詳細

櫻井　文教 （サクライ　フミノリ）

  • 薬学部 医療薬学科　教授
Last Updated :2024/05/15

コミュニケーション情報 byコメンテータガイド

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    ウイルスを利用してがんなどの難治性疾患を治療する遺伝子治療研究に取り組んでいます。特に、遺伝子治療薬の体内動態や治療効果、生体応答およびその相関について研究しています。

研究者情報

学位

  • 薬学博士（京都大学大学院）

ホームページURL

J-Global ID

研究キーワード

  • 遺伝子治療学、DDS、 生物薬剤学、薬物動態学、核酸医薬、ウイルス学、分子生物学   Gene Therapy   

現在の研究分野（キーワード）

    ウイルスを利用してがんなどの難治性疾患を治療する遺伝子治療研究に取り組んでいます。特に、遺伝子治療薬の体内動態や治療効果、生体応答およびその相関について研究しています。

研究分野

  • ライフサイエンス / 医療薬学
  • ライフサイエンス / 病態医化学
  • ライフサイエンス / 薬理学

経歴

  • 2024年04月 - 現在  近畿大学薬学部医療薬学科薬物動態学研究室教授
  • 2010年04月 - 2024年03月  大阪大学大学院薬学研究科分子生物学分野准教授
  • 2013年04月 - 2017年03月  大阪大学大学院薬学研究科 創薬臨床研究推進分野 核酸医薬評価学 准教授（兼任）
  • 2014年08月 - 2016年07月  文部科学省学術調査官（兼任）
  • 2005年04月 - 2010年03月  独立行政法人医薬基盤研究所遺伝子導入制御プロジェクト研究員
  • 2003年04月 - 2005年03月  国立医薬品食品衛生研究所研究官

学歴

  • 1996年04月 - 2001年03月   京都大学大学院   薬学研究科   薬品動態制御学分野
  •         - 2001年   京都大学   Graduate School of Phamacentical Sciences   Drug Delivery System
  • 1992年04月 - 1996年03月   京都大学   薬学部   製薬化学科
  •         - 1996年   京都大学   Faculty of Pharmaceutical Sciences

所属学協会

  • 日本免疫毒性学会   日本ウイルス学会   日本癌学会   日本DDS学会   日本薬学会   日本遺伝子細胞治療学会   アメリカ遺伝子治療学会   The Japanese Cancer Association   Japan Society of Drug Delivery System   The Japan Society of Gene Therapy   American Society of Gene Therapy   日本薬剤学会   

研究活動情報

論文

  • Kahori Shimizu; Moe Ono; Takenari Mikamoto; Yuya Urayama; Sena Yoshida; Tomomi Hase; Shotaro Michinaga; Hiroki Nakanishi; Miho Iwasaki; Tomoyuki Terada; Fuminori Sakurai; Hiroyuki Mizuguchi; Hideo Shindou; Koji Tomita; Toru Nishinaka
    The FASEB Journal 38 2 2024年01月 
    Abstract Postprandial hyperglycemia is an early indicator of impaired glucose tolerance that leads to type 2 diabetes mellitus (T2DM). Alterations in the fatty acid composition of phospholipids have been implicated in diseases such as T2DM and nonalcoholic fatty liver disease. Lysophospholipid acyltransferase 10 (LPLAT10, also called LPCAT4 and LPEAT2) plays a role in remodeling fatty acyl chains of phospholipids; however, its relationship with metabolic diseases has not been fully elucidated. LPLAT10 expression is low in the liver, the main organ that regulates metabolism, under normal conditions. Here, we investigated whether overexpression of LPLAT10 in the liver leads to improved glucose metabolism. For overexpression, we generated an LPLAT10‐expressing adenovirus (Ad) vector (Ad‐LPLAT10) using an improved Ad vector. Postprandial hyperglycemia was suppressed by the induction of glucose‐stimulated insulin secretion in Ad‐LPLAT10‐treated mice compared with that in control Ad vector‐treated mice. Hepatic and serum levels of phosphatidylcholine 40:7, containing C18:1 and C22:6, were increased in Ad‐LPLAT10‐treated mice. Serum from Ad‐LPLAT10‐treated mice showed increased glucose‐stimulated insulin secretion in mouse insulinoma MIN6 cells. These results indicate that changes in hepatic phosphatidylcholine species due to liver‐specific LPLAT10 overexpression affect the pancreas and increase glucose‐stimulated insulin secretion. Our findings highlight LPLAT10 as a potential novel therapeutic target for T2DM.
  • Sena Ikemoto; Fuminori Sakurai; Sora Tokuoka; Tomoki Yamashita; Kosuke Takayama; Kazuaki Hoshi; Takahiro Okabe; Issei Sumiyoshi; Shinsaku Togo; Kazuhisa Takahashi; Masashi Tachibana; Hiroyuki Mizuguchi
    PLOS ONE 18 10 e0286323 - e0286323 2023年10月 
    Circulating tumor cells (CTCs) are present in the blood of cancer patients from the early stage of cancer development, and their presence has been correlated with patient prognosis and treatment responses. Accordingly, CTCs have been attracting attention as a novel biomarker for early detection of cancer and monitoring of treatment responses. However, since patients typically have only a few CTCs per milliliter of blood, development of an accurate and highly sensitive CTC detection method is crucial. We previously developed a CTC detection method using a novel conditionally replicating adenovirus (Ad) that expresses green fluorescence protein (GFP) in a tumor cell-specific manner by expressing the E1 gene using a tumor-specific human telomerase reverse transcriptase (hTERT) promoter (rAdF35-142T-GFP). CTCs were efficiently detected using rAdF35-142T-GFP, but GFP expression levels in the CTCs and production efficiencies of rAdF35-142T-GFP were relatively low. In this study, in order to overcome these problems, we developed four types of novel GFP-expressing conditionally replicating Ads and examined their ability to visualize CTCs in the blood samples of lung cancer patients. Among the four types of novel recombinant Ads, the novel conditionally replicating Ad containing the 2A peptide and the GFP gene downstream of the E1A gene and the adenovirus death protein (ADP) gene in the E3 region (rAdF35-E1-2A-GFP-ADP) mediated the highest number of GFP-positive cells in the human cultured tumor cell lines. Titers of rAdF35-E1-2A-GFP-ADP were significantly higher (about 4-fold) than those of rAdF35-142T-GFP. rAdF35-E1-2A-GFP-ADP and rAdF35-142T-GFP efficiently detected CTCs in the blood of lung cancer patients at similar levels. GFP+/CD45- cells (CTCs) were found in 10 of 17 patients (58.8%) for both types of recombinant Ads.
  • Takuma Hotani; Kanako Nakagawa; Tomohito Tsukamoto; Hiroyuki Mizuguchi; Fuminori Sakurai
    Inflammation 2023年08月 [査読有り]
     
    Abstract Hypoxia inducible factor-1α (HIF-1α) is a crucial therapeutic target in various diseases, including cancer and fibrosis. We previously demonstrated that transfection with double-stranded RNA (dsRNA), including polyI:C and the dsRNA genome of mammalian orthoreovirus, resulted in significant reduction in HIF-1α protein levels in cultured cells; however, it remained to be elucidated how dsRNA induced down-regulation of HIF-1α protein levels. In this study, we examined the mechanism of dsRNA-mediated down-regulation of HIF-1α protein levels. We found that among the various cellular factors involved in dsRNA-mediated innate immunity, knockdown and knockout of protein kinase R (PKR) significantly restored HIF-1α protein levels in dsRNA-transfected cells, indicating that PKR was involved in dsRNA-mediated down-regulation of HIF-1α. Proteasome inhibitors significantly restored the HIF-1α protein levels in dsRNA-transfected cells. Ubiquitination levels of HIF-1α were increased by transfection with dsRNA. These findings indicated that degradation of HIF-1α in a ubiquitin–proteasome pathway was promoted in a PKR-dependent manner following dsRNA transfection. Expression of not only HIF-1α but also several proteins, including CDK4 and HER2, was down-regulated following dsRNA transfection. These data provide important clues for elucidation of the mechanism of dsRNA-mediated cellular toxicity, as well as for therapeutic application of dsRNA.
  • Eiko Sakai; Tsutomu Imaizumi; Ruruka Suzuki; Marcos Taracena-Gándara; Toshiki Fujimoto; Fuminori Sakurai; Hiroyuki Mizuguchi
    Communications biology 6 1 669 - 669 2023年06月 [査読有り]
     
    Non-alcoholic liver disease (NAFLD) is a condition caused by excessive fat accumulation in the liver and developed via multiple pathways. miR-27b has been suggested to play crucial roles in the development of NAFLD, assuming via targeting genes involved in lipid catabolism and anabolism. However, other pathways regulated by miR-27b are largely unknown. Here we show that lipid accumulation was induced in miR-27b-transfected human and mouse hepatic cells and that knockdowns of three miR-27b-target genes, β-1,4-galactosyltransferase 3 (B4GALT3), matrix AAA peptidase interacting protein 1 (MAIP1) and PH domain and leucine rich repeat protein phosphatase 2 (PHLPP2), induced lipid accumulation. We also show that B4GALT3 and MAIP1 were direct targets of miR-27b and overexpression of MAIP1 ameliorated miR-27b-induced lipid accumulation. In addition, we show that hepatic Maip1 expression declined in mice fed a high-fat diet, suggesting the involvement of decreased Maip1 expression in the condition of fatty liver. Overall, we identified MAIP1/miR-27b axis as a mediator of hepatic lipid accumulation, a potential therapeutic target for NAFLD.
  • Haoyang Zhou; Zhiqi Xie; Naosuke Morikawa; Fuminori Sakurai; Hiroyuki Mizuguchi; Daisuke Okuzaki; Naoki Okada; Masashi Tachibana
    Biochemistry and biophysics reports 33 101416 - 101416 2023年03月 [査読有り]
     
    Myeloid-derived suppressor cells (MDSCs), which accumulate in tumor bearers, are known to suppress anti-tumor immunity and thus promote tumor progression. MDSCs are considered a major cause of resistance against immune checkpoint inhibitors in patients with cancer. Therefore, MDSCs are potential targets in cancer immunotherapy. In this study, we modified an in vitro method of MDSC differentiation. Upon stimulating bone marrow (BM) cells with granulocyte-macrophage colony-stimulating factor in vitro, we obtained both lymphocyte antigen 6G positive (Ly-6G+) and negative (Ly-6G-) MDSCs (collectively, hereafter referred to as conventional MDSCs), which were non-immunosuppressive and immunosuppressive, respectively. We then found that MDSCs differentiated from Ly-6G- BM (hereafter called 6G- BM-MDSC) suppressed T-cell proliferation more strongly than conventional MDSCs, whereas the cells differentiated from Ly-6G+ BM (hereafter called 6G+ BM-MDSC) were non-immunosuppressive. In line with this, conventional MDSCs or 6G- BM-MDSC, but not 6G+ BM-MDSC, promoted tumor progression in tumor-bearing mice. Moreover, we identified that activated glutathione metabolism was responsible for the enhanced immunosuppressive ability of 6G- BM-MDSC. Finally, we showed that Ly-6G+ cells in 6G- BM-MDSC, which exhibited weak immunosuppression, expressed higher levels of Cybb mRNA, an immunosuppressive gene of MDSCs, than 6G+ BM-MDSC. Together, these data suggest that the depletion of Ly-6G+ cells from the BM cells leads to differentiation of immunosuppressive Ly-6G+ MDSCs. In summary, we propose a better method for MDSC differentiation in vitro. Moreover, our findings contribute to the understanding of MDSC subpopulations and provide a basis for further research on MDSCs.
  • Maho Eguchi; Seiya Hirata; Ikuho Ishigami; Naomi Shuwari; Ryosuke Ono; Masashi Tachibana; Masato Tanuma; Atsushi Kasai; Hitoshi Hashimoto; Ken-Ichi Ogawara; Hiroyuki Mizuguchi; Fuminori Sakurai
    Journal of controlled release : official journal of the Controlled Release Society 354 35 - 44 2023年02月 [査読有り]
     
    PEGylated liposomes (PEG-liposomes) are a promising drug delivery vehicle for tumor targeting because of their efficient tumor disposition profiles via the enhanced permeability and retention (EPR) effect. However, tumor targeting of PEG-liposomes, particularly their delivery inside the tumors, is often disturbed by physical barriers in the tumor, including tumor cells themselves, extracellular matrices, and interstitial pressures. In this study, B16 melanoma tumor-bearing mice were injected intravenously with oncolytic reovirus before administration of PEG-liposomes to enhance PEG-liposomes' tumor disposition. Three days after reovirus administration, significant expression of reovirus sigma 3 protein, elevation of apoptosis-related gene expression, and activation of caspase 3 in the tumors were found. Apoptotic cells were found inside the tumors. These data indicated that reovirus efficiently replicated in the tumors and induced apoptosis of tumor cells. The tumor disposition levels of PEG-liposomes were approximately doubled by reovirus pre-administration, compared with a PBS-pretreated group. PEG-liposomes were widely distributed in the tumors of reovirus-pretreated mice, whereas in the PBS-pretreated group, PEG-liposomes were found mainly around or inside the blood vessels in the tumors. Pre-treatment with reovirus also improved the tumor accumulation of PEG-liposomes in human pancreatic BxPC-3 tumors. 3D imaging analysis of whole BxPC-3 tumors demonstrated that pretreatment with reovirus led to the enhancement of PEG-liposome accumulation inside the tumors. Combination treatment with reovirus and paclitaxel-loaded PEG-liposomes (PTX-PEG-liposomes) significantly suppressed B16 tumor growth. These results provide important information for clinical use of combination therapy of reovirus and nanoparticle-based drug delivery system (DDS).
  • Ryosuke Ono; Kosuke Takayama; Rika Onishi; Sora Tokuoka; Fuminori Sakurai; Hiroyuki Mizuguchi
    Anticancer research 43 2 537 - 546 2023年02月 [査読有り]
     
    BACKGROUND/AIM: Oncolytic adenoviruses (Ads) (OAds) are gaining attention as an effective remedy for pancreatic cancer. Most OAds are based on human Ad serotype 5 (Ad5) (OAd5); however, two major drawbacks of OAd5 have been reported. Expression of coxsackievirus-adenovirus receptor, a primary infection receptor of Ad5, is often decreased on malignant tumor cells, including pancreatic cancers. More than 60% of adults have neutralizing antibodies against Ad5. Previously, we developed an OAd composed of Ad serotype 35 (Ad35) (OAd35). Ad35 recognizes CD46, which is often up-regulated on pancreatic cancers. In addition, only 20% or fewer adults have anti-Ad35 neutralizing antibodies. MATERIALS AND METHODS: We examined the tumor cell lysis activities of OAd35 in the four human pancreatic cancer cell lines in the presence and absence of human serum. The tumor growth suppression effects of OAd35 after local and systemic administration were evaluated in nude mice bearing human pancreatic tumors. RESULTS: OAd35 showed higher levels of tumor cell lysis activities than OAd5 in the human pancreatic cancer cell lines AsPC-1 and BxPC-3. Although the in vitro tumor cell lysis activities of OAd5 against MIA PaCa-2 and PANC-1 cells were strongly attenuated in the presence of human serum, OAd35 mediated comparable levels of tumor cell lysis in the presence and absence of human serum. Systemic administration of OAd5 did not mediate significant growth inhibition against the subcutaneous BxPC-3 tumor. On the other hand, OAd35 significantly suppressed tumor growth. CONCLUSION: OAd35 would be suitable as an alternative anticancer agent for pancreatic cancer.
  • Ryosuke Ono; Fumitaka Nishimae; Takuro Wakida; Fuminori Sakurai; Hiroyuki Mizuguchi
    Scientific reports 12 1 21560 - 21560 2022年12月 [査読有り]
     
    Oncolytic adenoviruses (OAds), most of which are based on species C human adenovirus serotype 5 (Ad5) (OAd5), have recently received much attention as potential anticancer agents. High seroprevalence of anti-Ad5 neutralizing antibodies is a major hurdle for Ad5-based gene therapy. However, the impacts of anti-Ad5 neutralizing antibodies on OAd5-mediated transgene expression in the tumor and antitumor effects remain to be fully elucidated. In this study, we examined the impact of anti-Ad5 neutralizing antibodies on the OAd5-mediated antitumor effects and OAd5-mediated transgene expression. The luciferase expression of OAd-tAIB-Luc, which contains the cytomegalovirus promoter-driven luciferase gene, was inhibited in human cultured cells in the presence of human serum. Although the inhibitory effects of human serum possessing the low anti-Ad5 neutralizing antibody titers were overcome by long-term infection, the in vitro tumor cell lysis activities of OAd-tAIB-Luc were entirely attenuated by human serum containing the high titers of anti-Ad5 neutralizing antibodies. OAd-tAIB-Luc-mediated luciferase expression in the subcutaneous tumors 3 days after administration and tumor growth suppression levels following intratumoral administration were significantly lower in mice possessing the high titers of anti-Ad5 neutralizing antibodies, compared to those in control mice. These results suggested that pre-existing anti-Ad5 antibodies attenuated both transgene expression and potential antitumor effects of OAd5 following intratumoral administration.
  • Kahori Shimizu; Syogo Nishimuta; Yuri Fukumura; Shotaro Michinaga; Yuka Egusa; Tomomi Hase; Tomoyuki Terada; Fuminori Sakurai; Hiroyuki Mizuguchi; Koji Tomita; Toru Nishinaka
    PLOS ONE 17 9 e0274297 - e0274297 2022年09月 [査読有り]
     
    The liver is the main organ that regulates lipid and glucose metabolism. Ectopic lipid accumulation in the liver impairs insulin sensitivity and glucose metabolism. Lipoprotein lipase (LPL), mainly expressed in the adipose tissue and muscle, is a key enzyme that regulates lipid metabolism via the hydrolysis of triglyceride in chylomicrons and very-low-density lipoproteins. Here, we aimed to investigate whether the suppression level of hepatic lipid accumulation via overexpression of LPL in mouse liver leads to improved metabolism. To overexpress LPL in the liver, we generated an LPL-expressing adenovirus (Ad) vector using an improved Ad vector that exhibited considerably lower hepatotoxicity (Ad-LPL). C57BL/6 mice were treated with Ad vectors and simultaneously fed a high-fat diet (HFD). Lipid droplet formation in the liver decreased in Ad-LPL-treated mice relative to that in control Ad vector-treated mice. Glucose tolerance and insulin resistance were remarkably improved in Ad-LPL-treated mice compared to those in control Ad vector-treated mice. The expression levels of fatty acid oxidation-related genes, such as peroxisome proliferator-activated receptor α, carnitine palmitoyltransferase 1, and acyl-CoA oxidase 1, were 1.7–2.0-fold higher in Ad-LPL-treated mouse livers than that in control Ad-vector-treated mouse livers. Furthermore, hepatic LPL overexpression partly maintained mitochondrial content in HFD-fed mice. These results indicate that LPL overexpression in the livers of HFD-fed mice attenuates the accumulation of lipid droplets in the liver and improves glucose metabolism. These findings may enable the development of new drugs to treat metabolic syndromes such as type 2 diabetes mellitus and non-alcoholic fatty liver disease.
  • Fumitaka Nishimae; Fuminori Sakurai; Ryosuke Ono; Rika Onishi; Kosuke Takayama; Hiroyuki Mizuguchi
    The Journal of general virology 103 6 2022年06月 [査読有り]
     
    Oncolytic adenoviruses (OAds) have attracted much attention as novel anticancer agents. Numerous studies have examined the antitumour effects of combinational use of an OAd and anticancer agents; however, few chemical compounds enhancing OAd infection have been reported. In this study, we screened a food and drug administration (FDA)-approved drug library containing 1134 small chemical compounds to identify chemical compounds that enhance OAd replication in human tumour cells. We found that domperidone, a dopamine D2 receptor antagonist, significantly enhanced the replication of an OAd in human tumour cells, including human pancreatic tumour cells, by two-fivefold, resulting in improvement of OAd-mediated tumour cell killing activities. The E1A mRNA levels were significantly increased in domperidone-pre-treated cells following OAd infection, which contributed to the promotion of OAd replication. However, mRNA levels of the dopamine D2 receptor (DRD2), which is known to be a target molecule of domperidone, were undetectable in most of the tumour cells by real-time reverse transcription (RT)-PCR analysis, indicating that domperidone promoted OAd replication by acting on a molecule other than DRD2. This study provides important clues for the improvement of OAd-mediated cancer therapy.
  • Hiroshi Tazawa; Kunitoshi Shigeyasu; Kazuhiro Noma; Shunsuke Kagawa; Fuminori Sakurai; Hiroyuki Mizuguchi; Hisataka Kobayashi; Takeshi Imamura; Toshiyoshi Fujiwara
    Cancer science 113 6 1919 - 1929 2022年06月 [査読有り]
     
    Conventional imaging techniques are available for clinical identification of tumor sites. However, detecting metastatic tumor cells that are spreading from primary tumor sites using conventional imaging techniques remains difficult. In contrast, fluorescence-based labeling systems are useful tools for detecting tumor cells at the single-cell level in cancer research. The ability to detect fluorescent-labeled tumor cells enables investigations of the biodistribution of tumor cells for the diagnosis and treatment of cancer. For example, the presence of fluorescent tumor cells in the peripheral blood of cancer patients is a predictive biomarker for early diagnosis of distant metastasis. The elimination of fluorescent tumor cells without damaging normal tissues is ideal for minimally invasive treatment of cancer. To capture fluorescent tumor cells within normal tissues, however, tumor-specific activated target molecules are needed. This review focuses on recent advances in tumor-targeted fluorescence labeling systems, in which indirect reporter labeling using tumor-specific promoters is applied to fluorescence labeling of tumor cells for the diagnosis and treatment of cancer. Telomerase promoter-dependent fluorescence labeling using replication-competent viral vectors produces fluorescent proteins that can be used to detect and eliminate telomerase-positive tumor cells. Tissue-specific promoter-dependent fluorescence labeling enables identification of specific tumor cells. Vimentin promoter-dependent fluorescence labeling is a useful tool for identifying tumor cells that undergo epithelial-mesenchymal transition (EMT). The evaluation of tumor cells undergoing EMT is important for accurately assessing metastatic potential. Thus, tumor-targeted fluorescence labeling systems represent novel platforms that enable the capture of tumor cells for the diagnosis and treatment of cancer.
  • Aoi Shiota; Fumitaka Nishimae; Fuminori Sakurai; Hiroyuki Mizuguchi
    Anticancer research 42 4 1719 - 1727 2022年04月 [査読有り]
     
    BACKGROUND/AIM: Efficient production of adenovirus vectors is crucial for their clinical use. Adenovirus death protein (ADP), which is encoded in the E3 region of the adenovirus genome, is involved in host-cell lysis and the subsequent release of progeny virus; however, the ADP gene is often removed from the adenovirus vector genome. MATERIALS AND METHODS: We have developed adenovirus vectors that possess the ADP gene and maintain a relatively large insertion capacity for foreign genes by deleting the partial E3 region. Adenovirus vector-mediated transgene expression levels and virus titers were examined. RESULTS: The adenovirus vectors maintaining the ADP gene showed cytopathic effect earlier than conventional adenovirus vector without the ADP gene following treatment of HEK293 cells, although there were no significant differences in total virus titers. CONCLUSION: The adenovirus vectors possessing the ADP gene showed efficient spread of progeny virus infection following transduction in HEK293 cells.
  • レオウイルス感染がん細胞由来細胞外小胞の抗腫瘍効果に関する検討
    種昂 なお実; 井上 智重子; 神宮司 健太郎; 辻川 和丈; 水口 裕之; 櫻井 文教
    日本薬学会年会要旨集 142年会 27J - am01S (公社)日本薬学会 2022年03月
  • Fuminori Sakurai; Masashi Tachibana; Hiroyuki Mizuguchi
    Drug metabolism and pharmacokinetics 42 100432 - 100432 2021年11月 [査読有り][招待有り]
     
    Replication-incompetent adenovirus (Ad) vectors have been widely used as gene delivery vehicles in both gene therapy studies and basic studies for gene function analysis due to their highly advantageous properties, which include high transduction efficiencies, relatively large capacities for transgenes, and high titer production. In addition, Ad vectors induce moderate levels of innate immunity and have relatively high thermostability, making them very attractive as potential vaccine vectors. Accordingly, it is anticipated that Ad vectors will be used in vaccines for the prevention of infectious diseases, including Ebola virus disease and acquired immune deficiency syndrome (AIDS). Much attention is currently focused on the potential use of an Ad vector vaccine for coronavirus disease 2019 (COVID-19). In this review, we describe the basic properties of an Ad vector, Ad vector-induced innate immunity and immune responses to Ad vector-produced transgene products. Development of novel Ad vectors which can overcome the drawbacks of conventional Ad vector vaccines and clinical application of Ad vector vaccines to several infectious diseases are also discussed.
  • Nozomi Kurisu; Tadataka Kaminade; Maho Eguchi; Ikuho Ishigami; Hiroyuki Mizuguchi; Fuminori Sakurai
    International journal of pharmaceutics 121269 - 121269 2021年11月 [査読有り]
     
    Oncolytic viruses, which mediate tumor cell-specific infection, resulting in efficient tumor cell killing, have attracted much attention as a novel class of anti-cancer biopharmaceutical agents. Cancer-associated fibroblasts (CAFs) are an important component of the tumor microenvironment that strongly supports the growth, survival, and metastasis of tumor cells, suggesting that CAFs would have influence to the antitumor effects of oncolytic viruses; however, it remains to be fully evaluated whether oncolytic viruses affect the viabilities and properties of CAFs following treatment. Oncolytic reovirus, which is a non-enveloped virus that contains 10-segmented double-stranded RNA genome, shows efficient tumor cell lysis without apparent cytotoxicity to normal cells and has been tested worldwide in clinical trials against various types of tumors. In this study, we demonstrated that reovirus exhibited cytotoxicity against mouse primary CAFs isolated from subcutaneous tumors, but not against tail-tip fibroblasts. Infection with reovirus resulted in activation of caspase 3 and up-regulation of apoptosis-related gene expression, indicating that reovirus induced apoptosis of mouse primary CAFs. Intratumoral administration of reovirus induced apoptosis of mouse CAFs in the tumor. Taken together, these results indicate that reovirus has the potential to mediate antitumor effects by killing not only cancer cells but also CAFs.
  • Jaeeun Lim; Eiko Sakai; Fuminori Sakurai; Hiroyuki Mizuguchi
    Scientific reports 11 1 19820 - 19820 2021年10月 [査読有り]
     
    Human induced pluripotent stem (hiPS) cells are feasible materials for studying the biological mechanisms underlying human embryogenesis. In early embryogenesis, definitive endoderm and mesoderm are differentiated from their common precursor, mesendoderm. Bone morphogenetic protein (BMP) signaling is responsible for regulating mesendoderm and mesoderm formation. Micro RNAs (miRNAs), short non-coding RNAs, broadly regulate biological processes via post-transcriptional repression. The expression of miR-27b, which is enriched in somatic cells, has been reported to increase through definitive endoderm and hepatic differentiation, but little is known about how miR-27b acts during early differentiation. Here, we used miR-27b-inducible hiPS cells to investigate the roles of miR-27b in the undifferentiated and early-differentiated stages. In undifferentiated hiPS cells, miR-27b suppressed the expression of pluripotency markers [alkaline phosphatase (AP) and nanog homeobox (NANOG)] and cell proliferation. Once differentiation began, miR-27b expression repressed phosphorylated SMAD1/5, the mediators of the BMP signaling, throughout definitive endoderm differentiation. Consistent with the above findings, miR-27b overexpression downregulated BMP-induced mesendodermal marker genes [Brachyury, mix paired-like homeobox 1 (MIXL1) and eomesodermin (EOMES)], suggesting that miR-27b had an inhibitory effect on early differentiation. Collectively, our findings revealed a novel antagonistic role of miR-27b in the BMP signaling pathway in the early differentiation of hiPS cells.
  • Ikuho Ishigami; Naomi Shuwari; Tadataka Kaminade; Hiroyuki Mizuguchi; Fuminori Sakurai
    Anticancer research 41 5 2431 - 2440 2021年05月 [査読有り]
     
    BACKGROUND/AIM: Oncolytic reovirus, which is a non-enveloped virus possessing a 10-segmented double-stranded RNA genome, has been anticipated as a novel class of antitumor agent. Hepatocellular carcinoma (HCC) is considered to be a target suitable for reovirus-mediated virotherapy. Transforming growth factor (TGF)-β plays an important role in the pathogenesis of HCC. TGF-β-signaling inhibitors have proceeded to clinical trials as potential antitumor agents for HCC. On the other hand, TGF-β is involved in induction of expression of cathepsins B and L, which are important for reovirus infection. It remains to be examined whether TGF-β signaling inhibitors affect reovirus-mediated lysis of HCC cells. The aim of this study was to evaluate the effects of TGF-β-signaling inhibitors on tumor cell lysis efficiency of reovirus in human HCC cells. MATERIALS AND METHODS: Reovirus was added to four types of human HCC cell lines pretreated with one of three TGF-β type I receptor inhibitors: SB431542, A-83-01, or galunisertib (LY2157299). Cell viability, virus genome copy numbers, and virus protein expression were evaluated following reovirus infection. RESULTS: SB431542 significantly inhibited reovirus-mediated killing of human HCC cell lines, while A-83-01 and galunisertib did not inhibit. CONCLUSION: These data indicate that SB431542 inhibited reovirus-mediated lysis of human HCC cells in a TGF-β signaling-independent manner.
  • Takamasa Hirai; Anna Sato; Naoya Koizumi; Yoh Kurioka; Yui Suzuki; Junpei Kano; Makie Yamakawa; Tetsuya Nomura; Makiko Fujii; Fuminori Sakurai; Hiroyuki Mizuguchi; Yoshiteru Watanabe; Naoki Utoguchi
    Journal of General Virology 102 4 2021年04月 [査読有り]
     
    Human adenoviruses (Ads), common pathogens that cause upper respiratory and gastrointestinal infections, are blocked by neutralizing antibodies (nAbs). However, Ads are not fully eliminated even in hosts with nAbs. In this study, we assessed the infectivity of progeny Ad serotype 5 (Ad5) in the presence of nAb. The infectivity of Ad5 was evaluated according to the expression of the Ad genome and reporter gene. Infection by wild-type Ad5 and Ad5 vector continued to increase until 3 days after infection even in the presence of nAb. We established an assay for determining the infection levels of progeny Ad5 using a sorting system with magnetic beads and observed little difference in progeny Ad5 counts in the presence and absence of nAb 1 day after infection. Moreover, progeny Ad5 in the presence of nAb more effectively infected coxsackievirus and adenovirus receptor (CAR)-positive cells than CAR-negative cells. We investigated the function of fiber proteins, which are the binding partners of CAR, during secondary infection, observing that fibre proteins spread from infected cells to adjacent cells in a CAR-dependent manner. In conclusion, this study revealed that progeny Ad5 could infect cells even in the presence of nAb, differing from the common features of the Ad5 infection cycle. Our findings may be useful for developing new therapeutic agents against Ad infection.
  • Kahori Shimizu; Yuya Ogiya; Kaede Yoshinaga; Hajime Kimura; Shotaro Michinaga; Moe Ono; Ayako Taketomi; Tomoyuki Terada; Fuminori Sakurai; Hiroyuki Mizuguchi; Koji Tomita; Toru Nishinaka
    Experimental and Clinical Endocrinology & Diabetes 2021年03月 [査読有り]
     
    AbstractGenome-wide association studies have identified more than 300 loci associated with type 2 diabetes mellitus; however, the mechanisms underlying their role in type 2 diabetes mellitus susceptibility remain largely unknown. Zinc finger AN1-type domain 3 (ZFAND3), known as testis-expressed sequence 27, is a type 2 diabetes mellitus-susceptibility gene. Limited information is available regarding the physiological role of ZFAND3 in vivo. This study aimed to investigate the association between ZFAND3 and type 2 diabetes mellitus. ZFAND3 was significantly upregulated in the liver of diabetic mice compared to wild-type mice. To overexpress ZFAND3, we generated a ZFAND3-expressing adenovirus (Ad) vector using an improved Ad vector exhibiting significantly lower hepatotoxicity (Ad-ZFAND3). Glucose tolerance was significantly improved in Ad-ZFAND3-treated mice compared to the control Ad-treated mice. ZFAND3 overexpression in the mouse liver also improved insulin resistance. Furthermore, gluconeogenic gene expression was significantly lower in primary mouse hepatocytes transduced with Ad-ZFAND3 than those transduced with the control Ad vector. The present results suggest that ZFAND3 improves glucose tolerance by improving insulin resistance and suppressing gluconeogenesis, serving as a potential novel therapeutic target for type 2 diabetes mellitus.
  • Ryosuke Ono; Kosuke Takayama; Fuminori Sakurai; Hiroyuki Mizuguchi
    Molecular therapy oncolytics 20 399 - 409 2021年03月 [査読有り]
     
    Oncolytic adenoviruses (OAds) are among the most promising oncolytic viruses. Almost all oncolytic adenoviruses are composed of human adenovirus serotype 5 (Ad5) (OAd5). However, expression of the primary infection receptor for Ad5, coxsackievirus-adenovirus receptor (CAR), often declines on malignant tumor cells, resulting in inefficient infection in CAR-negative tumor cells. In addition, at least 80% of adults have neutralizing antibodies against Ad5. In this study, we developed a novel OAd fully composed of OAd35. OAd35 recognizes CD46, which is ubiquitously expressed on almost all human cells and is often upregulated on malignant tumor cells, as an infection receptor. Moreover, 20% or fewer adults have neutralizing antibodies against Ad35. OAd35 mediated efficient cell lysis activities at levels similar to OAd5 in CAR-positive tumor cells, while OAd35 showed higher levels of cell lysis activities than OAd5 in CAR-negative tumor cells. Anti-Ad5 serum significantly inhibited in vitro tumor cell lysis activities of OAd5, whereas OAd35 exhibited comparable levels of in vitro tumor cell lysis activities in the presence of anti-Ad5 and naive serum. OAd35 significantly suppressed growth of the subcutaneous CAR-positive and CAR-negative tumors following intratumoral administration. These results indicated that OAd35 is a promising alternative oncolytic virus for OAd5.
  • Chieko Inoue; Ryosuke Negoro; Kazuo Takayama; Hiroyuki Mizuguchi; Fuminori Sakurai
    Virus research 296 198334 - 198334 2021年02月 [査読有り]
     
    The intestinal mucosa plays an important role as an immune barrier due to its continual exposure to invading pathogens, including viruses. It is thus highly important to evaluate virus infection profiles in the intestinal mucosa for prevention of virus infection and development of antivirus medicines; however, only a few enterocyte lines are available as in vitro intestinal models for the evaluation of virus infection. In this study, we evaluated profiles of infection and innate immune responses following infection with a mammalian orthoreovirus (hereafter reovirus), which has often been used as a tractable model for studies of viral pathogenesis, in human iPS cell-derived small intestinal epithelial-like cell (hiPS-SIEC) monolayers and cells of a human colon adenocarcinoma cell line, Caco-2. The levels of reovirus infection were similar between hiPS-SIEC and Caco-2 cell monolayers, which are often used as an intestinal model, after apical and basolateral infection. In hiPS-SIEC monolayers, more efficient replication of the virus genome was observed following basolateral infection than apical infection, while apical infection resulted in higher levels of virus protein expression and progeny virus production than basolateral infection. Reovirus significantly induced innate immune responses, including expression of type I and III interferons (IFNs), in hiPS-SIEC monolayers more efficiently than Caco-2 cells. Higher levels of type I and III interferon (IFN) expression were found in hiPS-SIEC monolayers following apical infection than basolateral infection. These results suggested that hiPS-SIECs are a promising in vitro model for the evaluation of virus infection.
  • Fuminori Sakurai; Fumitaka Nishimae; Kosuke Takayama; Hiroyuki Mizuguchi
    Anticancer research 41 2 773 - 782 2021年02月 [査読有り]
     
    BACKGROUND/AIM: Oncolytic adenoviruses (OAds) have attracted much attention as novel anticancer therapeutics. The proper design of an expression cassette containing the E1A gene, which is indispensable for self-replication of the Ad genome, is crucial for efficient tumor cell-specific infection of an OAd. Various types of oncolytic adenoviruses (OAds) possessing different types of the E1A gene expression cassettes have been developed, but their oncolytic activities and safety profiles have not been systematically evaluated. Herein we examined the oncolytic activities and safety profiles of five types of OAds possessing different types of the E1A gene expression cassette in order to optimize the E1A gene expression cassette for development of an efficient and safe OAd. MATERIALS AND METHODS: We prepared five types of OAds containing different types of E1 gene expression cassettes, and examined the oncolytic activities and safety profiles of the OAds. RESULTS: Among the OAds examined, OAd-Δ24, which had a 24-bp deletion in the E1A gene, mediated the most efficient oncolytic activities against the human tumor cell lines, although OAd-Δ24 showed slightly higher cytotoxicity to normal human cells than the other OAds. CONCLUSION: These results provide important clues for the development of safe and efficient OAds.
  • Kahori Shimizu; Fuminori Sakurai; Shunsuke Iizuka; Ryosuke Ono; Tomohito Tsukamoto; Fumitaka Nishimae; Shin-Ichiro Nakamura; Toru Nishinaka; Tomoyuki Terada; Yasushi Fujio; Hiroyuki Mizuguchi
    Journal of immunology (Baltimore, Md. : 1950) 206 2 410 - 421 2021年01月 [査読有り]
     
    Adenovirus (Ad) vector-mediated transduction can cause hepatotoxicity during two phases, at ∼2 and 10 days after administration. Early hepatotoxicity is considered to involve inflammatory cytokines; however, the precise mechanism remains to be clarified. We examined the mechanism of early Ad vector-induced hepatotoxicity by using a conventional Ad vector, Ad-CAL2, and a modified Ad vector, Ad-E4-122aT-CAL2. Ad-E4-122aT-CAL2 harbors sequences complementary to the liver-specific miR-122a in the 3' untranslated region of E4, leading to significant suppression of leaky Ad gene expression in the liver via posttranscriptional gene silencing and a significant reduction in late-phase hepatotoxicity. We found that Ad-E4-122aT-CAL2 transduction significantly attenuated acute hepatotoxicity, although Ad-E4-122aT-CAL2 and Ad-CAL2 induced comparable cytokine expression levels in the liver and spleen. IL-6, a major inflammatory cytokine induced by Ad vectors, significantly enhanced leaky Ad gene expression and cytotoxicity in primary mouse hepatocytes following Ad-CAL2 but not Ad-E4-122aT-CAL2 transduction. Furthermore, leaky Ad gene expression and cytotoxicity in Ad-CAL2-treated hepatocytes in the presence of IL-6 were significantly suppressed upon inhibition of JAK and STAT3. Ad vector-mediated acute hepatotoxicities and leaky Ad expression were significantly reduced in IL-6 knockout mice compared with those in wild-type mice. Thus, Ad vector-induced IL-6 promotes leaky Ad gene expression, leading to acute hepatotoxicity.
  • Fuminori Sakurai; Tomohito Tsukamoto; Ryosuke Ono; Fumitaka Nishimae; Aoi Shiota; Shunsuke Iizuka; Kahori Shimizu; Eiko Sakai; Yuji Ishida; Chise Tateno; Kazuaki Chayama; Hiroyuki Mizuguchi
    Biological & pharmaceutical bulletin 44 10 1506 - 1513 2021年 [査読有り]
     
