研究者総覧

久米 晃啓 (クメ アキヒロ)

  • 臨床研究センター 企画開発部 教授
メールアドレス: kumejichi.ac.jp
Last Updated :2023/05/31

研究者情報

学位

  • 博士(医学)(東北大学)

ホームページURL

J-Global ID

研究キーワード

  • レギュラトリーサイエンス   遺伝子治療   Gene Therapy   

研究分野

  • ライフサイエンス / 胎児医学、小児成育学
  • ライフサイエンス / 血液、腫瘍内科学
  • ライフサイエンス / 細胞生物学
  • ライフサイエンス / 分子生物学

経歴

  • 2016年 - 現在  自治医科大学教授
  • 2014年 - 2016年  独立行政法人医薬品医療機器総合機構再生医療製品等審査部主任専門員
  • 1996年 - 2016年  自治医科大学 講師/助教授/准教授
  • 2001年  - Jichi medical School, Assistant Professor
  • 1996年 - 2000年  Jichi medical School, Lecturer
  • 1994年 - 1996年  熊本大学 助手
  • 1994年 - 1996年  Kumamoto University, Research Assistant
  • 1992年 - 1994年  インディアナ大学 ポストドクトラルフェロー
  • 1992年 - 1994年  Indiana University, Post-doctoral Fellow

学歴

  •         - 1990年   東北大学   医学研究科   小児科学
  •         - 1990年   東北大学   Graduate School, Division of Medicine
  •         - 1984年   東北大学

所属学協会

  • 日本癌学会   アメリカ血液学会   アメリカ遺伝子治療学会   日本血液学会   日本遺伝子治療学会   日本人類遺伝学会   日本生化学会   日本小児科学会   

研究活動情報

論文

  • Fumio Kurosaki, Ryosuke Uchibori, Yoshihide Sehara, Yasushi Saga, Masashi Urabe, Hiroaki Mizukami, Koichi Hagiwara, Akihiro Kume
    Human gene therapy 29 11 1242 - 1251 2018年11月 [査読有り][通常論文]
     
    Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative disorder with limited therapeutic options. An aberrant wound healing process in response to repetitive lung injury has been suggested for its pathogenesis, and a number of cytokines including transforming growth factor β1 play pivotal roles in the induction and progression of fibrosis. Thus, the regulation of these pro-inflammatory conditions may reduce the progression of IPF and ameliorate its symptoms in patients. Interleukin-10 (IL-10), a pleiotropic cytokine, exerts anti-inflammatory and anti-fibrotic effects in numerous biological settings. In the present study, we investigated the preventive effects of IL-10 on bleomycin-induced pulmonary fibrosis in mice with the continuous expression of this cytokine via an adeno-associated virus serotype 6 vector. Mice were administered the adeno-associated virus serotype 6 vector encoding mouse IL-10 by intratracheal injection, and osmotic minipumps containing bleomycin were subcutaneously implanted seven days later. Lung histology and the expression levels of pro-inflammatory cytokines and fibrogenic cytokines were then analyzed. In mice exhibiting persistent IL-10 expression on day 35, the number of infiltrated inflammatory cells and the development of fibrosis in lung tissues were significantly reduced. Increases in transforming growth factor β1 and decreases in IFN-γ were also suppressed in treated animals, with changes in these cytokines playing important roles in the pathogenesis of pulmonary fibrosis. Furthermore, IL-10 significantly improved survival in bleomycin-induced mice. Our results provide insights into the potential benefit of the anti-fibrotic effects of IL-10 as a novel therapeutic approach for IPF.
  • Sato N, Saga Y, Uchibori R, Tsukahara T, Urabe M, Kume A, Fujiwara H, Suzuki M, Ozawa K, Mizukami H
    International journal of oncology 52 3 687 - 696 2018年03月 [査読有り][通常論文]
     
    The major causative agent of cervical cancer is human papilloma virus (HPV) the viral proteins E6 and E7 induce carcinogenesis through the inactivation of the host tumor-suppressor gene. Therefore, the stable expression of specific inhibitors of E6 and E7 in cancer cells is expected to provide effective treatment for cervical cancer without affecting normal tissue. In this study, we propose a novel therapeutic approach using an adeno-associated virus (AAV) vector encoding short hairpin RNA (shRNA) against the oncoproteins E6 and E7 (shE6E7) of HPV type 16 (HPV-16), termed AAV-shE6E7. Three different HPV-16-positive cervical cancer cell lines (BOKU, SiHa and SKG-IIIa cells) were tested for gene transfer efficiency using serotypes of AAV vectors. For in vitro analysis, the cells were transduced AAV-shE6E7 alternatively, in vivo studies were performed via the administration of a direct injection of AAV-shE6E7 into cervical cancer cellderived tumors in mice. The high gene transfer efficiency was observed using AAV2 in all three cervical cancer cell lines. Following transduction, we observed apoptosis, G1 phase arrest and cell growth inhibition. Additionally, in the transduced cells, the E6, E7 and p16 expression levels decreased, whereas the expression levels of p53, p21 and pRb levels were enhanced. The growth of subcutaneously transplanted tumors was markedly inhibited by the single administration of AAV2-shE6E7, and the tumors were almost completely eradicated without any adverse effects. These results provided evidence of the utility of AAV2-shE6E7 as a novel treatment approach for cervical cancer.
  • Takayuki Uehara, Takeharu Kanazawa, Hiroaki Mizukami, Ryosuke Uchibori, Tomonori Tsukahara, Masashi Urabe, Akihiro Kume, Kiyoshi Misawa, Thomas E. Carey, Mikio Suzuki, Keiichi Ichimura, Keiya Ozawa
    Cancer Science 105 1 72 - 80 2014年01月 [査読有り][通常論文]
     
    Galanin and its receptors, GALR1 and GALR2, are known tumor suppressors and potential therapeutic targets in head and neck squamous cell carcinoma (HNSCC). Previously, we demonstrated that, in GALR1-expressing HNSCC cells, the addition of galanin suppressed tumor proliferation via upregulation of ERK1/2 and cyclin-dependent kinase inhibitors, whereas, in GALR2-expressing cells, the addition of galanin not only suppressed proliferation, but also induced apoptosis. In this study, we first transduced HEp-2 and KB cell lines using a recombinant adeno-associated virus (rAAV)-green fluorescent protein (GFP) vector and confirmed a high GFP expression rate (> 90%) in both cell lines at the standard vector dose. Next, we demonstrated that GALR2 expression in the presence of galanin suppressed cell viability to 40-60% after 72 h in both cell lines. Additionally, the annexin V-positive rate and sub-G0/G1 phase population were significantly elevated in HEp-2 cells (mock vs GALR2: 12.3 vs 25.0% (P < 0.01) and 9.1 vs 32.0% (P < 0.05), respectively) after 48 h. These changes were also observed in KB cells, although to a lesser extent. Furthermore, in HEp-2 cells, GALR2-mediated apoptosis was caspase-independent, involving downregulation of ERK1/2, followed by induction of the pro-apoptotic Bcl-2 protein, Bim. These results illustrate that transient GALR2 expression in the presence of galanin induces apoptosis via diverse pathways and serves as a platform for suicide gene therapy against HNSCC. © 2013 The Authors.
  • Satsuki Miyata, Masashi Urabe, Akira Gomi, Mutsumi Nagai, Takashi Yamaguchi, Tomonori Tsukahara, Hiroaki Mizukami, Akihiro Kume, Keiya Ozawa, Eiju Watanabe
    NEUROLOGIA MEDICO-CHIRURGICA 53 10 645 - 654 2013年10月 [査読有り][通常論文]
     
