconditional shRNAi methods. STEM CELLS 2009;27:776-782.
Ji Young Yoo - One of the best experts on this subject based on the ideXlab platform.
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vegf specific Short Hairpin RNA expressing oncolytic adenovirus elicits potent inhibition of angiogenesis and tumor growth
Molecular Therapy, 2007Co-Authors: Ji Young Yoo, Joo Hang Kim, Young Guen Kwon, Eok Cheon Kim, Nam Kyu Kim, Hye Jin Choi, Chaeok YunAbstract:RNA interference is being developed to treat cancer. Although highly target specific, its use has been limited by its Short duration of expression. To overcome this Shortcoming, we constructed an oncolytic adenovirus (Ad)-based Short Hairpin RNA (shRNA) expression system (Ad-ΔB7-shVEGF) against vascular endothelial growth factor (VEGF), a key mediator in angiogenesis. To demonstrate the VEGF-specific nature of this Ad-based shRNA, replication-incompetent Ad expressing VEGF-specific shRNA (Ad-ΔE1-shVEGF) was also generated. Ad-ΔE1-shVEGF was highly effective in reducing VEGF expression, and elicited an antiangiogenic effect in vitro and in vivo. Similarly, Ad-ΔB7-shVEGF exhibited potent antiangiogenic effects in the matrigel plug assay. Moreover, Ad-ΔB7-shVEGF demonstrated a greater antitumor effect and enhanced survival compared to the cognate control oncolytic Ad, Ad-ΔB7. Ad-ΔB7-shVEGF induced significant reduction in tumor vasculature, verifying the antiangiogenic mechanism. Furthermore, both the duration and magnitude of gene silencing by Ad-ΔB7-shVEGF was greater than Ad-ΔE1-shVEGF. These results suggest that the combined effects of oncolytic viral therapy and cancer cell-specific expression of VEGF-targeted shRNA elicits greater antitumor effect than an oncolytic Ad alone.
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vegf specific Short Hairpin RNA expressing oncolytic adenovirus elicits potent inhibition of angiogenesis and tumor growth
Molecular Therapy, 2007Co-Authors: Ji Young Yoo, Joo Hang Kim, Young Guen Kwon, Eok Cheon Kim, Nam Kyu Kim, Hye Jin Choi, Chaeok YunAbstract:RNA interference is being developed to treat cancer. Although highly target specific, its use has been limited by its Short duration of expression. To overcome this Shortcoming, we constructed an oncolytic adenovirus (Ad)-based Short Hairpin RNA (shRNA) expression system (Ad-DeltaB7-shVEGF) against vascular endothelial growth factor (VEGF), a key mediator in angiogenesis. To demonstrate the VEGF-specific nature of this Ad-based shRNA, replication-incompetent Ad expressing VEGF-specific shRNA (Ad-DeltaE1-shVEGF) was also generated. Ad-DeltaE1-shVEGF was highly effective in reducing VEGF expression, and elicited an antiangiogenic effect in vitro and in vivo. Similarly, Ad-DeltaB7-shVEGF exhibited potent antiangiogenic effects in the matrigel plug assay. Moreover, Ad-DeltaB7-shVEGF demonstrated a greater antitumor effect and enhanced survival compared to the cognate control oncolytic Ad, Ad-DeltaB7. Ad-DeltaB7-shVEGF induced significant reduction in tumor vasculature, verifying the antiangiogenic mechanism. Furthermore, both the duration and magnitude of gene silencing by Ad-DeltaB7-shVEGF was greater than Ad-DeltaE1-shVEGF. These results suggest that the combined effects of oncolytic viral therapy and cancer cell-specific expression of VEGF-targeted shRNA elicits greater antitumor effect than an oncolytic Ad alone.
Timothy J Doran - One of the best experts on this subject based on the ideXlab platform.
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comparison of chicken 7sk and u6 RNA polymerase iii promoters for Short Hairpin RNA expression
BMC Biotechnology, 2007Co-Authors: Stephanie C Bannister, Terry G Wise, David M Cahill, Timothy J DoranAbstract:Background RNA polymerase III (pol III) type 3 promoters such as U6 or 7SK are commonly used to express Short-Hairpin RNA (shRNA) effectors for RNA interference (RNAi). To extend the use of RNAi for studies of development using the chicken as a model system, we have developed a system for expressing shRNAs using the chicken 7SK (ch7SK) promoter.
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suppression of bovine viral diarrhea virus replication by small interfering RNA and Short Hairpin RNA mediated RNA interference
Veterinary Microbiology, 2007Co-Authors: Luke S Lambeth, Robert J Moore, Morley Muralitharan, Timothy J DoranAbstract:Abstract Bovine viral diarrhea virus (BVDV) is a ubiquitous viral pathogen that affects cattle herds’ worldwide causing significant economic loss. The current strategies to control BVDV infection include vaccination (modified-live or killed) and control of virus spread by enhanced biosecurity management, however, the disease remains prevalent. With the discovery of the sequence-specific method of gene silencing known as RNA interference (RNAi), a new era in antiviral therapies has begun. Here we report the efficient inhibition of BVDV replication by small interfering (siRNA) and Short Hairpin RNA (shRNA)-mediated gene silencing. siRNAs were generated to target the 5′ non-translated (NTR) region and the regions encoding the C, NS4B and NS5A proteins of the BVDV genome. The siRNAs were first validated using an EGFP/BVDV reporter system and were then shown to suppress BVDV-induced cytopathic effects and viral titers in cell culture with surprisingly different activities compared to the reporter system. Efficient viral suppression was then achieved by bovine 7SK-expressed BVDV-specific shRNAs. Overall, our results demonstrated the use of siRNA and shRNA-mediated gene silencing to achieve efficient inhibition of the replication of this virus in cell culture.
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comparison of bovine RNA polymerase iii promoters for Short Hairpin RNA expression
Animal Genetics, 2006Co-Authors: Terry G Wise, Luke S Lambeth, Robert J Moore, Morley Muralitharan, Timothy J DoranAbstract:RNA interference (RNAi) mediated by DNA-based expression of Short Hairpin RNA (shRNA) is a powerful method of sequence-specific gene knockdown. A number of vectors for expression of shRNA have been developed that feature promoters from RNA polymerase III (pol III)-transcribed genes of mouse or human origin. To advance the use of RNAi as a tool for functional genomic research and for future development of specific therapeutics in the bovine species, we have developed shRNA expression vectors that feature novel bovine RNA pol III promoters. We characterized two bovine U6 small nuclear RNA (snRNA) promoters (bU6-2 and bU6-3) and a bovine 7SK snRNA promoter (b7SK). We compared the efficiency of each of these promoters to express shRNA molecules. Promoter activity was measured in the context of RNAi by targeting and suppressing the reporter gene encoding enhanced green fluorescent protein. Results show that the b7SK promoter induced the greatest level of suppression in a range of cell lines. The comparison of these bovine promoters in shRNA expression is an important component for the future development of bovine-specific RNAi-based research.