    Replication-incompetent adenovirus (Ad) vectors are promising gene delivery vehicles, especially for hepatocytes, due to their superior hepatic tropism; however, in vivo application of an Ad vector often results in hepatotoxicity, mainly due to the leaky expression of Ad genes from the Ad vector genome. In order to reduce the Ad vector-induced hepatotoxicity, we previously developed an Ad vector containing the sequences perfectly complementary to a liver-specific microRNA (miRNA), miR-122a, in the 3'-untranslated region (UTR) of the E4 gene. This improved Ad vector showed a significant reduction in the leaky expression of Ad genes and hepatotoxicity in the mouse liver and primary mouse hepatocytes; however, the safety profiles and transduction properties of this improved Ad vector in human hepatocytes remained to be elucidated. In this study, we examined the transgene expression and safety profiles of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene in human hepatocytes from chimeric mice with humanized liver. The transgene expression levels of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene were significantly higher than those of the conventional Ad vectors. The leaky expression levels of Ad genes of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene in the primary human hepatocytes were largely reduced, compared with the conventional Ad vectors, resulting in an improvement in Ad vector-induced cytotoxicity. These data indicated that this improved Ad vector was a superior gene delivery vehicle without severe cytotoxicity for not only mouse hepatocytes but also human hepatocytes.
  • Masashi Tachibana; Nobumasa Watanabe; Yuzo Koda; Yukako Oya; Osamu Kaminuma; Kazufumi Katayama; Zifei Fan; Fuminori Sakurai; Kenji Kawabata; Takachika Hiroi; Hiroyuki Mizuguchi
    International immunology 32 3 187 - 201 2020年03月 [査読有り]
     
    IL-10 is an immune regulatory cytokine and its genetic defect leads to gastrointestinal inflammation in humans and mice. Moreover, the IL-23/Th17 axis is known to be involved in these inflammatory disorders. IL-17A, a representative cytokine produced by Th17 cells, has an important role for the pathological process of inflammatory diseases. However, the precise function of IL-17A in inflammatory bowel disease (IBD) remains controversial. In this study, we evaluated the effect of IL-17A on colitis in IL-10-deficient (Il10-/-) mice. Mice lacking both IL-10 and IL-17A (Il10-/-Il17a-/-) suffered from fatal wasting and manifested more severe colitis compared with Il10-/-Il17a+/- mice. Moreover, we found that CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) accumulated in the bone marrow, spleen and peripheral blood of Il10-/-Il17a-/- mice. These MDSCs highly expressed inducible nitric oxide synthase (iNOS) (Nos2) and suppressed the T-cell response in vitro in a NOS-dependent manner. In correlation with these effects, the concentration of nitric oxide was elevated in the serum of Il10-/-Il17a-/- mice. Surprisingly, the severe colitis observed in Il10-/-Il17a-/- mice was ameliorated in Il10-/-Il17a-/-Nos2-/- mice. Our findings suggest that IL-17A plays suppressive roles against spontaneous colitis in Il10-/- mice in an iNOS-dependent manner and inhibits MDSC differentiation and/or proliferation.
  • Adenovirus Fiber can Distribute Itself to the Cell Surface without Membrane Damage
    Sato A; Hirai T; Koizumi N; Hatakeyama S; Watanabe A; Nomura T; Sakurai F; Mizuguchi H; Utoguchi N
    BPB Reports 2 6 113 - 118 2019年12月 [査読有り]
  • Tomohito Tsukamoto; Eiko Sakai; Fumitaka Nishimae; Fuminori Sakurai; Hiroyuki Mizuguchi
    Journal of biotechnology 304 1 - 9 2019年10月 [査読有り]
     
    Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas) 9 system is a powerful tool for genome editing and still being aggressively improved. Cas12a, a recently discovered Cas9 ortholog, is expected to become complementary to Cas9 due to its unique characteristics. Previously we attempted to establish an adenovirus (Ad) vector-mediated delivery of CRISPR-Cas12a system since Ad vector is widely used for gene transfer in basic researches and medical applications. However, we found difficulties preparing of Ad vectors at an adequate titer. In this study, we have developed Ad vectors that conditionally express Cas12a either by a tetracycline-controlled promoter or a hepatocyte specific promoter to avoid putative inhibitory effects of Cas12a. These vectors successfully proliferated in packaging cells, HEK293 cells, and were recovered at high titers. We have also developed packaging cells that express shRNA for Cas12a to suppress expression of Cas12a. Using the cells, the Ad vector directing constitutive expression of Cas12a proliferated efficiently and was successfully recovered at a high titer. Overall, we improved recovery of Ad vectors carrying CRISPR-Cas12a system, thus provided them as a tool in genome editing researches.
  • Fuminori Sakurai; Rina Hashimoto; Chieko Inoue; Keisaku Wakabayashi; Tomohito Tsukamoto; Tsutomu Imaizumi; Taracena Gandara Marcos Andres; Eiko Sakai; Kanae Itsuki; Naoya Sakamoto; Takaji Wakita; Hiroyuki Mizuguchi
    Virology journal 16 1 58 - 58 2019年05月 [査読有り]
     
    BACKGROUND: MicroRNAs (miRNAs) have gained much attention as cellular factors regulating hepatitis C virus (HCV) infection. miR-27b has been shown to regulate HCV infection in the hepatocytes via various mechanisms that have not been fully elucidated. In this study, therefore, we examined the mechanisms of miR-27b-mediated regulation of HCV infection. METHODS: In silico screening analysis, transfection with miR-27b mimic, and a cell-based reporter assay were performed to identify miR-27b target genes. Cell cultured-derived HCV (HCVcc) was added to Huh7.5.1 cells knocked down for aquaporin (AQP) 11 (AQP11) and overexpressing AQP11. HCV replication levels were evaluated by real-time RT-PCR analysis of HCVcc genome. RESULTS: Infection of Huh7.5.1 cells with HCVcc resulted in significant elevation in miR-27b expression levels. In silico analysis revealed that AQP11, which is an AQP family member and is mainly localized in the endoplasmic reticulum (ER), was a candidate for a target gene of miR-27b. Transfection of a miR-27b mimic significantly reduced AQP11 expression, but a cell-based reporter assay demonstrated that miR-27b did not suppress the expression of a reporter gene containing the 3'-untranslated region of the AQP11 gene, suggesting that miR-27b indirectly suppressed AQP11 expression. AQP11 expression levels were significantly reduced by infection with HCVcc in Huh7.5.1 cells. Knockdown and over-expression of AQP11 significantly reduced and increased HCVcc genome levels in the cells following infection, respectively, however, AQP11 knockdown did not show significant effects on the HCVcc titers in the culture supernatants. CONCLUSIONS: These results indicated that HCV infection induced a miR-27b-mediated reduction in AQP11 expression, leading to a modest reduction in HCV genome levels in the cells, not HCV titers in the culture supernatants.
  • Hotani T; Mizuguchi H; Sakurai F
    Molecular therapy oncolytics 12 162 - 172 2019年03月 [査読有り]
     
    Reovirus, which possesses a 10-segmented double-stranded RNA genome, mediates superior antitumor effects via not only virus replication in a tumor cell-specific manner but also other mechanisms distinct from virus replication. Several groups, including ours, reported the reovirus-mediated downregulation of hypoxia inducible factor-1α (HIF-1α) following infection in cultured tumor cells; however, it remained to be clarified whether reovirus downregulates the expression of HIF-1α and its target genes in tumor-bearing hosts. We found that reovirus induced significant downregulation of protein levels of HIF-1α and its target genes in the subcutaneous tumors at 120 h post-systemic administration. Expression of reovirus capsid protein σ3 was found in the pimonidazole-positive hypoxic area in the tumor. Significant levels of tumor cell apoptosis were not found in the tumors of reovirus-treated mice at this time point, suggesting that reovirus-mediated tumor cell killing did not largely contribute to the downregulation of HIF-1α protein levels in the tumors. UV-inactivated reovirus did not induce downregulation of HIF-1α expression in the tumors, indicating that virus replication was indispensable for downregulation of HIF-1α expression in the subcutaneous tumors. This study provides important information for the development of reovirus-mediated virotherapy against various types of tumors.
  • Yukiko Toba; Ayumi Kiso; Souichiro Nakamae; Fuminori Sakurai; Kazuo Takayama; Hiroyuki Mizuguchi
    Scientific reports 9 1 3713 - 3713 2019年03月 [査読有り]
     
    Human induced pluripotent stem cell-derived hepatocyte-like cells are expected to be utilized in pharmaceutical research and regenerative medicine. In general, human induced pluripotent stem (iPS) cells are differentiated into hepatocyte-like cells through definitive endoderm cells and hepatoblast-like cells using various growth factors that are essential for liver development. Although recombinant bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) are widely used in the hepatoblast differentiation, hepatoblast differentiation process has not been fully modified. In this study, we examined the roles of BMPs and FGFs in the hepatoblast differentiation from human iPS cells. Surprisingly, the gene expression levels of hepatoblast markers were upregulated by the removal of FGFs. In addition, the percentages of hepatoblast markers-positive cells were increased by the removal of FGFs. Furthermore, the hepatocyte differentiation potency was also significantly increased by the removal of FGFs. To examine whether FGF signals are completely unnecessary for the hepatoblast differentiation, the expression levels of endogenous FGF ligands and receptors were examined. The definitive endoderm cells highly expressed the FGF ligand, FGF2, and the FGF receptor, FGFR1. To examine the role of endogenous FGF signals, an FGFR inhibitor was treated during the hepatoblast differentiation. The hepatoblast differentiation was promoted by using FGFR inhibitor, suggesting that endogenous FGF signals are also unnecessary for the hepatoblast differentiation. In conclusion, we found that FGF signals are not essential for hepatoblast differentiation. We believe that our finding will be useful for generating functional hepatocyte-like cells for medical applications.
  • K Wakabayashi; M Machitani; M Tachibana; F Sakurai; H Mizuguchi
    Journal of virology 93 2 2019年01月 
    The adenovirus (Ad) serotype 5 genome encodes two noncoding small RNAs (virus-associated RNAs I and II [VA-RNAI and -II]), which are approximately 160-nucleotide (nt) RNAs transcribed by RNA polymerase III. It is well known that VA-RNAI supports Ad infection via the inhibition of double-stranded RNA-dependent protein kinase (PKR), which recognizes double-stranded RNA and acts as an antiviral system. Recent studies revealed that VA-RNAs are processed into VA-RNA-derived microRNAs (miRNAs) (mivaRNAI and -II); however, we and another group recently demonstrated that mivaRNAI does not promote Ad replication. On the other hand, the roles of VA-RNAII and mivaRNAII in Ad replication have remained to be clarified. In this study, we demonstrated mivaRNAII-mediated promotion of Ad replication. Transfection with chemically synthesized 3'-mivaRNAII-138, one of the most abundant forms of mivaRNAII, significantly enhanced Ad replication, while the other species of mivaRNAII did not. We identified 8 putative target genes of 3'-mivaRNAII-138 by microarray analysis and in silico analysis. Among the 8 candidates, knockdown of the cullin 4A (CUL4A) gene, which encodes a component of the ubiquitin ligase complex, most significantly enhanced Ad replication. CUL4A expression was significantly suppressed by 3'-mivaRNAII-138 via posttranscriptional gene silencing, indicating that CUL4A is a target gene of 3'-mivaRNAII-138 and mivaRNAII functions as a viral miRNA promoting Ad infection. It has been reported that CUL4A is involved in degradation of c-Jun, which acts as a transcription factor in the Jun-N-terminal kinase (JNK) signaling cascade. Treatment with JNK inhibitors dramatically suppressed Ad replication, suggesting that mivaRNAII-mediated downregulation of CUL4A enhanced JNK signaling and thereby promoted Ad infection.IMPORTANCE Several types of viruses encode viral miRNAs which regulate host and/or viral gene expression via posttranscriptional gene silencing, leading to efficient viral infection. Adenovirus (Ad) expresses miRNAs derived from VA-RNAs (mivaRNAI and -II); however, recent studies have revealed that processing of VA-RNAI into mivaRNAI inhibits Ad replication. Conversely, we demonstrate here that mivaRNAII significantly promotes Ad replication and that mivaRNAII-mediated suppression of CUL4A expression via posttranscriptional gene silencing induces accumulation of c-Jun, leading to promotion of Ad infection. These results exhibited the significance of VA-RNAII for supporting Ad infection through a mechanism complementary to that of VA-RNAI. These observations could provide important clues toward a new perspective on host-virus interaction. Moreover, Ad is widely used as a basic framework for viral vectors and oncolytic viruses. Our findings will help to regulate Ad infection and will promote the development of novel Ad vectors and oncolytic Ad.
  • Ryosuke Negoro; Kazuo Takayama; Kanae Kawai; Kazuo Harada; Fuminori Sakurai; Kazumasa Hirata; Hiroyuki Mizuguchi
    Stem cell reports 11 6 1539 - 1550 2018年12月 [査読有り]
     
    The small intestine plays an important role in the absorption and metabolism of oral drugs. In the current evaluation system, it is difficult to predict the precise absorption and metabolism of oral drugs. In this study, we generated small intestinal epithelial-like cells from human induced pluripotent stem cells (hiPS-SIECs), which could be applied to drug absorption and metabolism studies. The small intestinal epithelial-like cells were efficiently generated from human induced pluripotent stem cell by treatment with WNT3A, R-spondin 3, Noggin, EGF, IGF-1, SB202190, and dexamethasone. The gene expression levels of small intestinal epithelial cell (SIEC) markers were similar between the hiPS-SIECs and human adult small intestine. Importantly, the gene expression levels of colonic epithelial cell markers in the hiPS-SIECs were much lower than those in human adult colon. The hiPS-SIECs generated by our protocol exerted various SIEC functions. In conclusion, the hiPS-SIECs can be utilized for evaluation of drug absorption and metabolism.
  • Matoba N; Yamashita T; Takayama K; Sakurai F; Mizuguchi H
    Differentiation; research in biological diversity 104 13 - 21 2018年11月 [査読有り]
  • Kyoko Tomita; Fuminori Sakurai; Shunsuke Iizuka; Masahisa Hemmi; Keisaku Wakabayashi; Mitsuhiro Machitani; Masashi Tachibana; Kazufumi Katayama; Haruhiko Kamada; Hiroyuki Mizuguchi
    Scientific reports 8 1 12315 - 12315 2018年08月 [査読有り]
     
    Pre-existing anti-adenovirus (Ad) neutralizing antibodies (AdNAbs) are a major barrier in clinical gene therapy using Ad vectors and oncolytic Ads; however, it has not been fully elucidated which Ad capsid protein-specific antibodies are involved in AdNAb-mediated inhibition of Ad infection in vivo. In this study, mice possessing antibodies specific for each Ad capsid protein were prepared by intramuscular electroporation of each Ad capsid protein-expressing plasmid. Ad vector-mediated hepatic transduction was efficiently inhibited by more than 100-fold in mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid. An Ad vector pre-coated with FX before administration mediated more than 100-fold lower transduction efficiencies in the liver of warfarinized mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid, compared with those in the liver of warfarinized non-immunized mice. These data suggest that anti-fiber protein and anti-penton base antibodies bind to an Ad vector even though FX has already bound to the hexon, and inhibit Ad vector-mediated transduction. This study provides important clues for the development of a novel Ad vector that can circumvent inhibition with AdNAbs.
  • Junko Watanabe; Shinsaku Togo; Issei Sumiyoshi; Yukiko Namba; Kentaro Suina; Takafumi Mizuno; Kotaro Kadoya; Hiroaki Motomura; Moe Iwai; Tetsutaro Nagaoka; Shinichi Sasaki; Takuo Hayashi; Toshimasa Uekusa; Kanae Abe; Yasuo Urata; Fuminori Sakurai; Hiroyuki Mizuguchi; Shunsuke Kato; Kazuhisa Takahashi
    Oncotarget 9 35 24000 - 24013 2018年05月 [査読有り]
     
    Anti-anaplastic lymphoma kinase (ALK)-targeted therapy dramatically improves therapeutic responses in patients with ALK-rearranged lung adenocarcinoma (Ad-LC). A few cases of squamous cell lung carcinoma (Sq-LC) with ALK rearrangement have been reported; however, the clinicopathological features and clinical outcomes following treatment with ALK inhibitors are unknown. We addressed this in the present study by retrospectively comparing the clinical characteristics of five patients with ALK-rearranged Sq-LC with those of patients with ALK-rearranged Ad-LC and by evaluating representative cases of ALK inhibitor responders and non-responders. The prevalence of ALK rearrangement in Sq-LCs was 1.36%. Progression-free survival (PFS) after initial treatment with crizotinib was significantly shorter in Sq-LC than in Ad-LC with ALK rearrangement (p = 0.033). Two ALK rearrangements assayed by fluorescence in situ hybridization (FISH)-positive/immunohistochemistry-negative cases did not respond to crizotinb, and PFS decreased following alectinib treatment of ALK-rearranged Sq-LC (p = 0.045). A rebiopsy revealed that responders to ceritinib harbored the L1196M mutation, which causes resistance to other ALK inhibitors. However, non-responders were resistant to all ALK inhibitors, despite the presence of ALK rearrangement in FISH-positive circulating tumor cells and circulating free DNA and absence of the ALK inhibitor resistance mutation. These results indicate that ALK inhibitors remain a reasonable therapeutic option for ALK-rearranged Sq-LC patients who have worse outcomes than ALK-rearranged Ad-LC patients and that resistance mechanisms are heterogeneous. Additionally, oncologists should be aware of the possibility of ALK-rearranged Sq-LC based on clinicopathological features, and plan second-line therapeutic strategies based on rebiopsy results in order to improve patient outcome.
  • Yuki Katayama; Masashi Tachibana; Nozomi Kurisu; Yukako Oya; Yuichi Terasawa; Hiroshi Goda; Kouji Kobiyama; Ken J Ishii; Shizuo Akira; Hiroyuki Mizuguchi; Fuminori Sakurai
    Journal of immunology (Baltimore, Md. : 1950) 200 8 2987 - 2999 2018年04月 [査読有り]
     
    Oncolytic reovirus, which possesses 10 segments of dsRNA genome, mediates antitumor effects via not only virus replication in a tumor cell-specific manner, but also activation of antitumor immunity; however, the mechanism(s) of reovirus-induced activation of antitumor immunity have not been fully elucidated. Recent studies have demonstrated that overcoming an immunosuppressive environment in tumor-bearing hosts is important to achieve efficient activation of antitumor immunity. Among the various types of cells involved in immunosuppression, it has been revealed that myeloid-derived suppressor cells (MDSCs) are significantly increased in tumor-bearing hosts and play crucial roles in the immunosuppression in tumor-bearing hosts. In this study, we examined whether reovirus inhibits the immunosuppressive activity of MDSCs, resulting in efficient activation of immune cells after in vivo administration. The results showed that splenic MDSCs recovered from PBS-treated tumor-bearing mice significantly suppressed the Ag-specific proliferation of CD8+ T cells. In contrast, the suppressive activity of MDSCs on T cell proliferation was significantly reduced after reovirus administration. Reovirus also inhibited the immunosuppressive activity of MDSCs in IFN-β promoter stimulator-1 knockout (KO) mice and in wild-type mice. In contrast, the immunosuppressive activity of MDSCs in TLR-3 KO mice was not significantly altered by reovirus treatment. The activation levels of CD4+ and CD8+ T cells were significantly lower in TLR3 KO mice than in wild-type mice after reovirus administration. These results indicate that reovirus inhibits the immunosuppressive activity of MDSCs in a TLR3, but not IFN-β promoter stimulator-1, signaling-dependent manner.
  • Kazuo Takayama; Yasuko Hagihara; Yukiko Toba; Kiyotoshi Sekiguchi; Fuminori Sakurai; Hiroyuki Mizuguchi
    Biomaterials 161 24 - 32 2018年04月 [査読有り]
     
    Human iPS cell-derived hepatocyte-like cells are expected to be utilized in pharmaceutical research. However, the purity of high-functioning hepatocyte-like cells is not high enough. In particular, the purity of cytochrome P450 3A4 (CYP3A4), which is a representative hepatic drug-metabolizing enzyme, positive cells is still quite low (approximately 20%). To address this problem, we established the CYP3A4-NeoR-EGFP transgenic reporter human iPS cell line (CYP3A4-NeoR-EGFP iPS cells) by using genome editing technology. The CYP3A4-NeoR-EGFP iPS cells were differentiated into hepatocyte-like cells, and then the hepatocyte-like cells were treated with neomycin to concentrate the hepatocyte-like cells which strongly express CYP3A4. After the neomycin treatment, the percentage of CYP3A4-positive cells was higher than 80%. The gene expression levels of various drug-metabolizing enzymes, transporters, and hepatic transcription factors were significantly enhanced by neomycin treatment. In addition, the CYP1A2, 2C19, 2D6, and 3A4 activities and biliary excretion capacities were significantly increased by neomycin treatment. We also confirmed that the detection sensitivity of drug-inducing hepatotoxicity was enhanced by neomycin treatment. We succeeded in obtaining human iPS cell-derived hepatocyte-like cells that highly express CYP3A4 at high purity. We believe that our high-purity and high-functioning hepatocyte-like cells could be used to evaluate the risk of drug candidates.
  • Souichiro Nakamae; Yukiko Toba; Kazuo Takayama; Fuminori Sakurai; Hiroyuki Mizuguchi
    Stem cells and development 27 6 405 - 414 2018年03月 [査読有り]
     
    Human induced pluripotent stem cell-derived hepatocyte-like cells (HLCs) are expected to be utilized in pharmaceutical research, including drug screening. However, the hepatocyte functions of the HLCs are still lower than those of human hepatocytes. Therefore, we attempted to improve the hepatocyte differentiation method by modulating the DNA epigenetic status. We first examined the expression profiles of the maintenance DNA methyltransferase (DNMT) 1 and the de novo DNMTs DNMT3A and DNMT3B, all of which are essential for mammalian development. Among these DNMTs, the expression levels of DNMT3B were significantly decreased during the hepatoblast differentiation. To accelerate the hepatoblast differentiation, a DNMT3B-selective inhibitor, nanaomycin A, was treated during the hepatoblast differentiation. The gene expression levels of hepatoblast markers (such as alpha-fetoprotein and hepatocyte nuclear factor 4 alpha) were increased by the nanaomycin A treatment. On the other hand, the gene expression levels of hepatoblast markers were decreased by DNMT3B overexpression. These results suggest that it might be possible to promote the hepatoblast differentiation by DNMT3B inhibition using nanaomycin A. Importantly, we also confirmed that the hepatocyte differentiation potency of nanaomycin A-treated hepatoblast-like cells was higher than that of dimethyl sulfoxide-treated hepatoblast-like cells. Our findings should assist in the future generation of functional HLCs for pharmaceutical research.
  • Tomoki Yamashita; Kazuo Takayama; Fuminori Sakurai; Hiroyuki Mizuguchi
    Biochemical and biophysical research communications 496 4 1269 - 1275 2018年02月 [査読有り]
     
    Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells are expected to be utilized in drug screening and regenerative medicine. However, hepatocyte-like cells have not been fully used in such applications because it is difficult to produce such cells on a large scale. In this study, we tried to establish a method to mass produce hepatocyte-like cells using a three-dimensional (3D) cell culture bioreactor called the Rotary Cell Culture System (RCCS). RCCS enabled us to obtain homogenous hepatocyte-like cells on a billion scale (>109 cells). The gene expression levels of some hepatocyte markers (alpha-1 antitrypsin, cytochrome (CYP) 1A2, CYP2D6, and hepatocyte nuclear factor 4alpha) were higher in 3D-cultured hepatocyte-like cells than in 2D-cultured hepatocyte-like cells. This result suggests that RCCS could provide more suitable conditions for hepatocyte maturation than the conventional 2D cell culture conditions. In addition, more than 90% of hepatocyte-like cells were positive for albumin and could uptake low-density lipoprotein in the culture medium. We succeeded in the large-scale production of homogenous and functional hepatocyte-like cells from human iPS cells. This technology will be useful in drug screening and regenerative medicine, which require enormous numbers of hepatocyte-like cells.
  • Ryota Okamoto; Kazuo Takayama; Naoki Akita; Yasuhito Nagamoto; Daiki Hosokawa; Shunsuke Iizuka; Fuminori Sakurai; Hiroshi Suemizu; Kazuo Ohashi; Hiroyuki Mizuguchi
    Cell transplantation 27 2 299 - 309 2018年02月 [査読有り]
     
    Instead of liver transplantation or liver-directed gene therapy, genetic liver diseases are expected to be treated effectively using liver tissue engineering technology. Hepatocyte-like cells (HLCs) generated from human-induced pluripotent stem (iPS) cells are an attractive unlimited cell source for liver-like tissue engineering. In this study, we attempted to show the effectiveness of human iPS cell-based liver-like tissue engineering at an extrahepatic site for treatment of hemophilia B, also called factor IX (FIX) deficiency. HLCs were transplanted under the kidney capsule where the transplanted cells could be efficiently engrafted. Ten weeks after the transplantation, human albumin (253 μg/mL) and α-1 antitrypsin (1.2 μg/mL) could be detected in the serum of transplanted mice. HLCs were transplanted under the kidney capsule of FIX-deficient mice. The clotting activities in the transplanted mice were approximately 5% of those in wild-type mice. The bleeding time in transplanted mice was shorter than that in the nontransplanted mice. Taken together, these results indicate the success in generating functional liver-like tissues under the kidney capsule by using human iPS cell-derived HLCs. We also demonstrated that the human iPS cell-based liver-like tissue engineering technology would be an effective treatment of genetic liver disease including hemophilia B.
  • Masahiro Takakura; Takeo Matsumoto; Mitsuhiro Nakamura; Yasunari Mizumoto; Subaru Myojyo; Rena Yamazaki; Jyunpei Iwadare; Yukiko Bono; Shunsuke Orisaka; Takeshi Obata; Takashi Iizuka; Kyosuke Kagami; Kentaro Nakayama; Hideki Hayakawa; Fuminori Sakurai; Hiroyuki Mizuguchi; Yasuo Urata; Toshiyoshi Fujiwara; Satoru Kyo; Toshiyuki Sasagawa; Hiroshi Fujiwara
    Cancer science 109 1 231 - 240 2018年01月 [査読有り]
     
    Circulating tumor cells (CTC) are newly discovered biomarkers of cancers. Although many systems detect CTC, a gold standard has not yet been established. We analyzed CTC in uterine cervical cancer patients using an advanced version of conditionally replicative adenovirus targeting telomerase-positive cells, which was enabled to infect coxsackievirus-adenovirus receptor-negative cells and to reduce false-positive signals in myeloid cells. Blood samples from cervical cancer patients were hemolyzed and infected with the virus and then labeled with fluorescent anti-CD45 and anti-pan cytokeratin antibodies. GFP (+)/CD45 (-) cells were isolated and subjected to whole-genome amplification followed by polymerase chain reaction analysis of human papillomavirus (HPV) DNA. CTC were detected in 6 of 23 patients with cervical cancers (26.0%). Expression of CTC did not correlate with the stage of cancer or other clinicopathological factors. In 5 of the 6 CTC-positive cases, the same subtype of HPV DNA as that of the corresponding primary lesion was detected, indicating that the CTC originated from HPV-infected cancer cells. These CTC were all negative for cytokeratins. The CTC detected by our system were genetically confirmed. CTC derived from uterine cervical cancers had lost epithelial characteristics, indicating that epithelial marker-dependent systems do not have the capacity to detect these cells in cervical cancer patients.
  • Tomohito Tsukamoto; Eiko Sakai; Shunsuke Iizuka; Marcos Taracena-Gándara; Fuminori Sakurai; Hiroyuki Mizuguchi
    Biological & pharmaceutical bulletin 41 7 1089 - 1095 2018年 [査読有り]
     
    The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 system is now widely used as a genome editing tool. CRISPR-associated endonuclease in Prevotella and Francisella 1 (Cpf1) is a recently discovered Cas endonuclease that is designable and highly specific with efficiencies comparable to those of Cas9. Here we generated the adenovirus (Ad) vector carrying an Acidaminococcus sp. Cpf1 (AsCpf1) expression cassette (Ad-AsCpf1) for the first time. Ad-AsCpf1 was applied to primary human hepatocytes prepared from humanized mice with chimeric liver in combination with the Ad vector expressing the guide RNA (gRNA) directed to the Adeno-associated virus integration site 1 (AAVS1) region. The mutation rates were estimated by T7 endonuclease I assay around 12% of insertion/deletion (indel). Furthermore, the transduced human hepatocytes were viable (ca. 60%) at two weeks post transduction. These observations suggest that the Ad vector-mediated delivery of the CRISPR/AsCpf1 system provides a useful tool for genome manipulation of human hepatocytes.
  • Akikazu Asada; Hideki Hayakawa; Natsuki Yanase; Kanae Abe; Fuminori Sakurai; Hiroyuki Mizuguchi; Yasuo Urata
    Biological & pharmaceutical bulletin 41 10 1615 - 1619 2018年 [査読有り]
     
    In recent times, oncolytic viruses expressing an extraneous gene have attracted great interest; in fact, they have been engaged in multiple applications, such as medicine for cancer. Our group made an oncolytic adenovirus, namely, OBP-301, for use in treating solid cancers and press clinical trial to get approval for a pharmaceutical product. In this study, we applied a flow cytometry-based method to determine the titer of adenoviruses expressing an extraneous gene as well as assess their quality. We considered using the green fluorescent protein (GFP)50 titer as a measure of viral quality. The GFP50 titer (GFP50/mL) is the viral load required to render the HeLa S3 cell line 50% GFP-positive by analysing flow cytometry data. We measured the GFP50 titers for three types of recombinant adenoviruses (OBP-401, OBP-1101, and OBP-1106). We compared GFP50/mL and tissue culture infectious dose (TCID50/mL), a conventional titration index, and found that these titers showed a linear correlation, with a correlation coefficient of >0.9. Moreover, GFP50/mL showed high repetitive accuracy. We expect this flow cytometry-based method to be useful in case of clinically relevant viruses expressing an extraneous gene, in particular, to control viral quality.
  • Naosuke Morikawa; Masashi Tachibana; Yukio Ago; Hiroshi Goda; Fuminori Sakurai; Hiroyuki Mizuguchi
    Biological & pharmaceutical bulletin 41 12 1866 - 1869 2018年 [査読有り]
     
    Myeloid-derived suppressor cells (MDSCs) are immunosuppressive myeloid cells found in patients with cancer and in mouse tumor models. They suppress anti-tumor immunity, resulting in the promotion of tumor growth. The relationship between nutrition and cancer has recently been reported by several research groups. Tumor cells rely on glutaminolysis, in which glutamine is metabolized into glutamate for energy production, and hence, glutamate levels are elevated in tumor-bearing hosts. However, the mechanism of regulation of tumor progression by glutamate still remains unclear. In this study, we found that the metabotropic glutamate receptor (mGluR) 2/3 was expressed on MDSCs, and an mGluR2/3 antagonist LY341495 attenuated the immunosuppressive activity of MDSCs. Furthermore, we observed that LY341495 treatment inhibited B16-F10 melanoma growth in vivo. Taken together, our data suggest that glutamate signaling promotes tumor growth by increasing the potency of immune suppression.
  • Machitani M; Sakurai F; Wakabayashi K; Nakatani K; Tachibana M; Kato N; Fujiwara T; Mizuguchi H
    Molecular therapy oncolytics 7 76 - 85 2017年12月 [査読有り]
     
    Telomerase-specific replication-competent adenoviruses (Ads), i.e., TRADs, which possess an E1 gene expression cassette driven by the human telomerase reverse transcriptase promoter, are promising agents for cancer treatment. However, even though oncolytic Ads, including TRAD, are intratumorally administered, they are disseminated from the tumor to systemic circulation, causing concern about oncolytic Ad-mediated hepatotoxicity (due mainly to leaky expression of Ad genes in liver). We reported that inhibition of nuclear factor-κB (NF-κB) leads to the suppression of replication-incompetent Ad vector-mediated hepatotoxicity via reduction of the leaky expression of Ad genes in liver. Here, to develop a TRAD with an improved safety profile, we designed a TRAD that carries a liver-specific promoter-driven dominant-negative IκBα (DNIκBα) expression cassette (TRAD-DNIκBα). Compared with a conventional TRAD, TRAD-DNIκBα showed hepatocyte-specific inhibition of NF-κB signaling and significantly reduced Ad gene expression and replication in the normal human hepatocyte cell line. TRAD-induced hepatotoxicity was largely suppressed in mice following intravenous administration of TRAD-DNIκBα. However, the replication profiles and oncolytic activities of TRAD-DNIκBα were comparable with those of the conventional TRAD in human non-hepatic tumor cells. These results indicate that oncolytic Ads containing the liver-specific DNIκBα expression cassette have improved safety profiles without inhibiting oncolytic activities.
  • Kazuo Takayama; Naoki Akita; Natsumi Mimura; Rina Akahira; Yukimasa Taniguchi; Makoto Ikeda; Fuminori Sakurai; Osamu Ohara; Tomohiro Morio; Kiyotoshi Sekiguchi; Hiroyuki Mizuguchi
    Hepatology communications 1 10 1058 - 1069 2017年12月 [査読有り]
     
    Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells are expected to be applied for regenerative medicine. In this study, we attempted to generate safe and therapeutically effective human iPS-HLCs for hepatocyte transplantation. First, human iPS-HLCs were generated from a human leukocyte antigen-homozygous donor on the assumption that the allogenic transplantation might be carried out. Highly efficient hepatocyte differentiation was performed under a feeder-free condition using human recombinant laminin 111, laminin 511, and type IV collagen. The percentage of asialoglycoprotein receptor 1-positive cells was greater than 80%, while the percentage of residual undifferentiated cells was approximately 0.003%. In addition, no teratoma formation was observed even at 16 weeks after human iPS-HLC transplantation. Furthermore, harmful genetic somatic single-nucleotide substitutions were not observed during the hepatocyte differentiation process. We also developed a cryopreservation protocol for hepatoblast-like cells without negatively affecting their hepatocyte differentiation potential by programming the freezing temperature. To evaluate the therapeutic potential of human iPS-HLCs, these cells (1 × 106 cells/mouse) were intrasplenically transplanted into acute liver injury mice treated with 3 mL/kg CCl4 only once and chronic liver injury mice treated with 0.6 mL/kg CCl4 twice weekly for 8 weeks. By human iPS-HLC transplantation, the survival rate of the acute liver injury mice was significantly increased and the liver fibrosis level of chronic liver injury mice was significantly decreased. Conclusion: We were able to generate safe and therapeutically effective human iPS-HLCs for hepatocyte transplantation. (Hepatology Communications 2017;1:1058-1069).
  • Masahisa Hemmi; Masashi Tachibana; Natsuki Fujimoto; Masaki Shoji; Fuminori Sakurai; Kouji Kobiyama; Ken J. Ishii; Shizuo Akira; Hiroyuki Mizuguchi
    FRONTIERS IN IMMUNOLOGY 8 1456  2017年11月 [査読有り]
     
    Few current vaccines can establish antigen (Ag)-specific immune responses in both mucosal and systemic compartments. Therefore, development of vaccines providing defense against diverse infectious agents in both compartments is of high priority in global health. Intramuscular vaccination of an adenovirus vector (Adv) has been shown to induce Ag-specific cytotoxic T lymphocytes (CTLs) in both systemic and gut-mucosal compartments. We previously found that type I interferon (IFN) signaling is required for induction of gut-mucosal, but not systemic, CTLs following vaccination; however, the molecular mechanism involving type I IFN signaling remains unknown. Here, we found that T helper 17 (Th17)-polarizing cytokine expression was down-regulated in the inguinal lymph nodes (iLNs) of Ifnar(2-/-) mice, resulting in the reduction of Ag-specific Th17 cells in the iLNs and gut mucosa of the mice. We also found that prior transfer of Th17 cells reversed the decrease in the number of Ag-specific gut-mucosal CTLs in Ifnar2(-/-) mice following Adv vaccination. Additionally, prior transfer of Th17 cells into wild-type mice enhanced the induction of Ag-specific CTLs in the gut mucosa, but not in systemic compartments, suggesting a gut mucosa-specific mechanism where Th17 cells regulate the magnitude of vaccine-elicited Ag-specific CTL responses. These data suggest that Th17 cells translate systemic type I IFN signaling into a gut-mucosal CTL response following vaccination, which could promote the development of promising Adv vaccines capable of establishing both systemic and gut-mucosal protective immunity.
  • Daiki Nakamori; Hiroki Akamine; Kazuo Takayama; Fuminori Sakurai; Hiroyuki Mizuguchi
    SCIENTIFIC REPORTS 7 1 16675  2017年11月 [査読有り]
     
    Recently, it has been reported that human hepatocyte-like cells can be generated from fibroblasts by direct reprogramming technology. However, the conversion efficiency of human induced hepatocytelike cells (hiHeps) is not high enough. In addition, comparative analysis with the existing models of hepatocytes, such as human iPS cell-derived hepatocyte-like cells and primary human hepatocytes, has not been sufficiently carried out. In this study, we screened hepatic transcription factors for efficient direct hepatic reprogramming and compared hepatic functions between hiHeps and other existing hepatocyte models. We found that human fibroblasts were efficiently converted into hiHeps by using a combination of ATF5, PROX1, FOXA2, FOXA3, and HNF4A (albumin+/alpha-1 antitrypsin+cells = 27%, asialoglycoprotein receptor 1+ cells = 22%). The CYP expression levels and CYP activities in hiHeps were higher than those in human iPS cell-derived hepatocyte-like cells, but lower than those in short-term (4 hr) cultured primary human hepatocytes and primary human hepatocytes collected immediately after thawing. These results suggested that functional hiHeps could be efficiently generated by ATF5, PROX1, FOXA2, FOXA3, and HNF4A transduction. We believe that hiHeps generated by our method will be useful for the drug-discovery activities such as hepatotoxicity screening and drug metabolism tests.
  • Fuminori Sakurai; Takemaru Kunito; Kazuo Takayama; Rina Hashimoto; Masashi Tachibana; Naoya Sakamoto; Takaji Wakita; Hiroyuki Mizuguchi
    VIRUS RESEARCH 242 7 - 15 2017年10月 [査読有り]
     