    Cytosolic isocitrate dehydrogenase 1 (IDH1) with an R132H mutation in brain tumors loses its enzymatic activity for catalyzing isocitrate to alpha-ketoglutarate (alpha-KG) and acquires new activity whereby it converts alpha-KG to 2-hydroxyglutarate. The IDH1 mutation induces down-regulation of tricarboxylic acid cycle intermediates and up-regulation of lipid metabolism. Sterol regulatory element-binding proteins (SREBPs) regulate not only the synthesis of cholesterol and fatty acids but also acyclin-dependent kinase inhibitor p21 that halts the cell cycle at G1. Here we show that SREBPs were up-regulated in U87 human glioblastoma cells transfected with an IDH1(R132H)-expression plasmid. Small interfering ribonucleic acid (siRNA) for SREBP1 specifically decreased p21 messenger RNA (mRNA) levels independent of the p53 pathway. In IDH1(R132H)-expressing U87 cells, phosphorylation of Retinoblastoma (Rb) protein also decreased. We propose that metabolic changes induced by the IDH1 mutation enhance p21 expression via SREBP1 and inhibit phosphorylation of Rb, which slows progression of the cell cycle and may be associated with non-aggressive features of gliomas with an IDH1 mutation.
  • Kayoko Takahashi, Hiroaki Mizukami, Yasushi Saga, Yuji Takei, Masashi Urabe, Akihiro Kume, Shizuo Machida, Hiroyuki Fujiwara, Mitsuaki Suzuki, Keiya Ozawa
    CANCER SCIENCE 104 8 1107 - 1111 2013年08月 [査読有り][通常論文]
     
    Lymph node metastasis is the most important prognostic factor of endometrial cancer. However, effective therapy has not been established against lymph node metastasis. In this study, we explored the efficacy of gene therapy targeting lymph node metastasis of endometrial cancer by suppressing the action of vascular endothelial growth factor (VEGF)-C through soluble VEGF receptor-3 (sVEGFR-3) expression. For this purpose, we first conducted a model experiment by introducing sVEGFR-3 cDNA into an endometrial cancer cell line HEC1A and established HEC1A/sVEGFR-3 cell line with high sVEGFR-3 expression. The conditioned medium of HEC1A/sVEGFR-3 cells inhibited lymphatic endothelial cell growth in vitro, and sVEGFR-3 expression in HEC1A cells suppressed in vivo lymph node and lung metastases without inhibiting the growth of a subcutaneously inoculated tumor. To validate the therapeutic efficacy, adeno-associated virus vectors encoding sVEGFR-3 were injected into the skeletal muscle of mice with lymph node metastasis. Lymph node and lung metastases of HEC1A cells were completely suppressed by the muscle-mediated expression of sVEG-FR-3 using adeno-associated virus vectors. These results suggest the possibility of gene therapy against lymph node and lung metastases of endometrial cancer by using muscle-mediated expression of sVEGFR-3.
  • Tomonori Tsukahara, Ken Ohmine, Chihiro Yamamoto, Ryosuke Uchibori, Hiroyuki Ido, Takeshi Teruya, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Masataka Nakamura, Junichi Mineno, Kazutoh Takesako, Isabelle Riviere, Michel Sadelain, Renier Brentjens, Keiya Ozawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 438 1 84 - 89 2013年08月 [査読有り][通常論文]
     
    Adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) is promising for treatment of advanced B-cell malignancies. Tumor targeting of CAR-modified T-cells is likely to contribute therapeutic potency; therefore we examined the relationship between the ability of CD19-specific CAR (CD19-CAR)-transduced T-cells to accumulate at CD19(+) tumor lesions, and their ability to provide antitumor effects in xenograft mouse models. Normal human peripheral blood lymphocytes, activated with immobilized RetroNectin and anti-CD3 antibodies, were transduced with retroviral vectors that encode CD19-CAR. Expanded CD19-CAR T-cells with a high transgene expression level of about 75% produced IL-2 and IFN-gamma in response to CD19, and lysed both Raji and Daudi CD19(+) human B-cell lymphoma cell lines. Furthermore, these cells efficiently accumulated at Raji tumor lesions where they suppressed tumor progression and prolonged survival in tumor-bearing Rag2(-/-)gamma c(-/-) immunodeficient mice compared to control cohorts. These results show that the ability of CD19-CAR T-cells to home in on tumor lesions is pivotal for their anti-tumor effects in our xenograft models, and therefore may enhance the efficacy of adoptive T-cell therapy for refractory B-cell lymphoma. (C) 2013 Elsevier Inc. All rights reserved.
  • Uchibori R, Tsukahara T, Mizuguchi H, Saga Y, Urabe M, Mizukami H, Kume A, Ozawa K
    Cancer research 73 1 364 - 372 2013年01月 [査読有り][通常論文]
     
    Mesenchymal stem cells (MSC) accumulate at tumor sites when injected into tumor-bearing mice, perhaps offering cellular vectors for cancer-targeted gene therapy. However, the molecular mechanisms involved in MSC targeting the tumors are presently little understood. We focused on MSC-endothelial cell (EC) adhesion following TNF-alpha stimulation in an attempt to elucidate these mechanisms. Interestingly, stimulation of MSCs with TNF-alpha enhanced the adhesion of MSCs to endothelial cells in vitro. This adhesion was partially inhibited by blocking antibodies against vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4). It is well known that TNF-alpha induces VCAM-1 expression via the NF-kappa B signaling pathway. Parthenolide has an anti-inflammatory activity and suppressed NF-kappa B activity by inhibition of I kappa B alpha phosphorylation after TNF-a stimulation and strongly inhibited TNF-alpha-induced VCAM-1 expression on MSCs. In vivo imaging using luciferase-expressing MSCs revealed that the bioluminescent signal gradually increased at tumor sites in mice injected with untreated MSCs. In contrast, we observed very weak signals at tumor sites in mice injected with parthenolide-treated MSCs. Our results suggest that NF-kappa B activity regulates MSC accumulation at tumors, by inducing VCAM-1 and thereby its interaction with tumor vessel endothelial cells. These findings have implications for the ongoing development of efficient MSC-based gene therapies for cancer treatment. Cancer Res; 73(1); 364-72. (C)2012 AACR.
  • Hiroya Yagi, Sho Sanechika, Hiroshi Ichinose, Chiho Sumi-Ichinose, Hiroaki Mizukami, Masashi Urabe, Keiya Ozawa, Akihiro Kume
    NEUROREPORT 23 1 30 - 34 2012年01月 [査読有り][通常論文]
     