    Hepatitis C virus (HCV) infection is a major cause of liver-related morbidity and mortality. In order to develop effective remedies for hepatitis C, it is important to understand the HCV infection profile and host-HCV interaction. HCV-induced innate immune responses play a crucial role in spontaneous HCV clearance; however, HCV-induced innate immune responses have not been fully evaluated in hepatocytes, partly because there are few in vitro models of HCV-induced innate immunity. Recently, human induced pluripotent stem (iPS) cells have received much attention as an in vitro model of infection with various pathogens, including HCV. We previously established highly functional hepatocyte-like cells differentiated from human iPS cells (iPS-HLCs). Here, we examined the potential of iPS-HLCs as an in vitro HCV infection model, especially for evaluation of the relationship between HCV infection levels and HCV-induced innate immunity. Significant expressions of type I and III interferons (IFNs) and IFN-stimulated genes (ISGs) were induced following transfection with HCV genomic replicon RNA in iPS-HLCs. Following inoculation with the HCV JFH-1 strain in iPS-HLCs, peaks of HCV genome replication and HCV protein expression were observed on day 2, and then both the HCV genome and protein levels gradually declined, while the mRNA levels of type III IFNs and ISGs peaked at day 2 following inoculation. These results suggest that the HCV genome efficiently replicates in iPS-HLCs, resulting in HCV genome-induced up-regulation of IFNs and ISGs, and thereafter, HCV genome-induced up-regulation of IFNs and ISGs mediates a reduction in the HCV genome and protein levels in iPS-HLCs.
  • Shunsuke Iizuka; Fuminori Sakurai; Masashi Tachibana; Kazuo Ohashi; Hiroyuki Mizuguchi
    MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 6 183 - 193 2017年09月 [査読有り]
     
    Gene therapy during neonatal and infant stages is a promising approach for hemophilia B, a congenital disorder caused by deficiency of blood coagulation factor IX (FIX). An adenovirus (Ad) vector has high potential for use in neonatal or infant gene therapy for hemophilia B due to its superior transduction properties; however, leaky expression of Ad genes often reduces the transduction efficiencies by Ad protein-mediated tissue damage. Here, we used a novel Ad vector, Ad-E4122aT, which exhibits a reduction in the leaky expression of Ad genes in liver, in gene therapy studies for neonatal hemophilia B mice. Ad-E4-122aT exhibited significantly higher transduction efficiencies than a conventional Ad vector in neonatal mice. In neonatal hemophilia B mice, a single neonatal injection of Ad-E4-122aT expressing human FIX (hFIX) (Ad-E4-122aT-AHAFIX) maintained more than 6% of the normal plasma hFIX activity levels for approximately 100 days. Sequential administration of Ad-E4-122aT-AHAFIX resulted in more than 100% of the plasma hFIX activity levels for more than 100 days and rescued the bleeding phenotypes of hemophilia B mice. In addition, immunotolerance to hFIX was induced by Ad-E4-122aT-AHAFIX administration in neonatal hemophilia B mice. These results indicated that Ad-E4-122aT is a promising gene delivery vector for neonatal or infant gene therapy for hemophilia B.
  • Morifumi Hanawa; Kazuo Takayama; Fuminori Sakurai; Masashi Tachibana; Hiroyuki Mizuguchi
    STEM CELL REVIEWS AND REPORTS 13 4 542 - 551 2017年08月 [査読有り]
     
    Hepatocyte nuclear factor 4 alpha (HNF4 alpha) is a key transcription factor for liver development. Although HNF4 alpha is necessary for hepatoblast differentiation, the function of HNF4 alpha before the hepatoblast differentiation, such as in definitive endoderm differentiation, is not well known. In addition, it is known that there are nine HNF4 alpha isoforms, but the expression and function of each HNF4 alpha isoform during the definitive endoderm differentiation is also not clear. In this study, we examined the expression pattern of HNF4 alpha and its functions in the definitive endoderm differentiation from human induced pluripotent stem (iPS) cells. We found that the HNF4 alpha-1D isoform expression levels were significantly increased during the definitive endoderm differentiation, while the HNF4 alpha-1A isoform expression levels did not change. Therefore, we further examined the function of the HNF4 alpha-1D isoform in definitive endoderm differentiation. HNF4 alpha-1D overexpression or knockdown was found to promote or prevent the definitive endoderm differentiation, respectively. Interestingly, Lefty1 was directly regulated by HNF4 alpha-1D, and Lefty1 knockdown also prevented the definitive endoderm differentiation. These results suggest that HNF4 alpha-1D promotes definitive endoderm differentiation through the regulation of Lefty1. To our knowledge, this is the first report to clarify the expression pattern and function of HNF4 alpha during the definitive endoderm differentiation.
  • Eiko Sakai; Yusuke Miura; Emi Suzuki-Kouyama; Kengo Oka; Masashi Tachibana; Kenji Kawabata; Fuminori Sakurai; Hiroyuki Mizuguchi
    SCIENTIFIC REPORTS 7 1 5541  2017年07月 [査読有り]
     
    Angiogenesis, new vessel formation from pre-existing vessels, is a highly conserved event through vertebrates. However, the system for tuning angiogenesis by species-intrinsic factors is totally unknown. miR-1224 is a member of mammal-specific mirtrons, which were identified as non-canonical microRNAs. We found that the expression of miR-1224 was upregulated in capillary-like tube-forming human umbilical vein endothelial cells on Matrigel. Enforced expression of miR-1224 stimulated tube formation, whereas repression of endogenous miR-1224 inhibited formation. Enforced expression of miR-1224 enhanced VEGF signaling and repressed NOTCH signaling. The adaptor protein of clathrindependent endocytosis, epsin2, which has been shown to be a suppressor of angiogenesis, was a direct target of miR-1224. Knockdown of EPN2 stimulated tube formation, while overexpression of EPN2 repressed miR-1224-mediated stimulation. Our findings indicate that miR-1224 is a mammal specific modulator of angiogenesis.
  • Kahori Shimizu; Minako Okamoto; Tomoyuki Terada; Fuminori Sakurai; Hiroyuki Mizuguchi; Koji Tomita; Toru Nishinaka
    Biochemistry and biophysics reports 10 192 - 197 2017年07月 [査読有り]
     
    Japanese patients with type 2 diabetes mellitus present a different responsiveness in terms of insulin secretion to glucose and body mass index (BMI) from other populations. The genetic background that predisposes Japanese individuals to type 2 diabetes mellitus is under study. Recent genetic studies demonstrated that the locus mapped in macrophage erythroblast attacher (MAEA) increases the susceptibility to type 2 diabetes mellitus in East Asians, including Japanese individuals. MAEA encodes a protein that plays a role in erythroblast enucleation and in the normal differentiation of erythroid cells and macrophages. However, the contribution of MAEA to type 2 diabetes mellitus remains unknown. In this study, to overexpress MAEA in the mouse liver and primary mouse hepatocytes, we generated a MAEA-expressing adenovirus (Ad) vector using a novel Ad vector exhibiting significantly lower hepatotoxicity (Ad-MAEA). Blood glucose and insulin levels in Ad-MAEA-treated mice were comparable to those in control Ad-treated mice. Primary mouse hepatocytes transduced with Ad-MAEA showed lower levels of expression of gluconeogenesis genes than those transduced with the control Ad vector. Hepatocyte nuclear factor-4α (HNF-4α) mRNA expression in primary mouse hepatocytes was also suppressed by MAEA overexpression. These results suggest that MAEA overexpression attenuates hepatic gluconeogenesis, which could potentially lead to improvement of type 2 diabetes mellitus.
  • Mitsuhiro Machitani; Fuminori Sakurai; Keisaku Wakabayashi; Kosuke Nakatani; Masashi Tachibana; Hiroyuki Mizuguchi
    JOURNAL OF VIROLOGY 91 12 2017年06月 [査読有り]
     
    Recent studies have reported that host microRNAs (miRNAs) regulate infections by several types of viruses via various mechanisms and that inhibition of the miRNA processing factors enhances or prevents viral infection. However, it has not been clarified whether these effects of miRNAs extend to adenovirus (Ad) infection. Here we show that miR-27a and -b efficiently inhibit infection with an Ad via the downregulation of SNAP25 and TXN2, which are members of the SNARE proteins and the thioredoxin family, respectively. Approximately 80% reductions in Ad genomic copy number were found in cells transfected with miR-27a/b mimics, whereas there were approximately 2.5- to 5-fold larger copy numbers of the Ad genome following transfection with miR-27a/b inhibitors. Microarray gene expression analysis and in silico analysis demonstrated that SNAP25 and TXN2 are target genes of miR-27a/b. A reporter assay using plasmids containing the 3' untranslated regions of the SNAP25 and TXN2 genes showed that miR-27a/b directly suppressed SNAP25 and TXN2 expression through posttranscriptional gene silencing. Knockdown of SNAP25 led to a significant inhibition of Ad entry into cells. Knockdown of TXN2 induced cell cycle arrest at G(1) phase, leading to a reduction in Ad replication. In addition, overexpression of Ad-encoded small noncoding RNAs (VA-RNAs) restored the miR-27a/b-mediated reduction in infection level with a VA-RNA-lacking Ad mutant due to the VA-RNA-mediated inhibition of miR-27a/b expression. These results indicate that miR-27a and -b suppress SNAP25 and TXN2 expression via posttranscriptional gene silencing, leading to efficient suppression of Ad infection. IMPORTANCE Adenovirus (Ad) is widely used as a platform for replication-incompetent Ad vectors (Adv) and replication-competent oncolytic Ad (OAd) in gene therapy and virotherapy. Regulation of Ad infection is highly important for efficient gene therapies using both Adv and OAd. In this study, we demonstrate that miR-27a and -b, which are widely expressed in host cells, suppress SNAP25 and TXN2 expression through posttranscriptional gene silencing. Suppression of SNAP25 and TXN2 expression leads to inhibition of Ad entry into cells and to cell cycle arrest, respectively, leading to efficient suppression of Ad infection. Our findings provide important clues to the improvement of gene therapies using both Adv and OAd.
  • Seiji Mitani; Kazuo Takayama; Yasuhito Nagamoto; Kazuo Imagawa; Fuminori Sakurai; Masashi Tachibana; Ryo Sumazaki; Hiroyuki Mizuguchi
    MOLECULAR THERAPY 25 6 1420 - 1433 2017年06月 [査読有り]
     
    The function of hepatocytes largely depends on their position in the liver lobule. Although the method of differentiating hepatocytes from human pluripotent stem cells has been largely improved over the past decade, there remains no technique for generating hepatocyte-like cells (HLCs) with zone specific hepatic properties. In this study, we searched for the factors that promote acquisition of zone-specific properties of HLCs. Here, we identified that WNT7B and WNT8B secreted from hepatocytes and cholangiocytes play important roles in achieving perivenous zone-specific characteristics, such as the enhancement of glutamine secretion, citric acid cycle, cytochrome P450 (CYP) 1A2 metabolism, and CYP1A2 induction capacities. We also found that WNT inhibitory factor (WIF-1) secreted from cholangiocytes was necessary for achieving periportal zone-specific characteristics, such as the enhancement of urea secretion and gluconeogenesis capacities. Therefore, WNT signal modulators secreted from hepatocytes or cholangiocytes conferred zone specific hepatic properties onto HLCs.
  • Kazuo Takayama; Keisuke Igai; Yasuko Hagihara; Rina Hashimoto; Morifumi Hanawa; Tetsushi Sakuma; Masashi Tachibana; Fuminori Sakurai; Takashi Yamamoto; Hiroyuki Mizuguchi
    NUCLEIC ACIDS RESEARCH 45 9 5198 - 5207 2017年05月 [査読有り]
     
    Genome editing research of human ES/iPS cells has been accelerated by clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALEN) technologies. However, the efficiency of biallelic genetic engineering in transcriptionally inactive genes is still low, unlike that in transcriptionally active genes. To enhance the biallelic homologous recombination efficiency in human ES/iPS cells, we performed screenings of accessorial genes and compounds. We found that RAD51 overexpression and valproic acid treatment enhanced biallelic-targeting efficiency in human ES/iPS cells regardless of the transcriptional activity of the targeted locus. Importantly, RAD51 overexpression and valproic acid treatment synergistically increased the biallelic homologous recombination efficiency. Our findings would facilitate genome editing study using human ES/iPS cells.
  • Fuminori Sakurai; Shunsuke Inoue; Tadataka Kaminade; Takuma Hotani; Yuki Katayama; Eri Hosoyamada; Yuichi Terasawa; Masashi Tachibana; Hiroyuki Mizuguchi
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 524 1-2 238 - 247 2017年05月 [査読有り]
     
    Reovirus induces tumor cell death efficiently and specifically, and thus is currently undergoing clinical testing as an anticancer agent. In the intracellular trafficking of reovirus, proteolytic disassembly of reovirus capsid-proteins and subsequent penetration of viral particles into the cytosol are crucial steps. Cathepsins B and L are largely responsible for the proteolytic disassembly of reovirus. Reovirus efficiently lyses tumor cells exhibiting relatively high activities of cathepsins B and L, while tumor cells with low activities of cathepsins B and L are often refractory to reovirus, probably due to inefficient endo/lysosomal escape. In this study, in order to enhance the tumor cell-killing efficiencies of reovirus by promoting endo/lysosomal escape, especially in reovirus-resistant tumor cells, reovirus was complexed with a cationic liposome transfection reagent. Reovirus alone and reovirus-cationic liposome complex (reoplex) exhibited similar levels of tumor cell-killing efficiencies in reovirus-susceptible tumor cells, while reoplex mediated more than 30% higher levels of tumor cell-killing activities in reovirus-resistant tumor cells than reovirus alone. Reoplex-mediated tumor cell death was efficiently induced in the tumor cells pretreated with cathepsin inhibitors. The mRNA levels of interferon (IFN)-beta and apoptotic genes were significantly elevated following reoplex treatment. These results suggest that cationic liposomes efficiently promoted delivery of reovirus to the cytosol, leading to induction of apoptosis. (C) 2017 Elsevier B.V. All rights reserved.
  • Shinsaku Togo; Nobuyoshi Katagiri; Yukiko Namba; Miniwan Tulafu; Kumi Nagahama; Kotarou Kadoya; Kazuya Takamochi; Siaki Oh; Kenji Suzuki; Fuminori Sakurai; Hiroyuki Mizuguchi; Yasuo Urata; Kazuhisa Takahashi
    ONCOTARGET 8 21 34884 - 34895 2017年05月 [査読有り]
     
    Circulating tumor cells (CTCs) have a crucial role in the clinical outcome of cancer patients. Detection of non-small cell lung cancer (NSCLC) using an antibody against epithelial cell adhesion molecule (EpCAM) in captured CTCs has low sensitivity; the loss of epithelial markers leads to underestimation of CTCs with mesenchymal phenotype. We propose a new approach for detection of viable CTCs, including those with epithelial-mesenchymal transition status (EMT-CTCs), using the new telomerase-specific replication-selective adenovirus (OBP-1101), TelomeScan F35. Peripheral venous blood samples and clinicopathological data were collected from 123 NSCLC patients. The sensitivity of CTC detection was 69.1%, and for patients with stage I, II, III and IV, it was 59.6%, 40.0%, 85.7%, and 75.0%, respectively. Among the EMTCTC samples, 46% were vimentin positive and 39.0% of non-EMT-CTC samples were EpCAM positive. Patients testing positive for EMT-CTCs at baseline had poor response to chemotherapy (P = 0.025) and decreased progression-free survival (EMT-CTC positive vs. negative: 193 +/- 47 days vs. 388 +/- 47. days, P = 0.040) in comparison to those testing negative. TelomeScan F35 is a highly sensitive CTC detection system and will be a useful screening tool for early diagnosis of NSCLC patients. Mesenchymal-phenotype CTCs are crucial indicators of chemotherapeutic efficacy in NSCLC patients.
  • Fuminori Sakurai; Seiji Mitani; Tatsuro Yamamoto; Kazuo Takayama; Masashi Tachibana; Koichi Watashi; Takaji Wakita; Sayuki Iijima; Yasuhito Tanaka; Hiroyuki Mizuguchi
    SCIENTIFIC REPORTS 7 45698  2017年04月 [査読有り]
     
    In order to understand the life cycle of hepatitis B virus (HBV) and to develop efficient anti-HBV drugs, a useful in vitro cell culture system which allows HBV infection and recapitulates virus-host interactions is essential; however, pre-existing in vitro HBV infection models are often problematic. Here, we examined the potential of human induced-pluripotent stem (iPS) cell-derived hepatocyte-like cells (iPSHLCs) as an in vitro HBV infection model. Expression levels of several genes involved in HBV infection, including the sodium taurocholate cotransporting polypeptide (NTCP) gene, were gradually elevated as the differentiation status of human iPS cells proceeded to iPS-HLCs. The mRNA levels of these genes were comparable between primary human hepatocytes (PHHs) and iPS-HLCs. Following inoculation with HBV, we found significant production of HBV proteins and viral RNAs in iPS-HLCs. The three major forms of the HBV genome were detected in iPS-HLCs by Southern blotting analysis. Anti-HBV agents entecavir and Myrcludex-B, which are a nucleoside analogue reverse transcriptase inhibitor and a synthetic pre-S1 peptide, respectively, significantly inhibited HBV infection in iPS-HLCs. These data demonstrate that iPS-HLCs can be used as a promising in vitro HBV infection model.
  • Mitsuhiro Machitani; Fuminori Sakurai; Keisaku Wakabayashi; Kosuke Nakatani; Kazuo Takayama; Masashi Tachibana; Hiroyuki Mizuguhi
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 40 3 272 - 277 2017年03月 [査読有り]
     
    Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome engineering technology is a powerful tool for generation of cells and animals with engineered mutations in their genomes. In order to introduce the CRISPR/Cas9 system into target cells, nonviral and viral vectors are often used; however, such vectors trigger innate immune responses associated with production of type I interferons (IFNs). We have recently demonstrated that type I IFNs inhibit short-hairpin RNA-mediated gene silencing, which led us to hypothesize that type I IFNs may also inhibit CRISPR/Cas9-mediated genome mutagenesis. Here we investigated this hypothesis. A single-strand annealing assay using a reporter plasmid demonstrated that CRISPR/Cas9-mediated cleavage efficiencies of the target double -stranded DNA were significantly reduced by IFN alpha. A mismatch recognition nuclease-dependent genotyping assay also demonstrated that IFNa reduced insertion or deletion (indel) mutation levels by approximately half. Treatment with IFNa did not alter Cas9 protein expression levels, whereas the copy numbers of guide RNA (gRNA) were significantly reduced by IFNa stimulation. These results indicate that type I IFNs significantly reduce gRNA expression levels following introduction of the CRISPR/Cas9 system in the cells, leading to a reduction in the efficiencies of CRISPR/Cas9-mediated genome mutagenesis. Our findings provide important clues for the achievement of efficient genome engineering using the CRISPR/Cas9 system.
  • Mitsuhiro Machitani; Fuminori Sakurai; Keisaku Wakabayashi; Kosuke Takayama; Masashi Tachibana; Hiroyuki Mizuguchi
    MOLECULAR THERAPY-NUCLEIC ACIDS 6 173 - 182 2017年03月 [査読有り]
     
    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-alpha significantly inhibited shRNA-mediated knockdown of target genes, while it did not inhibit small interfering RNA (siRNA)-mediated knockdown. Following treatment with IFN-alpha, 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-alpha. These results indicate that type I IFNs inhibit shRNA-mediated RNAi via inhibition of dicermediated 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.
  • Kazuo Imagawa; Kazuo Takayama; Shigemi Isoyama; Ken Tanikawa; Masato Shinkai; Kazuo Harada; Masashi Tachibana; Fuminori Sakurai; Emiko Noguchi; Kazumasa Hirata; Masayoshi Kage; Kenji Kawabata; Ryo Sumazaki; Hiroyuki Mizuguchi
    Scientific Reports 7 41806  2017年02月 [査読有り]
     
    Bile salt export pump (BSEP) plays an important role in hepatic secretion of bile acids and its deficiency results in severe cholestasis and liver failure. Mutation of the ABCB11 gene encoding BSEP induces BSEP deficiency and progressive familial intrahepatic cholestasis type 2 (PFIC2). Because liver transplantation remains standard treatment for PFIC2, the development of a novel therapeutic option is desired. However, a well reproducible model, which is essential for the new drug development for PFIC2, has not been established. Therefore, we attempted to establish a PFIC2 model by using iPSC technology. Human iPSCs were generated from patients with BSEP-deficiency (BD-iPSC), and were differentiated into hepatocyte-like cells (HLCs). In the BD-iPSC derived HLCs (BD-HLCs), BSEP was not expressed on the cell surface and the biliary excretion capacity was significantly impaired. We also identified a novel mutation in the 5'-untranslated region of the ABCB11 gene that led to aberrant RNA splicing in BD-HLCs. Furthermore, to evaluate the drug efficacy, BD-HLCs were treated with 4-phenylbutyrate (4PBA). The membrane BSEP expression level and the biliary excretion capacity in BD-HLCs were rescued by 4PBA treatment. In summary, we succeeded in establishing a PFIC2 model, which may be useful for its pathophysiological analysis and drug development.
  • Takamasa Hirai; Yoshiaki Yamagishi; Naoya Koizumi; Miwa Nonaka; Rina Mochida; Kenta Shida; Tetsuya Nomura; Makiko Fujii; Fuminori Sakurai; Hiroyuki Mizuguchi; Yoshiteru Watanabe; Naoki Utoguchi
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 40 2 195 - 204 2017年02月 [査読有り]
     
    Cell-penetrating peptides (CPPs) have been highly anticipated as an efficient delivery system due to their ability to cross biological membranes and transport various cargoes into cells. In the present study, we have identified adenovirus-derived CPPs using various capsid-mutant adenovirus (Ad) vectors. First, we examined the endocytosis-inducing ability of these vectors. A fiber-shaft substituted Ad vector, Ad type 5 vector with the fiber shaft domain replaced by that derived from Ad type 35, induced the highest fluorescein isothiocyanate (FITC)-dextran uptake into a human liver cell line, HepG2 cells. In contrast, the FITC-dextran uptake in HepG2 cells was not significantly different between coxsackievirus and adenovirus receptor (CAR)-binding-ablated Ad vector, integrin-binding-ablated Ad vector or conventional Ad vector. Next, we produced a recombinant Ad type 35 shaft protein using the Escherichia coli recombinant system. The recombinant Ad type 35 shaft protein retained the ability for FITC-dextran uptake and efficient gene delivery by plasmid transfection reagent. Furthermore, we identified 26 C-terminal amino acids in the Ad type 35 shaft protein as the cell membrane binding domain. The 26 amino-acid peptides also have the potential to be internalized into cultured cells. The internalization ability of the peptide was dependent on degree and was inhibited by an actin polymerization inhibitor (Latrunculin B) and by a lipid raft formation inhibitor (methyl-beta-cyclodextrin). The results of the present study indicate that Ad type 35-derived peptides induce endocytosis in cultured cells and have the ability to cross biological membranes. This report is the first paper to identify Ad-derived CPPs.
  • Mitsuhiro Machitani; Fuminori Sakurai; Keisaku Wakabayashi; Masashi Tachibana; Toshiyoshi Fujiwara; Hiroyuki Mizuguchi
    MOLECULAR CANCER THERAPEUTICS 16 1 251 - 259 2017年01月 [査読有り]
     
    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 shorthairpin 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. (C) 2016 AACR.
  • Teppei Onishi; Hiroshi Tazawa; Yuuri Hashimoto; Makoto Takeuchi; Takeshi Otani; Shuji Nakamura; Fuminori Sakurai; Hiroyuki Mizuguchi; Hiroyuki Kishimoto; Yuzo Umeda; Yasuhiro Shirakawa; Yasuo Urata; Shunsuke Kagawa; Toshiyoshi Fujiwara
    Scientific Reports 6 38060  2016年11月 [査読有り]
     
    "Cell-in-cell" denotes an invasive phenotype in which one cell actively internalizes in another. The novel human T-cell line HOZOT, established from human umbilical cord blood, was shown to penetrate a variety of human cancer cells but not normal cells. Oncolytic viruses are emerging as biological therapies for human cancers; however, efficient viral delivery is limited by a lack of tumor-specific homing and presence of pre-existing or therapy-induced neutralizing antibodies. Here, we report a new, intriguing approach using HOZOT cells to transmit biologics such as oncolytic viruses into human cancer cells by cell-in-cell invasion. HOZOT cells were successfully loaded via human CD46 antigen with an attenuated adenovirus containing the fiber protein of adenovirus serotype 35 (OBP-401/F35), in which the telomerase promoter regulates viral replication. OBP-401/F35-loaded HOZOT cells were efficiently internalized into human cancer cells and exhibited tumor-specific killing by release of viruses, even in the presence of anti-viral neutralizing antibodies. Moreover, intraperitoneal administration of HOZOT cells loaded with OBP-401/F35 significantly suppressed peritoneally disseminated tumor growth in mice. This unique cell-in-cell property provides a platform for selective delivery of biologics into human cancer cells, which has important implications for the treatment of human cancers.
  • Mitsuhiro Machitani; Fuminori Sakurai; Keisaku Wakabayashi; Kyoko Tomita; Masashi Tachibana; Hiroyuki Mizuguchi
    SCIENTIFIC REPORTS 6 27598  2016年06月 [査読有り]
     
    In various organisms, including nematodes and plants, RNA interference (RNAi) is a defense system against virus infection; however, it is unclear whether RNAi functions as an antivirus system in mammalian cells. Rather, a number of DNA viruses, including herpesviruses, utilize post-transcriptional silencing systems for their survival. Here we show that Dicer efficiently suppresses the replication of adenovirus (Ad) via cleavage of Ad-encoding small RNAs (VA-RNAs), which efficiently promote Ad replication via the inhibition of eIF2 alpha phosphorylation, to viral microRNAs (mivaRNAs). The Dicer knockdown significantly increases the copy numbers of VA-RNAs, leading to the efficient inhibition of eIF2 alpha phosphorylation and the subsequent promotion of Ad replication. Conversely, overexpression of Dicer significantly inhibits Ad replication. Transfection with mivaRNA does not affect eIF2 alpha phosphorylation or Ad replication. These results indicate that Dicer-mediated processing of VA-RNAs leads to loss of activity of VA-RNAs for enhancement of Ad replication and that Dicer functions as a defence system against Ad in mammalian cells.
  • Yasuhito Nagamoto; Kazuo Takayama; Kazuo Ohashi; Ryota Okamoto; Fuminori Sakurai; Masashi Tachibana; Kenji Kawabata; Hiroyuki Mizuguchi
    JOURNAL OF HEPATOLOGY 64 5 1068 - 1075 2016年05月 [査読有り]
     
    Background & Aims: Hepatocyte transplantation is one of the most attractive approaches for the treatment of patients with liver failure. Because human induced pluripotent stem cell derived hepatocyte-like cells (iPS-HLCs) can be produced on a large scale and generated from a patient with liver failure, they are expected to be used for hepatocyte transplantation. However, when using conventional transplantation methods, i.e., intrasplenic or portal venous infusion, it is difficult to control the engraftment efficiency and avoid unexpected engraftment in other organs because the transplanted cells are delivered into blood circulation before their liver engraftment. Methods: In this study, to resolve these issues, we attempted to employ a cell sheet engineering technology for experimental hepatocyte transplantation. The human iPS-HLC sheets were attached onto the liver surfaces of mice with liver injury. Results: This method reduced unexpected engraftment in organs other than the liver compared to that by intrasplenic transplantation. Human albumin levels in the mice with human iPS-HLC sheets were significantly higher than those in the intrasplenically-transplanted mice, suggesting the high potential for cell engraftment of the sheet transplantation procedure. In addition, human iPS-HLC sheet transplantation successfully ameliorated lethal acute liver injury induced by the infusion of carbon tetrachloride (CCI4). Moreover, we found that the hepatocyte growth factor secreted from the human iPS-HLC sheet played an important role in rescuing of mice from acute hepatic failure. Conclusions: Human iPS-HLC sheet transplantation would be a useful and reliable therapeutic approach for a patient with severe liver diseases. (C) 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
  • Kazuo Takayama; Seiji Mitani; Yasuhito Nagamoto; Fuminori Sakurai; Masashi Tachibana; Yukimasa Taniguchi; Kiyotoshi Sekiguchi; Hiroyuki Mizuguchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 474 1 91 - 96 2016年05月 [査読有り]
     
    The drug discovery research for cholestatic liver diseases has been hampered by the lack of a well-established human cholangiocyte model. Functional cholangiocyte-like cells differentiated from human induced pluripotent stem (iPS) cells are expected to be a promising candidate for such research, but there remains no well-established method for differentiating cholangiocytes from human iPS cells. In this study, we searched for a suitable extracellular matrix to promote cholangiocyte differentiation from human iPS cells, and found that both laminin 411 and laminin 511 were suitable for this purpose. The gene expression levels of the cholangiocyte markers, aquaporin I (AQP1), SRY-box 9 (SOX9), cystic fibrosis transmembrane conductance regulator (CFTR), G protein-coupled bile acid receptor 1 (GPBAR1), Jagged 1 (JAG1), secretin receptor (SUR), and gamma-glutamyl transferase (GGTI) were increased by using laminin 411 or laminin 511 as a matrix. In addition, the percentage of AQP1-positive cells was increased from 61.8% to 92.5% by using laminin 411 or laminin 511. Furthermore, the diameter and number of cysts consisted of cholangiocyte-like cells were increased when using either matrix. We believe that the human iPS cell derived cholangiocyte-like cells, which were generated by using our differentiation technology, would be useful for the drug discovery research of cholestatic liver diseases. (C) 2016 Elsevier Inc. All rights reserved.
  • Ryosuke Negoro; Kazuo Takayama; Yasuhito Nagamoto; Fuminori Sakurai; Masashi Tachibana; Hiroyuki Mizuguchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 472 4 631 - 636 2016年04月 [査読有り]
     
    Many drugs have potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in small intestinal enterocytes. Therefore, a model that can accurately evaluate drug-mediated CYP3A4 induction is urgently needed. In this study, we overlaid Matrigel on the human induced pluripotent stem cells-derived enterocyte-like cells (hiPS-ELCs) to generate the mature hiPS-ELCs that could be applied to drug-mediated CYP3A4 induction test. By overlaying Matrigel in the maturation process of enterocyte-like cells, the gene expression levels of intestinal markers (VILLIN, sucrase-isomaltase, intestine-specific homeobox, caudal type homeobox 2, and intestinal fatty acid-binding protein) were enhanced suggesting that the enterocyte-like cells were maturated by Matrigel overlay. The percentage of VILLIN-positive cells in the hiPS-ELCs found to be approximately 55.6%. To examine the CYP3A4 induction potential, the hiPS-ELCs were treated with various drugs. Treatment with dexamethasone, phenobarbital, rifampicin, or alpha,25-dihydroxyvitamin D3 resulted in 5.8-fold, 13.4-fold, 9.8 fold, or 95.0-fold induction of CYP3A4 expression relative to that in the untreated controls, respectively. These results suggest that our hiPS-ELCs would be a useful model for CYP3A4 induction test. (C) 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
  • Sayaka Tsuzuki; Masashi Tachibana; Masahisa Hemmi; Tomoko Yamaguchi; Masaki Shoji; Fuminori Sakurai; Kouji Kobiyama; Kenji Kawabata; Ken J. Ishii; Shizuo Akira; Hiroyuki Mizuguchi
    INTERNATIONAL IMMUNOLOGY 28 3 105 - 115 2016年03月 [査読有り]
     
    Adenovirus vectors (Adv) elicit innate immune responses via several pattern-recognition receptors. Although it has been suggested that various Adv-induced mechanisms play important roles in the induction of innate immunity in vitro, the impacts of these mechanisms in vivo remain unclear. Viral nucleic acids elicit innate immune responses through the recognition of cytosolic nucleic acid sensors and transduce intracellular signals to TANK-binding kinase (TBK) 1. In this study, to determine the impacts of viral nucleic acids on innate immune responses in vivo, we administered transgene-expressing Adv to Tbk1-deficient mice. The systemic Adv administration failed to induce type I interferons (type I IFNs) in the spleen, but not the liver, of Tbk1-deficient mice, resulting in the increase of transgene-expressing cells in the spleen, but not the liver. Moreover, Adv failed to induce type I IFNs in the bone-marrow-derived dendritic cells, but not the mouse embryonic fibroblasts, from Tbk1-deficient mice in vitro. These results support the idea that Adv elicit innate immunity in immune cells and non-immune cells in a TBK1-dependent and TBK1-independent manner, respectively.
  • Machitani M; Sakurai F; Wakabayashi K; Nakatani K; Shimizu K; Tachibana M; Mizuguchi H
    Scientific Reports 6 19922 2016年01月 [査読有り]
     
    The replication-incompetent adenovirus (Ad) vector is one of the most promising vectors for gene therapy; however, systemic administration of Ad vectors results in severe hepatotoxicities, partly due to the leaky expression of Ad genes in the liver. Here we show that nuclear factor-kappa B (NF-kappa B) mediates the leaky expression of Ad genes from the Ad vector genome, and that the inhibition of NF-kappa B leads to the suppression of Ad gene expression and hepatotoxicities following transduction with Ad vectors. Activation of NF-kappa B by recombinant tumor necrosis factor (TNF)-alpha significantly enhanced the leaky expression of Ad genes. More than 50% suppression of the Ad gene expression was found by inhibitors of NF-kappa B signaling and siRNA-mediated knockdown of NF-kappa B. Similar results were found when cells were infected with wild-type Ad. Compared with a conventional Ad vector, an Ad vector expressing a dominant-negative I kappa B alpha (Adv-CADNI kappa B alpha), which is a negative regulator of NF-kappa B, mediated approximately 70% suppression of the leaky expression of Ad genes in the liver. Adv-CADNI kappa B alpha did not induce apparent hepatotoxicities. These results indicate that inhibition of NF-kappa B leads to suppression of Ad vector-mediated tissue damages via not only suppression of inflammatory responses but also reduction in the leaky expression of Ad genes.
  • Nakamori D; Takayama K; Nagamoto Y; Mitani S; Sakurai F; Tachibana M; Mizuguchi H
    Biochemical and biophysical research communications 469 3 424 - 429 2016年01月 [査読有り]
     
    Hepatocyte-like cells differentiated from human iPS cells (human iPS-HLCs) are expected to be utilized in drug development and research. However, recent hepatic characterization of human iPS-HLCs showed that these cells resemble fetal hepatocytes rather than adult hepatocytes. Therefore, in this study, we aimed to develop a method to enhance the hepatic function of human iPS-HLCs. Because the gene expression levels of the hepatic transcription factors (activating transcription factor 5 (ATF5), CCAAT/enhancer-binding protein alpha (c/EBP alpha), and prospero homeobox protein 1 (PROX1)) in adult liver were significantly higher than those in human iPS-HLCs and fetal liver, we expected that the hepatic functions of human iPS-HLCs could be enhanced by adenovirus (Ad) vector-mediated ATF5, c/EBPa, and PROX1 transduction. The gene expression levels of cytochrome P450 (CYP) 2C9, 2E1, alpha-1 antitrypsin, transthyretin, Na+/taurocholate cotransporting polypeptide, and uridine diphosphate glucuronosyl transferase 1A1 and protein expression levels of CYP2C9 and CYP2E1 were upregulated by ATF5, c/EBP alpha, and PROX1 transduction. These results suggest that the hepatic functions of the human iPS-HLCs could be enhanced by ATF5, c/EBP alpha, and PROX1 transduction. Our findings would be useful for the hepatic maturation of human iPS-HLCs. (C) 2015 The Authors. Published by Elsevier Inc.
  • Fuminori Sakurai; Nobuhiro Narii; Kyoko Tomita; Shinsaku Togo; Kazuhisa Takahashi; Mitsuhiro Machitani; Masashi Tachibana; Masaaki Ouchi; Nobuyoshi Katagiri; Yasuo Urata; Toshiyoshi Fujiwara; Hiroyuki Mizuguchi
    MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 3 16001  2016年 [査読有り]
     
    Circulating tumor cells (CTCs) are promising biomarkers in several cancers, and thus methods and apparatuses for their detection and quantification in the blood have been actively pursued. A novel CTC detection system using a green fluorescence protein (GFP)-expressing conditionally replicating adenovirus (Ad) (rAd-GFP) was recently developed; however, there is concern about the production of false-positive cells (GFP-positive normal blood cells) when using rAd-GFP, particularly at high titers. In addition, CTCs lacking or expressing low levels of coxsackievirus-adenovirus receptor (CAR) cannot be detected by rAd-GFP, because rAd-GFP is constructed based on Ad serotype 5, which recognizes CAR. In order to suppress the production of false-positive cells, sequences perfectly complementary to blood cell-specific microRNA, miR-142-3p, were incorporated into the 3'-untranslated region of the E1B and GFP genes. In addition, the fiber protein was replaced with that of Ad serotype 35, which recognizes human CD46, creating rAdF35-142T-GFP. rAdF35-142T-GFP efficiently labeled not only CAR-positive tumor cells but also CAR-negative tumor cells with GFP. The numbers of false-positive cells were dramatically lower for rAdF35-142T-GFP than for rAd-GFP. CTCs in the blood of cancer patients were detected by rAdF35-142T-GFP with a large reduction in false-positive cells.
  • Hiroyuki Hanayama; Kazuo Ohashi; Rie Utoh; Hirofumi Shimizu; Kazuya Ise; Fuminori Sakurai; Hiroyuki Mizuguchi; Hiroyuki Tsuchiya; Teruo Okano; Mitsukazu Gotoh
    Cell medicine 8 1-2 31 - 8 2015年12月 [査読有り]
     
    To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells.
  • NASH/NAFLD関連マイクロRNA miR-27bの脂質蓄積促進能を担う標的遺伝子の解析
    鈴木 瑠々香; 酒井 英子; 立花 雅史; 櫻井 文教; 結束 貴臣; 中島 淳; 和田 孝一郎; 水口 裕之
    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [1P1207] - [1P1207] (公社)日本生化学会 2015年12月
  • Tatsuya Ozawa; Kazuo Takayama; Ryota Okamoto; Ryosuke Negoro; Fuminori Sakurai; Masashi Tachibana; Kenji Kawabata; Hiroyuki Mizuguchi
    SCIENTIFIC REPORTS 5 16479  2015年11月 [査読有り]
     
    Enterocytes play an important role in drug absorption and metabolism. However, a widely used enterocyte model, Caco-2 cell, has difficulty in evaluating both drug absorption and metabolism because the expression levels of some drug absorption and metabolism-related genes in these cells differ largely from those of human enterocytes. Therefore, we decided to generate the enterocyte-like cells from human induced pluripotent stem (iPS) cells (hiPS-ELCs), which are applicable to drug absorption and metabolism studies. The efficiency of enterocyte differentiation from human iPS cells was significantly improved by using EGF, SB431542, and Wnt3A, and extending the differentiation period. The gene expression levels of cytochrome P450 3A4 (CYP3A4) and peptide transporter 1 in the hiPS-ELCs were higher than those in Caco-2 cells. In addition, CYP3A4 expression in the hiPS-ELCs was induced by treatment with 1, 25-dihydroxyvitamin D3 or rifampicin, which are known to induce CYP3A4 expression, indicating that the hiPS-ELCs have CYP3A4 induction potency. Moreover, the transendothelial electrical resistance (TEER) value of the hiPS-ELC monolayer was approximately 240 Omega*cm(2), suggesting that the hiPS-ELC monolayer could form a barrier. In conclusion, we succeeded in establishing an enterocyte model from human iPS cells which have potential to be applied for drug absorption and metabolism studies.
  • Hotani T; Tachibana M; Mizuguchi H; Sakurai F
    Biochemical and biophysical research communications 460 4 1041 - 1046 2015年05月 [査読有り]
     
    Reovirus has genomes consisting of 10-segmented double-stranded RNAs, and have received much attention as an oncolytic virus. A previous study reported that reovirus down-regulates hypoxiainducible factor 1 alpha (HIF-1 alpha) protein levels following infection in tumor cells, which contributes to the antitumor effects of reovirus; however, the mechanism remains to be elucidated. In this study, we examined which virus component was involved in reovirus-mediated down-regulation of HIF-1 alpha. Reovirus induced significant down-regulation of HIF-1 alpha protein levels in not only reovirus-permissive tumor cells but also reovirus resistant tumor cells. UV-inactivated reovirus also induced a reduction in HIF-1 alpha protein levels. These data indicate that reovirus induces HIF-1 alpha down-regulation independently of virus replication. Furthermore, transfection with not only reovirus genomes but also polyI:C efficiently induced HIF-1 alpha down-regulation in a manner similar to reovirus, indicating that doublestranded reovirus RNA genomes are a key component for HIF-1 alpha down-regulation. Reovirus-mediated HIF-1 alpha down-regulation was inhibited when tumor cells were pretreated with inhibitors of cathepsins B and L, which play a crucial role in endo-lysosomal escape of virions to the cytoplasm. These data suggest that endo-lysosomal escape of reovirus genome into the cytoplasm is crucial for HIF-1 alpha down-regulation; however, the retinoic acid-inducible gene-I (RIG-I) or interferon-beta promoter stimulator-1 (IPS-1), which are involved in reovirus genome-induced innate immunity in the cytoplasm, did not play a crucial role in reovirus-mediated HIF-1 alpha reduction. (C) 2015 Elsevier Inc. All rights reserved.
  • Y. Terasawa; T. Hotani; Y. Katayama; M. Tachibana; H. Mizuguchi; F. Sakurai
    CANCER GENE THERAPY 22 4 188 - 197 2015年04月 [査読有り]
     
    Reovirus has gained much attention as an anticancer agent; however, the mechanism of the tumor cell-specific replication of reovirus is not fully understood. Although Ras activation is known to be crucial for tumor cell-specific replication of reovirus, it remains controversial which cellular factors are required for the reovirus-mediated tumor cell killing. In this study, we systematically investigated which cellular factors determined the efficiencies of reovirus-mediated tumor cell killing in various human cultured cell lines. The efficiency of reovirus-mediated cell killing varied widely among the cell lines. Junction adhesion molecule-A, a reovirus receptor, was highly expressed in almost all cell lines examined. Ras activation levels were largely different between the cell lines; however, there were no apparent correlations among the reovirus-mediated cell killing efficiencies and Ras activation status. On the other hand, activity levels of the cysteine proteases cathepsins B and L, which are crucial for proteolytic disassembly of the outer capsid proteins of reovirus, showed a tendency to be correlated with the efficiency of reovirus-mediated cell killing. These results indicate that the activity of cathepsins B and L is the most suitable as a bionnarker for the efficacy of reovirus-mediated oncolysis among the factors examined in this study.
  • NAFLD/NASH関連マイクロRNA miR-27bの肝脂肪化促進能を担う新規標的遺伝子の探索
    今泉 務; 酒井 英子; 立花 雅史; 櫻井 文教; 結束 貴臣; 中島 淳; 和田 孝一郎; 水口 裕之
    日本薬学会年会要旨集 135年会 3 68 - 68 (公社)日本薬学会 2015年03月
  • Yasuhito Nagamoto; Kazuo Takayama; Katsuhisa Tashiro; Chise Tateno; Fuminori Sakurai; Masashi Tachibana; Kenji Kawabata; Kazuo Ikeda; Yasuhito Tanaka; Hiroyuki Mizuguchi
    Cell transplantation 24 6 1127 - 38 2015年 [査読有り]
     
    Human liver chimeric mice are expected to be applied for drug toxicity tests and human hepatitis virus research. Human induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs) are a highly attractive donor source for the generation of human liver chimeric mice because they can be produced on a large scale and established from an individual. Although these cells have been successfully used to generate human liver chimeric mice, there is still room for improvement in the repopulation efficiency. To enhance the repopulation efficacy, the human iPSC-HLCs were transduced with an adenovirus vector (Ad-FNK) expressing FNK, a hyperactive mutant gene from Bcl-xL, which was expected to inhibit apoptosis in the process of integration into liver parenchyma. We then transplanted Ad-FNK-transduced human iPSC-HLCs into urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice (FNK mice) and evaluated the repopulation efficacy. The antiapoptotic effects of the human iPSC-HLCs were enhanced by FNK overexpression in vitro. Human albumin levels in the transplanted mice were significantly increased by transplantation of Ad-FNK-transduced human iPSC-HLCs (about 24,000 ng/ml). Immunohistochemical analysis with an anti-human αAT antibody revealed greater repopulation efficacy in the livers of FNK mice than control mice. Interestingly, the expression levels of human hepatocyte-related genes in the human iPSC-HLCs of FNK mice were much higher than those in the human iPSC-HLCs before transplantation. We succeeded in improving the repopulation efficacy of human liver chimeric mice generated by transplanting the Ad-FNK-transduced human iPSC-HLCs into uPA/SCID mice. Our method using ectopic expression of FNK was useful for generating human chimeric mice with high chimerism.
  • Quantitative Analysis of Virus-associated RNAI Expression following Transduction with a Replication-incompetent Adenovirus Vector In Vitro and In Vivo.
    Wakabayashi K; Machitani M; Shimizu K; Tachibana M; Sakurai F; Mizuguchi H
    J. Mol. Genet. Med. 9 2 2015年 [査読有り]
  • Yasuhito Nagamoto; Kazuo Takayama; Katsuhisa Tashiro; Chise Tateno; Fuminori Sakurai; Masashi Tachibana; Kenji Kawabata; Kazuo Ikeda; Yasuhito Tanaka; Hiroyuki Mizuguchi
    CELL TRANSPLANTATION 24 6 1127 - 1138 2015年 [査読有り]
     
    Human liver chimeric mice are expected to be applied for drug toxicity tests and human hepatitis virus research. Human induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs) are a highly attractive donor source for the generation of human liver chimeric mice because they can be produced on a large scale and established from an individual. Although these cells have been successfully used to generate human liver chimeric mice, there is still room for improvement in the repopulation efficiency. To enhance the repopulation efficacy, the human iPSC-HLCs were transduced with an adenovirus vector (Ad-FNK) expressing FNK, a hyperactive mutant gene from Bcl-xL, which was expected to inhibit apoptosis in the process of integration into liver parenchyma. We then transplanted Ad-FNK-transduced human iPSC-HLCs into urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice (FNK mice) and evaluated the repopulation efficacy. The antiapoptotic effects of the human iPSC-HLCs were enhanced by FNK overexpression in vitro. Human albumin levels in the transplanted mice were significantly increased by transplantation of Ad-FNK-transduced human iPSC-HLCs (about 24,000 ng/ml). Immunohistochemical analysis with an anti-human alpha AT antibody revealed greater repopulation efficacy in the livers of FNK mice than control mice. Interestingly, the expression levels of human hepatocyte-related genes in the human iPSC-HLCs of FNK mice were much higher than those in the human iPSC-HLCs before transplantation. We succeeded in improving the repopulation efficacy of human liver chimeric mice generated by transplanting the Ad-FNK-transduced human iPSC-HLCs into uPA/SCID mice. Our method using ectopic expression of FNK was useful for generating human chimeric mice with high chimerism.
  • Yuki Katayama; Yuichi Terasawa; Masashi Tachibana; Hiroyuki Mizuguchi; Fuminori Sakurai
    BIOMED RESEARCH INTERNATIONAL 2015 468457  2015年 [査読有り]
     
    Oncolytic reovirus induces innate immune responses, which contribute to the antitumor activity of reovirus, following in vivo application. Reovirus-induced innate immune responses have been relatively well characterized in immune cells and mouse embryonic fibroblasts cells; however, the mechanisms and profiles of reovirus-induced innate immune responses in human tumor cells have not been well understood. In particular, differences in reovirus-induced innate immune responses between reovirus-susceptible and reovirus-refractory tumor cells remain unknown, although the intracellular trafficking of reovirus differs between these tumor cells. In this study, we examined reovirus-induced upregulation of interferon- (IFN-) beta and of the proapoptotic gene, Noxa, in reovirus-susceptible and -refractory tumor cells. IFN-beta and Noxa were significantly induced by reovirus via the IFN-beta promoter stimulator-1 (IPS-1) signaling in both types of tumor cells. Inhibition of cathepsins B and L, which are important for disassembly of reovirus outer capsid proteins and escape into cytoplasm, largely suppressed reovirus-induced upregulation of IFN-beta and Noxa expression in not only reovirus-susceptible but also reovirus-refractory tumor cells. These results indicated that in both reovirus-susceptible and reovirus-refractory tumor cells, disassembly of the outer capsid proteins by cathepsins and the escape into the cytoplasm were crucial steps for reovirus-induced innate immunity.
  • Shunsuke Iizuka; Fuminori Sakurai; Kahori Shimizu; Kazuo Ohashi; Shin-ichiro Nakamura; Masashi Tachibana; Hiroyuki Mizuguchi
    BIOMED RESEARCH INTERNATIONAL 2015 685374  2015年 [査読有り]
     
    In gene therapy for congenital disorders, treatments during neonate and infant stages are promising. Replication-incompetent adenovirus (Ad) vectors have been used in gene therapy studies of genetic disorders; however, the transduction properties of Ad vectors in neonates and infants have not been fully examined. Accordingly, this study examined the properties of Ad vector-mediated transduction in neonatal mice. A first-generation Ad vector containing a cytomegalovirus (CMV) promoter-driven luciferase expression cassette was administered to neonatal mice on the second day of life via retro-orbital sinus. The highest Ad vector genome copy numbers and transgene expression were found in the neonatal liver. The neonatal heart exhibited the second highest levels of transgene expression among the organs examined. There was an approximately 1500-fold difference in the transgene expression levels between the adult liver and heart, while the neonatal liver exhibited only an approximately 30-fold higher level of transgene expression than the neonatal heart. A liver-specific promoter for firefly luciferase expression conferred a more than 100-fold higher luciferase expression in the liver relative to the other organs. No apparent hepatotoxicity was observed in neonatal mice following Ad vector administration. These findings should provide valuable information for gene therapy using Ad vectors in neonates and infants.
  • Kazuo Takayama; Yuta Morisaki; Shuichi Kuno; Yasuhito Nagamoto; Kazuo Harada; Norihisa Furukawa; Manami Ohtaka; Ken Nishimura; Kazuo Imagawa; Fuminori Sakurai; Masashi Tachibana; Ryo Sumazaki; Emiko Noguchi; Mahito Nakanishi; Kazumasa Hirata; Kenji Kawabata; Hiroyuki Mizuguchi
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 111 47 16772 - 16777 2014年11月 [査読有り]
     
    Interindividual differences in hepatic metabolism, which are mainly due to genetic polymorphism in its gene, have a large influence on individual drug efficacy and adverse reaction. Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells have the potential to predict interindividual differences in drug metabolism capacity and drug response. However, it remains uncertain whether human iPSC-derived HLCs can reproduce the interindividual difference in hepatic metabolism and drug response. We found that cytochrome P450 (CYP) metabolism capacity and drug responsiveness of the primary human hepatocytes (PHH)-iPS-HLCs were highly correlated with those of PHHs, suggesting that the PHH-iPS-HLCs retained donor-specific CYP metabolism capacity and drug responsiveness. We also demonstrated that the interindividual differences, which are due to the diversity of individual SNPs in the CYP gene, could also be reproduced in PHH-iPS-HLCs. We succeeded in establishing, to our knowledge, the first PHH-iPS-HLC panel that reflects the interindividual differences of hepatic drug-metabolizing capacity and drug responsiveness.
  • Shuichi Kuno; Fuminori Sakurai; Kahori Shimizu; Naoya Matsumura; Soonih Kim; Hitoshi Watanabe; Katsuhisa Tashiro; Masashi Tachibana; Tsuyoshi Yokoi; Hiroyuki Mizuguchi
    DRUG METABOLISM AND PHARMACOKINETICS 29 4 296 - 304 2014年08月 [査読有り]
     
    Cytochrome P450 3A4 (CYP3A4) plays a crucial role in the pharmacokinetic and safety profiles of drugs. However, it is difficult to properly predict the pharmacokinetics and hepatotoxicity of drugs in humans using data from experimental animals, because the catalytic activities of CYP3A4 and other drug-metabolizing enzymes differ between human and animal organs. In order to easily generate an animal model for proper evaluation of human CYP3A4-mediated drug metabolism, we developed a human CYP3A4-expressing adenovirus (Ad) vector based on our novel Ad vector exhibiting significantly lower hepatotoxicity (Ad-E4-122aT-hCYP3A4). Intravenous administration of Ad-E4-122aT-hCYP3A4 at a dose of 2 x 1011 virus particles/mouse produced a mouse exhibiting human CYP3A4 activity at a level similar to that in the human liver, as shown in the dexamethasone metabolic experiment using liver microsomes. The area under the curve (AUC) of 6 beta OHD was 2.7-fold higher in the Ad-E4-122aT-hCYP3A4-administered mice, compared with the mice receiving a control Ad vector. This Ad vector-expressing human CYP3A4 would thus be a powerful tool for evaluating human CYP3A4-mediated drug metabolism in the livers of experimental animals.
  • F. Sakurai; Y. Nanjo; S. Okamoto; M. Tachibanal; H. Mizuguchi
    CANCER GENE THERAPY 21 4 164 - 170 2014年04月 [査読有り]
     
    Recent studies have demonstrated that small double-stranded RNAs (dsRNAs) complementary to the promoter region of target genes enhance the expression of those genes following transfection into cells. Here we show that expression of the matrix metalloproteinase (MMP) inhibitor RECK is activated in the cultured tumor cell lines by transfection with dsRNA complementary to the promoter of the RECK gene, leading to suppression of the expression of MMPs and it inhibited tumor cell invasion. These results support the suggestion that dsRNA complementary to the promoter region of tumor suppressor genes would have potential as a novel antitumor agent.
  • Hitoshi Watanabe; Kazuo Takayama; Mitsuru Inamura; Masashi Tachibana; Natsumi Mimura; Kazufumi Katayama; Katsuhisa Tashiro; Yasuhito Nagamoto; Fuminori Sakurai; Kenji Kawabata; Miho Kusuda Furue; Hiroyuki Mizuguchi
    PLOS ONE 9 3 e90791  2014年03月 [査読有り]
     
    Human embryonic stem cells (hESCs) could provide a major window into human developmental biology, because the differentiation methods from hESCs mimic human embryogenesis. We previously reported that the overexpression of hematopoietically expressed homeobox (HHEX) in the hESC-derived definitive endoderm (DE) cells markedly promotes hepatic specification. However, it remains unclear how HHEX functions in this process. To reveal the molecular mechanisms of hepatic specification by HHEX, we tried to identify the genes directly targeted by HHEX. We found that HHEX knockdown considerably enhanced the expression level of eomesodermin (EOMES). In addition, HHEX bound to the HHEX response element located in the first intron of EOMES. Loss-of-function assays of EOMES showed that the gene expression levels of hepatoblast markers were significantly upregulated, suggesting that EOMES has a negative role in hepatic specification from the DE cells. Furthermore, EOMES exerts its effects downstream of HHEX in hepatic specification from the DE cells. In conclusion, the present results suggest that HHEX promotes hepatic specification by repressing EOMES expression.
  • Takayama K; Kawabata K; Nagamoto Y; Inamura M; Ohashi K; Okuno H; Yamaguchi T; Tashiro K; Sakurai F; Hayakawa T; Okano T; Furue MK; Mizuguchi H
    Development (Cambridge, England) 141 1 91 - 100 2014年01月 [査読有り]
     
    Human embryonic stem cells (hESCs) and their derivatives are expected to be used in drug discovery, regenerative medicine and the study of human embryogenesis. Because hepatocyte differentiation from hESCs has the potential to recapitulate human liver development in vivo, we employed this differentiation method to investigate the molecular mechanisms underlying human hepatocyte differentiation. A previous study has shown that a gradient of transforming growth factor beta (TGF beta) signaling is required to segregate hepatocyte and cholangiocyte lineages from hepatoblasts. Although CCAAT/enhancer binding proteins (c/EBPs) are known to be important transcription factors in liver development, the relationship between TGF beta signaling and c/EBP-mediated transcriptional regulation in the hepatoblast fate decision is not well known. To clarify this relationship, we examined whether c/EBPs could determine the hepatoblast fate decision via regulation of TGF beta receptor 2 (TGFBR2) expression in the hepatoblast-like cells differentiated from hESCs. We found that TGFBR2 promoter activity was negatively regulated by c/EBP alpha and positively regulated by c/EBP beta. Moreover, c/EBP alpha overexpression could promote hepatocyte differentiation by suppressing TGFBR2 expression, whereas c/EBP beta overexpression could promote cholangiocyte differentiation by enhancing TGFBR2 expression. Our findings demonstrated that c/EBP alpha and c/EBP beta determine the lineage commitment of hepatoblasts by negatively and positively regulating the expression of a common target gene, TGFBR2, respectively.
  • Kahori Shimizu; Fuminori Sakurai; Kyoko Tomita; Yasuhito Nagamoto; Shin-ichiro Nakamura; Kazufumi Katayama; Masashi Tachibana; Kenji Kawabata; Hiroyuki Mizuguchi
    MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 1 14035  2014年 [査読有り]
     
    Leaky expression of adenovirus (Ad) genes occurs following transduction with a conventional replication-incompetent Ad -vector, leading to an induction of cellular immunity against Ad proteins and Ad protein-induced toxicity, especially in the late phase -following administration. To suppress the leaky expression of Ad genes, we developed novel Ad vectors by incorporating four tandem copies of sequences with perfect complementarity to miR-122a or miR-142-3p into the 3'-untranslated region (UTR) of the E2A, E4, or pIX gene, which were mainly expressed from the Ad vector genome after transduction. These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional 293 cells. The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2-to 100-fold, compared with a conventional Ad vector, by insertion of the miRNA-targeted sequences. Notably, the Ad vector carrying the miR-122a-targeted sequences into the 3'-UTR of the E4 gene expressed higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver than a conventional Ad vector. miR-122a-mediated suppression of the E4 gene expression in the liver significantly reduced the hepatotoxicity which an Ad vector causes via both adaptive and non-adaptive immune responses.
  • Masahisa Hemmi; Masashi Tachibana; Sayaka Tsuzuki; Masaki Shoji; Fuminori Sakurai; Kenji Kawabata; Kouji Kobiyama; Ken J. Ishii; Shizuo Akira; Hiroyuki Mizuguchi
    BIOMED RESEARCH INTERNATIONAL 2014 158128  2014年 [査読有り]
     
    Few of the vaccines in current use can induce antigen-(Ag-) specific immunity in both mucosal and systemic compartments. Hence, the development of vaccines that realize both mucosal and systemic protection against various pathogens is a high priority in global health. Recently, it has been reported that intramuscular (i.m.) vaccination of an adenovirus vector (Adv) can induce Ag-specific cytotoxic T lymphocytes (CTLs) in both systemic and gut mucosal compartments. We previously revealed that type I IFN signaling is required for the induction of gut mucosal CTLs, not systemic CTLs. However, the molecular mechanism via type I IFN signaling is largely unknown. Here, we report that type I IFN signaling following i.m. Adv vaccination is required for the expression of type I IFN in the inguinal lymph nodes (iLNs), which are the draining lymph nodes of the administration site. We also showed that the type I IFN signaling is indispensable for the early activation of CTLs in iLNs. These data suggested that type I IFN signaling has an important role in the translation of systemic innate immune response into mucosal adaptive immunity by amplifying the innate immune signaling and activating CTLs in the iLN.
  • M. Machitani; F. Sakurai; K. Katayama; M. Tachibana; T. Suzuki; H. Matsui; T. Yamaguchi; H. Mizuguchi
    VIRUS RESEARCH 178 2 357 - 363 2013年12月 [査読有り]
     
    Several studies have reported that short hairpin RNA (shRNA)-mediated RNA interference (RNAi) was competitively inhibited by the expression of adenovirus (Ad)-encoded small RNAs (VA-RNAs), which are expressed from a replication-incompetent Ad vector, as well as a wild-type Ad; however, it remained to be clarified whether an shRNA-expressing Ad vector-mediated knockdown was inhibited by VA-RNAs transcribed from the same Ad vector genome. In this study, we demonstrated that a lack of VA-RNA expression from the Ad vector leads to an increase in knockdown efficiencies of Ad vector-mediated RNAi. In the cells transduced with a first-generation Ad vector (FG-Ad) expressing shRNA (FG-Ad-shRNA), the copy numbers of shRNA and VA-RNAs incorporated into the RNA-induced silencing complex (RISC) was comparable. In contrast, higher amounts of shRNA were found in the RISC when the cells were transduced with an shRNA-expressing helper-dependent Ad (HD-Ad) vector, in which all viral genes, including VA-RNAs, were deleted (HD-Ad-shRNA), compared with FG-Ad-shRNA. HD-Ad vectors expressing shRNA against luciferase and p53 showed 7.4% and 37.3% increases in the knockdown efficiencies compared to the corresponding FG-Ad-shRNA, respectively, following in vitro transduction. Furthermore, higher levels of knockdown efficiencies were also found by the transduction with shRNA-expressing Ad vectors lacking VA-RNA expression (Ad Delta VR-shRNA) than by transduction with FG-Ad-shRNA. These results indicate that VA-RNAs expressed from an Ad vector inhibit knockdown by the shRNA-expressing Ad vector and that HD-Ad-shRNA and Ad Delta VR-shRNA are a powerful framework for shRNA-mediated knockdown. (C) 2013 H. Mizuguchi. Published by Elsevier B.V. All rights reserved.
  • Kazuo Takayama; Yasuhito Nagamoto; Natsumi Mimura; Katsuhisa Tashiro; Fuminori Sakurai; Masashi Tachibana; Takao Hayakawa; Kenji Kawabata; Hiroyuki Mizuguchi
    STEM CELL REPORTS 1 4 322 - 335 2013年10月 [査読有り]
     
    The establishment of self-renewing hepatoblast-like cells (HBCs) from human pluripotent stem cells (PSCs) would realize a stable supply of hepatocyte-like cells for medical applications. However, the functional characterization of human PSC-derived HBCs was not enough. To purify and expand human PSC-derived HBCs, human PSC-derived HBCs were cultured on dishes coated with various types of human recombinant laminins (LN). Human PSC-derived HBCs attached to human laminin-111 (LN111)-coated dish via integrin alpha 6 and beta 1 and were purified and expanded by culturing on the LN111-coated dish, but not by culturing on dishes coated with other laminin isoforms. By culturing on the LN111-coated dish, human PSC-derived HBCs were maintained for more than 3 months and had the ability to differentiate into both hepatocyte-like cells and cholangiocyte-like cells. These expandable human PSC-derived HBCs would be manageable tools for drug screening, experimental platforms to elucidate mechanisms of hepatoblasts, and cell sources for hepatic regenerative therapy.
  • Hanayama Hiroyuki; Ohashi Kazuo; Utoh Rie; Ise Kazuya; Shimizu Tatsuya; Yamato Masayuki; Mizuguchi Hiroyuki; Sakurai Fuminori; Okano Teruo; Gotoh Mitsukazu
    TRANSPLANTATION 96 6 S22  2013年09月 [査読有り]
  • Hayato Matsui; Fuminori Sakurai; Kazufumi Katayama; Yasuhiro Abe; Mitsuhiro Machitani; Shinnosuke Kurachi; Masashi Tachibana; Hiroyuki Mizuguchi
    Biomaterials 34 16 4191 - 4201 2013年05月 [査読有り]
     
    A major drawback of adenovirus (Ad) vectors is their nonspecific transduction into various types of cells or tissue after in vivo application, which might lead to unexpected toxicity and tissue damage. To overcome this problem, we developed a fiber-mutant Ad vector displaying a monobody specific for epidermal growth factor receptor (EGFR) or vascular endothelial growth factor receptor 2 (VEGFR2) in the C-terminus of the knobless fiber protein derived from T4 phage fibritin. A monobody, which is a single domain antibody mimic based on the tenth human fibronectin type III domain scaffold with a structure similar to the variable domains of antibodies, would be suitable as a targeting molecule for display on the Ad capsid proteins because of its highly stable structure even under reducing conditions and low molecular weight (approximately 10 kDa). Surface plasmon resonance (SPR) analysis revealed that the monobody-displaying Ad vector specifically bound to the targeted molecules, leading to significant increases in cellular binding and transduction efficiencies in the targeted cells. Transduction with the monobody-displaying Ad vectors was significantly inhibited in the presence of the Fc-chimera protein of EGFR and VEGFR2. This monobody-displaying Ad vector would be a crucial resource for targeted gene therapy.
  • Kazuo Takayama; Kenji Kawabata; Yasuhito Nagamoto; Keisuke Kishimoto; Katsuhisa Tashiro; Fuminori Sakurai; Masashi Tachibana; Katsuhiro Kanda; Takao Hayakawa; Miho Kusuda Furue; Hiroyuki Mizuguchi
    Biomaterials 34 7 1781 - 9 2013年02月 [査読有り]
     
    Although it is expected that hepatocyte-like cells differentiated from human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells will be utilized in drug toxicity testing, the actual applicability of hepatocyte-like cells in this context has not been well examined so far. To generate mature hepatocyte-like cells that would be applicable for drug toxicity testing, we established a hepatocyte differentiation method that employs not only stage-specific transient overexpression of hepatocyte-related transcription factors but also a three-dimensional spheroid culture system using a Nanopillar Plate. We succeeded in establishing protocol that could generate more matured hepatocyte-like cells than our previous protocol. In addition, our hepatocyte-like cells could sensitively predict drug-induced hepatotoxicity, including reactive metabolite-mediated toxicity. In conclusion, our hepatocyte-like cells differentiated from human ES cells or iPS cells have potential to be applied in drug toxicity testing.
  • 櫻井 文教; 近藤 昌夫; Aono H.
    藥學雜誌 133 3 289 - 289 公益社団法人 日本薬学会 2013年 [査読有り]
  • Fuminori Sakurai; Toshiyoshi Fujiwara; Hiroyuki Mizuguchi
    Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan 133 3 291 - 6 2013年 [査読有り]
     
    An easy and sensitive detection method for circulating tumor cells (CTC) is expected to be developed because CTC are a promising biomarker for early diagnosis of tumors and prognosis prediction of tumor patients. Our group has already developed a CTC detection method using a conditionally replicating adenovirus (Ad) which efficiently replicates and expresses GFP in telomerase-positive tumor cells. However, malignant tumor cells express much low levels of coxsackievirus and adenovirus receptor (CAR), leading to inefficient infection with a conventional conditionally replicating Ad. In addition, a tiny fraction of normal blood cells, including lymphocytes, express GFP following infection. To overcome these problems, we have developed a next-generation conditionally replicating Ad. The next-generation conditionally replicating Ad possesses the fiber protein derived from Ad serotype 35, leading to efficient infection in both CAR-positive and -negative tumor cells, because the fiber protein of Ad serotype 35 binds to CD46, which is expressed on almost all human cells. Furthermore, sequences complementary to blood cell-specific miRNA (miR-142-3p) were inserted into the 3' untranslated region of the E1 gene and GFP gene, leading to the suppression of GFP expression in normal blood cells. In this symposium, we will not only introduce the importance of CTC as a biomarker and conventional CTC detection methods but also show our data of the novel CTC detection using the next-generation conditionally replicating Ad.
  • Katsuhisa Tashiro; Miyuki Omori; Kenji Kawabata; Nobue Hirata; Tomoko Yamaguchi; Fuminori Sakurai; Satoshi Takaki; Hiroyuki Mizuguchi
    STEM CELLS AND DEVELOPMENT 21 18 3381 - 3390 2012年12月 [査読有り]
     
    Embryonic stem (ES) cell-and induced pluripotent stem (iPS) cell-derived hematopoietic stem/progenitor cells (HSPCs) are considered as an unlimited source for HSPC transplantation. However, production of immature hematopoietic cells, especially HSPCs, from ES and iPS cells has been challenging. The adaptor protein Lnk has been shown to negatively regulate HSPC function via the inhibition of thrombopoietin (TPO) and stem cell factor signaling, and Lnk-deficient HSPCs show an enhanced self-renewal and repopulation capacity. In this study, we examined the role of Lnk on the hematopoietic differentiation from mouse ES and iPS cells by the inhibition of Lnk using a dominant-negative mutant of the Lnk (DN-Lnk) gene. We generated mouse ES and iPS cells stably expressing a DN-Lnk, and found that enforced expression of a DN-Lnk in ES and iPS cells led to an enhanced generation of Flk-1-positive mesodermal cells, thereby could increase in the expression of hematopoietic transcription factors, including Scl and Runx1. We also showed that the number of both total hematopoietic cells and immature hematopoietic cells with colony-forming potential in DN-Lnk-expressing cells was significantly increased in comparison with that in control cells. Furthermore, Lnk inhibition by the overexpression of the DNLnk gene augmented the TPO-induced phosphorylation of Erk1/2 and Akt, indicating the enhanced sensitivity to TPO. Adenovirus vector-mediated transient DN-Lnk gene expression in ES and iPS cells could also increase the hematopoietic cell production. Our data clearly showed that the inhibition of Lnk in ES and iPS cells could result in the efficient generation and expansion of hematopoietic cells.
  • David Bennett; Fuminori Sakurai; Kahori Shimizu; Hayato Matsui; Kyoko Tomita; Takayuki Suzuki; Kazufumi Katayama; Kenji Kawabata; Hiroyuki Mizuguchi
    MOLECULAR PHARMACEUTICS 9 12 3452 - 3463 2012年12月 [査読有り]
     
    In order to detarget undesirable transduction in the liver by an adenovirus (Ad) vector, we previously demonstrated that insertion of sequences perfectly complementary to liver-specific miR-122a into the 3'-untranslated region (UTR) of transgene specifically reduced the transgene expression in the liver by approximately 100-fold; however, a certain level of residual transgene expression was still found in the liver. In order to further suppress the hepatic transduction, we developed a two-Ad vector system that uses the microRNA (miRNA)-regulated transgene expression system and the Cre-loxP recombination system, i.e., insertion of miR-122a target sequences and loxP sites into the transgene expression cassette and coadministration of a Cre recombinase-expressing Ad vector. In addition, to maintain as much as possible the transgene expression in the spleen, which is the target organ of this study, spleen-specific miR-142-3p target sequences were inserted into the 3'-UTR of the Cre recombinase gene to suppress Cre recombinase expression in the spleen. The spleen is an attractive target for immunotherapy because the spleen plays important roles in the immune system. Coadministration of Ad vector possessing CMV promoter-driven Cre recombinase expression cassette with miR-142-3p target sequences resulted in a further 24-fold reduction in the hepatic transgene expression by the Ad vector containing miR-122a target sequences and loxP sites, compared with coadministration of control Ad vector. On the other hand, there was no significant reduction of transgene expression in the spleen.
  • Kahori Shimizu; Fuminori Sakurai; Masashi Tachibana; Hiroyuki Mizuguchi
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 132 12 1407 - 1412 2012年12月 [査読有り]
     
    Replication-incompetent adenovirus (Ad) vectors are widely used in gene therapy studies because they are beneficial as a gene delivery vehicle enabling high-titer production and highly efficient gene transfer into a wide spectrum of dividing and non-dividing cells in vitro and in vivo. Theoretically, Ad genes should not be expressed following transduction with a replication-incompetent Ad vector. However, leaky expression of viral genes is known to occur following transduction with a conventional Ad vector, which leads to a cellular immunity against Ad proteins as well as Ad protein-induced toxicity. Such Ad protein-induced cellular immunity and toxicity frequently cause both an elimination of Ad vector-transduced cells and tissue damage, leading to short-lived transgene expression. To date, no detailed analysis of the leaky expression profile of Ad genes has been performed. First, we systematically examined the expression profiles of Ad genes in cells using real-time RT-PCR following transduction with a conventional Ad vector. The results revealed that significant expression was found for E2A, E4, and pIX genes. Next, in order to suppress the leaky expression of Ad genes, complementary sequences for microRNA (miRNA) were inserted into the 3'-untranslated region of the E2A, E4, or pIX genes. miRNAs are an approximately 22-nt length non-coding RNA, and bind to imperfectly complementary sequences in the 3'-untranslated region of target mRNA, leading to suppression of gene expression via post-transcriptional regulation. Incorporation of the miRNA-targeted sequences significantly suppressed the leaky expression of Ad genes in an miRNA-dependent manner.
  • Masaki Shoji; Kazufumi Katayama; Masashi Tachibana; Kyoko Tomita; Fuminori Sakurai; Kenji Kawabata; Hiroyuki Mizuguchi
    VACCINE 30 50 7278 - 7285 2012年11月 [査読有り]
     
    Mucosal delivery of antigens induces antigen-specific immune responses in both systemic and mucosal compartments, and is an attractive approach for preventing initial infection with mucosal pathogens. It has been shown that the intramuscular (i.m.) immunization of plasmid DNA by in vivo electroporation (DNA e.p.) induces both cellular and humoral immune responses in the airway-mucosal compartment as well as in the systemic compartment, implying there is a mechanism that bridges between the systemic and mucosal immune responses. An important question is whether the i.m. DNA e.p.-immunization alone can induce antigen-specific immune responses in the gut-mucosal compartment. Here, we investigated the induction of antigen-specific CD8(+) T cells and antibodies in both systemic and gut-mucosal compartments following i.m. DNA e.p.-immunization to mice. Surprisingly, the i.m. DNA e.p.-immunization induced the antigen-specific CD8(+) T cells and antigen-specific antibodies in the gut-mucosal as well as the systemic compartment. These results suggest that the i.m. DNA e.p.-immunization should be considered as an effective vaccine strategy for the prevention of gut-mucosal infectious diseases. (C) 2012 Elsevier Ltd. All rights reserved.
  • Takayama K; Inamura M; Kawabata K; Sugawara M; Kikuchi K; Higuchi M; Nagamoto Y; Watanabe H; Tashiro K; Sakurai F; Hayakawa T; Furue MK; Mizuguchi H
    Journal of hepatology 57 3 628 - 636 2012年09月 [査読有り]
     
    Background & Aims: Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can be utilized as a tool for screening for hepatotoxicity in the early phase of pharmaceutical development. We have recently reported that hepatic differentiation is promoted by sequential transduction of SOX17, HEX, and HNF4 alpha into hESC- or hiPSC-derived cells, but further maturation of hepatocyte-like cells is required for widespread use of drug screening. Methods: To screen for hepatic differentiation-promoting factors, we tested the seven candidate genes related to liver development. Results: The combination of two transcription factors, FOXA2 and HNF1 alpha, promoted efficient hepatic differentiation from hESCs and hiPSCs. The expression profile of hepatocyte-related genes (such as genes encoding cytochrome P450 enzymes, conjugating enzymes, hepatic transporters, and hepatic nuclear receptors) achieved with FOXA2 and HNF1 alpha transduction was comparable to that obtained in primary human hepatocytes. The hepatocyte-like cells generated by FOXA2 and HNF1 alpha transduction exerted various hepatocyte functions including albumin and urea secretion, and the uptake of indocyanine green and low density lipoprotein. Moreover, these cells had the capacity to metabolize all nine tested drugs and were successfully employed to evaluate drug-induced cytotoxicity. Conclusions: Our method employing the transduction of FOXA2 and HNF1 alpha represents a useful tool for the efficient generation of metabolically functional hepatocytes from hESCs and hiPSCs, and the screening of drug-induced cytotoxicity. (C) 2012 European Association for the Study of the Liver. Published by Elsevier B. V. All rights reserved.
  • Masaki Shoji; Masashi Tachibana; Kazufumi Katayama; Kyoko Tomita; Sayaka Tsuzuki; Fuminori Sakurai; Kenji Kawabata; Ken J. Ishii; Shizuo Akira; Hiroyuki Mizuguchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 425 1 89 - 93 2012年08月 [査読有り]
     
    Adenovirus vector (Adv) vaccination at a systemic site, such as intramuscular (i.m.) immunization, can induce antigen-specific CD8(+) T cell responses in both systemic and mucosal compartments. It remains unclear, however, how antigen-specific CD8(+) T cell response is induced in the mucosa. In this study, we found that type-I IFN signaling is required for the induction of mRNA expression of retinal dehydrogenase in the draining lymph nodes following the i.m. Adv vaccination. We show that type-I IFN signaling is required for the induction of antigen-specific CD8(+) T cell response in the gut-mucosal compartment following the i.m. Adv vaccination. (C) 2012 Elsevier Inc. All rights reserved.
  • Yasuhito Nagamoto; Katsuhisa Tashiro; Kazuo Takayama; Kazuo Ohashi; Kenji Kawabata; Fuminori Sakurai; Masashi Tachibana; Takao Hayakawa; Miho Kusuda Furue; Hiroyuki Mizuguchi
    BIOMATERIALS 33 18 4526 - 4534 2012年06月 [査読有り]
     
    Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are known to be a useful cell source for drug screening. We recently developed an efficient hepatic differentiation method from hESCs and hiPSCs by sequential transduction of FOXA2 and HNF1 alpha. It is known that the combination of three-dimensional (3D) culture and co-culture, namely 3D co-culture, can maintain the functions of primary hepatocytes. However, hepatic maturation of hESC- or hiPSC-derived hepatocyte-like cells (hEHs or hiPHs, respectively) by 3D co-culture systems has not been examined. Therefore, we utilized a cell sheet engineering technology to promote hepatic maturation. The gene expression levels of hepatocyte-related markers (such as cytochrome P450 enzymes and conjugating enzymes) and the amount of albumin secretion in the hEHs or hiPHs, which were 3D co-cultured with the Swiss 3T3 cell sheet, were significantly up-regulated in comparison with those in the hEHs or hiPHs cultured in a monolayer. Furthermore, we found that type I collagen synthesized in Swiss 3T3 cells plays an important role in hepatic maturation. The hEHs or hiPHs that were 3D co-cultured with the Swiss 3T3 cell sheet would be powerful tools for medical applications, such as drug screening. (C) 2012 Elsevier Ltd. All rights reserved.
  • Fuminori Sakurai; Norihisa Furukawa; Maiko Higuchi; Sayuri Okamoto; Kaori Ono; Takeshi Yoshida; Masuo Kondoh; Kiyohito Yagi; Naoya Sakamoto; Kazufumi Katayama; Hiroyuki Mizuguchi
    VIRUS RESEARCH 165 2 214 - 218 2012年05月 [査読有り]
     