    Phenylketonuria (PKU) is a common genetic disorder arising from a deficiency of phenylalanine hydroxylase. If left untreated, the accumulation of phenylalanine leads to brain damage and neuropsychological dysfunction. One of the abnormalities found in hyperphenylalaninemic patients and a mouse model of PKU is an aminergic deficit in the brain. We previously showed correction of hyperphenylalaninemia and concomitant behavioral recovery in PKU mice after liver-targeted gene transfer with a viral vector. Here, we addressed whether such a functional recovery was substantiated by an improved amine metabolism in the brain. After gene transfer, brain dopamine, norepinephrine, and serotonin levels in the PKU mice were significantly elevated to normal or near-normal levels, along with systemic improvement of phenylalanine catabolism. The results of biochemical analyses validated the efficacy of PKU gene therapy in the central nervous system. NeuroReport 23:30-34 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.
  • Kayoko Takahashi, Yasushi Saga, Hiroaki Mizukami, Yuji Takei, Masashi Urabe, Akihiro Kume, Mitsuaki Suzuki, Keiya Ozawa
    CANCER SCIENCE 102 12 2272 - 2277 2011年12月 [査読有り][通常論文]
     
    Controlling lymph node metastasis is currently a key issue in cancer therapy. Lymph node metastasis is one of the most important prognostic factors in various types of cancers, including endometrial cancer. Vascular endothelial growth factor-C (VEGF-C) plays a crucial role in lymphangiogenesis, and is implicated to play an important role in lymph node metastasis. To evaluate the role of VEGF-C in lymph node metastasis, we developed an animal model by using an endometrial cancer cell line, HEC1A. This cell line is not invasive by nature and secretes moderate amounts of VEGF-C; intrauterine injection of HEC1A cells into Balb/c nude mice resulted in uterine cancer with lymph node metastasis after 8 weeks. To analyze the contribution of VEGF-C to lymph node metastasis, its corresponding gene was stably introduced into HEC1A cells (HEC1A/VEGF-C), which then produced more than 10 times the amount of VEGF-C. The number of lymph node metastases was significantly higher in HEC1A/VEGF-C cells than in HEC1A cells (3.2 vs 1.1 nodes/animal, respectively). Augmented lymphangiogenesis was observed within tumors when HEC1A/VEGF-C cells were inoculated. These results indicate that VEGF-C plays a critical role in lymph node metastasis, in addition to serving as a platform to test the efficacy of various therapeutic modalities against lymph node metastasis. (Cancer Sci 2011; 102: 22722277)
  • Hiroya Yagi, Tsuyoshi Ogura, Hiroaki Mizukami, Masashi Urabe, Hiromi Hamada, Hiroyuki Yoshikawa, Keiya Ozawa, Akihiro Kume
    JOURNAL OF GENE MEDICINE 13 2 114 - 122 2011年02月 [査読有り][通常論文]
     
    Background Classical phenylketonuria (PKU) arises from a deficiency of phenylalanine hydroxylase (PAH) that catalyses phenylalanine oxidation in the liver. Lack of PAH activity causes massive hyperphenylalaninemia and consequently severe brain damage. Preclinical studies showed that conventional adeno-associated virus (AAV) vectors could correct hyperphenylalaninemia in a mouse model of PKU, although limitations such as very large dose requirement and relative inefficiency in female animals were recognized. Method An AAV8-pseudotyped vector was constructed with a self-complementary AAV (scAAV) genome for efficient liver transduction and expression. Following vector injection to PKU mice, blood Phe was periodically measured by an enzymatic fluorometric assay. In vivo Phe oxidation was evaluated by a non-invasive breath test using [1-(13)C] Phe. Vector copy number in the host tissues was determined by quantitative polymerase chain reaction. Results A single injection of 1 x 10(11)-1 x 10(12) particles of the scAAV8 vector resulted in a reduction of blood Phe to normal or near-normal levels for more than 1 year in both genders. The treated animals showed normal level of in vivo Phe oxidation. The presence of > 1 copy of vector DNA per diploid genome in the liver was associated with normal blood Phe in the AAV-treated PKU mice. Conclusions Complete phenotypic correction of PKU mice was achieved by the scAAV8 vector for the longest duration reported to date. The vector overcame the female-specific disadvantage in AAV-mediated liver transduction; thus, it offers a promising platform of long-lasting gene therapy for PKU. Copyright (C) 2011 John Wiley & Sons, Ltd.
  • Shin-ichi Muramatsu, Ken-ichi Fujimoto, Seiya Kato, Hiroaki Mizukami, Sayaka Asari, Kunihiko Ikeguchi, Tadataka Kawakami, Masashi Urabe, Akihiro Kume, Toshihiko Sato, Eiju Watanabe, Keiya Ozawa, Imaharu Nakano
    MOLECULAR THERAPY 18 9 1731 - 1735 2010年09月 [査読有り][通常論文]
     
    Gene transfer of dopamine-synthesizing enzymes into the striatal neurons has led to behavioral recovery in animal models of Parkinson's disease (PD). We evaluated the safety, tolerability, and potential efficacy of adenoassociated virus (AAV) vector-mediated gene delivery of aromatic l-amino acid decarboxylase (AADC) into the putamen of PD patients. Six PD patients were evaluated at baseline and at 6 months, using multiple measures, including the Unified Parkinson's Disease Rating Scale (UPDRS), motor state diaries, and positron emission tomography (PET) with 6-[(18)F] fluoro-l-m-tyrosine (FMT), a tracer for AADC. The short-duration response to levodopa was measured in three patients. The procedure was well tolerated. Six months after surgery, motor functions in the OFF-medication state improved an average of 46% based on the UPDRS scores, without apparent changes in the short-duration response to levodopa. PET revealed a 56% increase in FMT activity, which persisted up to 96 weeks. Our findings provide class IV evidence regarding the safety and efficacy of AADC gene therapy and warrant further evaluation in a randomized, controlled, phase 2 setting.
  • Muramatsu S, Fujimoto K, Kato S, Mizukami H, Asari S, Ikeguchi K, Kawakami T, Urabe M, Kume A, Sato T, Watanabe E, Ozawa K, Nakano I: A phase I study of aromatic L-amino acid decarboxylase gene therapy for Parkinson’s disease
    KUME Akihiro
    Molecular Therapy 18 9 1081 - 1086 2010年09月 [査読有り][通常論文]
  • Yasuomi Horiuchi, Masafumi Onodera, Yoshitaka Miyagawa, Ban Sato, Keiko Onda, Yohko U. Katagiri, Hajime Okita, Mayumi Okada, Makoto Otsu, Akihiro Kume, Torayuki Okuyama, Junichiro Fujimoto, Tadatoshi Kuratsuji, Nobutaka Kiyokawa
    HUMAN GENE THERAPY 20 7 777 - 783 2009年07月 [査読有り][通常論文]
     
    The CH-296 recombinant fragment of human fibronectin is essential for murine leukemia virus (MLV)-derived retroviral transduction of CD34(+) cells for the purpose of stem cell gene therapy. Although the major effect of CH-296 is colocalization of the MLV-derived retrovirus and target cells at specific adhesion domains of CH-296 mediated by integrins expressed on CD34(+) cells, the precise roles of the integrins are unclear. We examined the kinetics of integrin expression on CD34(+) cells during the course of MLV-derived retrovirus-mediated gene transduction with CH-296. Flow cytometry revealed that the levels of both very late activation protein (VLA)-4 and VLA-5 on CD34(+) cells freshly isolated from cord blood were insufficient for effective MLV-derived retroviral transduction. However, increases were achieved during culture for preinduction and MLV-derived retrovirus-mediated gene transduction in the presence of a cocktail of cytokines. In addition, we confirmed by using specific antibodies that inhibition of the cell adhesion mediated by the integrins significantly reduced transduction efficiency, indicating that integrin expression is indeed important for CH-296-based MLV-derived retroviral transduction. Only a few cytokines are capable of inducing integrin expression, and stem cell factor plus thrombopoietin was found to be the minimal combination that was sufficient for effective transduction of an MLV-derived retrovirus based on CH-296. Our findings should be useful for improving the culture conditions for CH-296-based MLV-derived retroviral transduction in stem cell gene therapy.
  • Ryosuke Uchibori, Takashi Okada, Takayuki Ito, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Keiya Ozawa
    JOURNAL OF GENE MEDICINE 11 5 373 - 381 2009年05月 [査読有り][通常論文]
     
    Background Mesenchymal stein cells (MSCs) are a promising vehicle for targeted cancer gene therapy because of their potential of tumor tropism. For efficient therapeutic application, we developed retroviral vector-producing MSCs that enhance tumor transduction via progeny vector production. Methods Rat bone marrow-derived MSCs were nucleofected with the proviral plasmids (vesicular stomatitis virus-G protein-pseudotyped retroviral vector components) (VP-MSCs) or pLTR plasmid alone (non-VP-MSCs). The luciferase-based in vivo imaging system was used to assess gene expression periodically. To evaluate the anticancer effects, we administered MSCs expressing herpes simplex virus-thymidine kinase (HSV-tk) into the left ventricular cavity of nude mice engrafted with 9L glioma cells subcutaneously. Results In vivo imaging revealed that administration of luciferase-expressing non-VP-MSCs enhanced the bioluminescence signal at the inoculation sites of 9L cells, whereas no accumulation was observed in juice at the site of the control Rat-1 fibroblasts. Compared to non-VP-MSCs, the administration of VP-MSCs resulted in significant augmentation of the signal with an increase in transgene copy number. Immunohistochemical analysis showed marked luciferase expression at the tumor periphery in mice injected with VP-MSCs, whereas little expression was detected in those in injected. with non-VP-MSCs. Under the continuous infusion of ganciclovir systemic, administration of VP-MSCs expressing HSV-tk Suppressed tumor growth more effectively than non-VP-MSC administration, whereas no anticancer effect was observed Without ganciclovir treatment. Furthermore, VP-MSC administration caused no transgene transduction in the normal tissues and organs. Conclusions VP-MSCs accumulated at the site of tumors after intravascular injection in tumor-bearing mice, followed by in situ gene transfer to tumors without transduction of normal organs. When applied to the HSV-tk/ganciclovir suicide gene therapy, more efficient tumor growth Suppression was observed using VP-MSCs compared to non-VP-MSCs. This VP-MSC-based system has great potential for improved cancer gene therapy. Copyright (C) 2009 John Wiley & Sons, Ltd.
  • Hiroshi Kawaguchi, Soshi Okamoto, Dwaipayan Sikdar, Akihiro Kume, Fang Li, Omar Mahmoud Mohamed Mohafez, Mohammed Hassan Shehata, Koichi Hiraga
    GENE 432 1-2 7 - 18 2009年03月 [査読有り][通常論文]
     
    Regions required for chicken glycine decarboxylase gene transcription were examined. A region between -82 and +22 (-82/+22) with motifs similar to binding sites for Sp1, NF-Y and CP2 was assigned to the proximal promoter active in both chicken hepatoma cell line, LMH, and hepatocytes in primary culture. In LMH cells, a genomic region, KX, between Kpn1 (-4155) and Xbal (-2113) sites changed promoter activity with the aid of four additional genomic regions termed upstream regulator regions for suppression (UpRS) and activation (UpRA) of transcription. Those precise segments are UpR1S (-376/-346), UpR1A (-345/-291), UpR2S (-137/-108) and UpR2A (-107/-83). Within KX, -4155/-3605 activates and -3604/-3367 suppresses the promoter. -3366/-3024 activates or suppresses the promoter, probably with different UpR counterparts. -2197/-2113 restores the actions of -3366/-3024. While in LMH cells, the upstream UpRs abrogate the functions of immediately downstream UpRs, UpR1S or UpR2S or both may be at least less active in hepatocytes than in LMH cells. Nuclear extracts from various chicken tissues and LMH cells had UpR2A binding proteins in different populations, suggesting that together with the UpRs, the segments in KX are involved in the regulation of cell type-specific transcription of this gene. (c) 2008 Elsevier B.V. All rights reserved.
  • Keiya Ozawa, Kazuya Sato, Iekuni Oh, Katsutoshi Ozaki, Ryosuke Uchibori, Yoko Obara, Yuji Kikuchi, Takayuki Ito, Takashi Okada, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume
    JOURNAL OF AUTOIMMUNITY 30 3 121 - 127 2008年05月 [査読有り][通常論文]
     
    Mesenchymal stem cells (MSCs) are considered to be a promising platform for cell and gene therapy for a variety of diseases. First, in the field of hematopoietic stem cell transplantation, there are two applications of MSCs: 1) the improvement of stem cell engrafting and the acceleration of hematopoietic reconstitution based on the hematopoiesis-supporting ability; and 2) the treatment of severe graft-versus-host disease (GVHD) based on the immunomodulatory ability. Regarding the immunosuppressive ability, we found that nitric oxide (NO) is involved in the MSC-mediated suppression of T cell proliferation. Second, tumor-bearing nude mice were injected with luciferase-expressing MSCs. An in vivo imaging analysis showed the significant accumulation of the MSCs at the site of tumors. The findings suggest that MSCs can be utilized to target metastatic tumors and to deliver anti-cancer molecules locally. As the third application, MSCs may be utilized as a cellular vehicle for protein-supplement gene therapy. When long-term transgene expression is needed, a therapeutic gene should be introduced with a minimal risk of insertional mutagenesis. To this end, site-specific integration into the AAVS 1 locus on the chromosome 19 (19q13.4) by using the integration machinery of adeno-associated virus (AAV) would be particularly valuable. There will be wide-ranging applications of MSCs to frontier medical treatments in the near future. (C) 2008 Elsevier Ltd. All rights reserved.
  • Mutsuko Nonaka-Sarukawa, Takashi Okada, Takayuki Ito, Keiji Yamamoto, Toru Yoshioka, Tatsuya Nomoto, Yukihiro Hojo, Masahisa Shimpo, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Uichi Keda, Kazuyuki Shimada, Keiya Ozawa
    JOURNAL OF GENE MEDICINE 10 4 368 - 374 2008年04月 [査読有り][通常論文]
     