    Recent studies have demonstrated that the liver-specific microRNA (miRNA) miR-122a plays an important role in the replication of hepatitis C virus (HCV). Antisense nucleotides against miR-122a, including locked nucleic acid (LNA), have shown promising results for suppression of HCV replication; however, a liver-specific delivery system of antisense nucleotides has not been fully developed. In this study, an adenovirus (Ad) vector that expresses tough decoy (TuD)-RNA against miR-122a (TuD-122a) was developed to suppress the HCV replication in the liver hepatocytes. Ad vectors have been well established to exhibit a marked hepatotropism following systemic administration. An in vitro reporter gene expression assay demonstrated that Ad vector-mediated expression of TuD-122a efficiently blocked the miR-122a in Huh-7 cells. Furthermore, transduction with the Ad vector expressing TuD-122a in HCV replicon-expressing cells resulted in significant reduction in the HCV replicon levels. These results indicate that Ad vector-mediated expression of TuD-122a would be a promising tool for treatment of HCV infection. (C) 2012 Elsevier B.V. All rights reserved.
  • Hayato Matsui; Fuminori Sakurai; Kazufumi Katayama; Tomoko Yamaguchi; Sayuri Okamoto; Kohdai Takahira; Masashi Tachibana; Shinsaku Nakagawa; Hiroyuki Mizuguchi
    BIOMATERIALS 33 14 3743 - 3755 2012年05月 [査読有り]
     
    We previously developed a hexon-specific PEGylated adenovirus (Ad) vector by utilizing avidin-biotin interaction. However, the Ad vector was aggregated due to the multiple interactions between avidin and biotin, resulting in a reduction in the transduction efficiencies in the organs following systemic administration. In this study, we developed a new method for hexon-specific PEGylation by mixing Ad vectors with PEGylated blood coagulation factor X (FX) (PEG-FX). FX specifically binds to the hexon protein, suggesting that FX serves as an adaptor molecule for hexon-specific modification. Intravenous administration of the PEG-FX-associated Ad (PEG-FX-Ad) vector into conventional mice resulted in prolonged blood retention. However, the transduction efficiencies in the liver were not reduced by PEG-FX. On the other hand, in the warfarinized mice, the PEG-FX-Ad vectors exhibited a significant reduction in the liver transduction. In addition, incubation of the PEG-FX-Ad vector with unmodified FX resulted in dissociation of PEG-FX from the Ad vector, indicating that a substitution of PEG-FX with endogenous FX occurs in the blood following administration. This study demonstrates that FX can be used as an adaptor molecule for hexon-specific modification; however, modified FX might be substituted with endogenous FX in the blood. (C) 2012 Elsevier Ltd. All rights reserved.
  • Kyoko Tomita; Fuminori Sakurai; Masashi Tachibana; Hiroyuki Mizuguchi
    ANTICANCER RESEARCH 32 4 1145 - 1152 2012年04月 [査読有り]
     
    Pre-existing anti-adenovirus neutralizing antibodies (AdNAbs) are a major barrier in clinical gene therapy using adenovirus vectors; however, the transduction profile of adenovirus vectors in the presence of AdNAbs following intratumoral injection has not been fully examined, although such vectors are often intratumorally injected in clinical studies. In this study, we evaluated the correlation between the titer of AdNAbs in the serum and the transduction profiles in the tumor and the liver following intratumoral administration into mice possessing various titers of AdNAbs. Adeno virus vector-mediated transduction in the tumor was inhibited by AdNAbs; however, when the titer of AdNAbs was less than 200, the levels of inhibition in the transduction efficiencies within the tumor ranged from approximately 2- to 100-fold. A more than 2500-fold reduction of adenovirus vector-mediated transduction was found in most of the mice when the titers of AdNAbs were >200. On the other hand, the transduction efficiencies in the liver were largely reduced almost to the levels of the mock-transduced mice even at the low titers of AdNAbs. These results provide crucial information for the clinical use of adenovirus vectors.
  • Katsuhisa Tashiro; Kenji Kawabata; Miyuki Omori; Tomoko Yamaguchi; Fuminori Sakurai; Kazufumi Katayama; Takao Hayakawa; Hiroyuki Mizuguchi
    STEM CELL RESEARCH 8 2 300 - 311 2012年03月 [査読有り]
     
    Ectopic expression of HoxB4 in embryonic stem (ES) cells leads to an efficient production of hematopoietic cells, including hematopoietic stem/progenitor cells. Previous studies have utilized a constitutive HoxB4 expression system or tetracycline-regulated HoxB4 expression system to induce hematopoietic cells from ES cells. However, these methods cannot be applied therapeutically due to the risk of transgenes being integrated into the host genome. Here, we report the promotion of hematopoietic differentiation from mouse ES cells and induced pluripotent stem (iPS) cells by transient HoxB4 expression using an adenovirus (Ad) vector. Ad vector could mediate efficient HoxB4 expression in ES cell-derived embryoid bodies (ES-EBs) and iPS-EBs, and its expression was decreased during cultivation, showing that Ad vector transduction was transient. A colony-forming assay revealed that the number of hematopoietic progenitor cells with colony-forming potential in HoxB4-transduced cells was significantly increased in comparison with that in non-transduced cells or LacZ-transduced cells. HoxB4-transduced cells also showed more efficient generation of CD41-, CD45-, or Sca-1-positive cells than control cells. These results indicate that transient, but not constitutive, HoxB4 expression is sufficient to augment the hematopoietic differentiation of ES and iPS cells, and that our method would be useful for clinical applications, such as cell transplantation therapy. (C) 2011 Elsevier B.V. All rights reserved.
  • K. Iguchi; F. Sakurai; K. Tomita; K. Katayama; T. Yamaguchi; K. Kawabata; M. Tagawa; M. Kawabata; T. Shirakawa; H. Mizuguchi
    CANCER GENE THERAPY 19 2 118 - 125 2012年02月 [査読有り]
     
    Carrier cells delivering a conditionally replicating adenovirus (CRAd), which selectively replicates in tumor cells and induces tumor cell lysis, have promising potential for treatment of cancer because CRAd-loaded carrier cells evade inhibition by neutralizing anti-adenovirus (Ad) antibodies and because the carrier cells are locally retained at the injection point after local injection. A previous study by Hamada et al. demonstrated that carrier cells (CRAd-containing cell fragments derived from the carrier cells) are engulfed into the target cells, probably through a pathway independent of the primary receptor for Ad, the coxsackievirus and Ad receptor (CAR) (Mol Ther, 15: 1121-1128; 2007); however, it remains to be elucidated whether carrier cells infected with a conventional CRAd, which is composed of subgroup-C Ad serotype-5 (Ad5), mediate antitumor effects on CAR-negative cells. In order to examine whether carrier cells delivering a conventional CRAd (Carrier-F5) induce lysis of CAR-negative tumor cells, CAR-positive and CAR-negative tumor cells were incubated with Carrier-F5. Carrier-F5 mediated efficient killing of CAR-positive tumor cells; however, CAR-negative tumor cells were almost refractory to Carrier-F5. On the other hand, carrier cells loaded with a fiber-substituted CRAd containing fiber proteins of Ad serotype-35 (Ad35) (CRAd-F35), which binds to human CD46 for infection, showed efficient killing of both CAR-positive and CAR-negative tumor cells. Intra-tumoral injection of carrier cells loaded with CRAd-F35 (Carrier-F35) also resulted in efficient regression of both CAR-positive and CAR-negative tumors. These results demonstrated that the expression levels of receptors for Ad are an important factor for CRAd-loaded carrier cell-mediated cancer therapy, and that Carrier-F35 would have potential as a cancer treatment for not only CAR-positive tumors but also CAR-negative tumors. Cancer Gene Therapy (2012) 19, 118-125; doi: 10.1038/cgt.2011.74; published online 11 November 2011
  • 高山 和雄; 稲村 充; 川端 健二; 菅原 道子; 菊池 きよ美; 櫻井 文教; 古江(楠田) 美保; 水口 裕之
    日本毒性学会学術年会 39 P - 10 日本毒性学会 2012年 
    【目的】肝臓は多くの薬物を代謝する臓器であり、創薬過程における候補薬物の肝毒性を正確に予測することが、安全な医薬品開発には重要である。無限増殖能と多分化能を有するヒト胚性幹細胞（ES細胞）およびヒト人工多能性幹細胞（ヒトiPS細胞）から分化誘導した肝細胞は、創薬過程における候補薬物の毒性評価などへの応用が期待されている。我々はこれまでに、SOX17、HEX、HNF4α遺伝子を分化過程の適切な時期に導入することにより、ヒトES/iPS細胞から成熟肝細胞を効率良く分化誘導できることを報告した（Mol Ther. 2012 Jan;20(1): 127-37）。ヒトES/iPS細胞由来の肝細胞を薬物の毒性評価へ応用するには、ヒト初代培養肝細胞と同程度の薬剤代謝能を有している必要がある。そこで本研究では、非常に高い遺伝子導入効率を示すアデノウイルスベクターを用いて肝関連転写因子を遺伝子導入し、さらに高い薬剤代謝能を有する肝細胞の作製を試みた。【方法】ヒトES/iPS細胞から肝細胞の分化過程において７種類の肝関連転写因子を導入し、最も効率良く肝分化を促進できる転写因子を探索した。分化誘導肝細胞の薬剤代謝能をヒト初代培養肝細胞と比較し、さらに肝臓で毒性を示すベンゾブロマロンなどの薬剤を分化誘導肝細胞に作用させた後の細胞毒性についても検討した。【結果・考察】検討した７種類の肝関連転写因子のうち、FOXA2およびHNF1α遺伝子を組み合わせて導入することにより、最も効率良く成熟肝細胞が分化誘導された。またシトクロムP450酵素などで代謝される9種類の薬物の代謝プロファイルを調べたところ、分化誘導肝細胞の薬物代謝能はヒト初代培養肝細胞より低いものの、いずれの薬物に対しても代謝能を有していることが確認された。さらに、分化誘導肝細胞は肝毒性を示す薬剤に対してヒト初代培養肝細胞と同様に細胞毒性を呈した。以上のことから、FOXA2およびHNF1α遺伝子を導入することにより、ヒトES/iPS細胞から薬物代謝能を有する肝細胞を効率良く分化誘導できるだけでなく、薬物の毒性スクリーニングに使用可能であることが示唆された。
  • Takayama K; Inamura M; Kawabata K; Katayama K; Higuchi M; Tashiro K; Nonaka A; Sakurai F; Hayakawa T; Furue MK; Mizuguchi H
    Molecular therapy : the journal of the American Society of Gene Therapy 20 1 127 - 137 2012年01月 [査読有り]
     
    Hepatocyte-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are expected to be a useful source of cells drug discovery. Although we recently reported that hepatic commitment is promoted by transduction of SOX17 and HEX into human ESC- and iPSC-derived cells, these hepatocyte-like cells were not sufficiently mature for drug screening. To promote hepatic maturation, we utilized transduction of the hepatocyte nuclear factor 4 alpha (HNF4 alpha) gene, which is known as a master regulator of liver-specific gene expression. Adenovirus vector-mediated overexpression of HNF4 alpha in hepatoblasts induced by SOX17 and HEX transduction led to upregulation of epithelial and mature hepatic markers such as cytochrome P450 (CYP) enzymes, and promoted hepatic maturation by activating the mesenchymal-to-epithelial transition (MET). Thus HNF4 alpha might play an important role in the hepatic differentiation from human ESC-derived hepatoblasts by activating the MET. Furthermore, the hepatocyte like-cells could catalyze the toxication of several compounds. Our method would be a valuable tool for the efficient generation of functional hepatocytes derived from human ESCs and iPSCs, and the hepatocyte-like cells could be used for predicting drug toxicity.
  • Takeshi Yoshida; Kazuo Takayama; Masuo Kondoh; Fuminori Sakurai; Hideki Tani; Naoya Sakamoto; Yoshiharu Matsuura; Hiroyuki Mizuguchi; Kiyohito Yagi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 416 1-2 119 - 124 2011年12月 [査読有り]
     
    Host tropism of hepatitis C virus (HCV) is limited to human and chimpanzee. HCV infection has never been fully understood because there are few conventional models for HCV infection. Human induced pluripotent stem cell-derived hepatocyte-like (iPS-Hep) cells have been expected to use for drug discovery to predict therapeutic activities and side effects of compounds during the drug discovery process. However, the suitability of iPS-Hep cells as an experimental model for HCV research is not known. Here, we investigated the entry and genomic replication of HCV in iPS-Hep cells by using HCV pseudotype virus (HCVpv) and HCV subgenomic replicons, respectively. We showed that iPS-Hep cells, but not iPS cells, were susceptible to infection with HCVpv. The iPS-Hep cells expressed HCV receptors, including CD81, scavenger receptor class B type I (SR-BI), claudin-1, and occludin; in contrast, the iPS cells showed no expression of SR-BI or claudin-1. HCV RNA genome replication occurred in the iPS-Hep cells. Anti-CD81 antibody, an inhibitor of HCV entry, and interferon, an inhibitor of HCV genomic replication, dose-dependently attenuated HCVpv entry and HCV subgenomic replication in iPS-Hep cells, respectively. These findings suggest that iPS-Hep cells are an appropriate model for HCV infection. (C)2011 Elsevier Inc. All rights reserved.
  • Mitsuhiro Machitani; Kazufumi Katayama; Fuminori Sakurai; Hayato Matsui; Tomoko Yamaguchi; Takayuki Suzuki; Hiroyuki Miyoshi; Kenji Kawabata; Hiroyuki Mizuguchi
    JOURNAL OF CONTROLLED RELEASE 154 3 285 - 289 2011年09月 [査読有り]
     
    A major limitation of the use of adenovirus (Ad) vectors is the innate immune response, which causes inflammatory cytokine production and tissue damages. To overcome this limitation, it is necessary to develop safer Ad vectors that are less likely to induce innate immunity. The Ad genome encodes two non-coding small RNAs, virus-associated (VA)-RNA I and VA-RNA II, which are transcribed by RNA polymerase III and promote Ad replication. Recently, we reported that VA-RNAs are produced in the cells transduced with a conventional first-generation (El-deleted) Ad vector (FG-Ad) and trigger innate immune responses through intracellular nucleic acid sensors. In the present study, we have developed a VA-RNA-deleted Ad (Ad Delta VR) vector, in which the transcriptional control elements of the VA-RNA-expression were deleted. Although conventional HEK293 cells did not support the propagation of the Ad Delta VR vectors, HEK293 transformants inducibly expressing VA-RNA I (VR293 cells) with appropriate induction of VA-RNA I expression allowed the propagation of the Ad Delta VR vector. The Ad Delta VR vector showed high transduction efficiency comparable to that of the conventional FG-Ad vector in the cultured cells. The Ad Delta VR vector may be a safer alternative to the FG-Ad vector. (C) 2011 Elsevier B.V. All rights reserved.
  • Dongbo Yu; Fuminori Sakurai; David R. Corey
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 21 18 5202 - 5205 2011年09月 [査読有り]
     
    Rett Syndrome is an X-linked progressive neurological disorder caused by inactivation of one allele of the MECP2 gene. There are no curative treatments, and activation of wild-type MECP2 expression is one strategy for stabilizing or reversing the disease. We isolated fibroblast clones that express exclusively either the wild-type or a 32-bp-deletion mutant form of MECP2. We developed a sensitive assay for measuring wild-type MECP2 mRNA levels and tested small molecule epigenetic activators for their ability to activate gene expression. Although our pilot screen did not identify activators of MECP2 expression, it established the value of using clonal cells and defined challenges that must be overcome. (C) 2011 Elsevier Ltd. All rights reserved.
  • Mitsuhiro Machitani; Tomoko Yamaguchi; Kahori Shimizu; Fuminori Sakurai; Kazufumi Katayama; Kenji Kawabata; Hiroyuki Mizuguchi
    Pharmaceutics 3 3 338 - 53 2011年07月 [査読有り]
     
    The major limitation of the clinical use of replication-incompetent adenovirus (Ad) vectors is the interference by innate immune responses, including induction of inflammatory cytokines and interferons (IFN), following in vivo application of Ad vectors. Ad vector-induced production of inflammatory cytokines and IFNs also results in severe organ damage and efficient induction of acquired immune responses against Ad proteins and transgene products. Ad vector-induced innate immune responses are triggered by the recognition of Ad components by pattern recognition receptors (PRRs). In order to reduce the side effects by Ad vector-induced innate immune responses and to develop safer Ad vectors, it is crucial to clarify which PRRs and which Ad components are involved in Ad vector-induced innate immune responses. Our group previously demonstrated that myeloid differentiating factor 88 (MyD88) and toll-like receptor 9 (TLR9) play crucial roles in the Ad vector-induced inflammatory cytokine production in mouse bone marrow-derived dendritic cells. Furthermore, our group recently found that virus associated-RNAs (VA-RNAs), which are about 160 nucleotide-long non-coding small RNAs encoded in the Ad genome, are involved in IFN production through the IFN-β promoter stimulator-1 (IPS-1)-mediated signaling pathway following Ad vector transduction. The aim of this review is to highlight the Ad vector-induced innate immune responses following transduction, especially VA-RNA-mediated innate immune responses. Our findings on the mechanism of Ad vector-induced innate immune responses should make an important contribution to the development of safer Ad vectors, such as an Ad vector lacking expression of VA-RNAs.
  • Yukari Motegi; Kazufumi Katayama; Fuminori Sakurai; Takuya Kato; Tomoko Yamaguchi; Hayato Matsui; Masahide Takahashi; Kenji Kawabata; Hiroyuki Mizuguchi
    JOURNAL OF CONTROLLED RELEASE 153 2 149 - 153 2011年07月 [査読有り]
     
    Viral vectors expressing short hairpin RNA (shRNA) are attractive for efficient and tissue-specific RNA interference (RNAi) delivery. We and others previously reported that recombinant adenovirus (Ad) vector-mediated RNAi has great potential for a variety of applications in molecular biology studies and gene therapy. In the present study, we have developed an efficient Ad vector-mediated RNAi system, in which an Ad vector carries four shRNA-expression cassettes (Ad-multi-shRNA vector), a simple and effective strategy for enhancing the RNAi response per Ad vector particle. The data demonstrated that the Ad-multi-shRNA vectors showed an enhanced RNAi effect compared to conventional Ad vectors containing a single shRNA-expression cassette. An application of the Ad-multi-shRNA vector carrying four same shRNA.-sequences against the RET finger protein, an oncogene known to desensitize cells to oxidative stress and cisplatin, resulted in an enhanced cytotoxic effect of cisplatin, demonstrating the advantages of the Ad-multi-shRNA vector for silencing target genes. Furthermore, an Ad-multi-shRNA carrying four different shRNA-sequences efficiently silenced the multiple target genes simultaneously. These data suggest the potential usefulness of the Ad-multi-shRNA vector not only in basic research but also in clinical gene therapy. (C) 2011 Elsevier B.V. All rights reserved.
  • Kahori Shimizu; Fuminori Sakurai; Mitsuhiro Machitani; Kazufumi Katayama; Hiroyuki Mizuguchi
    MOLECULAR PHARMACEUTICS 8 4 1430 - 1435 2011年07月 [査読有り]
     
    Theoretically, adenovirus (Ad) genes should not be expressed following transduction with a replication-incompetent Ad vector because the E1A gene, which is essential for the expression of other viral gene, is deleted in a replication-incompetent Ad vector. However, leaky expression of viral genes is known to occur following transduction with an E1-deleted Ad vector, leading to an induction of cellular immunity against Ad proteins. To date, no detailed analysis of the leaky expression profiles of Ad genes has been performed. In this study, we systematically examined the expression profiles of Ad genes in cells following transduction with a replication-incompetent Ad vector (Ad-L2) at multiplicities of infection (MOIs) of 10 and 100 using real-time RT-PCR Significant expression was found for the E4 and pIX genes following transduction with Ad-L2 in cultured cells. The expression levels of the E4 and pIX genes were approximately 30- to 600-fold lower than those of the transgene (firefly luciferase), and 50- to 5000-fold lower than those of the E4 and pIX genes following transduction at the same MOI with the wild-type Ad. Unexpectedly, expression levels of the major capsid proteins were approximately the same as, or even slightly above, the background levels (Ad gene expression levels in mock-transduced cells). This study provides valuable information for the design of a safe and efficient replication-incompetent Ad vector.
  • Kazuo Takayama; Mitsuru Inamura; Kenji Kawabata; Katsuhisa Tashiro; Kazufumi Katayama; Fuminori Sakurai; Takao Hayakawa; Miho Kusuda Furue; Hiroyuki Mizuguchi
    PLOS ONE 6 7 e21780  2011年07月 [査読有り]
     
    The establishment of methods for directive differentiation from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is important for regenerative medicine. Although Sry-related HMG box 17 (SOX17) overexpression in ESCs leads to differentiation of either extraembryonic or definitive endoderm cells, respectively, the mechanism of these distinct results remains unknown. Therefore, we utilized a transient adenovirus vector-mediated overexpression system to mimic the SOX17 expression pattern of embryogenesis. The number of alpha-fetoprotein-positive extraembryonic endoderm (ExEn) cells was increased by transient SOX17 transduction in human ESC- and iPSC-derived primitive endoderm cells. In contrast, the number of hematopoietically expressed homeobox (HEX)-positive definitive endoderm (DE) cells, which correspond to the anterior DE in vivo, was increased by transient adenovirus vector-mediated SOX17 expression in human ESC- and iPSC-derived mesendoderm cells. Moreover, hepatocyte-like cells were efficiently generated by sequential transduction of SOX17 and HEX. Our findings show that a stage-specific transduction of SOX17 in the primitive endoderm or mesendoderm promotes directive ExEn or DE differentiation by SOX17 transduction, respectively.
  • Kazufumi Katayama; Rie Furuki; Hideaki Yokoyama; Makoto Kaneko; Masashi Tachibana; Ichiro Yoshida; Hisamitsu Nagase; Keiichi Tanaka; Fuminori Sakurai; Hiroyuki Mizuguchi; Shinsaku Nakagawa; Tsuyoshi Nakanishi
    BIOMATERIALS 32 17 4185 - 4193 2011年06月 [査読有り]
     
    Among viral vectors, the fiber-mutant adenovirus vector carrying the Arg-Gly-Asp (RGD) peptide sequence (Ad-RGD) seems to have potential for both clinical gene therapy and basic research. As a part of a thorough evaluation of Ad-RGD in preclinical studies, we designed an experiment to investigate in detail the distribution of Ad-RGD compared with conventional adenovirus vector (WT-Ad) in pregnant mice. Surprisingly, Ad-RGD had substantial placental tropism, at 10-100 times that of WT-Ad. Transgene expression was sustained for at least 7 days, and Ad-RGD expressing firefly luciferase or red fluorescent protein has so far caused no placental dysfunction leading to fetal death. Ad-RGD showed high levels of transduction efficiency in in vitro-differentiated trophoblast stem cells, in which higher expression of alpha v beta 3 integrin than in undifferentiated cells was observed. Our results suggest that the use of Ad-RGD or another RGD-mediated targeting strategy holds promise for drug delivery to the placenta. (C) 2011 Elsevier Ltd. All rights reserved.
  • Takayuki Suzuki; Tomomi Sasaki; Koyori Yano; Fuminori Sakurai; Kenji Kawabata; Masuo Kondoh; Takao Hayakawa; Kiyohito Yagi; Hiroyuki Mizuguchi
    VIRUS RESEARCH 158 1-2 154 - 160 2011年06月 [査読有り]
     
    In a conventional adenovirus (Ad) vector production method using 293 cells, homologous recombination between Ad vector DNA and 293 cell-derived Ad El DNA occurs with low efficiency, resulting in the generation of replication-competent adenovirus (RCA). RCA can induce the spread of replication-incompetent Ad vectors, leading to unexpected tissue damage. In order to overcome this problem, we developed an Ad vector production system free of RCA generation by utilizing the Ad packaging size limit of the viral genome. It is well known that up to approximately 105% (37.7 kb) of the wild-type genome (35.9 kb) can be packaged in the Ad virion. We designed the Ad vector genome by insertion of a transgene expression cassette into the E3 region, such that homologous recombination between the Ad vector DNA and 293 cell-derived Ad E1 DNA would produce an Ad vector genome that exceeds in the size of the packaging limit. In accord with our strategy, no RCA generation was observed during the passages when we used the E1 (3.2 kb)-deleted Ad vectors containing a more than 3.0-kb transgene expression cassette in the E3 region. In contrast, the E1 (3.2 kb)-deleted Ad vectors, which retain 37.7 kb of the viral genome and have an insertion of a 2.1-kb transgene expression cassette in the E3 region, generated RCA, although RCA derived from this Ad vector exceeded the packaging size limit (105.0%). These results suggest that RCA generation can be avoided when the genome size of RCA is more than 108.3% (38.9 kb) of the wild-type Ad genome. This Ad vector production system generates safe, easy, and efficient Ad vector stock for both basic study as well as clinical research. (C) 2011 Elsevier B.V. All rights reserved.
  • Fuminori Sakurai; Kazufumi Katayama; Hiroyuki Mizuguchi
    FRONTIERS IN BIOSCIENCE-LANDMARK 16 2389 - 2401 2011年06月 [査読有り]
     
    For safe and effective gene therapy, targeted tissue-restricted transgene expression is desirable. Various methods have been developed to achieve such expression, including the use of tissue-specific promoters. In addition to these approaches, a new system which can regulate transgene expression, including viral gene expression, by exploiting microRNAs (miRNAs) has recently been developed. miRNAs are approximately 22-nucleotide (nt)-long non-coding RNAs that translationally suppress or catalytically degrade target mRNA through binding to imperfectly complementary sequences in the 3'-untranslated region (UTR). In miRNA-regulated transgene expression systems, tandem copies of sequences perfectly complementary to the miRNAs are usually incorporated into the 3'-UTR of the transgene expression cassette, leading to the suppression of transgene expression in cells expressing the corresponding miRNAs. miRNA-mediated regulation of transgene expression was first demonstrated for lentivirus vectors, and subsequently this technology was applied to replication-incompetent adenovirus vectors, tumor-specific oncolytic viruses for cancer therapy, and recombinant live attenuated viruses for vaccine therapy. The aim of this review is to highlight the applications of miRNA-regulated transgene expression systems for gene therapy and virotherapy.
  • Kumiko Sugio; Fuminori Sakurai; Kazufumi Katayama; Katsuhisa Tashiro; Hayato Matsui; Kenji Kawabata; Atsushi Kawase; Masahiro Iwaki; Takao Hayakawa; Toshiyoshi Fujiwara; Hiroyuki Mizuguchi
    CLINICAL CANCER RESEARCH 17 9 2807 - 2818 2011年05月 [査読有り]
     
    Purpose: Oncolytic adenoviruses (Ad) have been actively pursued as potential agents for cancer treatment. Among the various types of oncolytic Ads, the telomerase-specific replication-competent Ad (TRAD), which possesses an E1 gene expression cassette driven by the human telomerase reverse transcriptase promoter, has shown promising results in human clinical trials; however, the E1 gene is also slightly expressed in normal cells, leading to replication of TRAD and cellular toxicity in normal cells. Experimental Design: To overcome this problem, we utilized a microRNA (miRNA)-regulated gene expression system. Four copies of complementary sequences for miR-143, -145, -199a, or let-7a, which have been reported to be exclusively downregulated in tumor cells, were incorporated into the 3'-untranslated region of the E1 gene expression cassette. Results: Among the TRAD variants (herein called TRADs) constructed, TRADs containing the sequences complementary to miR-143, -145, or -199a showed efficient oncolytic activity comparable to the parental TRAD in the tumor cells. On the other hand, replication of the TRADs containing the miRNA complementary sequences was at most 1,000-fold suppressed in the normal cells, including primary normal cells. In addition, to suppress the replication of the TRADs in hepatocytes as well as other normal cells, we constructed a TRAD containing 2 distinct complementary sequences for miR-199a and liver-specific miR-122a (TRAD-122a/199aT). TRAD-122a/199aT exhibited more than 10-fold reduction in viral replication in all the normal cells examined, including primary hepatocytes. Conclusions: This study showed that oncolytic Ads containing the sequences complementary to normal cell-specific miRNAs showed significantly improved safety profiles without altering tumor cell lysis activity. Clin Cancer Res; 17(9); 2807-18. (C)2011 AACR.
  • Mitsuru Inamura; Kenji Kawabata; Kazuo Takayama; Katsuhisa Tashiro; Fuminori Sakurai; Kazufumi Katayama; Masashi Toyoda; Hidenori Akutsu; Yoshitaka Miyagawa; Hajime Okita; Nobutaka Kiyokawa; Akihiro Umezawa; Takao Hayakawa; Miho K. Furue; Hiroyuki Mizuguchi
    MOLECULAR THERAPY 19 2 400 - 407 2011年02月 [査読有り]
     
    Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed a-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.
  • Emi Suzuki-Kouyama; Kazufumi Katayama; Fuminori Sakurai; Tomoko Yamaguchi; Shinnosuke Kurachi; Kenji Kawabata; Shinsaku Nakagawa; Hiroyuki Mizuguchi
    BIOMATERIALS 32 6 1724 - 1730 2011年02月 [査読有り]
     
    PEGylation of recombinant adenovirus (Ad) vectors is a promising approach for not only evasion from neutralizing anti-Ad antibodies and uptake by phagocytic cells, but also prolongation of the blood retention time of Ad vectors after systemic administration. However, the conventional PEGylation leads to significant reduction in the transduction activity of Ad vectors, probably because PEG is nonspecifically conjugated to the Ad capsid protein and inhibits the binding of Ad vectors to the primary receptor, coxsackievirus-adenovirus receptor (CAR). In order to PEGylate an Ad vector without significant reduction in the transduction activity, the biotin-binding peptide (BAP) was inserted into the hypervariable region (HVR) 5 of the hexon, which is not involved in the binding to CAR, and PEG was then specifically conjugated to the hexon HVR5 via avidin-biotin interaction. In vitro transduction experiments demonstrated that the hexon-specific PEGylation did not cause an apparent reduction in the transduction efficiency of the Ad vector, although the insertion of the BAP into the HVR5 itself reduced the transduction efficiency by 50-fold, compared with the conventional Ad vector, in the absence of anti-Ad serum. In the presence of anti-Ad serum, the transduction with the Ad vector with the BAP in the hexon HVR5 was significantly blocked; however, anti-Ad serum only slightly inhibited the transduction with the hexon-specifically PEGylated Ad vector (Ad-BAP/Bio/Avi/Bio-PEG-L2). Intravenous administration of Ad-BAP/Bio/Avi/Bio-PEG-L2 resulted in prolonged blood retention, significant reduction in the transduction in the liver, and accumulation in the tumor; however, unexpectedly, the transduction efficiency of Ad-BAP/Bio/Avi/Bio-PEG-L2 in the tumor was almost at the background level. (C) 2010 Elsevier Ltd. All rights reserved.
  • Norihisa Furukawa; Fuminori Sakurai; Kazufumi Katayama; Naohiko Seki; Kenji Kawabata; Hiroyuki Mizuguchi
    JOURNAL OF CONTROLLED RELEASE 150 1 94 - 101 2011年02月 [査読有り]
     
    MicroRNAs (miRNAs) are small regulatory non-coding RNAs endogenously expressed in a tissue-type specific pattern. Recent studies have demonstrated that miRNAs are involved in almost all cellular biological processes, including cellular development, differentiation, apoptosis, and proliferation. To elucidate the function of miRNAs in biological processes, it is crucial that we develop miRNA-expressing vectors for the efficient expression of miRNAs in cultured cells and animals. At the present time, however, no fully optimized miRNA-expressing vectors have been developed, since such vectors require consideration of the choice of promoters and several other complex factors. In this study, we constructed various types of plasmid vectors expressing human miR-199a. There are two genes encoding miR-199a in the different chromosomes, resulting in expression of two different precursors which produce the two mature miRNAs from the 5'- and 3'-strands (miR-199a-5p and -3p). When the miR-199a precursors containing the genomic sequences flanking the hairpin were expressed, the cytomegalovirus (CMV) promoter, CMV promoter/enhancer containing the intron A (CMVi). and CMV enhancer/beta-actin (CA) promoter were more effective than the human phosphoglycerate kinase (PGK) promoter. The reduction levels by the human U6 promoter-transcribed miR-199a were different between the cell lines. The suppressive effects of miR-199a on the reporter gene expression were different between miR-199a-5p and -3p, especially when miR-199a was expressed as a stem-loop structure under the control of the U6 promoter. Expression of miR-199a as a short-hairpin RNA (shRNA) with an artificial hairpin sequence and independent expression of the mature miR-199a and its complementary strand were effective for distinguishing the function of the 5'- and 3'-strand of the miRNA. In addition, expression of miRNAs as an shRNA and separate expression of mature miRNAs and their complementary strand would be promising methods under conditions in which the processing steps of miRNAs are impaired. The results of this study provide important information on the construction of miRNA-expressing vectors for miRNA function analysis as well as for gene therapy using miRNA-expressing vectors. (C) 2010 Elsevier B.V. All rights reserved.
  • Matsui H; Sakurai F; Katayama K; Mizuguchi H
    Nihon yakurigaku zasshi. Folia pharmacologica Japonica 137 2 70 - 74 公益社団法人 日本薬理学会 2011年02月 [査読有り]
     
    1970年代後半からの分子生物学の顕著な進歩を背景に，これまで不明であった様々な疾患の原因が，遺伝子やタンパク質のレベルで解き明かされる時代となってきた．そして今日では，明らかとなった疾患関連遺伝子情報を元に，今までに効果的な治療が望めなかったような難治性疾患に対する遺伝子治療研究が精力的に行われている．しかし，遺伝子治療の実用化には依然多くの問題が残されており，特に安全に効率よく治療用遺伝子を標的細胞に導入可能なベクターの開発が最重要研究課題となっている．アデノウイルス（Ad）ベクターは，既存の遺伝子導入用ベクターの中では最も高い遺伝子導入効率を示すなど，遺伝子治療用ベクターとして多くの長所を有することから，遺伝子治療臨床研究で広く用いられている．しかしながら，Adベクターの抱える問題点として，1）Coxsackievirus and adenovirus receptor（CAR）を受容体とし感染するため，CAR陰性細胞（樹状細胞や悪性度の高いがん細胞など）への遺伝子導入が困難であること，2）生体内に投与した場合，Adベクターからわずかに産生されるウイルスタンパク質に対する細胞障害性T細胞が誘導され，組織障害が生じることなどが挙げられる．これらの問題を解決するために，これまでに様々な改良型Adベクターが開発されてきた．本総説の前半では，主な改良型Adベクターを紹介し，後半では，Adベクターを用いた神経系細胞への遺伝子導入ならびに神経疾患・神経膠腫（グリオーマ）の遺伝子治療について最近の報告を織り交ぜて述べることにする．
  • Hayato Matsui; Fuminori Sakurai; Kazufumi Katayama; Shinnosuke Kurachi; Katsuhisa Tashiro; Kumiko Sugio; Kenji Kawabata; Hiroyuki Mizuguchi
    VIRUS RESEARCH 155 1 48 - 54 2011年01月 [査読有り]
     
    Fiber-substituted Ad serotype 5 vectors containing the fiber protein from Ad serotype 35 (Ad5F35) exhibit properties that render them suitable as a platform for targeted Ad vectors. Ad5F35 vectors do not show apparent tropism in certain organs, including the liver, and they elicit less innate immunity than other vectors after intravenous administration. In order to develop a targeted Ad vector, we previously developed fiber-mutant Ad5F35 vectors containing the integrin binding Arg-Gly-Asn (RGD) motif in the FG or HI loop of the Ad35 fiber knob. Mutant Ad5F35 vectors containing the RGD peptide in the FG or HI loop transduced CD46-negative cells more efficiently in an RGD-dependent manner, as compared to the efficiency achieved with unmodified Ad5F35 vectors (Matsui et al., 2009. Gene Therapy 16, 1050-1057). However, the transduction efficiency of the mutant Ad5F35 vectors in CD46-negative cells remained lower than had been expected. Ad5F35 vectors containing the RGD peptide in the HI or FG loop enabled a 6-fold higher transduction efficiency than that achieved with unmodified Ad5F35 vectors in CD46-negative cells, although this cell type abundantly expresses alpha(v)-integrins. In the present study, we aimed to enhance the transduction efficiency of fiber-mutant Ad5F35 vectors. To this end, we developed an Ad5F35-vector system in which foreign peptides could be incorporated into regions of FG and HI loops of the Ad35 fiber knob by means of in vitro ligation. Using this Ad5F35-vector system, firefly luciferase-expressing mutant Ad5F35 vectors containing the RGD peptides in both loops (Ad5F35-2xRGD-L2) were constructed. In CD46-negative cells, Ad5F35-2xRGD-L2 showed 12-fold and 3-fold greater transduction efficiency than unmodified Ad5F35 vectors and mutant Ad5F35 vectors containing only one copy of the RGD peptide in the FG or the HI loop. In addition, transduction with Ad5F35-2xRGD-L2 in CD46-negative cells was RGD peptide-dependent. These results indicate that fiber-mutant Ad5F35 vectors, by which foreign peptides can be simultaneously incorporated into both the FG and the HI loops of the Ad35 fiber knob, could be a promising gene delivery vehicle for various gene therapies, and could facilitate basic research efforts such as analyses of gene function. (C) 2010 Elsevier B.V. All rights reserved.
  • Fuminori Sakurai; Kazuko Nakashima; Tomoko Yamaguchi; Takako Ichinose; Kenji Kawabata; Takao Hayakawa; Hiroyuki Mizuguchi
    JOURNAL OF CONTROLLED RELEASE 148 2 212 - 218 2010年12月 [査読有り]
     
    Recently, much attention has focused on replication-incompetent adenovirus (Ad) vectors containing fiber proteins derived from species B Ad serotype 35 (Ad35) (Ad5F35) and Ad vectors fully constructed from Ad35 as vaccine vectors expressing antigens. However, differences in the transduction properties, including the induction of innate immunity, of Ad5F35 and Ad35 vectors have not been properly and fully examined, partly because the transduction properties of these Ad vectors should be evaluated using nonhuman primates or human CD46-transgenic (CD46TG) mice, which ubiquitously express the primary receptor of Ad35, human CD46, in a pattern similar to that of humans. In the present study, we evaluated innate immune responses of mouse dendritic cells (mDCs) derived from bone marrow cells of wild-type (WT) and CD46TG mice following transduction with Ad serotype 5 (Ad5), fiber-substituted Ad5F35, or Ad35 vectors. Ad5F35 and Ad35 vectors mediated more efficient transduction in mDCs derived from CD46TG mice (CD46TG-mDCs) than did Ad5 vectors. Upregulation of costimulatory molecules and inflammatory cytokine induction by Ad5F35 and Ad35 vectors were significantly higher than those by Ad5 vectors in CD46TG-mDCs. However, the induction properties of the innate immune responses were different between Ad5F35 and Ad35 vectors. Ad35 vectors induced higher levels of costimulatory molecule expression and inflammatory cytokine production than did Ad5F35 vectors in CD46TG-mDCs. Furthermore, intravenous administration of Ad35 vectors in WT and CD46TG mice resulted in higher levels of serum interleukin (IL)-6 and IL-12 compared with administration of Ad5F35 vectors, which exhibited almost mock-transduced levels of these inflammatory cytokines. This study indicates that innate immune responses by Ad35 and Ad5F35 vectors are distinct even although both Ad vectors recognize human CD46 as a receptor. (C) 2010 Elsevier B.V. All rights reserved.
  • Masayuki Matsui; Fuminori Sakurai; Sayda Elbashir; Donald J. Foster; Muthiah Manoharan; David R. Corey
    CHEMISTRY & BIOLOGY 17 12 1344 - 1355 2010年12月 [査読有り]
     