    Background Inflammation plays an important role in the pathogenesis of hypertension and hypertensive organ damage. Interleukin (IL)-10, a pleiotropic anti-inflammatory cytokine, exerts vasculoprotective effects in many animal models. In the present study, we examined the preventive effects of adeno-associated virus (AAV) vector-mediated sustained IL-10 expression against hypertensive heart disease and renal dysfunction in Dahl salt-sensitive rats. Methods We injected the rats intramuscularly with an AAV type I-based vector encoding rat IL-10 or enhanced green fluorescent protein (EGFP) at 5 weeks of age; subsequently, the rats were fed a high-sodium diet from 6 weeks of age. Results Sustained IL-10 expression significantly improved survival rate of Dahl salt-sensitive rats compared with EGFP expression (62.5% versus 0%, p < 0.001); it also caused 26.0% reduction in systolic blood pressure at 15 weeks (p < 0.0001). Echocardiography exhibited a 22.0% reduction in hypertrophy (p < 0.0001) and a 26.3% improvement in fractional shortening (p < 0.0001) of the rat left ventricle in the IL-10 group compared to the EGFP group. IL-10 expression also caused a 21.7% decrease in the heart weight/body weight index and cardiac atrial natriuretic peptide levels. Histopathological studies revealed that IL-10 decreased inflammatory cell infiltration, fibrosis, and transforming growth factor-P, levels in the failing heart. Furthermore, IL-10 expression significantly reduced urine protein excretion with increased glomerular filtration rates. Conclusions This is the first study to demonstrate that the anti-inflammatory cytokine IL-10 has a significant anti-hypertensive effect. AAV vector-mediated IL-10 expression potentially prevents the progression of refractory hypertension and hypertensive organ damage in humans. Copyright (c) 2008 John Wiley & Sons, Ltd.
  • Yuhe Liu, Takashi Okada, Kuniko Shimazaki, Kianoush Sheykholeslami, Tatsuya Nomoto, Shin-Ichi Muramatsu, Hiroaki Mizukami, Akihiro Kume, Shuifang Xiao, Keiichi Ichimura, Keiya Ozawa
    MOLECULAR THERAPY 16 3 474 - 480 2008年03月 [査読有り][通常論文]
     
    Since standard aminoglycoside treatment progressively causes hearing disturbance with hair cell degeneration, systemic use of the drugs is limited. Adeno-associated virus (AAV)-based vectors have been of great interest because they mediate stable transgene expression in a variety of postmitotic cells with minimal toxicity. In this study, we investigated the effects of regulated AAV1-mediated glial cell line-derived neurotrophic factor (GDNF) expression in the cochlea on aminoglycoside-induced damage. AAV1-based vectors encoding GDNF or vectors encoding GDNF with an rtTA2s- S2 Tet- on regulation system were directly microinjected into the rat cochleae through the round window at 5 x 10(10) genome copies/body. Seven days after the virus injection, a dose of 333 mg/kg of kanamycin was subcutaneously given twice daily for 12 consecutive days. GDNF expression in the cochlea was confirmed and successfully modulated by the Tet-on system. Monitoring of the auditory brain stem response revealed an improvement of cochlear function after GDNF transduction over the frequencies tested. Damaged spiral ganglion cells and hair cells were significantly reduced by GDNF expression. Our results suggest that AAV1-mediated expression of GDNF using a regulatedexpression system in the cochlea is a promising strategy to protect the cochlea from aminoglycoside-induced damage.
  • Yuhe Liu, Takashi Okada, Kuniko Shimazaki, Kianoush Sheykholeslami, Tatsuya Nomoto, Shin-Ichi Muramatsu, Hiroaki Mizukami, Akihiro Kume, Shuifang Xiao, Keiichi Ichimura, Keiya Ozawa
    MOLECULAR THERAPY 16 3 474 - 480 2008年03月 [査読有り][通常論文]
     
    Since standard aminoglycoside treatment progressively causes hearing disturbance with hair cell degeneration, systemic use of the drugs is limited. Adeno-associated virus (AAV)-based vectors have been of great interest because they mediate stable transgene expression in a variety of postmitotic cells with minimal toxicity. In this study, we investigated the effects of regulated AAV1-mediated glial cell line-derived neurotrophic factor (GDNF) expression in the cochlea on aminoglycoside-induced damage. AAV1-based vectors encoding GDNF or vectors encoding GDNF with an rtTA2s- S2 Tet- on regulation system were directly microinjected into the rat cochleae through the round window at 5 x 10(10) genome copies/body. Seven days after the virus injection, a dose of 333 mg/kg of kanamycin was subcutaneously given twice daily for 12 consecutive days. GDNF expression in the cochlea was confirmed and successfully modulated by the Tet-on system. Monitoring of the auditory brain stem response revealed an improvement of cochlear function after GDNF transduction over the frequencies tested. Damaged spiral ganglion cells and hair cells were significantly reduced by GDNF expression. Our results suggest that AAV1-mediated expression of GDNF using a regulatedexpression system in the cochlea is a promising strategy to protect the cochlea from aminoglycoside-induced damage.
  • Takayuki Ito, Takashi Okada, Jun Mimuro, Hiroshi Miyashita, Ryosuke Uchibori, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Masafumi Takahashi, Uichi Ikeda, Yoichi Sakata, Kazuyuki Shimada, Keiya Ozawa
    HYPERTENSION 50 3 531 - 536 2007年09月 [査読有り][通常論文]
     