    Low-density lipoprotein receptor (LDLR) is a cell-surface receptor that plays a central role in regulating cholesterol levels. Increased levels of LDLR would lead to reduced cholesterol levels and contribute to strategies designed to treat hypercholesterolemia. We have previously shown that duplex RNAs complementary to transcription start sites can associate with noncoding transcripts and activate gene expression. Here we show that duplex RNAs complementary to the promoter of LDLR activate expression of LDLR and increase the display of LDLR on the surface of liver cells. Activation requires complementarity to the LDLR promoter and can be achieved by chemically modified duplex RNAs. Promoter-targeted duplex RNAs can overcome repression of LDLR expression by 25-hydroxycholesterol and do not interfere with activation of LDLR expression by lovastatin. These data demonstrate that small RNAs can activate LDLR expression and affect LDLR function.
  • Fuminori Sakurai; Makiya Nishikawa
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 130 11 1487 - 1488 2010年11月 [査読有り]
  • Fuminori Sakurai; Hiroyuki Mizuguchi
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 130 11 1497 - 1504 2010年11月 [査読有り]
     
    Target tissue-specific delivery and transcription of foreign genes are desirable for safe and effective gene therapy. Two approaches for this purpose, "Targeted Delivery" and "Targeted Expression", have been mainly reported. Among "Targeted Expression" approaches, microRNA (miRNA) -mediated "post-transcriptional de-targeting" has been recently demonstrated, and much attention has been focused on this approach. MiRNAs are an approximately 22-nt length non-coding RNA, and bind to imperfectly complementary sequences in the 3`-untranslated region (UTR) of target mRNA, leading to suppression of gene expression via post-transcriptional regulation. First, in order to reduce the hepatic transduction by Ad vectors, complementary sequences of liver-specific miRNA, miR-122a, were inserted into the 3`-UTR of the transgene expression cassette. Intratumor injection of this Ad vector resulted in approximately 100-fold lower hepatic expression than that of the conventional Ad vector, without reducing gene expression in the tumor. Second, complementary sequences for miRNAs selectively down-regulated in tumor cells were inserted into the El gene expression cassette in oncolytic Ads, which exhibit tumor cell-specific replication and antitumor effects. Recent studies demonstrated that expression of several miRNAs is exclusively reduced in tumor cells. Oncolytic Ads containing the miRNA complementary sequences showed reduced replication in the normal cells, without altering the antitumor effects. MiRNA-regulated gene expression system mediates "post-transcriptional de-targeting", in which translation of transgene is suppressed in a tissue-specific manner; however, tissue-specific transgene expression can be achieved by taking tropism of gene delivery vehicles into consideration and reducing the transgene expression in untargeted organs via miRNA-regulated gene expression system.
  • Katsuhisa Tashiro; Kenji Kawabata; Mitsuru Inamura; Kazuo Takayama; Norihisa Furukawa; Fuminori Sakurai; Kazufumi Katayama; Takao Hayakawa; Miho Kusuda Furue; Hiroyuki Mizuguchi
    CELLULAR REPROGRAMMING 12 5 501 - 507 2010年10月 [査読有り]
     
    We examined the transduction efficiency in human embryonic stem (ES) and induced pluripotent stem (iPS) cells using an adenovirus (Ad) vector. RT-PCR analysis revealed the expression of the coxsackievirus and adenovirus receptor, a receptor for Ad, in these cells. However, gene expression after the transduction with an Ad vector was observed only in the periphery of ES and iPS cell colonies, when human ES and iPS cells were passaged as small colonies. This suggests that the Ad vector could not enter inside the ES and iPS cell colonies by their tight connection. We thus attempted to transduce foreign genes into the dissociated form of human ES and iPS cells, which were passaged using Rho-associated kinase inhibitor. In this condition, transduction efficiency in human ES and iPS cells was markedly increased and transgene expression was observed even inside the colonies by using Ad vectors. Furthermore, Ad vector-mediated transduction did not alter the expression of undifferentiated markers such as Oct-3/4, Nanog, and SSEA-4. Our results indicate that Ad vectors are effective tools for transduction into human ES and iPS cells.
  • Tomoko Yamaguchi; Kenji Kawabata; Emi Kouyama; Ken J. Ishii; Kazufumi Katayama; Takayuki Suzuki; Shinnosuke Kurachi; Fuminori Sakurai; Shizuo Akira; Hiroyuki Mizuguchi
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 107 40 17286 - 17291 2010年10月 [査読有り]
     
    Transduction with replication-incompetent recombinant adenovirus (Ad) vectors results in a rapid activation of innate immune responses, such as inflammatory cytokine production and subsequent tissue damage. The precise mechanisms of the innate immune responses induced by Ad vectors remain to be clarified. Possible components of Ad vectors that activate innate immune responses are the capsid protein, the viral genome (DNA), and viral transcripts. In the present study, we demonstrate that virus-associated RNAs (VA-RNAs), which are small RNAs transcribed by RNA polymerase III, induce the production of type I IFN (IFN-alpha and IFN-beta), but they do not induce the production of inflammatory cytokines (IL-6 and IL-12), in mouse embryonic fibroblasts (MEFs) and granulocyte-macrophage colony-stimulating factor-generated bone marrow-derived dendritic cells (GM-DCs). We also show that IFN-beta promoter stimulator-1 is involved in VA-RNA-dependent IFN-beta production in MEFs and is partially involved in type I IFN production in GM-DCs. This study provides important insight into the mechanisms of Ad vector-triggered innate immune responses, which may lead to more advanced and rational Ad vector designs for gene therapies and vaccine applications.
  • Masahiro Ushitora; Fuminori Sakurai; Tomoko Yamaguchi; Shin-ichiro Nakamura; Masuo Kondoh; Kiyohito Yagi; Kenji Kawabata; Hiroyuki Mizuguchi
    JOURNAL OF CONTROLLED RELEASE 142 3 431 - 437 2010年03月 [査読有り]
     
    Liver ischemia/reperfusion (I/R) injury, which is mainly caused by the generation of reactive oxygen species (ROS) during the reperfusion, remains an important clinical problem associated with liver transplantation and major liver surgery. Therefore, ROS should be detoxified to prevent hepatic I/R-induced injury. Delivery of antioxidant genes into liver is considered to be promising for prevention of hepatic I/R injury: however, therapeutic effects of antioxidant gene transfer to the liver have not been fully examined. The aim of this study was to examine whether adenovirus (Ad) vector-mediated catalase gene transfer in the liver is an effective approach for scavenging ROS and preventing hepatic I/R injury. Intravenous administration of Ad vectors expressing catalase. which is an antioxidant enzyme scavenging H(2)O(2), resulted in a significant increase in catalase activity in the liver. Pre-injection of catalase-expressing Ad vectors dramatically prevented I/R-induced elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and hepatic necrosis. The livers were also protected in another liver injury model. CCl(4)-induced liver injury, by catalase-expressing Ad vectors. Furthermore, the survival rates of mice subjected to both partial hepatectomy and I/R treatment were improved by pre-injection of catalase-expressing Ad vectors. On the other hand, control Ad vectors expressing p,galactosidase did not show any significant preventive effects in the liver on the models of I/R-induced or CCl(4)-induced hepatic injury described above. These results indicate that hepatic delivery of the catalase gene by Ad vectors is a promising approach for the prevention of oxidative stress-induced liver injury. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.
  • Kenji Kawabata; Haruna Sakurai; Katsuhisa Tashiro; Tomoko Yamaguchi; Fuminori Sakurai; Shinsaku Nakagawa; Hiroyuki Mizuguchi
    JOURNAL OF GENE MEDICINE 11 12 1170 - 1171 2009年12月 [査読有り]
  • H. Matsui; F. Sakurai; S. Kurachi; K. Tashiro; K. Sugio; K. Kawabata; K. Yamanishi; H. Mizuguchi
    GENE THERAPY 16 8 1050 - 1057 2009年08月 [査読有り]
     
    Fiber-substituted adenovirus (Ad) vectors containing fibers of Ad serotype 35 (AdF35) efficiently transduce a variety of human cells because their receptor, human CD46, is ubiquitously expressed on almost all nucleated cells. However, the ubiquitous expression of CD46 might lead to unexpected transduction in untargeted organs. In this study, we developed fiber-modified AdF35 vectors with an integrin-binding Arg-Gly-Asn (RGD) peptide incorporated into the FG, HI or IJ loop, which have been identified as important regions for binding to CD46. Incorporation of foreign peptides into these loops does not inhibit trimerization of the fibers. In CD46-negative cells, fiber-mutant AdF35 vectors containing an RGD peptide in the FG or HI loop showed 6- to 30-fold higher transduction efficiencies in an RGD-peptide-dependent manner than the unmodified AdF35 vectors. In contrast, in CD46-positive cells, insertion of foreign peptides markedly reduced the transduction efficiencies of the AdF35 vectors, indicating that insertion of foreign peptides significantly inhibits binding to CD46. In particular, CD46-mediated transduction was completely diminished by insertion of foreign peptides into the HI loop. Our findings indicate that HI loop is the most suitable domain to mediate a foreign peptide-dependent and CD46-independent transduction by incorporation of foreign peptides into the Ad35 fiber knob. Gene Therapy (2009) 16, 1050-1057; doi: 10.1038/gt.2009.65; published online 11 June 2009
  • Katsuhisa Tashiro; Mitsuru Inamura; Kenji Kawabata; Fuminori Sakurai; Koichi Yamanishi; Takao Hayakawa; Hiroyuki Mizuguchi
    STEM CELLS 27 8 1802 - 1811 2009年08月 [査読有り]
     
    Induced pluripotent stem (iPS) cells, which are generated from somatic cells by transducing four genes, are expected to have broad application to regenerative medicine. Although establishment of an efficient gene transfer system for iPS cells is considered to be essential for differentiating them into functional cells, the detailed transduction characteristics of iPS cells have not been examined. Previously, by using an adenovirus (Ad) vector containing the elongation factor-1 alpha (EF-1 alpha) and the cytomegalovirus enhancer/beta-actin (CA) promoters, we developed an efficient transduction system for mouse embryonic stem (ES) cells and their aggregate form, embryoid bodies (EBs). In this study, we applied our transduction system to mouse iPS cells and investigated whether efficient differentiation could be achieved by Ad vector-mediated transduction of a functional gene. As in the case of ES cells, the Ad vector containing EF-1 alpha and the CA promoter could efficiently transduce transgenes into mouse iPS cells. At 3,000 vector particles/cell, 80%-90% of iPS cells expressed transgenes by treatment with an Ad vector containing the CA promoter, without a decrease in pluripotency or viability. We also found that the CA promoter had potent transduction ability in iPS cell-derived EBs. Moreover, exogenous expression of a PPAR gamma gene or a Runx2 gene into mouse iPS cells by an optimized Ad vector enhanced adipocyte or osteoblast differentiation, respectively. These results suggest that Ad vector-mediated transient transduction is sufficient to increase cellular differentiation and that our transduction methods would be useful for therapeutic applications based on iPS cells. STEM CELLS 2009;27:1802-1811
  • F. Sakurai; S. -I Nakamura; K. Akitomo; H. Shibata; K. Terao; K. Kawabata; T. Hayakawa; H. Mizuguchi
    GENE THERAPY 16 2 297 - 302 2009年02月 [査読有り]
     
    Adenovirus (Ad) serotype 35 (Ad35) vectors have attracted remarkable attention as alternatives to conventional Ad serotype 5 (Ad5) vectors. In a previous study, we showed that intravenously administered Ad35 vectors exhibited a safer profile than Ad5 vectors in cynomolgus monkeys, which ubiquitously express CD46, an Ad35 receptor, in a pattern similar to that in humans. However, the Ad35 vectors poorly transduced the organs. In this study, we examined the transduction properties of Ad35 vectors after local administration into organs of cynomolgus monkeys. The vectors transduced different types of cells depending on the organ. Hepatocytes and microglia were mainly transduced after the vectors were injected into the liver and cerebrum, respectively. Injection of the vectors into the femoral muscle resulted in the transduction of cells that appeared to be fibroblasts and/or macrophages. Conjunctival epithelial cells showed transgene expression following infusion into the vitreous body of the eyeball. Transgene expression was limited to areas around the injection points in most of the organs. In contrast, Ad35 vector-mediated transgene expression was not detected in any of the organs not injected with Ad35 vectors. These results suggest that Ad35 vectors are suitable for gene delivery by direct administration to organs.
  • Haiying Huang; Fuminori Sakurai; Yuriko Higuchi; Shigeru Kawakami; Mitsuru Hashida; Kenji Kawabata; Hiroyuki Mizuguchi
    JOURNAL OF CONTROLLED RELEASE 133 2 139 - 145 2009年01月 [査読有り]
     
    To date, no countermeasures have been developed to overcome adenovirus (Ad) vector-induced innate immune responses in gene therapies that utilize these vectors. In the present study, we attempted to suppress Ad vector-induced innate immune responses and hepatotoxicity by delivering in NF-kappa B decoy to splenic macrophages and dendritic cells, as well as to Kupffer cells in the liver, using sugar-modified cationic liposomes. Pre-injection of the sugar-modified cationic liposome/NF-kappa B decoy complexes reduced serum levels of interleukin (IL)-12, which is a typical inflammatory cytokine induced by Ad vectors, and also reduced hepatotoxicity following Ad vector injection. Electrophoretic mobility shift assay demonstrated a reduction of Ad vector-induced NF-kappa B activation by pre-injection of the sugar-modified cationic liposome/NF-kappa B decoy complexes. Two types of sugar-modified cationic liposomes, fucosylated cationic liposomes (Fuc-liposomes) and mannosylated cationic liposomes (Man-liposomes), were tested in this study. Man-liposomes. which are considered to have greater affinity than Fuc-liposomes for splenocytes, appeared to have Superior Suppressive effects to those of Fuc-liposomes. Pre-injection Of the sugar-modified cationic liposome/NF-kappa B decoy complexes did not inhibit Ad vector-mediated transduction ill the organs. This Study provides important findings for overcoming Ad vector-induced innate immune responses. (C) 2008 Elsevier B.V. All rights reserved.
  • Katsuhisa Tashiro; Asami Kondo; Kenji Kawabata; Haruna Sakurai; Fuminori Sakurai; Koichi Yamanishi; Takao Hayakawa; Hiroyuki Mizuguchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 379 1 127 - 132 2009年01月 [査読有り]
     
    Bone marrow stromal cells (BMSCs) are expected to be a source for tissue regeneration because they call differentiate into Multiple cell types. Establishment of efficient gene transfer systems for BMSCs is essential for their application to regenerative medicine. In this Study, we compared the transduction efficiency in mouse primary BMSCs by using fiber-modified adenovirus (Ad) vectors, and demonstrated that AdK7, which harbors a polylysin (K7) peptide in the C-terminus of the fiber knob, could efficiently express a transgene in BMSCs. Notably, AdK7 robustly drove trans,gene expression it) more than 90% of the BMSCs at 3,000 Vector particles/cell. Furthermore, we showed that in vitro and in vivo osteogenic potential of BMSCs was dramatically promoted by the transduction of Runx2 gene using AdK7. These results indicate that this transduction system could be a powerful tool for therapeutic applications based oil BMSCs. (C) 2008 Elsevier Inc. All rights reserved.
  • Fuminori Sakurai
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 128 12 1751 - 1761 2008年12月 [査読有り]
     
    Properties of gene delivery vehicles, including gene transfer efficiencies and toxicities, are a key parameter for successful gene therapy. Among various types of gene delivery vehicles that have been developed so far, adenovirus (Ad) vectors have promising potentials as a vector for gene therapy because they can easily be grown to high titers and can efficiently deliver genes to both dividing and non-dividing cells. However, recent studies demonstrated some drawbacks of conventional Ad vectors, which are composed of subgroup C Ad serotype 5 (Ad5). First, Ad5 vectors poorly transduce cells lacking the primary receptor for Ad5, coxsackievirus and adenovirus receptor (CAR). Second, majority of adults have neutralizing antibodies to Ad5. In order to overcome these drawbacks, we developed a novel Ad vector which is fully composed of subgroup B Ad serotype 35 (Ad35). Ad35 vectors can infect a variety of human cells because the primary receptor for Ad35, CB46, is ubiquitously expressed in human cells. Furthermore, Ad35 vectors efficiently transduce in the presence of anti-Ad5 antibodies, and seroprevalence of Ad35 in adults is much lower than that of Ad5. In the current review, I introduce our recent work on development and evaluation of Ad35 vectors, and I also discuss the potential of Ad35 vectors as gene delivery vehicles.
  • Takayuki Suzuki; Fuminori Sakurai; Shin-Ichiro Nakamura; Emi Kouyama; Kenji Kawabata; Masuo Kondoh; Kiyohito Yagi; Hiroyuki Mizuguchi
    MOLECULAR THERAPY 16 10 1719 - 1726 2008年10月 [査読有り]
     
    The combined use of adenovirus (Ad) vectors expressing herpes simplex virus thymidine kinase (HSVtk) and ganciclovir (GCV) offers a potential therapeutic strategy against cancer. However, intratumorally injected Ad vectors are disseminated into the systemic circulation and efficiently transduce the liver, resulting in severe hepatotoxicity. In order to overcome this problem, an Ad vector carrying a microRNA (miRNA)-regulated expression system was developed by inserting into the 3'-untranslated region (3'-UTR) of the expression cassette four tandem copies of sequences with perfect complementarity to miR-122a, which exhibits liver-specific expression. Transgene expression from the Ad vector carrying the miR-122a target sequences was 7- to 70-fold lower in cells with high miR-122a expression as compared to expression from a conventional Ad vector. Intratumoral injection of the Ad vector containing the miR-122a target sequences resulted in a 130- to 1,500-fold reduction in hepatic transgene products (without affecting the transgene expression in the tumor) when compared with those from a conventional Ad vector. In suicide gene therapy, the inclusion of the miR-122a target sequences in the HSVtk expression cassette achieved not only significant antitumor effects, but also a dramatic reduction in HSVtk/GCV-induced hepatotoxicity. These results indicate that Ad vectors that mediate miR-122a-regulated HSVtk expression provide a safe and efficient suicide gene therapy strategy.
  • Fuminori Sakurai
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 31 10 1819 - 1825 2008年10月 [査読有り]
     
    The capacity of gene delivery vehicles is considered to be a critical factor determining the success of gene therapy. To date, various types of gene delivery vehicle have been developed. Among them, recombinant adenovirus (Ad) vectors have potential that has favored their worldwide use in vitro and in vivo. Conventional Ad vectors are composed of subgroup C Ad serotype 5 (Ad5), although it has been clarified that the drawbacks of Ad5 vectors are a high seroprevalence of Ad5 in adults and low transduction efficiencies in cells lacking the primary receptor for Ad5. coxsackievirus and adenovirus receptor. To overcome these problems, we developed a novel Ad vector fully composed of Ad serotype 35 (Ad35). Ad35 vectors show a wide tropism for human cells because Ad35 binds to human CD46, which is ubiquitously expressed on almost all human cells, as a primary receptor. In addition. anti-Ad5 antibodies do not inhibit Ad35 vector-mediated transduction and the seroprevalence of Ad35 in adults is lower than that of Ad5. This paper reviews our studies on the development and evaluation of Ad35 vectors. Ad vectors derived from other Ad serotypes different from Ad5, including Ad5, are expected to be gene delivery vehicles alternative to conventional Ad5 vectors.
  • Kazuko Nakashima; Fuminori Sakurai; Kenji Kawabata; Hiroyuki Mizuguchi
    JOURNAL OF CONTROLLED RELEASE 129 3 215 - 222 2008年08月 [査読有り]
     
    Mast cells (MCs) have been shown to play an important role in immunoglobulin E (IgE)-associated immediate hypersensitivity and innate immunity by producing a variety of lipid mediators and cytokines. An efficient gene-delivery system is indispensable for elucidation of these mechanisms. In the present study, human and rodent MCs were transduced with various types of modified adenovirus (Ad) vectors. Fiber modification in Ad vectors significantly improved the transduction efficiencies in MCs. A fiber-substituted Ad serotype 5 (Ad5) vector containing Ad serotype 35 (Ad35) fiber proteins (Ad5F35) and an Ad35 vector, both of which transduce cells via human CD46, mediated 9.9-fold and 10.1-fold higher transduction efficiencies than conventional Ad5 vectors in the human mast cell line LAD2 among the Ad vectors. Ad5F35 and Ad35 vectors also efficiently transduced bone marrow-derived MCs (BMMCs) prepared from human CD46-transgenic (CD46TG) mice. The rat mast cell line RBL-2H3 were most efficiently transduced with a fiber-mutant Ad5 vector containing the Arg-Gly-Asp (RGD) peptide in the HI loop (Ad-RGD) of the fiber knob. Transduction with the Ad vectors did not induce degranulation or inflammatory cytokine production in the MCs. These results indicate that Ad vectors, including fiber-mutant Ad vectors, are effective gene-delivery tools for MCs. (C) 2008 Elsevier B.V. All rights reserved.
  • Naoko Kanagawa; Ryosuke Koretomo; Sayaka Murakami; Fuminori Sakurai; Hiroyuki Mizuguchi; Shinsaku Nakagawa; Takuya Fujita; Akira Yamamoto; Naoki Okada
    VIROLOGY 374 2 411 - 420 2008年05月 [査読有り]
     
    Adenoviral vector (Ad)-mediated gene transfer is an attractive method for manipulating the immunostimulatory properties of dendritic cells (DCs) for cancer immunotherapy. DCs treated with Ad have phenotype alterations (maturation) that facilitate T cell sensitization. We investigated the mechanisms of DC maturation with Ad transduction. Expression levels of a maturation marker (CD40) on DCs treated with conventional Ad, fiber-modified Ads (AdRGD, AdF35, AdF35 Delta RGD), or a different serotype Ad (Ad35) were correlated with their transduction efficacy. The alpha(v)-integrin directional Ad, AdRGD, exhibited the most potent ability to enhance both foreign gene expression and CD40 expression, and induced secretion of interleukin-12, tumor necrosis factor-alpha, and interferon-alpha in DCs. The presence of a foreign gene expression cassette in AdRGD was not necessary for DC maturation. Maturation of DCs treated with AdRGD was suppressed by destruction of the Ad genome, inhibition of endocytosis, or endosome acidification, whereas proteasome inhibition increased CD40 expression levels on DCs. Moreover, inhibition of a,integrin signal transduction and blockade of cytokine secretion affected the maturation of DCs treated with AdRGD only slightly or not at all, respectively. Thus, our data provide evidence that Ad-induced DC maturation is due to Ad invasion of the DCs, followed by nuclear transport of the Ad genome, and not to the expression of foreign genes. (c) 2008 Elsevier Inc. All rights reserved.
  • Katsuhisa Tashiro; Kenji Kawabata; Haruna Sakurai; Shinnosuke Kurachi; Fuminori Sakurai; Koichi Yamanishi; Hiroyuki Mizuguchi
    JOURNAL OF GENE MEDICINE 10 5 498 - 507 2008年05月 [査読有り]
     
    Background Establishment of a transient gene delivery system, such as adenovirus (Ad) vectors, into embryonic stem (ES) cells and their aggregation form, embryoid bodies (EBs), is essential for its application in regenerative medicine because the transgene should not be integrated in the host genome. In this study, we optimized Ad vector-mediated transduction into EBs, and examined whether Ad vector-mediated transduction of adipogenesis-related gene into EBs could promote the adipocyte differentiation. Methods We prepared beta-galactosidase-expressing Ad vectors under the control of four different promoters (cytomegalovirus (CMV), rouse sarcoma virus, human elongation factor-la, and CMV enhancer/beta-actin promoter (CA)) to estimate the transduction efficiency. Adipocyte differentiation efficiency by transduction of the PPAR gamma or C/EBP alpha gene into EBs was examined. Results Of the four promoters tested, the CA promoter exhibited the highest transduction efficiency in the EBs. However, Ad vector-mediated transduction was observed only in the periphery of the EBs. When repeated transduction by Ad vector was performed, gene expression was observed even in the interior of EBs as well. When EB-derived single cells were transduced by an Ad vector containing the CA promoter, more than 90% of the cells were transduced. Furthermore, Ad vector-mediated PPAR gamma gene transduction into EBs led to more efficient differentiation into adipocytes than could untransduced EBs, examined in terms of lipogenic enzyme activities and accumulation of the lipid droplets. Conclusions Ad vector-mediated transduction into EBs could be a valuable tool for molecular switching of cell differentiation and could be applied to regenerative medicine. Copyright (c) 2008 John Wiley & Sons, Ltd.
  • Shinnosuke Kurachi; Katsuhisa Tashiro; Haruna Sakurai; Fuminori Sakurai; Kenji Kawabata; Katsutoshi Yayama; Hiroshi Okamoto; Shinsaku Nakagawa; Hiroyuki Mizuguchi
    JOURNAL OF GENE MEDICINE 10 4 452 - 452 2008年04月 [査読有り]
  • Haruna Sakurai; Katsuhide Igarashi; Katsuhisa Tashiro; Kenji Kawabata; Fuminori Sakurai; Shinnosuke Kurachi; Shinsaku Nakagawa; Ken-ichi Aisaki; Jun Kanno; Hiroyuki Mizuguchi
    JOURNAL OF GENE MEDICINE 10 4 468 - 468 2008年04月 [査読有り]
  • Fuminori Sakurai; Shin-ichiro Nakamura; Kimiyo Akitomo; Hiroaki Shibata; Keiji Terao; Kenji Kawabata; Takao Hayakawa; Hiroyuki Mizuguchi
    MOLECULAR THERAPY 16 4 726 - 733 2008年04月 [査読有り]
     
    Adenovirus serotype 35 (Ad35) vectors have shown promise as effective gene delivery vehicles. However, the transduction profiles of Ad35 vectors in conventional mice allow only a limited estimation of transduction properties of these vectors, because the mouse analog of the subgroup B Ad receptor, CD46, is restricted to the testis. In order to assess the transduction properties of Ad35 vectors more completely, we performed transduction experiments using cynomolgus monkeys, which ubiquitously express CD46 in a pattern similar to that in humans. In vitro transduction experiments demonstrated that cultured cells from the cynomolgus monkey were efficiently transduced with Ad35 vectors. In contrast, after intravenous administration into live monkeys hardly any evidence of Ad35 vector mediated transduction was found in any of the organs, although Ad35 vector genomes were detected in various organs. Less severe histopathological abnormalities were found in the Ad35 vector-infused monkeys than in the conventional Ad5 vector-injected monkeys. In the latter, serious tissue damage and inflammatory responses, such as hepatocyte necrosis and lymphatic hyperplasia in the colon, were induced. Both Ad35 and Ad5 vectors caused similar hematological changes (increase in CD3(+) cells, and decrease in CD16(+) cells and CD20(+) cells) in peripheral blood cells. These results should provide valuable information for the clinical application of Ad35 vectors.
  • Haruna Sakurai; Kenji Kawabata; Furninori Sakurai; Shinsaku Nakagawa; Hiroyuki Mizuguchi
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 354 1-2 9 - 15 2008年04月 [査読有り]
     
    Gene therapy is a clinical strategy that has the potential to treat an array of genetic and nongenetic diseases. Vectors for gene transfer are the essential tools of gene therapy. For gene therapy to be successful, an appropriate amount of the therapeutic gene must be delivered into the target cells without substantial toxicity. A major limitation of the use of gene therapy vectors is the innate immune responses triggered by systemic administration of such vectors. It is essential to overcome vector-mediated innate immune responses, such as production of inflammatory cytokines, the maturation of antigen-presenting cells and tissue damage, because the induction of these responses not only shortens the period of gene expression but also leads to serious side effects. Viral vectors (for example, adenovirus (Ad) vectors) have been assumed to be more potent in inducing innate immune responses in spite of their high transduction efficiency since they contain pathogenic proteins. However, recent studies have demonstrated that not only viral vectors but also nonviral vectors, such as lipoplex (liposome/plasmid DNA complex), can induce innate immune responses. Indeed, nonviral vectors including lipoplex induce comparable or larger levels of innate immune response than viral vectors. In this review, we present an overview of the innate immune responses induced by Ad vector and lipoplex, which are used primarily for in vivo gene transfer. (C) 2007 Elsevier B.V. All rights reserved.
  • Shinnosuke Kurachi; Naoya Koizumi; Katsuhisa Tashiro; Haruna Sakurai; Fuminori Sakurai; Kenji Kawabata; Shinsaku Nakagawa; Hiroyuki Mizuguchi
    JOURNAL OF CONTROLLED RELEASE 127 1 88 - 95 2008年04月 [査読有り]
     
    Adenovirus (Ad) vector application in gene therapy is limited by its naive tropism. We previously developed protein IX (pIX)-modified and hexon-modified Ad vectors in order to alter Ad vector tropism. However, these modified Ad vectors failed to infect cells with the foreign ligands displayed in the pIX or hexon. We hypothesized that steric hindrance by fiber proteins might have prevented the ligand-mediated transduction, as fibers are the outmost capsid proteins of Ad vectors. Therefore, we generated a series of fiberless Ad vectors and investigated their gene expression properties. Unexpectively, however, pIX- or hexon-modified fiberless Ad vector did not achieve any gene expression (the gene expression level by these vectors was similar to the background level). These results might be caused by the fact that the fiberless particles were weaker against physical burdens. To the best of our knowledge, this study is the first reported attempt to develop fiberless Ad vectors containing foreign ligands, in the pIX or hexon region. The drawback of the lower stability of fiberless Ad vectors must be overcome to develop targeted Ad vectors based on such vectors. This study could provide basic information for the development of effective targeted Ad vectors. (c) 2008 Elsevier B.V. All rights reserved.
  • Haruna Sakurai; Katsuhisa Tashiro; Kenji Kawabata; Tomoko Yamaguchi; Fuminori Sakurai; Shinsaku Nakagawa; Hiroyuki Mizuguchi
    JOURNAL OF IMMUNOLOGY 180 7 4931 - 4938 2008年04月 [査読有り]
     
    Adenovirus (Ad) vectors are among the most commonly used viral vectors in gene therapy clinical trials. However, the application of Ad vectors has been limited to local injection in many cases, because the systemic administration of Ad vectors triggers innate immune responses such as inflammatory cytokine production and tissue damage. To overcome this limitation, it will be necessary to develop safer Ad vectors less likely to induce the innate immune response. In the present study, we demonstrated that a suppressor of cytokine signaling-1 (SOCS1)-expressing Ad vector, Ad-SOCS1, reduces the innate immune response induced by Ad vectors. RAW264.7-SOCS1, a macrophage-like cell line that stably expresses SOCS1, was shown to produce lower levels of inflammatory cytokines after the transduction of Ad vectors. The systemic administration of Ad-SOCS1 into mice elicited the reduced production of inflammatory cytokines, as compared with that elicited by control Ad vectors, i.e., luciferase-expressing Ad vector, Ad-L2. Furthermore, the coadministration of Ad-L2 with Ad-SOCS1 attenuated inflammatory cytokine production and liver toxicity as compared with injection with Ad-L2 alone, and this was achieved without the suppression of luciferase production in various organs. The JAK/STAT pathway was involved in Ad vector-mediated cytokine production, which was impaired by the overexpression of SOCS1. These findings indicate that Ad-SOCS1 could be useful for reducing Ad vector-mediated innate immunity.
  • Fuminori Sakurai; Shin-ichiro Nakamura; Kimiyo Akitomo; Hiroaki Shibata; Keiji Terao; Kenji Kawabata; Takao Hayakawa; Hiroyuki Mizuguchi
    MOLECULAR THERAPY 16 4 726 - 733 2008年04月 [査読有り]
     
    Adenovirus serotype 35 (Ad35) vectors have shown promise as effective gene delivery vehicles. However, the transduction profiles of Ad35 vectors in conventional mice allow only a limited estimation of transduction properties of these vectors, because the mouse analog of the subgroup B Ad receptor, CD46, is restricted to the testis. In order to assess the transduction properties of Ad35 vectors more completely, we performed transduction experiments using cynomolgus monkeys, which ubiquitously express CD46 in a pattern similar to that in humans. In vitro transduction experiments demonstrated that cultured cells from the cynomolgus monkey were efficiently transduced with Ad35 vectors. In contrast, after intravenous administration into live monkeys hardly any evidence of Ad35 vector mediated transduction was found in any of the organs, although Ad35 vector genomes were detected in various organs. Less severe histopathological abnormalities were found in the Ad35 vector-infused monkeys than in the conventional Ad5 vector-injected monkeys. In the latter, serious tissue damage and inflammatory responses, such as hepatocyte necrosis and lymphatic hyperplasia in the colon, were induced. Both Ad35 and Ad5 vectors caused similar hematological changes (increase in CD3(+) cells, and decrease in CD16(+) cells and CD20(+) cells) in peripheral blood cells. These results should provide valuable information for the clinical application of Ad35 vectors.
  • Takayuki Suzuki; Fuminori Sakurai; Shin-Ichiro Nakamura; Emi Kouyama; Kenji Kawabata; Masuo Kondoh; Kiyohito Yagi; Hiroyuki Mizuguchi
    Molecular Therapy 16 10 1719 - 1726 2008年 [査読有り]
     
    The combined use of adenovirus (Ad) vectors expressing herpes simplex virus thymidine kinase (HSVtk) and ganciclovir (GCV) offers a potential therapeutic strategy against cancer. However, intratumorally injected Ad vectors are disseminated into the systemic circulation and efficiently transduce the liver, resulting in severe hepatotoxicity. In order to overcome this problem, an Ad vector carrying a microRNA (miRNA)-regulated expression system was developed by inserting into the 3′-untranslated region (3′-UTR) of the expression cassette four tandem copies of sequences with perfect complementarity to miR-122a, which exhibits liver-specific expression. Transgene expression from the Ad vector carrying the miR-122a target sequences was 7- to 70-fold lower in cells with high miR-122a expression as compared to expression from a conventional Ad vector. Intratumoral injection of the Ad vector containing the miR-122a target sequences resulted in a 130- to 1,500-fold reduction in hepatic transgene products (without affecting the transgene expression in the tumor) when compared with those from a conventional Ad vector. In suicide gene therapy, the inclusion of the miR-122a target sequences in the HSVtk expression cassette achieved not only significant antitumor effects, but also a dramatic reduction in HSVtk/GCV-induced hepatotoxicity. These results indicate that Ad vectors that mediate miR-122a-regulated HSVtk expression provide a safe and efficient suicide gene therapy strategy.
  • S. Murakami; F. Sakurai; K. Kawabata; N. Okada; T. Fujita; A. Yamamoto; T. Hayakawa; H. Mizuguchi
    GENE THERAPY 14 21 1525 - 1533 2007年11月 [査読有り]
     
    Most subgroup B adenoviruses (Ads), including adenovirus (Ad) serotype 35 (Ad35), bind to human CD46 as a receptor; however, the infection processes of subgroup B Ads following attachment to CD46 remain to be elucidated. Subgroup B Ads possess Arg-Gly-Asp (RGD) motifs in the penton base, similarly to subgroup C Ad serotypes 2 and 5. In this study, we examined the role of penton base RGD motifs in Ad35 vector-mediated transduction in human hematopoietic cells. Inhibition of interaction between integrins and the RGD motifs by divalent cation chelation and a synthetic RGD peptide reduced the transduction efficiencies of Ad35 vectors; however, the amounts of cell-associated vector DNA of Ad35 vectors at 4 or 37 degrees C were not decreased by divalent cation chelation or the RGD peptide. Mutation of penton base RGD motifs reduced the transduction efficiencies of Ad35 vectors, although the amounts of cell- associated vector DNA of Ad35 vectors at 4 or 37 degrees C were not altered by mutation of penton base RGD motifs in Ad35 vectors. Furthermore, preincubation with several types of anti- integrin antibodies significantly inhibited Ad35 vectormediated transduction. These results suggest that interaction between integrins and penton base RGD motifs plays a crucial role in Ad35 vector-mediated transduction in hematopoietic cells, probably in the post-internalization steps.
  • Manabu Yamashita; Asami Ino; Kenji Kawabata; Fuminori Sakurai; Hiroyuki Mizuguchi
    INTERNATIONAL JOURNAL OF CANCER 121 8 1690 - 1696 2007年10月 [査読有り]
     
    The coxsackie and adenovirus receptor (CAR) is involved in the epithelial cell tight junction, the downregulated expression of which is observed in different cancer types. In the present study, we examined CAR's role in tumor metastasis using a B16 melanoma and CT26 colon adenocarcinoma model of experimental metastasis. In lung metastasis, the colony number of B16 cells stably expressing CAR (B16CAR) was significantly lower than that of the control CAR-negative B16 cells. B16 and CT26 cells transiently expressing CAR, which were transduced with adenovirus (Ad) vector expressing CAR, also reduced lung metastasis, suggesting that CAR plays a role in the early stage of metastasis. CAR expression significantly decreased the accumulation of B16 cells in the lung after i.v. injection and the migration in vitro. CAR expression reduced expression of alpha(v), alpha(4), beta(3) and beta(1) integrin, which play important roles in attachment to cells or basement membrane. Thus, CAR expression likely acts as a metastatic suppressor.
  • Jian-Qing Gao; Yusuke Eto; Yasuo Yoshioka; Fumiko Sekiguchi; Shinnosuke Kurachi; Tomohiro Morishige; Xinglei Yao; Hikaru Watanabe; Ratima Asavatanabodee; Fuminori Sakurai; Hiroyuki Mizuguchi; Yuka Okada; Yohei Mukai; Yasuo Tsutsumi; Tadanori Mayumi; Naoki Okada; Shinsaku Nakagawa
    JOURNAL OF CONTROLLED RELEASE 122 1 102 - 110 2007年09月 [査読有り]
     
    Conjugation of polyethylene glycol to protein or particles (PEGylation) prolongs their plasma half-lives and promotes their accumulation in tumors due to enhanced permeability and retention (EPR) effect. Although PEGylation of adenovirus vectors (Ads) is an attractive strategy to improve the in vivo kinetics of conventional Ads, the EPR effect of PEGylated Ad (PEG-Ad) had not previously been reported. In this study, we prepared PEG-Ads with PEG at various modification ratios, injected them intravenously into tumor-bearing mice, and determined the blood kinetics, viral distribution, and gene expression patterns, respectively. In addition, we conducted a cancer therapeutic study of PEG-Ad encoding tumor necrosis factor (TNF)-alpha. The plasma half-life of PEG-Ad was longer than that of unmodified-Ad, and accumulation of PEG-Ad in tumor tissue increased as the PEG modification ratio increased. In particular, PEG-Ad with about 90% modification ratio showed higher (35 times) gene expression in turner and lower (6%) in liver, compared with values for unmodified Ad. Moreover, PEG-Ad encoding TNF-alpha demonstrated not only stronger tumor-suppressive activity but also fewer hepatotoxic side effects compared with unmodified-Ad. PEGylation of Ad achieved turner targeting through the EPR effect, and these attributes suggest that systemic injection of PEG-Ad has great potential as an anti-tumor treatment. (c) 2007 Elsevier B.V. All rights reserved.
  • K. Kawabata; K. Tashiro; F. Sakurai; N. Osada; J. Kusuda; T. Hayakawa; K. Yamanishi; H. Mizuguchi
    GENE THERAPY 14 16 1199 - 1207 2007年08月 [査読有り]
     
    Coxsackievirus and adenovirus receptor (CAR) is a member of the immunoglobulin (lg) superfamily and a component of epithelial tight junction. CAR also functions as a primary receptor for coxsackievirus B and adenovirus (Ad) infection. In this study, we report the identification of a novel protein, CAR-like soluble protein (CLSP), which is closely related to CAR. Mouse CLSP (mCLSP) was composed of 390 amino acids, including three lg domains, and showed strong homology to the lgV domain of CAR. Interestingly, mCLSP lacks a transmembrane domain, indicating that this is a soluble protein. mCLSP mRNA was detected primarily in the brain and ovary. When mCLSP cDNA was introduced into SK HEP-1 cells, which were known to be CAR positive and easily infected with Ad vector, the infection with Ad vector was severely inhibited. On the other hand, mCLSP promoted the infection with Ad vector in CAR-negative NIH3T3 cells. Furthermore, recombinant CLSP directly bound to Ad and inhibited the Ad vector-mediated transduction in SK HEP-1 cells. Computational analysis for a genome database showed that the CLSP gene is rodent-specific, and that human and bovine lack this gene. These results suggest that CLSP may play a role in the antiviral defense of the host in rodent animals.
  • Tomoko Yamaguchi; Kenji Kawabata; Naoya Koizumi; Fuminori Sakurai; Kazuko Nakashima; Haruna Sakurai; Tomomi Sasaki; Naoki Okada; Koichi Yamanishi; Hiroyuki Mizuguchi
    HUMAN GENE THERAPY 18 8 753 - 762 2007年08月 [査読有り]
     
    A replication-incompetent adenoviral ( Ad) vector is generating interest for both gene therapy and immunotherapy. A major limitation of the use of Ad vectors is the innate immune response, which causes inflammatory cytokine production and tissue damage; however, the precise mechanism of the innate immune response remains to be clarified. Here, we show that serotype 5 human Ad vectors elicit innate immune responses through a myeloid differentiating factor 88 ( MyD88)/ Toll-like receptor ( TLR)-9-dependent and/ or -independent manner according to cell type. After stimulation with Ad vectors, the production of interleukin ( IL)-6 and IL-12 was significantly decreased in MyD88-or TLR9-deficient dendritic cells ( DCs), compared with wild-type DCs. In addition, the surface expression of maturation marker proteins, such as CD40, CD80, CD86, and MHC class II, in MyD88-or TLR9-deficient granulocyte-macrophage colony-stimulating factor ( GM-CSF)-DCs was similar to that in wild-type DCs. On the other hand, MyD88-or TLR9-deficient peritoneal macrophages produced the same level of IL-6 as wild-type macrophages after infection with Ad vectors. We did not find any differences in the mRNA expression levels of the molecules involved in innate immunity, such as MyD88, TLR3, TLR7, and TLR9, between DCs and macrophages. The intravenous injection of luciferase-expressing Ad vectors into MyD88-or TLR9-deficient mice resulted in almost comparable levels of IL-6 and IL-12 production and luciferase expression with wild-type mice. These results suggest that Ad vectors can activate innate immunity via MyD88/ TLR9-dependent and -independent mechanisms.
  • Fuminori Sakurai; Kenji Kawabata; Hiroyuki Mizuguchi
    CURRENT GENE THERAPY 7 4 229 - 238 2007年08月 [査読有り]
     
    Recombinant adenovirus (Ad) vectors have gained attention as gene delivery vehicles because they efficiently introduce foreign DNA into host cells, can be produced in high titers, and are able to transduce terminally differentiated cells. Conventional Ad vectors commonly used in the world, including clinical trials, are derived from subgroup C Ad serotype 5 (Ad5). Although Ad5 vector-mediated transduction provides encouraging results, preclinical and clinical applications have revealed several disadvantages of Ad5 vectors, such as high seroprevalence of anti-Ad5 antibodies in adults and low transduction efficiencies of Ad5 vectors in cells lacking the primary receptor for AM, the coxsackievirus and adenovirus receptor (CAR). To overcome these problems, novel recombinant Ad vectors, which are derived entirely from subgroup B Ads, including Ad serotypes 3, 7, 11, and 3 5, have been developed. These subgroup B Ad vectors can infect cells via human CD46 (membrane complement protein), which is ubiquitously expressed in almost all human cells, and/or via unidentified receptors other than CAR, leading to efficient transduction of subgroup B Ad vectors in most human cells, including CAR-negative cells. In addition, transduction efficiencies of subgroup B Ad vectors do not decrease in the presence of anti-Ad5 antibodies, and seroprevalences of most subgroup B Ads are lower than that of Ad5, indicating that transduction with subgroup B Ad vectors is unlikely to be hampered by preexisting anti-Ad antibodies. In this paper, we review the advances in subgroup B Ad vector research.
  • F. Sakurai; K. Akitomo; K. Kawabata; T. Hayakawa; H. Mizuguchi
    GENE THERAPY 14 11 912 - 919 2007年06月 [査読有り]
     
    Human CD46 (membrane cofactor protein), which serves as a receptor for a variety of pathogens, including strains of measles virus, human herpesvirus type 6 and Neisseria, is rapidly downregulated from the cell surface following infection by these pathogens. Here, we report that replication-incompetent adenovirus (Ad) serotype 35 (Ad35) vectors, which belong to subgroup B and recognize human CD46 as a receptor, downregulate CD46 following infection. A decline in the surface expression of CD46 in human peripheral blood mononuclear cells was detectable 6 h after infection, and reached maximum (72%) 12 h after infection. Ad35 vectorinduced downregulation of surface CD46 levels gradually recovered after the removal of Ad35 vectors, however, complete recovery of CD46 expression was not observed even at 96 h after removal. The surface expression of CD46 was also reduced after incubation with fiber-substituted Ad serotype 5 (Ad5) vectors bearing Ad35 fiber proteins, ultraviolet-irradiated Ad35, vectors and recombinant Ad35 fiber knob proteins; in contrast, conventional Ad5 vectors did not induce surface CD46 downregulation, suggesting that the fiber knob protein of Ad35 plays a crucial role in the downregulation of surface CD46 density. These results have important implications for gene therapy using CD46-utilizing Ad vectors and for the pathogenesis of Ads that interact with CD46.
  • Eri Mukai; Shimpei Fujimoto; Fuminori Sakurai; Kenji Kawabata; Manabu Yamashita; Nobuya Inagaki; Hiroyuki Mizuguchi
    JOURNAL OF CONTROLLED RELEASE 119 1 136 - 141 2007年05月 [査読有り]
     
    We investigated the efficiency of gene transduction into murine pancreatic islets using the adenovirus (Ad) vector. Western blotting analysis showed that mouse pancreatic islets express coxsackievirus and adenovirus receptor, a receptor for Ad. Nevertheless, gene expression after transduction of the Ad vector in vitro was observed only in the periphery of the islets, probably due to physical obstruction against Ad infection of the cells in the inside of islets. Ca2+-free treatment before the Ad vector transduction enhanced transduction efficiency in the islets, but not the cells in the inside of islets. The Ad vector transduction through the celiac artery in vivo and then cultivation of islets in vitro resulted in efficient transduction even in the inside of islets. Thus we propose a new strategy for efficient gene transfer to pancreatic beta-cells. (c) 2007 Elsevier B.V All rights reserved.
  • Naoya Koizumi; Tomoko Yamaguchi; Kenji Kawabata; Fuminori Sakurai; Tomomi Sasaki; Yoshiteru Watanabe; Takao Hayakawa; Hiroyuki Mizuguchi
    JOURNAL OF IMMUNOLOGY 178 3 1767 - 1773 2007年02月 [査読有り]
     
    Adenovirus (Ad) vectors are one of the most commonly used viral vectors in gene therapy clinical trials. However, they elicit a robust innate immune response and inflammatory responses. Improvement of the therapeutic index of Ad vector gene therapy requires elucidation of the mechanism of Ad vector-induced inflammation and cytokine/chemokine production as well as development of the safer vector. In the present study, we found that the fiber-modified Ad vector containing poly-lysine peptides in the fiber knob showed much lower serum IL-6 and aspartate aminotransferase levels (as a maker of liver toxicity) than the conventional Ad vector after i.v. administration, although the modified Ad vector showed higher transgene production in the liver than the conventional Ad vector. RT-PCR analysis showed that spleen, not liver, is the major site of cytokine, chemokine, and IFN expression. Splenic CD11c(+) cells were found to secret cytokines. The tissue distribution of Ad vector DNA showed that spleen distribution was much reduced in this modified Ad vector, reflecting reduced IL-6 levels in serum. Liver toxicity by the conventional Ad vector was reduced by anti-IL-6R Ab, suggesting that IL-6 signaling is involved in liver toxicity and that decreased liver toxicity of the modified Ad vector was due in part to the reduced IL-6 production. This study contributes to an understanding of the biological mechanism in innate immune host responses and liver toxicity toward systemically administered Ad vectors and will help in designing safer gene therapy methods that can reduce robust innate immunity and inflammatory responses.
  • Haruna Sakurai; Fuminoni Sakurai; Kenji Kawabata; Tomomi Sasaki; Naoya Koizumi; Haiying Huang; Katsuhisa Tashiro; Shinnosuke Kurachi; Shinsaku Nakagawa; Hiroyuki Mizuguchi
    JOURNAL OF CONTROLLED RELEASE 117 3 430 - 437 2007年02月 [査読有り]
     
    Vectors for gene expression are the essential tools for both gene therapy and basic research. There are two groups of gene therapy vectors, viral and non-viral vectors. At present, toxicity triggered by vectors is one of the major concerns for clinical trials. In general, non-viral vectors, such as plasmid DNA-cationic liposome complex (lipoplex), are thought to be safer than viral vectors, such as adenovirus (Ad) vector, although lipoplex is less efficient in term of gene expression than the Ad vector. However, there has been no study directly comparing the gene expression efficiency and safety of viral and non-viral vectors. Here, we present evidence that the Ad vector shows much more efficient gene expression and is safer than lipoplex, at least with respect to the innate immune response. After being systemically administered to mice, the Ad vector showed a transduction efficiency that was 2 to 5 log orders higher than that of lipoplex, depending on the organ. On the other hand, surprisingly, the administration of lipoplex produced a greater amount of inflammatory cytokines such as interleukin-6, interleukin-12, and tumor necrosis factor-alpha than did the administration of the Ad vector, whereas a comparable level of hepatotoxicity was induced by these vectors. The production of inflammatory cytokines induced by the injection of lipoplex was reduced when the CpG motifs were removed completely from plasmid DNA. Thus, care should be taken to ensure the innate immune response induced by gene therapy vectors, especially lipoplex. (c) 2007 Elsevier B.V. All rights reserved.
  • Kenji Kawabata; Fuminori Sakurai; Hiroyuki Mizuguchi
    Drug Delivery System 22 2 148 - 154 2007年 [査読有り]
     
    The application of adenovirus (Ad) vectors, which are widely used in gene therapy, depends on CAR (coxsackievirus and adenovirus receptor) expression on the cells. To overcome this problem, the capsid proteins of Ad vectors have been genetically modified. Here, we introduce several types of capsid-modified Ad vectors. Furthermore, we describe the application of capsid-modified Ad vectors into some kinds of stem cells for regenerative medicine. © 2007, THE JAPAN SOCIETY OF DRUG DELIVERY SYSTEM. All rights reserved.
  • Hiroyuki Mizuguchi; Naoko Funakoshi; Tetsuji Hosono; Fuminori Sakurai; Kenji Kawabata; Teruhide Yamaguchi; Takao Hayakawa
    HUMAN GENE THERAPY 18 1 74 - 80 2007年01月 [査読有り]
     
    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 El deletion region of the Ad vector plasmid by in vitro ligation or homologous recombination in Escherichia cola, 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. Luciferase and p53 genes were efficiently knocked down by Ad vectors generated by the new method and expressing siRNA against the target gene. This method should be useful for RNA interference-based experiments, and should make it easy to construct an siRNA-expressing Ad vector library for functional screening.
  • Fuminori Sakurai; Kenji Kawabata; Hiroyuki Mizuguchi
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 126 11 1013 - 1019 2006年11月 [査読有り]
     
    Recombinant Adenovirus (Ad) vectors are considered to be a promising gene delivery vehicle of high utility because they are easy to construct, can be produced at high titers, and efficiently transduce various types of cells. Ad vectors commonly used in the world, including clinical trials, are composed of Ad serotype 5 (AM), which belongs to subgroup C. In recent years, however, it has become apparent that Ad5 vectors have some drawbacks, such as high seroprevalence of anti-Ad5 antibodies in adults and low transduction efficiencies of Ad5 vectors in cells lacking a primary receptor for Ad5, coxsackievirus and adenovirus receptor (CAR). To overcome these limitations of Ad5 vectors, we have developed a novel type of Ad vector, which is composed of Ad serotype 35 (AM), belonging to subgroup B. Ad35 vectors recognize human CD46, not CAR, as a cellular receptor for infection. Human CD46 is expressed in almost all of human cells, leading to a broad tropism of Ad35 vectors to human cells, in contrast, expression of rodent CD46 is limited to the testis. Therefore, in vivo transduction properties of Ad35 vectors are not appropriately evaluated in normal mice. In order to evaluate the in vivo transduction properties of Ad35 vectors, Ad35 vectors were applied to human CD46-transgenic mice and nonhuman primates, which express CD46 in a similar pattern to humans. The data obtained using CD46-transgenic mice and nonhuman primates would provide valuable information towards clinical applications of Ad35 vectors.
  • Fuminori Sakurai; Sayaka Murakami; Kenji Kawabata; Naoki Okada; Akira Yamamoto; Tsukasa Seya; Takao Hayakawa; Hiroyuki Mizuguchi
    Journal of controlled release : official journal of the Controlled Release Society 113 3 271 - 8 2006年07月 [査読有り]
     
    Human CD46 (membrane cofactor protein) has recently been identified to be an attachment receptor for subgroup B adenoviruses (Ads); however, the precise interaction between human CD46 and subgroup B Ads are just beginning to be understood. In this study, to characterize the interaction between human CD46 and subgroup B Ads, varieties of mutant CD46 were tested for their ability to act as a receptor for Ad serotype 35 (Ad35), which belongs to subgroup B. In addition, we determined Ad35 vector-mediated transgene expression and cellular uptake of Ad35 vectors in the presence of a set of anti-CD46 antibodies. Our data demonstrated that the short consensus repeats (SCRs) 1 and 2 in human CD46 are important for interaction with Ad35, whereas the cytoplasmic domain of human CD46 was found not to be required for the function as an Ad35 receptor. Rather, a complete deletion of the cytoplasmic domain of human CD46 increased the transduction efficiencies of Ad35 vectors. This information should help in elucidation of the mechanism of subgroup B Ad infection, as well in the improvement of the subgroup B Ad vectors.
  • F Sakurai; K Kawabata; N Koizumi; N Inoue; M Okabe; T Yamaguchi; T Hayakawa; H Mizuguchi
    GENE THERAPY 13 14 1118 - 1126 2006年07月 [査読有り]
     
    We previously demonstrated that systemic administration of adenovirus serotype 35 ( Ad35) vectors to mice does not mediate efficient transduction in organs, probably because expression of the mouse analog of the subgroup B Ad receptor, human CD46 ( membrane cofactor protein), is limited to the testis. Here, we describe the in vitro and in vivo transduction characteristics of Ad35 vectors by using homozygous and hemizygous human CD46-transgenic ( CD46TG) mice, which ubiquitously express human CD46. An Ad35 vector more efficiently transduced the primary dendritic cells and macrophages prepared from CD46TG mice than those from wild-type mice. In vivo transduction experiments demonstrated that CD46TG mice are more susceptible to Ad35 vector-mediated in vivo transduction than are wild-type mice. In particular, homozygous CD46TG mice, which express higher levels of CD46 in the organs than hemizygous CD46TG mice, tend to exhibit higher transduction efficiencies after intraperitoneal administration than hemizygous CD46TG mice. Intraperitoneal administration of Ad35 vectors resulted in efficient transduction into the mesothelial cells of the peritoneal organs in homozygous CD46TG mice. These results indicate that an Ad35 vector recognizes human CD46 as a cellular receptor in CD46TG mice. However, the in vivo transduction efficiencies of Ad35 vectors in CD46TG mice are much lower than those of conventional Ad5 vectors in wild-type mice.
  • N Koizumi; K Kawabata; F Sakurai; Y Watanabe; T Hayakawa; H Mizuguchi
    HUMAN GENE THERAPY 17 3 264 - 279 2006年03月 [査読有り]
     
    Coxsackievirus and adenovirus receptor (CAR), alpha(v) integrins, and heparan sulfate glycosaminoglycans (HSGs) are the tropism determinants of adenoviral (Ad) vectors in vivo. For the development of a targeted Ad vector, its broad tropism needs to be blocked (or reduced). We have previously developed Ad vectors with ablation of CAR, alpha(v) integrin, and HSG binding by mutation of the FG loop in the fiber knob (deletion of T489, A490, Y491, and T492 of the fiber protein), deletion of the RGD motif of the penton base, and substitution of the fiber shaft domain for that derived from Ad type 35, respectively, and have shown that this triple-mutant Ad vector [Ad/Delta F(FG)Delta P-S35-L2] exhibits significantly lower transduction in mouse liver compared with the conventional Ad vector [Koizumi, N., Mizuguchi, H., Sakurai, F., Yamaguchi, T., Watanabe, Y., and Hayakawa, T. ( 2003). J. Virol. 77, 13062-13072]. In the present study, we optimized the fiber knob mutation for further reduced in vivo transduction and examined toxicity of the modified Ad vectors. Ad/Delta F(AB)Delta P-S35-L2, a triple-mutant Ad vector containing a mutation of the AB loop in the fiber knob (R412S, A415G, E416G, and K417G), mediated approximately 15,000- and 500-fold lower mouse liver transduction by intravenous and intraperitoneal administration, respectively, than the conventional Ad vector, and mediated 10-fold lower mouse liver transduction than did Ad/Delta F(FG)Delta P-S35-L2. Ad/Delta F(AB)Delta P-S35-L2 also exhibited lower transduction of other organs compared with Ad/Delta F(FG)Delta P-S35-L2 and the conventional Ad vector. Levels of both liver serum enzymes (aspartate transferase [AST] and alanine transferase (ALT)] and interleukin (IL)-6 in mouse serum after intravenous administration of Ad/Delta F(AB)Delta P-S35-L2 were similar to those in the nontreatment mouse serum, whereas the conventional Ad vector led to high levels of AST, ALT, and IL-6. We therefore succeeded in further improving the mutant Ad vector, abolishing both viral natural tropism and toxicity. This new Ad vector appears to be a fundamental vector for targeted gene delivery.
  • Kenji Kawabata; Funlinori Sakurai; Naoya Koizumi; Takao Hayakawa; Hiroyuki Mizuguchi
    MOLECULAR PHARMACEUTICS 3 2 95 - 103 2006年03月 [査読有り]
     
    Stem cells, including embryonic stem (ES) cells, mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs), are defined by their capacity for self-renewal and multilineage differentiation. Efficient gene transfer into stem cells is essential for the basic research in developmental biology and for therapeutic applications in gene-modified regenerative medicine. Adenovirus (Ad) vectors, based on Ad type 5, can efficiently and transiently introduce the exogenous gene into many cell types via the primary receptor, coxsackievirus, and adenovirus receptor (CAR). However, some kinds of stem cells, such as MSCs and HSCs, cannot be efficiently transduced with conventional Ad vectors based on Ad serotype 5 (Ad5), because of the lack of CAR expression. To overcome this problem, fiber-modified Ad vectors and an Ad vector based on another serotype of Ad have been developed. Here, we review the advances in the development of Ad vectors suitable for stem cells and discuss their application in basic biology and clinical medicine.
  • H Mizuguchi; ZL Xu; F Sakurai; K Kawabata; T Yamaguchi; T Hayakawa
    JOURNAL OF CONTROLLED RELEASE 110 1 202 - 211 2005年12月 [査読有り]
     
    Previously, we developed single adenovirus (Ad) vectors that contained the gene of interest in the E I deletion region and the transactivator gene for the tetracycline-controllable expression system in the E3 deletion region. In the present study, we improved the Ad vector-mediated tetracycline-control table expression system by the fiber modification of Ad. We developed fiber-modified Ad vectors containing the tet-off system, which are effective in overcoming the limitations of conventional Ad vectors, specifically their inefficient gene transfer into cells lacking the primary receptor, the coxsackievirus and adenovirus receptor (CAR). Ad vectors containing the tet-off system with an Arg-Gly-Asp (RGD) peptide in the HI loop of the fiber knob or the Ad type 35 fiber greatly improved transduction efficiency (more than 1-2-log orders) into the cells lacking CAR expression but expressing alpha v integrin or CD46, respectively. They exhibited vastly higher regulation of gene expression by doxycycline. The combination of fiber-modified Ad vectors and the tetracycline-controllable expression system should offer a powerful tool for gene therapy and gene transfer experiment. (c) 2005 Elsevier B.V. All rights reserved.
  • F Sakurai; K Kawabata; T Yamaguchi; T Hayakawa; H Mizuguchi
    GENE THERAPY 12 19 1424 - 1433 2005年10月 [査読有り]
     
    Adenoviral gene transfer to hematopoietic stem cells (HSCs)/ progenitors would provide a new approach to the treatment of hematopoietic diseases and study of the hematopoietic system. We have previously reported that an adenovirus ( Ad) vector composed of whole Ad serotype 35 (Ad35), which belongs to subgroup B, shows efficient gene transfer into human bone marrow CD34(+) cells. However, Ad35 vector-mediated transduction into human HSCs/progenitors has not yet been fully optimized. In the present study, we have systematically examined promoter activity in the context of Ad35 vectors in human bone marrow CD34+ cells and primitive CD34+ subsets to optimize the transduction efficiency in human hematopoietic stem/progenitor cells. In the first of the transduction experiments, the improved in vitro ligation method was applied to Ad35 vector construction to allow for simple and efficient production of an E1/E3-deleted Ad35 vector. Using this method, we constructed a series of Ad35 vectors encoding the enhanced green fluorescence protein (GFP) under the control of a variety of strong viral and cellular promoters. Of the six types of promoters tested, significantly higher transduction efficiencies were achieved with the human elongation factor 1 alpha promoter (EF1 alpha promoter), the human cytomegalovirus (CMV) immediateearly 1 gene enhancer/beta-actin promoter with beta-actin intron ( CA promoter), and the CMV promoter/enhancer with the largest intron of CMV ( intron A) (CMVi promoter) in the human CD34(+) cells and the immature subsets (CD34(+)CD38(low)/ and CD34(+) AC133(+) subsets). In particular, the CA promoter was found to allow for the highest transduction efficiencies in both the whole human CD34+ cells and the immature hematopoietic subsets. Furthermore, the CA promoter-mediated GFP-expressing cells differentiated into progenitor cells of all lineages. These results indicate the construction of an optimized Ad35 vector backbone for efficient transduction into HSCs/progenitors.
  • 水口 裕之; 川端 健二; 櫻井 文教; 早川 堯夫
    炎症・再生 : 日本炎症・再生医学会雑誌 = Inflammation and regeneration 25 5 447 - 451 The Japanese Society of Inflammation and Regeneration 2005年09月 
    Efficient gene transfer into stem cells which are able to self-renew and differenciate into certain type of cell is essential for not only difining the precise molecular mechanism of self-renewal and differenciation, but the supplying of the cells for regenerative medicine. In this paper, we review our approach to the efficient gene transfer into hematopoietic stem cell, mesenchymal stem cell, and ES cell by modified adenovirus vectors.
  • K Kawabata; F Sakurai; T Yamaguchi; T Hayakawa; H Mizuguchi
    MOLECULAR THERAPY 12 3 547 - 554 2005年09月 [査読有り]
     
    Efficient and transient gene transfer into embryonic stem (ES) cells is expected to be of use for basic studies in developmental biology and for applications in regenerative medicine. Here, we report the development of an adenovirus (Ad) vector that efficiently expresses foreign genes in mouse ES (mES) cells. We prepared four LacZ-expressing Ad vectors, each of which contained one of the following: Rous sarcoma virus (RSV), cytomegalovirus (CMV), beta-actin promoter/CMV enhancer (CA), or EF-1 alpha promoter. While the RSV and CMV promoters were inactive in mES cells, the CA and EF-1 alpha promoters strongly drove LacZ expression in more than 90% of the mES cells. The EF-1 alpha promoter was found to be slightly more efficient than the CA promoter. mES cells were found to express the Ad primary receptor, coxsackievirus and adenovirus receptor, suggesting that while Ad vectors could introduce the exogenous gene into mES cells, the choice of a suitable promoter was critical for efficient gene expression. Fiber-mutant Ad vectors containing RGD or polylysine peptide on the fiber knob mediated efficient LacZ expression, not only in mES cells, but also in feeder cells. Exogenous expression of Oct-3/4 or the dominant-negative mutant of STAT3 (STAT3F) by conventional Ad vectors containing the EF-1 alpha, promoter promoted the differentiation of mES cells into the cells of three germ layers, and STAT3F-mediated differentiation was rescued by the coexpression of Nanog. These results suggest that Ad vectors can be used for basic research using ES cells and that they may be of great utility for therapeutic applications in gene-modified regenerative medicine based on ES cells.
  • H Mizuguchi; T Sasaki; K Kawabata; F Sakurai; T Hayakawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 332 4 1101 - 1106 2005年07月 [査読有り]
     
    Human mesenchymal stem cells (hMSCs) are considered a source of cells for regenerative medicine, and cell and gene therapy. Efficient gene transfer into hMSCs is essential for basic investigations into cellular differentiation and developmental biology, and for therapeutic applications in gene-modified regenerative medicine. In the present study, we optimized the transduction of hMSCs by means of fiber-modified adenovirus (Ad) vectors. Among the various types of Ad vectors tested, the polylysine modification of the C-terminal of the fiber knob most markedly improved the efficiency of hMSC transduction. At 300 vector particles per cell of polylysine-modified Ad vectors, more than 95% of the hMSCs expressed transgene. In this condition, polylysine-modified Ad vectors mediated 460-fold more transgene activity than the conventional Ad vectors. Ad vectors containing the Ad type 35 fiber or an Arg-Gly-Asp (RGD) peptide in the fiber knob mediated 130 or 16 timed, respectively, the transgene activity mediated by the conventional Ad vectors. We also examined the efficiency of transduction into adipogenic-differentiated hMSCs. In this latter case, only Ad vectors containing the Ad type 35 fiber showed efficient gene expression. These results showed that fiber-modified Ad vectors could become a potent tool for basic research into, and the therapeutic application of, hMSCs and adipogenic-differentiated hMSCs. (c) 2005 Elsevier Inc. All rights reserved.
  • ZL Xu; H Mizuguchi; F Sakurai; C Koizumi; T Hosono; K Kawabata; Y Watanabe; T Yamaguchi; T Hayakawa
    ADVANCED DRUG DELIVERY REVIEWS 57 5 781 - 802 2005年04月 [査読有り]
     
    Adenovirus (Ad) vectors have been expected to play a great role in gene therapy because of their extremely high transduction efficiency and wide tropism. However, due to the intrinsic deficiency of their immunogenic toxicities, Ad vectors are rapidly cleared from the host, transgene expression is transient, and readministration of the same serotype Ad vectors is problematic. As a result, Ad vectors are continually undergoing refinement to realize their potential for gene therapy application. Even after 1999, when a patient fatally succumbed to the toxicity associated with Ad vector administration at a University of Pennsylvania (U.S.) experimental clinic, enthusiasm of gene therapists for Ad vectors has not waned. With great efforts from various research groups, significant advances have been achieved through comprehensive approaches to improving the kinetics of Ad vector-delivered genes and gene products. (c) 2005 Elsevier B.V. All rights reserved.
  • Fuminori Sakurai; Yoshinobu Takakura; Mitsuru Hashida
    Non-viral Gene Therapy: Gene Design and Delivery 339 - 347 2005年 [査読有り]
     
    The success of gene therapy is largely dependent on the development of gene transfer vector systems. So far, various types of gene delivery vectors, both viral and nonviral, have been developed.Among these, synthetic non-viral vectors, such as cationic lipids and cationic polymers, which are electrostatically complexed with plasmid DNA, are particularly suitable for in vivo gene delivery due to their simplicity of preparation and the absence of a specific immune response. Some of these non-viral vectors have shown promise in in-vivo gene delivery settings (Dow et al. 1999 Hood et al. 2002, Sakurai et al. 2003 Yamada et al. 2003).
  • T Hosono; H Mizuguchi; K Katayama; ZL Xu; F Sakurai; A Ishii-Watabe; K Kawabata; T Yamaguchi; S Nakagawa; T Mayumi; T Hayakawa
    HUMAN GENE THERAPY 15 8 813 - 819 2004年08月 [査読有り]
     
    RNA interference (RNAi) is a powerful tool for the knockdown of gene expression. Here, we report on the development of an adenovirus (Ad)vector-mediated doxycycline (Dox)-inducible small interfering RNA (siRNA) expression system. We used this siRNA system to control the expression of p53 and c-Myc in human cancer cells. Coinfection of Ad vectors containing the siRNA expression system under the control of the Dox-inducible H1 promoter and Ad vectors expressing a tetracycline repressor inhibited the expression levels of p53 and c-Myc in a dose-dependent manner with both Dox and viral dose. Regulated silencing of p53 and c-Myc expression was obtained. Because an Ad vector-mediated inducible RNAi system can efficiently transduce a variety of cell types in vitro and in vivo, and the degree of loss of gene expression can be modulated according to the dose of Dox, this expression system should be a useful tool for both basic research on the analysis of gene function and therapeutic applications of RNAi.
  • N Koizumi; H Mizuguchi; F Sakurai; T Yamaguchi; Y Watanabe; T Hayakawa
    JOURNAL OF VIROLOGY 77 24 13062 - 13072 2003年12月 [査読有り]
     
    The primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, alphav integrins, are the tropism determinants of adenovirus (Ad) type 5. Inhibition of the interaction of both the fiber with CAR and the penton base with the alphav integrin appears to be crucial to the development of targeted Ad vectors, which specifically transduce a given cell population. In this study, we developed Ad vectors with ablation of both CAR and alphav integrin binding by mutating the fiber knob and the RGD motif of the penton base. We also replaced the fiber shaft domain with that derived from Ad type 35. High transduction efficiency in the mouse liver was suppressed approximately 130- to 270-fold by intravenous administration of the double-mutant Ad vectors, which mutated two domains each of the fiber knob and shaft and the RGD motif of the penton base compared with those of conventional Ad vectors (type 5). Most significantly, the triple-mutant Ad vector containing the fiber knob with ablation of CAR binding ability, the fiber shaft of Ad type 35, and the penton base with a deletion of the RGD motif mediated a >30,000-fold lower level of mouse liver transduction than the conventional Ad vectors. This triple-mutant Ad vector also mediated reduced transduction in other organs (the spleen, kidney, heart, and lung). Viral DNA analysis showed that systemically delivered triple-mutant Ad vector was primarily taken up by liver nonparenchymal cells and that most viral DNAs were easily degraded, resulting in little gene expression in the liver. These results suggest that the fiber knob, fiber shaft, and RGD motif of the penton base each plays an important role in Ad vector-mediated transduction to the mouse liver and that the triple-mutant Ad vector exhibits little tropism to any organs and appears to be a fundamental vector for targeted Ad vectors.
  • F Sakurai; H Mizuguchi; T Yamaguchi; T Hayakawa
    MOLECULAR THERAPY 8 5 813 - 821 2003年11月 [査読有り]
     
    We have recently developed a replication-defective, recombinant adenovirus (Ad) vector composed of the whole Ad serotype 35 (Ad35), a member of subgroup B. We describe herein the in vitro and in vivo gene transfer properties of Ad35 vector in comparison with Ad serotype 5 (Ad5) and the Ad5F35 vector, which is a fiber-substituted Ad5 vector containing Ad35 fiber proteins. In vitro, Ad35 vector efficiently transduced not only human CAR-positive cells but also CAR-negative cells. Following intravenous administration into mice, both Ad5 and Ad35 vectors were rapidly cleared from the bloodstream with a half-life of approximately 3 min. Ad5 vector-mediated transgene expression predominantly occurred in liver parenchymal cells, although the Ad5 vector was delivered to both liver parenchymal and nonparenchymal cells. In contrast, Ad35 vector was efficiently taken up by liver nonparenchymal cells and mediated transduction efficiency in the liver on a level 4 log orders lower than the Ad5 vector. These findings demonstrate that Ad35 vector is an attractive vehicle for gene transfer into human cells, while the biodistribution profile of Ad35 vector in mice is much different from that of the Ad5 vector.
  • F Sakurai; T Terada; M Maruyama; Y Watanabe; F Yamashita; Y Takakura; M Hashida
    CANCER GENE THERAPY 10 9 661 - 668 2003年09月 [査読有り]
     
    We have evaluated and compared the efficacy of systemic administration of lipoplex formulations containing plasmids encoding IFN-beta or IFN-gamma, and a synthetic double-strand RNA poly I: poly C (pI: pC), a type I IFN inducer, in a lung metastasis model in which colon carcinoma CT-26 cells were inoculated intravenously into immunocompatible mice. Injection of lipoplexes containing plasmid DNA, regardless of IFN gene insertion, stimulated a transient increase in the serum concentration of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and IFN-gamma, while injection of lipoplexes containing pI: pC led to a low level of TNF-alpha and undetectable IFN-gamma production. Furthermore, injection of these lipoplexes containing plasmids resulted in the production of a mixture of type I and type II IFNs, partly derived from the inserted IFN genes, in lung tissue cultures. In tumor-prophylactic experiments, intravenous injection of lipoplexes containing plasmid, regardless of IFN gene insertion, showed a significant reduction in lung metastatic nodules probably due to proinflammatory cytokines such as TNF-alpha and IFN-gamma nonspecifically induced by the CpG motifs in the plasmid and the type I IFNs produced. On the other hand, the antimetastatic effect of pI: pC-lipoplex seemed to be due mainly to IFN-beta induced by pI: pC. In established lung metastasis experiments, a single intravenous administration of lipoplexes containing IFN-beta gene or pI: pC, but not other lipoplexes, showed a significant therapeutic effect on the tumor metastasis: reduction in tumor nodules and prolongation of survival time of tumor-burden mice. The therapeutic effects were specifically impaired by anti-IFN-beta antibody treatment, indicating that IFN-beta produced by the lipoplexes played an important role in the suppression of established metastatic lung tumors. Thus, the local IFN-beta in the lung delivered by intravenous administration of lipoplex containing IFN-beta gene or pI: pC may be a convenient and useful method of inhibiting established metastatic lung tumors.
  • H Mizuguchi; ZL Xu; F Sakurai; T Mayumi; T Hayakawa
    HUMAN GENE THERAPY 14 13 1265 - 1277 2003年09月 [査読有り]
     
    We previously developed single adenovirus ( Ad) vectors that contained the components for a tetracycline-regulatable gene-expression system in the E1 and E3 deletion regions, and showed that the Ad vectors containing the tet-on system exhibit a much inferior regulation of transgene expression than those containing the tet-off system. In many cases, the tet-on system may be preferable because of its positive regulation of transgene expression. To this end, in the present study, by introducing the latest generation reverse tetracycline-responsive transcriptional activator (rtTA2(s)-M2 or rtTA2(s)-S2) and the tetracycline-controlled transcriptional silencer (tTS) into the original tet-on system, we constructed various modified Ad-mediated tet-on systems. Among them, the novel single Ad vector, which contained three heterologous gene-expression cassettes of the gene of interest, rtTA2(s)-S2, and tTS in the E1 deletion region, the E3 deletion region, and the region between E4 and 3'ITR, respectively, displayed vastly improved doxycycline-inducible gene expression in terms of low basal expression, high induced expression, and high responsiveness to doxycycline both in vitro and in vivo. These results also suggest that the low responsiveness to doxycycline may explain why the original tet-on system in the context of the Ad vector is not effective in vivo. This is the first report describing the cloning of three heterologous gene-expression cassettes into three separate regions of the E1/E3-deleted Ad genome. This improved Ad-mediated tet-on system might be useful for gene therapy and greatly facilitate the analyses of gene function.
  • F Sakurai; H Mizuguchi; T Hayakawa
    GENE THERAPY 10 12 1041 - 1048 2003年06月 [査読有り]
     
    Efficient gene transfer into human hematopoietic stem cells (HSCs) is the most important requirement for gene therapy of hematopoietic disorders and for study of the hematopoietic system. An adenovirus (Ad) vector based on the Ad serotype 5 (Ad5) is known to transduce HSCs, including CD34(+) cells, with very low efficiency because of low-level expression of its primary receptor, coxsackievirus and adenovirus receptor (CAR). In the present study, we developed a recombinant Ad vector composed of the whole Ad serotype 35 (Ad35), which recognizes an unidentified receptor different from CAR for its infection. A transduction study showed that the Ad35-based vectors exhibit a higher transduction efficiency in human CD34(+) cells than the conventional Ad5 vectors and the Ad5F35 vectors, which are fiber-substituted Ad5 vectors containing Ad35 fiber proteins. The mean of fluorescence intensity in the CD34+ cells transduced with the Ad35 vectors was 12-76 and 1.4-3 times higher than that in the cells transduced with the Ad5 and Ad5F35 vectors, respectively. The percentages of green fluorescent protein (GFP)-positive CD34(+) cells by transduction with Ad35, Ad5, and Ad5F35 vectors expressing GFP at 300 PFU/cell were 53%, 5%, and 52%, respectively, suggesting that Ad35 vectors mediate a more efficient gene transfer into human CD34(+) cells than Ad5 and Ad5F35 vectors, although the percentage of transduced cells was similar between Ad35 and Ad5F35 vectors. The Ad vector based on Ad35 could be very useful in gene therapy for blood disorders and gene transfer experiments using HSCs. Gene Therapy (2003) 10, 1041-1048. doi: 10.1038/sj.gt.3301959.
  • F Sakurai; T Terada; K Yasuda; F Yamashita; Y Takakura; M Hashida
    GENE THERAPY 9 16 1120 - 1126 2002年08月 [査読有り]
     