    Prostacyclin synthase (PGIS) is the final committed enzyme in the metabolic pathway of prostacyclin production. The therapeutic option of intravenous prostacyclin infusion in patients with pulmonary arterial hypertension is limited by the short half-life of the drug and life-threatening catheter-related complications. To develop a better delivery system for prostacyclin, we examined the feasibility of intramuscular injection of an adenoassociated virus (AAV) vector expressing PGIS for preventing monocrotaline-induced pulmonary arterial hypertension in rats. We developed an AAV serotype 1-based vector carrying a human PGIS gene (AAV-PGIS). AAV-PGIS or the control AAV vector expressing enhanced green fluorescent protein was injected into the anterior tibial muscles of 3-week-old male Wistar rats; this was followed by the monocrotaline administration at 7 weeks. Eight weeks after injecting the vector, the plasma levels of 6-keto-prostaglandin F-1 alpha increased in a vector dose-dependent manner. At this time point, the PGIS transduction (1x10(10) genome copies per body) significantly decreased mean pulmonary arterial pressure (33.9 +/- 2.4 versus 46.1 +/- 3.0 mm Hg; P < 0.05), pulmonary vascular resistance (0.26 +/- 0.03 versus 0.41 +/- 0.03 mm Hg . mL(-1) . min(-1) . kg(-1); P < 0.05), and medial thickness of the peripheral pulmonary artery (14.6 +/- 1.5% versus 23.5 +/- 0.5%; P < 0.01) as compared with the controls. Furthermore, the PGIS-transduced rats demonstrated significantly improved survival rates as compared with the controls (100% versus 50%; P < 0.05) at 8 weeks postmonocrotaline administration. An intramuscular injection of AAV-PGIS prevents monocrotaline-pulmonary arterial hypertension in rats and provides a new therapeutic alternative for preventing pulmonary arterial hypertension in humans.
  • Takayuki Ito, Takashi Okada, Hiroshi Miyashita, Tatsuya Nomoto, Mutsuko Nonaka-Sarukawa, Ryosuke Uchibori, Yoshikazu Maeda, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Masafumi Takahashi, Uichi Ikeda, Kazuyuki Shimada, Keiya Ozawa
    CIRCULATION RESEARCH 101 7 734 - 741 2007年09月 [査読有り][通常論文]
     
    Pulmonary arterial hypertension (PAH) is a fatal disease associated with inflammation and pathological remodeling of the pulmonary artery (PA). Interleukin (IL)-10 is a pleiotropic antiinflammatory cytokine with vasculoprotective properties. Here, we report the preventive effects of IL-10 on monocrotaline-induced PAH. Three-week-old Wistar rats were intramuscularly injected with an adeno-associated virus serotype 1 vector expressing IL-10, followed by monocrotaline injection at 7 weeks old. IL-10 transduction significantly improved survival rates of the PAH rats 8 weeks after monocrotaline administration compared with control gene transduction (75% versus 0%, P < 0.01). IL-10 also significantly reduced mean PA pressure (22.8 +/- 1.5 versus 29.7 +/- 2.8 mm Hg, P < 0.05), a weight ratio of right ventricle to left ventricle plus septum (0.35 +/- 0.04 versus 0.42 +/- 0.05, P < 0.05), and percent medial thickness of the PA (12.9 +/- 0.3% versus 21.4 +/- 0.4%, P < 0.01) compared with controls. IL-10 significantly reduced macrophage infiltration and vascular cell proliferation in the remodeled PA in vivo. It also significantly decreased the lung levels of transforming growth factor-beta(1) and IL-6, which are indicative of PA remodeling. In addition, IL-10 increased the lung level of heme oxygenase-1, which strongly prevents PA remodeling. In vitro analysis revealed that IL-10 significantly inhibited excessive proliferation of cultured human PA smooth muscle cells treated with transforming growth factor-beta(1) or the heme oxygenase inhibitor tin protoporphyrin IX. Thus, IL-10 prevented the development of monocrotaline-induced PAH, and these results provide new insights into the molecular mechanisms of human PAH.
  • A Kume, R Xu, Y Ueda, M Urabe, K Ozawa
    GENE THERAPY 7 14 1193 - 1199 2000年07月 [査読有り][通常論文]
     
    Hematopoietic stem cells (HSCs) are attractive targets for gene therapy, but current gene transfer methodologies are inadequate for efficient HSC transduction and perpetual transgene expression. To improve gene transfer vectors and transduction protocols, it is vital to establish a system to evaluate transgene expression and the long-term behavior of transduced cells in vivo. For this purpose, we constructed a bicistronic retrovirus encoding the human CD24 (as the first cistron) and the enhanced green fluorescent protein (EGFP; as the second cistron). Murine bone marrow cells were transduced with this vector and the transgene expression was monitored along with hematopoietic reconstitution. Stable expression of CD24 and EGFP was demonstrated in the long-term repopulating cells for at least 6 months, and multi-parameter flow cytometry illustrated expression of both markers in all the lymphohematopoietic lineages examined (B and T lymphoid, erythroid and myeloid). Sustained expression was also shown in the secondary transplants for 6 months, suggesting that self-renewing HSCs were transduced by this vector. Overall, EGFP-tagged bicistronic retroviruses would provide powerful tools for detailed in vivo analysis of transduced hematopoietic cells, such as transgene expression in conjunction with lineage differentiation.

書籍

  • CD34 Workshop : Cross - linking of CD34 on hematopoietic cells induces homotypic cell adhesion via clustering of CD34 and mobilization of adhesion molecules. (共著)
    ()
    Lenkocyte Typing VI , Garland Puhilishing Inc . , New York and London 1997年

作品等

  • 造血幹細胞を用いる遺伝子治療技術の開発:遺伝子導入細胞の選択*増幅法に関する研究
    1999年 -2000年
  • Selevtive Expansion of Gene Tically Modified Hemutopoietic stom cells
    1999年 -2000年

MISC

  • T Hara, A Kume, Y Hanazono, H Mizukami, T Okada, H Tsurumi, H Moriwaki, Y Ueda, M Hasegawa, K Ozawa GENE THERAPY 11 (18) 1370 -1377 2004年09月 [査読無し][通常論文]
     
    Hematopoietic stem cell gene therapy has not provided clinical success in disorders such as chronic granulomatous disease (CGD), where genetically corrected cells do not show a selective advantage in vivo. To facilitate selective expansion of transduced cells, we have developed a fusion receptor system that confers drug-induced proliferation. Here, a 'selective amplifier gene (SAG)' encodes a chimeric receptor (GcRER) that generates a mitotic signal in response to estrogen. We evaluated the in vivo efficacy of SAG-mediated cell expansion in a mouse disease model of X-linked CGD (X-CGD) that is deficient in the NADPH oxidase gp91(phox) subunit. Bone marrow cells from X-CGD mice were transduced with a bicistronic retrovirus encoding GcRER and gp91(phox), and transplanted to lethally irradiated CGD recipients. Estrogen was administered to a cohort of the transplants, and neutrophil superoxide production was monitored. A significant increase in oxidase-positive cells was observed in the estrogen-treated mice, and repeated estrogen administration maintained the elevation of transduced cells for 20 weeks. In addition, oxidase-positive neutrophils were increased in the X-CGD transplants given the first estrogen even at 9 months post-transplantation. These results showed that the SAG system would enhance the therapeutic effects by boosting genetically modified, functionally corrected cells in vivo.
  • S Mochizuki, H Mizukami, T Ogura, S Kure, A Ichinohe, K Kojima, Y Matsubara, E Kobayahi, T Okada, A Hoshika, K Ozawa, A Kume GENE THERAPY 11 (13) 1081 -1086 2004年07月 [査読無し][通常論文]
     
    Classical phenylketonuria (PKU) is a metabolic disorder caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). If untreated, accumulation of phenylalanine will damage the developing brain of affected individuals, leading to severe mental retardation. Here, we show that a liver-directed PAH gene transfer brought about long-term correction of hyperphenylalaninmia and behavioral improvement in a mouse model of PKU. A recombinant adeno-associated virus (AAV) vector carrying the murine PAH cDNA was constructed and administered to PAH-deficient mice ( strain PAH(enu2)) via the portal vein. Within 2 weeks of treatment, the hyperphenylalaninemic phenotype improved and completely normalized in the animals treated with higher vector doses. The therapeutic effect persisted for 40 weeks in male mice, while serum phenylalanine concentrations in female animals gradually returned to pretreatment levels. Notably, this long-term correction of hyperphenylalaninemia was associated with a reversal of hypoactivity observed in PAH(enu2) mice. While locomotory activity over 24 h and exploratory behavior were significantly decreased in untreated PAHenu2 mice compared with the age-matched controls, these indices were completely normalized in 12-month- old male PKU mice with lowered serum phenylalanine. These results demonstrate that AAV-mediated liver transduction ameliorated the PKU phenotype, including central nervous system dysfunctions.
  • A Kume, M Koremoto, RF Xu, T Okada, H Mizukami, Y Hanazono, M Hasegawa, K Ozawa JOURNAL OF GENE MEDICINE 5 (3) 175 -181 2003年03月 [査読無し][通常論文]
     
    Background Hematopoietic stem-cell-directed gene transfer has achieved limited success in transducing clinically relevant levels of target cells. The expansion of gene-modified cells is one way to circumvent the problem of inefficient transduction with current vectors. To this end, we have developed 1 selective amplifier genes' (SAGS) that encode chimeric proteins that are a fusion of granulocyte colony-stimulating factor receptor and the steroid-binding domain. Prototype SAGS conferred estrogen-responsive growth on murine hematopoietic progenitors. Methods We constructed a retroviral vector coexpressing an SAG for 4-hydroxytamoxifen (Tm)-specific proliferation and the enhanced green fluorescent protein (EGFP). Murine bone marrow cells were transduced with this vector and transplanted into myeloablated mice. Subsequently, recipients were challenged with Tm, and EGFP(+) cells were enumerated. Results The challenge induced a significant increase in EGFP(+) leukocytes (21 +/- 4% to 27 +/- 5%), while EGFP(+) cells decreased in untreated animals (21 +/- 5% to 10 +/- 3%). Three months later, bone marrow cells were transplanted from the unchallenged mice to secondary hosts. Again the administration of Tm resulted in an increase of EGFP(+) cells (16 +/- 4% to 35 +/- 3%), contrasting to a decrease in controls (22 +/- 4% to 12 +/- 4%), and the difference was significant for more than 3 months. A detailed study of lineage showed a preferential expansion of EGFP(+) cells in granulocytes and monocytes following Tm administration. Conclusions Long-term repopulating cells were transduced with the SAG, and the transduced granulocyte/monocyte precursors were most likely to be expandable in vivo upon Tm stimulation. Copyright (C) 2002 John Wiley Sons, Ltd.
  • Cancer Science 94 (7) 639 -643 2003年 [査読無し][通常論文]
  • Bone Warrow Transplantion 30 (2) 113 2002年 [査読無し][通常論文]
  • Gene Therapy 9 (16) 1055 2002年 [査読無し][通常論文]
  • Long-term tracking of murine hemutopoietic cells with a bicistronic retrovirus containing CD24 and EGFP genes.(共著)
    Gene Therapy 7 (14) 1193 -1199 2000年 [査読無し][通常論文]
  • Gene theapy for chronic granulomafous disease(共著)
    Journal of Clinical and Laboratory Medicine 135 (2) 122 -128 2000年 [査読無し][通常論文]
  • A Kume, Y Hanazono, H Mizukami, M Urabe, K Ozawa INTERNATIONAL JOURNAL OF HEMATOLOGY 69 (4) 227 -233 1999年06月 [査読無し][通常論文]
     
    Retrovirus-mediated gene transfer into murine hematopoietic stem cells and reconstitution of syngeneic mice have demonstrated persistence and functioning of the transgenes over extended periods of time. In contrast. clinically relevant levels of gene transfer into large animal and human stem cells have not been widely achieved. Results of current clinical gene transfer studies have raised fundamental questions about the physiology of primitive human hematopoietic cells and gene therapy vectors. Efforts are being undertaken to answer these problems and to develop more efficient gene therapy strategies. (C) 1999 The Japanese Society of Hematology.
  • A Kume, K Ito, Y Ueda, M Hasegawa, M Urabe, H Mano, K Ozawa BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 260 (1) 9 -12 1999年06月 [査読無し][通常論文]
     
    We have developed a novel system for expansion of transduced hematopoietic cells. This system involves "selective amplifier genes" encoding fusion proteins between the granulocyte colony-stimulating factor receptor (Gcr) and the estrogen receptor (Er). The GcrEr chimeric gene conferred estrogen-dependent growth ability on murine hematopoietic cells. Here, we constructed a modified "selective amplifier gene" to circumvent possible concerns with the Er/estrogen switching system. The bacterial gyrase B (Gyr) gene was fused to the Gcr gene, and the GcrGyr fusion construct was introduced into interleukin-3 (IL-3)-dependent Ba/F3 cells. The dimeric antibiotic coumermycin induced IL-3-independent growth in Ba/F3 cells expressing GcrGyr, This stimulatory effect was antagonized by an excess amount of novobiocin, a monomeric form of coumermycin. These results suggest the feasibility of using Gyr as a molecular switch to regulate a growth signal in hematopoietic cells. (C) 1999 Academic.
  • KM Matsuda, A Kume, Y Ueda, M Urabe, M Hasegawa, K Ozawa GENE THERAPY 6 (6) 1038 -1044 1999年06月 [査読無し][通常論文]
     
    We have proposed a novel concept, ie selective expansion of transduced cells, to overcome the low efficiency of gene transfer into hematopoietic stem cells. Previously, a fusion gene encoding a chimeric receptor (Delta GCRER) between the mouse granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain of rat estrogen receptor was constructed as a 'selective amplifier gene'. Although the chimeric gene conferred estrogen-inducible proliferation on the transduced Ba/F3 cells, it also mediated differentiation of the retrovirally transduced 32D cells upon estrogen treatment. Since only a growth signal is required for our purpose, we further modified the Delta GCRER gene to attenuate its differentiation signal. Based on the observation that tyrosine-703 in wild-type G-CSFR plays a pivotal role in transmitting the differentiation signal, phenylalanine was substituted for this residue in Delta GCRER. When the resultant selective amplifier gene (Delta Y703F-GCRER gene) was expressed in 32D cells, sustained growth was supported by estrogen, while differentiation was suppressed. These cells ceased to grow upon estrogen withdrawal and differentiated with G-CSF treatment. The present findings suggested that Delta Y703F-GCRER may have desirable properties as a selective amplifier for hematopoietic stem cell expansion and gene therapy.
  • A selective amplifier gene for tamoxifen-induced expansion of hematopoietic cells(共著)
    Journal of Gene Medicine 1 (4) 236 -244 1999年 [査読無し][通常論文]
  • H Kodaira, A Kume, Y Ogasawara, M Urabe, K Kitano, A Kakizuka, K Ozawa JAPANESE JOURNAL OF CANCER RESEARCH 89 (7) 741 -747 1998年07月 [査読無し][通常論文]
     