    Recent studies have demonstrated that intravenous administration of a plasmid DNA-cationic liposome complex (lipoplex) induced significant proinflammatory cytokine production in blood and inhibited transgene expression in pulmonary endothelial cells. In this study, we examined the effects of gadolinium chloride (GdCl3) pretreatment on the biodistribution and induction of proinflammatory cytokine production and transgene expression after intravenous injection of a lipoplex In mice. GdCl3 is known to transiently deplete liver Kupffer cells and spleen macrophages after intravenous administration. Intravenous administration of a lipoplex triggers high levels of proinflammatory cytokine production, such as TNF-alpha, IFN-gamma and IL-12 in serum and a large amount of P-32-labeled lipoplex accumulates in the liver 1 h after intravenous administration. However, pretreatment with GdCl3 dramatically reduces serum levels of these cytokines and liver accumulation of the lipoplex. RT-PCR analysis showed that mRNA expression of TNF-alpha greatly increases in the liver and spleen after lipoplex injection and that pretreatment with GdCl3 reduces mRNA expression in these organs. Messenger RNA expression of TNF-alpha in the liver occurs in non-parenchymal cells (sinusoidal endothelial cells and/or Kupffer cells). Inhibition of cytokine production by pretreatment with GdCl3 leads to recovery of transgene expression in the lung following the second injection of lipoplex, which was reduced following the first injection of lipoplex. Thus, the present study demonstrates that tissue macrophages involving liver Kupffer cells and spleen macrophages are closely involved in TNF-alpha production following i.v. administration of the lipoplex. It is also suggested that avoiding lipoplex uptake and subsequent cytokine production by these cells would be a useful method of maintaining a high level of gene expression in the lung after repeated injections.
  • F Sakurai; T Nishioka; F Yamashita; Y Takakura; M Hashida
    EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS 52 2 165 - 172 2001年09月 [査読有り]
     
    Plasmid DNA-cationic liposome complexes (lipoplexes) accumulate in the lung to a great extent immediately after intravenous administration, and gene expression occurs predominantly in the lung. However, the detailed mechanisms underlying the lung accumulation of lipoplexes are not fully understood. In this study, we investigated the effect of blood components on the lung accumulation of lipoplexes using a single-pass rat lung perfusion system. Two types of lipoplexes, Chol-containing lipoplex ([P-32]DNA-DOTMA/Chol liposome complex) and DOPE-containing lipoplex ([P-32]DNA-DOTMA/DOPE liposome complex), pre-incubated with whole blood, serum, or erythrocytes, were injected into the perfused lung via an artery. Similarly to in vivo observations, extensive lung accumulation was observed for both types of lipoplexes after incubation with whole blood during a single passage. The P-32-labeled lipoplexes pre-incubated with erythrocytes showed similar lung accumulation, whereas their lung accumulation after incubation with serum was significantly reduced, suggesting that erythrocytes would be more responsible blood components for extensive uptake by the perfused lung. However, there was a clear difference in the amounts of the accumulated erythrocytes after intra-arterial injection between the two lipoplex formulations. A significant degree of erythrocyte accumulation was observed when the DOPE-containing lipoplex was injected, whereas the Chol-containing lipoplex failed to induce any significant erythrocyte accumulation in the lung. In vitro experiments showed that the major fraction of both lipoplexes was bound to erythrocytes. These data suggested that Chol-containing lipoplexes bound to erythrocytes before injection dissociate from the erythrocytes and are transferred to the lung capillary endothelial cells during their passage through the lung. In contrast, DOPE-containing lipoplexes bound to erythrocytes cause aggregation and are embolized in the lung capillary with erythrocytes. Thus, the present study demonstrated that the interaction with erythrocytes plays an important role in the lung accumulation of lipoplexes and that neutral helper lipid significantly affects this interaction. (C) 2001 Elsevier Science B.V. All rights reserved.
  • F Sakurai; T Nishioka; H Saito; T Baba; A Okuda; O Matsumoto; T Taga; F Yamashita; Y Takakura; M Hashida
    GENE THERAPY 8 9 677 - 686 2001年05月 [査読有り]
     
    Recent studies have indicated that there are many barriers to successful systemic gene delivery via cationic lipid vectors using the intravenous route. The purpose of this study was to investigate the effect of binding and interaction between erythrocytes, a major constituent of blood cells, and the complexes, in relation to the role of the helper lipid, on the in vivo gene delivery to the lung following intravenous injection. We used three types of cationic lipid vectors, DNA-DOTMA/Chol liposome complexes, DNA-DOTMA liposome complexes, and DNA-DOTMA/DOPE liposome complexes. Although the three types of vectors bind to murine blood cells in vivo and in vitro. DOTMA/Chol and DOTMA complexes with a higher in vivo transfection activity do not induce fusion between erythrocytes, whereas DOTMA/DOPE complexes, a less efficient vector in vivo, induce fusion between the erythrocytes after a short incubation period. Pre-incubation of DOTMA/DOPE complexes with erythrocytes significantly reduced the transfection efficiency while DOTMA/Chol- and DOTMA complexes were more resistant to such treatment. The differences in the physicochemical and structural properties of these complexes could explain the differences in interaction with erythrocytes and subsequent gene expression. Lipids in DOTMA/Chol and DOTMA complexes have a stable lamellar structure. However, lipids in DOTMA/DOPE complexes have a highly curved structure with high fluidity. These results indicate that the interaction with erythrocytes depends on the properties of the cationic lipid vectors and this is an important factor for intravenous gene delivery using cationic lipid vectors.
  • F. Sakurai; R. Inoue; Y. Nishino; A. Okuda; O. Matsumoto; T. Taga; F. Yamashita; Y. Takakura; M. Hashida
    Journal of Controlled Release 69 1 219 - 220 2000年10月 [査読有り]
  • F Sakurai; R Inoue; Y Nishino; A Okuda; O Matsumoto; T Taga; F Yamashita; Y Takakura; M Hashida
    JOURNAL OF CONTROLLED RELEASE 66 2-3 255 - 269 2000年05月 [査読有り]
     
    In order to identify the important factors involved in cationic liposome-mediated gene transfer, in vitro transfection efficiencies by plasmid DNA complexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios were evaluated using four types of cultured cells with respect to their physicochemical properties. Significant changes were observed in the particle size and zeta potential of the complexes as well as in their structures, assessed by atomic force microscopy, which depended on the mixing ratio. In transfection experiments, except for RAW 264.7 cells (mouse macrophages), efficient gene expression was obtained in MET-2 cells (mouse bladder tumor), NLH3T3 cells (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) at an optimal ratio of 1:5, 1:7.5 or 1:5, respectively. On the other hand, cellular uptake of the [P-32]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected by the transfection efficiency. The cellular damage determined by MTT assay was minimal even at the highest DNA/liposome ratio (1:10), indicating that the lower gene expression level at the higher ratio was not due to cytotoxicity induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition of negatively charged liposomes, was restricted as the mixing ratio increased. Furthermore, confocal microscopic studies using HUVEC showed that the 1:5 complexes exhibited a dispersed distribution in the cytoplasm whereas a punctuate intracellular distribution was observed for the 1:10 complexes. This suggests that there was a significant difference in intracellular trafficking, probably release from the endosomes or lysosomes, of the plasmid DNA/cationic liposome complexes between these mixing ratios. Taken together, these findings suggest that the DNA/liposome mixing ratio significantly affects the intracellular trafficking of plasmid DNA complexed with the cationic liposomes, which is an important determinant of the optimal mixing ratio in cationic liposome-mediated transfection. (C) 2000 Elsevier Science B.V. All rights reserved.
  • 櫻井 文教; 山下 富義; 高倉 喜信; 橋田 充
    薬物動態 15 112 - 113 The Japanese Society for the Study of Xenobiotics 2000年 
    Cationic liposome-mediated transfection is a useful method for in vitro and in vivo gene transfer. However, there is little information on the relationship between physicochemical and structural properties of DNA-cationic liposome complexes and their transfection efficiency. In this study, we have systemically studied the relationship between various properties of DNA-cationic liposome complexes and their transfection efficiency in vivo and in vitro. Our results demonstrated that for both in vitro and in vivo transfection, structural and physicochemical characteristics of the complexes were significantly related to their transfection efficiency. In in vitro transfection, the charge ratio between DNA and cationic liposome significantly affects the release of plasmid DNA complexed with the cationic liposomes from the complexes, which is an important determinant of the optimal charge ratio. For in vivo intravenous transfection, Chol-containing complexes with a higher transfection activity showed a stable lamellar structure and was more stable to the interaction with erythrocytes. DOPE-containing complexes, a less efficient vector, induced the fusion between erythrocytes due to its fusogenic property and lost their transfection activity. These results indicate that the interaction with erythrocytes is an important factor for intravenous gene delivery.
  • Hiromitsu Aoki; Tsuneaki Tottori; Fuminori Sakurai; Kaoru Fuji; Koichiro Miyajima
    International Journal of Pharmaceutics 156 2 163 - 174 1997年10月 [査読有り]
     
    The effects of positive charge density on the liposomal surface on the disposition kinetics of liposomes in rats were investigated. The cationic liposomes with zeta potentials of about +15 mV remained in the blood longer than did the neutral liposomes, and the hepatic uptake of these liposomes decreased. The blood clearance of the liposomes with zeta potentials under +10 mV was comparable to that of the neutral liposomes. In contrast, the blood circulation of the liposomes with a higher positive charge density, above +25 mV, was shortened and their hepatic uptake was almost the same as that of the neutral liposomes. The optimum value of positive charge density on the liposomal surface to prolong the residency of liposomes in the blood circulation was thus determined. A liver perfusion experiment showed that the uptake of cationic liposomes with a zeta potential of about +15 mV was effectively suppressed in the presence of erythrocytes, while that of liposomes with a higher zeta potential were little affected. Thus, above +15 mV, the suppressive effect of erythrocytes on the hepatic uptake of cationic liposomes decreased with the increase of the positive charge on the liposomal surface. These results cannot be explained by the binding model, and we therefore propose the ionic atmosphere model: Cationic liposomes surround the erythrocytes with a negative surface charge like the ion atmosphere of the Debye-Huckel theory. The cationic liposomes with a suitable positive charge surround the erythrocytes as an ionic atmosphere and could then escape the reticuloendothelial system (RES). The higher positively charged liposomes were taken up by the liver probably due to the shield of the negative charge of erythrocytes provided by the cationic liposomal atmosphere.

書籍

  • 腫瘍溶解性ウイルスによる抗腫瘍免疫の活性化とがん免疫療法との併用
    櫻井文教; 水口裕之 Drug delivery system 2023年01月
  • アデノウイルスベクターワクチンの現状と展望
    水口裕之; 立花雅史; 櫻井文教 Drug delivery system 2022年11月
  • ヒト35型アデノウイルスを基本骨格とした腫瘍溶解性アデノウイルスの開発
    櫻井文教; 小野良輔; 水口裕之 (担当:共著範囲:)BIO Clinica 2022年06月
  • アデノウイルスってどんなものでしたっけ？
    櫻井文教 日経メディカル（オンライン掲載） 2021年04月
  • ヒトiPS細胞由来肝細胞のB型肝炎ウイルス感染評価系への応用
    櫻井文教; 水口裕之 ファルマシア 2020年12月
  • CHOPCHOP v3 を用いた CRISPR Cas9 の標的配列検 索方法
    塚本智仁; 酒井英子; 櫻井文教; 水口裕之 Drug Delivery System 2020年07月
  • アデノウイルス由来小分子RNAによる ウイルス増殖促進機構の解明と遺伝子組換えウイルスへの応用
    若林圭作; 櫻井文教; 水口裕之 生産と技術 2019年10月
  • ウイルスを基盤とした遺伝子治療薬の臨床開発の現状と今後の展望
    櫻井文教 Drug Delivery System 2019年03月
  • microRNAによる遺伝子発現制御システムを搭載した遺伝子発現ベクター
    櫻井文教; 水口裕之 (担当:共著範囲:)ドラッグデリバリーシステムの新展開II 2012年
  • microRNAによる遺伝子発現制御システムを搭載したアデノウイルスベクターの開発
    櫻井文教; 水口裕之 (担当:共著範囲:)遺伝子医薬MOOK．臨床・創薬利用が見えてきたmicroRNA 2012年
  • ウイルスベクターと遺伝子治療
    水口裕之; 櫻井文教 (担当:共著範囲:)医薬ジャーナル 2012年
  • 改良型アデノウイルスベクターの開発と神経系細胞への遺伝子導入
    松井勇人; 櫻井文教; 形山和史; 水口裕之 日薬理誌 2011年02月
  • 組織特異的かつ長期安定的な遺伝子発現を目指した新規アデノウイルスベクター の開発
    櫻井文教 Drug Delivery System 2011年
  • 遺伝子組換えウイルスの安全性向上に向けた遺伝子改変～microRNAによる遺伝子発現制御システムを搭載した組換えウイルスの開発～
    櫻井文教; 川端健二; 水口裕之 Drug Delivery System 2010年01月
  • Evaluation of immune response after administration of plasmid DNA-nonviral vector complex.
    櫻井文教; 高倉喜信; 橋田充 (担当:共著範囲:)Non-viral Gene Therapy: Gene Design and Delivery. 2005年

講演・口頭発表等

  • 腫瘍溶解性ウイルス製剤であるレオウイルスによる脱線維化効果  [招待講演]
    石神育歩; 水口裕之; 櫻井文教
    第29回日本遺伝子細胞治療学会学術集会 2023年09月
  • 35型アデノウイルスを基盤とした新規腫瘍溶解性ウイルスの開発  [招待講演]
    櫻井文教; 小野良輔; 水口裕之
    第29回日本遺伝子細胞治療学会学術集会 2023年09月
  • miRNAによるpost-transcriptional de-targeting systemを搭載したアデノウイルスベクターおよび腫瘍溶解性アデノウイルスの開発  [招待講演]
    櫻井文教; 水口裕之
    日本薬物動態学会第37年会 2022年11月 口頭発表（招待・特別）
  • アデノウイルスベクターの作製・増幅・精製法  [招待講演]
    櫻井文教
    千里ライフサイエンス技術講習会（第６９回） 2021年09月
  • 35型アデノウイルスを基盤とした新規腫瘍溶解性アデノウイルスの開発  [招待講演]
    櫻井文教; 小野良輔; 西前文敬; 水口裕之
    第27回日本遺伝子細胞治療学会学術集会 2021年09月
  • miRNAによる遺伝子発現制御システムを搭載した次世代型アデノウイルスベクターの開発と肝障害誘導機構の解明  [招待講演]
    櫻井文教; 水口裕之
    第48回日本毒性学会学術年会 2021年07月
  • Roles of virus-associated RNAs in adenovirus infection and their applications for development of novel oncolytic adenoviruses  [招待講演]
    櫻井 文教; 若林 圭作; 町谷 充洋; 水口裕之
    第25回日本遺伝子細胞治療学会学術集会 2019年07月
  • 腫瘍溶解性ウイルスであるレオウイルスによるHIF-1aの発現量低下  [招待講演]
    櫻井 文教; 宝谷拓磨; 水口裕之
    日本薬学会 第139年会 2019年03月
  • Neonatal Gene Therapy for Hemophilia B by a Novel Adenovirus Vector Showing Reduced Leaky Expression of Viral Genes  [招待講演]
    櫻井 文教
    第24回日本遺伝子細胞治療学会学術集会 2018年07月 口頭発表（招待・特別）
  • 腫瘍溶解性ウイルスであるレオウイルスの ドラックリポジショニングによる 組織線維化治療  [通常講演]
    櫻井 文教
    第10回関西バイオ創薬研究会 2018年04月 口頭発表（招待・特別）
  • ウイルスを基盤とした遺伝子治療薬の臨床開発の現状と今後の展望  [招待講演]
    櫻井 文教
    日本薬学会第138年会 2018年03月 シンポジウム・ワークショップパネル（指名）
  • Post-transcriptional Gene Silencing機構を利用した高機能型遺伝子組換えアデノウイルスの開発  [招待講演]
    櫻井 文教
    第16回阪大薬-基盤研ランチミーティング 2018年02月
  • 組織線維化を治療可能なウイルス製剤の開発  [招待講演]
    櫻井 文教
    第5回阪大マルホ研究交流会 2017年06月
  • レオウイルスを基盤とした新規免疫アジュバントの開発  [招待講演]
    櫻井 文教
    第1回阪大微生物病研究会・薬学研究科研究交流会 2016年11月
  • 遺伝子組換えアデノウイルスを利用した末梢循環腫瘍細胞検出法の開発と臨床応用  [招待講演]
    櫻井 文教
    公益財団法人臨床薬理財団第8回研究大賞受賞講演 2016年02月
  • Development of a potent oncolytic adenovirus via regulation of dicer-mediated processing of virus-associated RNAs  [招待講演]
    櫻井 文教
    第21回日本遺伝子治療学会年会シンポジウム 2015年07月 シンポジウム・ワークショップパネル（指名）
  • Post-transcriptional Gene Silencing機構を利用した高機能型遺伝子組換えアデノウイルスの開発  [招待講演]
    櫻井 文教
    日本薬学会第135年会シンポジウム 2015年03月 シンポジウム・ワークショップパネル（指名）
  • 非コードRNAによる遺伝子発現制御機構を利用した遺伝子組換えアデノウイルスの開発  [招待講演]
    櫻井 文教
    第124関西動物研究会 2014年12月 シンポジウム・ワークショップパネル（指名）
  • アデノウイルスベクターの応用について  [招待講演]
    櫻井 文教
    千里ライフサイエンス技術講習 2013年06月

MISC

受賞

  • 2018年07月 日本遺伝子細胞治療学会 タカラバイオ賞
     
    受賞者: 櫻井 文教
  • 2017年03月 大阪大学 薬友会賞（研究部門）
     
    受賞者: 櫻井文教
  • 2016年07月 大阪大学 総長奨励賞
     
    受賞者: 櫻井 文教
  • 2015年07月 大阪大学 総長奨励賞
     
    受賞者: 櫻井 文教
  • 2015年06月 公益財団法人臨床薬理研究振興財団 研究大賞
     
    受賞者: 櫻井 文教
  • 2014年07月 大阪大学 総長奨励賞
     
    受賞者: 櫻井 文教
  • 2011年06月 日本DDS学会 奨励賞
     
    受賞者: 櫻井 文教
  • 2008年03月 日本薬学会 奨励賞
     
    受賞者: 櫻井 文教

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年04月 -2025年03月 
    代表者 : 向井 英史; 櫻井 文教; 片岡 洋祐; 坂本 啓
  • 血管を経由する新発想での臓器の脱線維化技術の開発
    日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2022年06月 -2024年03月 
    代表者 : 岡田 欣晃; 藤尾 慈; 櫻井 文教; 立花 雅史
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年07月 -2024年03月 
    代表者 : 櫻井 文教
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 櫻井 文教; 水口 裕之; 立花 雅史
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 岡田 欣晃; 青枝 大貴; 櫻井 文教
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 櫻井 文教; 大河原 賢一
     
    癌関連線維芽細胞（Cancer-associated fibroblasts; CAF)は、癌治療の重要な標的である。本研究では、腫瘍溶解性ウイルスであるレオウイルスがCAFに対し殺細胞効果を示すかどうか検討した。その結果、レオウイルスはマウスCAFに対しアポトーシスを誘導することが明らかとなった。さらにレオウイルスは癌細胞およびCAFをアポトーシスを誘導することで、その後に投与されたナノ粒子製剤の腫瘍集積性を向上させることが示された。またレオウイルスは、癌組織での低酸素誘導因子1alpha (HIF-1alpha)の発現を低減させることで、癌組織の悪性化を抑制していることが明らかとなった。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2015年04月 -2018年03月 
    代表者 : 櫻井 文教; 藤原 俊義
     
    癌関連線維芽細胞（Cancer-associated fibroblasts; CAF)は、癌の増殖・生存・悪性化に関与することから、癌治療における重要な標的である。本研究では、腫瘍溶解性ウイルスであるレオウイルスが、CAFに対し殺細胞効果を示すか検討した。マウス皮下腫瘍より単離したCAFの生存率は、レオウイルス作用後、50-60％まで減少したことから、レオウイルスはマウスCAFに対し殺細胞効果を有することが明らかとなった。一方で、レオウイルスは通常の線維芽細胞に対しては殺細胞効果を示さなかった。さらに、担癌マウスにレオウイルスを静脈内投与したところ、アポトーシスを起こしたCAFが検出された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2014年04月 -2018年03月 
    代表者 : 櫻井 文教; 水口 裕之; 立花 雅史
     
    アデノウイルス（Ad)は、そのゲノムに2種類の小分子非コードRNA（VA-RNA)をコードしている。VA-RNAはAd増殖を促進すること、Dicerによりプロセシングされ、miRNA様の小分子RNAを産生する。しかし、VA-RNAによるAdの増殖促進と、Dicerによるプロセシングの関係は不明である。そこで本研究では、DicerによるVA-RNAのプロセンシングがAd増殖に及ぼす影響について検討した。その結果、VA-RNAはDicerによってプロセシングされることでAdの増殖促進能を失うことを明らかにした。さらにその結果を基に、高い抗腫瘍効果を示す新規腫瘍溶解性Adを開発した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2014年04月 -2018年03月 
    代表者 : 水口 裕之; 櫻井 文教; 立花 雅史
     
    研究成果の概要（和文）：本研究では、我々が先駆的に開発を進めてきたアデノウイルス(Ad)ベクター改変技術やマイクロRNAによる遺伝子発現制御技術を駆使して、遺伝子治療やワクチン、癌の診断等に利用可能な革新的な組換えAdの開発とその応用を進めた。具体的には、ゲノム編集を目的としたCas9発現Adベクター、Adベクターワクチンを全身投与した場合の粘膜面でのCTL誘導のメカニズム解明、末梢循環腫瘍細胞（CTC）を高感度に検出可能なGFP発現制限増殖型Adの改良を行い、良好な結果を得た。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2013年04月 -2016年03月 
    代表者 : 櫻井 文教; 鎌田 春彦
     
    本研究では、RNA干渉に必須の分子であるArgonaute (Ago)2の核移行メカニズムの解明を試みた。その結果、Ago2のある部位に変異を導入することで核移行が抑制されることが明らかとなった。さらに、その部位はあるタンパク質（タンパク質X)との結合に重要な領域であった。そこでタンパク質Xとの結合を免疫沈降法により検討したところ、タンパク質Xとの結合性を欠失した変異型Ago2は核への移行性を消失していた。またタンパク質Xをノックダウンすることで、Ago2の核移行が減弱した。従って、Ago2はタンパク質Xとともに核に移行していることが示唆された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2013年04月 -2016年03月 
    代表者 : 丸山 正人; 加瀬 政彦; トリフォノフ ステファン; 櫻井 文教
     
    グリオーマは、脳腫瘍の中で最も多くみられる悪性腫瘍であり、治療後の再発率が高く、予後が悪い。近年、腫瘍治療後の転移・再発の原因として、癌幹細胞が注目されている。これまでに実施されたグリオーマに対する様々な臨床試験は、腫瘍全体を標的としており、治療抵抗性を有する癌幹細胞には有効でなかったため、治療効果が得られなかったと考えられている。そこで本研究では、ヒトグリオーマの癌幹細胞株を樹立し、グリオーマ癌幹細胞株に特異的に発現する遺伝子を同定したことで、グリオーマ癌幹細胞選択的に治療用遺伝子を発現させるための分子基盤を構築した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2011年04月 -2015年03月 
    代表者 : 櫻井 文教; 水口 裕之; 立花 雅史; 中村 紳一朗; 藤原 俊義; 清水 かほり; 岡本 小百合; 細山田 衣里; 町谷 充洋; 林 晃平; 飯塚 俊輔
     
    遺伝子治療の実用化に向けては、標的組織特異的に高効率に遺伝子発現可能な遺伝子導入ベクターの開発が必要不可欠である。そこで本研究では、特にmRNAの安定性・分解を制御することにより遺伝子発現効率（治療効果）の改善を試みた。HuRやAU-rich配列を利用した検討については期待されたほどの遺伝子発現効率の向上は観察されなかった。一方、Adベクターゲノムに非コードRNA標的配列を組み込み、Ad遺伝子の発現を抑制したところ、搭載遺伝子の発現を劇的に改善することに成功した。また本Adベクターは成体マウスのみならず新生仔マウスにおいても有用であった。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2011年 -2012年 
    代表者 : 田代 克久; 川端 健二; 水口 裕之; 櫻井 文教; 山口 朋子; 大森 美幸
     
    本研究では、造血幹細胞の増殖・機能を負に制御しているアダフター蛋白質Lnkの機能を阻害することによりマウスES/ips細胞から効率的な血液細胞の産生、そして造血幹細胞の作製を試みた。その結果、Lnkの阻害により、(1)中胚葉細胞への分化効率が向上するとともに、(2)造血系サイトカインへの応答能が充進しており、これらの作用により効率的に血液細胞が産生されることを明らかにした。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2011年 -2012年 
    代表者 : 櫻井 文教; 小比賀 聡; 水口 裕之; 立花 雅史; 形山 和史; 後藤 平; 南條 有紀; 谷野 文仁
     
    標的遺伝子のプロモーター領域に相同な配列を有する二本鎖RNAを導入することにより、標的遺伝子の発現を活性化させるRNA activationは、治療への応用が期待されている。本研究では、マトリックスメタロプロテアーゼ(MMP)を阻害することで癌の転移を抑制するRECKの発現をRNA activationにより活性化することを試みた。癌細胞に二本鎖RNAを導入したところ、RECKの発現が約6倍に上昇した。さらに、RNA activationによるRECK遺伝子の発現活性化により、MMPの発現および活性が有意に抑制されるとともに、癌細胞の侵潤が約1/5に抑制された。本結果より、RNA activationによるRECK遺伝子の活性化は、癌の転移抑制に向けて有用であることが示唆された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2009年 -2011年 
    代表者 : 川端 健二; 櫻井 文教
     
    ヒトiPS細胞の内胚葉分化誘導系を用いて内胚葉分化に関与する新規遺伝子の単離を試みた。その結果、Runx1遺伝子の発現が中内胚葉から内胚葉への分化過程で消失し、内胚葉特異的遺伝子であるFoxa2およびSox17の転写調節領域(プロモーター領域)に実際に結合し、その転写を負に制御していることが示された。同時に、Runx1遺伝子の転写能を阻害することにより、Foxa2、Sox17、GATA4等の内胚葉関連遺伝子の転写が活性化された。したがって、Runx1は中胚葉形成に重要であることは既に知られているが、本研究により内胚葉への分化も積極的に抑制していることも明らかとなった。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2009年 -2010年 
    代表者 : 水口 裕之; 川端 健二; 櫻井 文教
     
    本年度は、初年度に樹立に成功したmir-877、mir-1225ノックアウトマウスの解析を行った。当初予測された血管形成に関しては、新生仔網膜ならびに動脈の器官培養系などにおける血管新生能への関与を検討したところ、予想に反して異常のないことが判明した。一方、in silicoでのmir-877標的候補遺伝子の探索を行ったところ、いずれもノルアドレナリン・ドーパミン合成の制御に関わる転写因子PHOX2B、HAND2が挙がった。これを受け、sh877(mir-877)の強制発現実験を行ったところ、3'UTRを挿入したレポーター遺伝子の発現、内因性遺伝子の発現について、いずれにおいても抑制効果が確認された。さらに、新生仔の脳幹や成体の副腎において、Phox2bの発現がノックアウトマウスでは野生型に比べて上昇していることがタンパク質レベルで確認された。これらの知見より、Phox2bならびにHand2の発現制御を介して、中枢神経システムの構築や副腎髄質からのアドレナリン産生の調節に、mir-877が関わる可能性が明らかとなった。現在、中枢性ドーパミン、セロトニン神経の構築がどのような影響を受けているかについて組織学的解析を進めている。両神経系のバランスが行動・情動に及ぼす影響は重大であり、ヒトの精神疾患との関わりについて今後強い興味が持たれる。一方、中枢性の表現型の他に、ノックアウトマウスでは、アドレナリンの過剰産生によりもたらされたと説明できる骨量の低下が観察されている。また、Hand2は子宮上皮細胞の増殖制御により着床機能に深く関わることが最近明らかとなっており、このHand2の脱制御に基づくと説明できる妊娠機能の低下が、ノックアウトマウスの雌では観察されている。以上のように、本研究により、mir-877機能の意外な全体像が浮かび上がるとともに、mirtronを介した遺伝子調節システムの解明に大きく貢献する成果が挙げられた。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2008年 -2010年 
    代表者 : 櫻井 文教; 水口 裕之; 川端 健二; 形山 和史; 田代 克久; 近藤 昌夫; 八木 清仁; 中村 紳一朗; 川瀬 篤史; 岩城 正宏; 早川 尭夫; 藤原 俊義; 鈴木 孝幸; 杉尾 久美子; 清水 かほり; 富田 恭子
     
    本研究では、アデノウイルス(Ad)ベクターに、マイクロRNA(miRNA)による遺伝子発現制御システムを搭載することにより、miRNAの発現依存的に遺伝子の発現を制御可能な安全性の高いAdベクターを開発することに成功した。具体的には、(1)肝臓もしくは脾臓における導入遺伝子の発現を抑制可能なAdベクター、(2)Ad遺伝子の非特異的な発現を抑制可能なAdベクター、(3)正常細胞での増殖を大きく抑制した制限増殖型Adを開発した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2007年 -2008年 
    代表者 : 川端 健二; 櫻井 文教
     
    マウスCLSP (mCLSP; CAR-like soluble protein) は390アミノ酸から成っており、3つの免疫グロブリン様(IgV)領域を有する可溶性タンパク質であった。これら3つのIgV領域は、Ad受容体であるCARのIgV領域と強い相同性を示した。そこで、CAR陽性細胞株にmCLSPを安定発現させ、その後アデノウイルス(Ad)ベクターを作用させた結果、Adベクターの感染が親株と比較し著明に阻害された。また、遺伝子組換えmCLSPも同様にAdベクターの感染を有意に阻害した。一方、CAR陰性細胞株にmCLSPを発現させると、Adベクターの感染効率が親株と比較し上昇した。したがって、CLSPはCAR発現の有無によりAdの感染を正または負に制御するタンパク質であることが明らかとなった。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2006年 -2007年 
    代表者 : 水口 裕之; 川端 健二; 櫻井 文教
     
    間葉系幹細胞(mesenchymal stem cells;MSC)は成体骨髄に存在し,骨芽細胞,脂肪細胞,軟骨細胞等,多彩な細胞に分化できること,患者本人から細胞の調製が可能であり倫理的問題が少ないこと,細胞をinvitroで容易に増幅可能であることから,細胞治療や遺伝子治療への利用が期待されている。また,MSCは腫瘍組織への遊走能を有し,腫瘍間質細胞へと分化することが報告されている。即ち,MSCは本来的に腫瘍組織への集積性を示すことから,腫瘍へのターゲティングキャリアとして利用できると考えられる。そこで,B16BL6メラノーマ肺転移モデルに対し,サイトカイン遺伝子を導入したMSCを用いて腫瘍局所にサイトカインを産生させることで,治療効果が得られるか否かを検討した。本年度は以下の結果を得た(1)本実験で用いた初代培養マウス骨髄由来MSCが,脂肪細胞や骨芽細胞への分化能を有していることを確認した。(2)マウスMSCの腫瘍組織へのホーミング能の検証を目的に,初代培養MSCにGFPやルシフェラーゼ発現Adベクターを作用させることでGFPやルシフェラーゼ標識したMSCを調製した。B16メラノーマ肺転移モデルにおいて,これらMSCを全身投与し,腫瘍部位へのMSCの生着についてGFPやルシフェラーゼ発現を指標に組織学的に評価した。しかしながら,腫瘍部位におけるGFPやルシフェラーゼ発現はわずかであり,またコントロール群との有意差も認められず,MSCの腫瘍部位への正着量は極めて少ないことが判明した。(3)MSCへの遺伝子導入活性・転写活性・翻訳活性に優れたTNF-αやIL-12発現Adベクターを作製した。現在,これらのべクターをMSCに作用させ,このMSCを全身投与した場合の抗腫瘍効果について,B16メラノーマの肺転移モデルにおいて検討中である。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2005年 -2007年 
    代表者 : 水口 裕之; 川端 健二; 櫻井 文教; 寺尾 惠治; 中村 紳一朗
     
    35型アデノウイルス(Ad)ベクターは従来の5型Adベクター同様、遺伝子導入ベクターとして期待されている。しかしながら、35型Adベクターの受容体であるCD46はマウスでは精巣でしか発現していないことから、通常のマウスでは35型Adベクターの機能を適正に評価できない。そこで本研究ではヒトと同様にCD46をほぼ全ての臓器で発現している霊長類(カニクイザル)を用いて35型Adベクターの機能評価を行った。まず35型AdのCD46結合部位を明らかにするとともに、カニクイザル由来培養細胞を用いて35型Adベクターがカニクイザル由来の細胞に感染可能であることを示した。次にカニクイザルに対し5型および35型Adベクターを静脈内投与したところ、5型Adベクターでは主に肝臓および脾臓で遺伝子発現が見られた。しかし35型Adベクター投与群では、各臓器より35型Adベクターゲノムが検出されているにもかかわらず、ほとんど遺伝子発現しなかった。一方Adベクター投与後の血清組織障害性マーカーの濃度を測定したところ、5型Adベクター投与群と比較し35型Adベクター投与群では総じて低い値を示す傾向が見られた。また解剖所見においても5型Adベクター投与個体では全身のリンパ節で顕著な肥大が認められたのに対し、35型Adベクター投与個体ではほとんどそのような反応は見られなかった。従って35型Adベクターの高い安全性が示された。 さらに35型Adベクターの臓器局所投与による遺伝子導入実験を行った。その結果、投与部位周辺に限局した遺伝子発現が見られ、投与していない臓器においては遺伝子発現は観察されなかった。従って、局所投与した35型Adベクターが投与部位から漏れ出て他臓器に分布したとしても、投与部位以外で遺伝子発現・毒性を示す可能性が低いことから、35型Adベクターは局所投与に適したベクターとなりうることが示された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2005年 -2005年 
    代表者 : 水口 裕之; 川端 健二; 櫻井 文教
     
    高効率な遺伝子治療用ベクターとして繁用されているアデノウイルス(Ad)ベクターは、細胞表面受容体であるCAR(coxsackievirus and adenovirus receptor)を介して感染することが知られている。CARは免疫グロブリンスーパーファミリーに属する細胞接着分子であり、このファミリーに属する分子は互いに相同性を有することが知られている。そこで、CARと相同性を有する新規タンパク質を探索するため、ESTデータベースを検索したところ、CARと非常に相同性の高い新規タンパク質sCAR(similar to CAR)を同定することができた。sCARは3つの免疫グロブリン様領域を有した390アミノ酸から成る可溶性タンパク質であった。マウスにおけるsCARの発現をRT-PCRにより検討したところ、肺や心臓、腎臓など多くの組織で発現が見られ、特に脳および卵巣で高い発現が見られた。sCARはCARのAdベクター結合部位とほぼ同様の一次構造を有していたため、次に、Adベクター感染におよぼすsCARの影響を調べた。SK HEP-1細胞にsCAR遺伝子を導入しAdベクターを感染させたところ、親株に比較し有意に感染が阻害された。また、sCAR導入SK HEP-1細胞の培養上清にもAdベクター感染阻害活性が見られた。したがって、sCARはAdベクターおよび野生型Adの生理的感染阻害タンパク質である可能性が示唆された。この可能性をさらに確認するため、現在、sCARのリコンビナントタンパク質を大腸菌あるいはバキュロウイルス等で作成し、このタンパク質によるAdベクターの感染におよぼす影響を検討しているところである。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2005年 -2005年 
    代表者 : 櫻井 文教
     
    まず本年度は、ヒト造血幹細胞を含む画分である骨髄由来CD34陽性細胞において最も高い転写活性を示したCAプロモーター(cytomegarovirus由来エンハンサーおよびβ-actinプロモーターにβ-actinのイントロンを加えたプロモーター)を搭載した35型アデノウイルス(Ad)ベクターを用いて検討を行った。CAプロモーターを搭載した35型Adベクターにより遺伝子発現を示したCD34陽性細胞が自己増殖能ならびに分化能を有しているかどうかについてcolony-formingアッセイを用いて検討したところ、遺伝子発現細胞は未処理細胞とほぼ同程度のコロニーを形成した。また遺伝子発現細胞においては、最も未分化な細胞由来のコロニーであるCFU-Mixコロニーの形成も観察された。従って35型ベクターは造血前駆細胞にも遺伝子導入可能であること、また遺伝子発現細胞は自己増殖能および分化能を有していることが明らかとなった。 35型Adベクターを用いることでヒト造血幹細胞に対し高効率な遺伝子導入が可能になったことから、自己複製関連遺伝子を導入し、造血幹細胞の自己複製を試みた。自己複製関連遺伝子としては、HoxB4およびBmi1を選択した。ヒト骨髄由来CD34陽性細胞に対し、35型Adベクターを用いてこれらの遺伝子を導入したところ、有意な自己増殖促進は見られなかった。現在、これらの遺伝子の発現レベルを調製することにより自己増殖促進が観察されないかどうか検討するとともに、その他の自己複製関連遺伝子について検討中である。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2003年 -2004年 
    代表者 : 水口 裕之; 川端 健二; 櫻井 文教
     
    組織特異性を有したアデノウイルスベクターの開発には、nativeのアデノウイルスの受容体であるCAR(coxsackievirus-adenovirus receptor :第1の受容体)やαvインテグリン(第2の受容体)、ヘパラン硫酸(第3の受容体)を認識せず、ファイバーノブのHIループやC末端コード領域などに挿入した外来ペプチドを介して細胞特異的受容体を認識してのみ感染するベクターシステムの開発が必要である。本年度は、昨年度までに基本ユニットを開発済みのファイバーノブのFGループとペントンベース、ファイバーシャフトの3領域を同時に改変することで、上記の3つの受容体とは結合しないトリプルミュータントアデノウイルスベクターの改良を行った。CARとの結合能を除去するためのファイバーノブの変異として、FGループに変異(アミノ酸欠損)をもったベクター(昨年度に開発済み)に加え、ABループに変異(アミノ酸変異)を有したトリプルミュータントアデノウイルスベクター(ペントンベースのRGDモチーフの欠損、およびファイバーシャフト領域を35型アデノウイルス由来に置換)を作製した。これらのベクターの遺伝子導入・発現特性を検討したところ、ABループに変異を加えたベクターでは更なる遺伝子発現能の減弱が認められ、in vitroとin vivo(マウス)の両条件化でmock群に近いレベルの活性しか認められなかった。従って、特定の臓器で目的遺伝子の発現を起こさないアデノウイルスベクターの更なる改良に成功し、ターゲティングアデノウイルスベクターのための基盤ベクターになりうるものと期待された。

委員歴

  • 2022年08月 - 現在   日本遺伝子細胞治療学会 幹事
  • 2022年04月 - 現在   文部科学省 科学技術・学術政策研究所 科学技術予測センター 専門調査員
  • 2018年07月 - 現在   日本遺伝子細胞治療学会   評議員
  • 2017年08月 - 現在   日本DDS学会   評議員
  • 2016年07月 - 現在   日本遺伝子細胞治療学会   若手ワーキンググループメンバー
  • 2011年04月 - 現在   日本薬学会   関西支部委員
  • 2019年04月 - 2021年03月   日本薬学会   代議員
  • 2009年03月 - 2019年03月   日本薬剤学会   フォーカスグループ委員
  • 2013年04月 - 2017年03月   日本薬学会   代議員
  • 2010年04月 - 2015年03月   日本薬剤学会   英語セミナ-委員

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