    Several cancer gene therapy strategies involve suicide genes to kill the neoplasm, or to regulate effector cells such as lymphocytes, We have developed an inducible apoptosis system with a Fas-estrogen receptor fusion protein (MfasER) for rapid elimination of transduced cells. In the present study, we further improved this molecular switch for estrogen-inducible apoptosis to overcome concerns with the wild-type estrogen receptor and its natural ligand, 17 beta-estradiol (E-2). The ligand-binding domain of MfasER was replaced with that of a mutant estrogen receptor which is unable to bind estrogen yet retains affinity for a synthetic ligand, 4-hydroxytamoxifen (Tm), The resultant fusion protein (MfasTmR) and MfasER were expressed in L929 cells for examination of their ligand specificities. Tm induced apoptosis in MfasTmR-expressing cells (L929MfasTmR) at 10(-8) M or higher concentrations, but induced no apoptosis in MfasER-expressing cells (L929MfasER) at up to 10(-6) M, On the other hand, E-2 induced apoptosis in L929MfasER at concentrations as low as 10(-10)-10(-9) M, while it did so partially in L929MfasTmR at concentrations greater than 10(-7) M, Thus, L929MfasTmR cells were highly susceptible to Tm, but refractory to E-2, with 100-1,000 times more tolerance than L929MfasER, These results suggest that the MfasTmR/Tm system would induce apoptosis in the target cells more safely in vitro, working independently of endogenous estrogen.
  • Apoptosis - mediated regulation of recoarbinant human granulocyte colony - Stimulating factor production by genetically engineered fibroblasts. (共著)
    Gene Therapy 5 (7) 923 -929 1998年 [査読無し][通常論文]
  • A Kume, H Nishiura, J Suda, T Suda BLOOD 89 (9) 3434 -3442 1997年05月 [査読無し][通常論文]
     
    The involvement of focal adhesion kinase (FAK) in myeloid differentiation was investigated in primary murine bone marrow (BM) cells, In unstimulated BM, FAK mRNA was defected in myeloid and lymphoid cells, but not in erythroid precursors. When the BM cells were incubated with granulo-cyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), FAK expression showed a remarkable difference depending on the cytokine. Although FAK was upregulated in the cells stimulated by GM-CSF (GM-treated cells), the kinase was barely detectable in the cells cultured with IL-3 (IL-3-treated cells). Morphology and flow cytometry analysis showed GM-CSF promoted the growth and differentiation of monocyte/macrophage lineage stronger than IL-3. In addition, motility of the cytokine-differentiated cells showed an overt distinction between the cultures, which was closely correlated with FAK expression. After 7 days of stimulation, GM-treated cells showed active migration and chemoattractant-induced morphologic polarization. In contrast, IL-3-treated cells showed minimal migration and polarization. These results suggest an important role of GM-CSF in the terminal differentiation of monocytes/macrophages, and possible involvement of FAK in functional maturity of this lineage. (C) 1997 by The American Society of Hematology.
  • CJ Ding, A Kume, H Bjorgvinsdottir, RG Hawley, N Pech, MC Dinauer BLOOD 88 (5) 1834 -1840 1996年09月 [査読無し][通常論文]
     
    The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91(phox), a 91-kD membrane glycoprotein that is the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a new retroviral vector for expression of human gp91(phox), MSCV-h91Neo, based on murine stem cell virus vectors, was evaluated using a human X-CGD myeloid cell line (X-CGD PLB-985 cells) and murine bone marrow cells. Expression of recombinant gp91(phox) in transduced X-CGD PLB-985 cells was substantially improved compared with levels achieved previously using a different retroviral construct, and respiratory burst oxidase activity was fully reconstituted in the majority of clones analyzed. Expression of gp91(phox) transcripts was also observed in primary and secondary murine colony-forming unit-spleen derived from transduced bone marrow cells. Furthermore, respiratory burst activity was restored to granulocyte-monocyte progeny of transduced X-CGD mice bone marrow cells cultured in vitro. This observation is the first reported use of gene transfer to correct the enzymatic defect in murine CGD phagocytes and is also consistent with the high conservation of the oxidase complex among different species. Taken together, these data suggest that the MSCV-h91Neo vector may be useful for gene replacement therapy in X-linked CGD, in which high-level reconstitution of phagocyte oxidase activity may be important for full correction of phagocyte microbicidal function. (C) 1996 by The American Society of Hematology.
  • Retrovirus-mediated Reconstitution of Respiratory Burst Activity in X-linked Chronic Granulomatous Disease Cells(共著)
    Blood 84 (10) 3311 1994年 [査読無し][通常論文]
  • Non-Ketotic Hyperglycinemia : A Life-threatening Disorder in Neonate(共著)
    Early Human Development 29 (1) 75 1992年 [査読無し][通常論文]
  • The Glycine Cleavage System : the Coupled Expression of the Glycine Decarboxylase Gene and the H-protein Gene in the Chicken(共著)
    The Journal of Biological Chemistry 266 (5) 3330 1991年 [査読無し][通常論文]
  • The Glycine Cleavage System : Molecular Cloning of the Chicken and Human Glycine Decarboxylase cDNAs and Some Characteristics Involved in the Deduced Protein Structures(共著)
    The Journal of Biological Chemistry 266 (5) 3323 1991年 [査読無し][通常論文]
  • Genomic Analysis of Nonketotic Hyperglycinemia : A Partial Deletion of P-protein Gene(共著)
    Journal of Inherited Metabolic Disease 13 (5) 766 1991年 [査読無し][通常論文]
  • Biochemical and Biophysical Research Communications 173 (3) 801 -806 1990年 [査読無し][通常論文]
  • Biochemical and Biophysical Research Communications 154 (1) 292 -297 1988年 [査読無し][通常論文]
  • Green fluorescent protein as a sele ctable marker of retroairally transduced hematopoietic progenitors(共著)
    Stem Cells 17 (4) 226 -232 [査読無し][通常論文]

共同研究・競争的資金等の研究課題

  • Development of New Vectors for Gene Therapy
    Grant-in-Aid for Scientific Research
    研究期間 : 1996年 -2023年
  • Proliferation and Differentiation of Hemutopoietic Stem cells
    Grant-in-Aid for Scientific Research
    研究期間 : 1994年 -2023年
  • 遺伝子治療ベクターの開発
    科学研究費補助金
    研究期間 : 1996年 -2013年
  • 造血幹細胞の増殖および分化
    科学研究費補助金
    研究期間 : 1994年 -2003年
  • Department of Gene Therapy Vectors

委員歴

  • 2012年 - 現在   日本遺伝子治療学会   理事
  • 1998年   日本遺伝子治療学会   評議員   日本遺伝子治療学会


Copyright © MEDIA FUSION Co.,Ltd. All rights reserved.