Researchers Database

mizukami hiroaki

    CenterforMolelucarMedicine(DivisionofGeneticTherapeutics) Professor
Last Updated :2021/12/08

Researcher Information

Degree

  • Ph.D.
  • M.D.

URL

J-Global ID

Research Interests

  • 分子生物学   ウィルス学   血液内科学   Molecular Biology   Virology   Hematology   

Research Areas

  • Life sciences / Internal medicine - General

Academic & Professional Experience

  • 2014 - Today  同 教授
  • 2011 - 2014  同准教授
  • 2004 - 2011  同 講師
  • 1998 - 2003  Jichi Medical University
  • 1995 - 1998  Chief academic administrater. school for
  • 1993 - 1995  米国国立保健研究所 客員研究員
  • 1993 - 1995  National Institutes of Health, Visiting
  • 1988 - 1990  潜水医学実験隊(防衛庁)実験部員
  • emergency medical corp. JSDF Hospital Yokosuka
  • Associate
  • Jichi Medical UniversityProfessor

Education

  •        - 1986  防衛医科大学校  医学部

Association Memberships

  • 欧州遺伝子細胞治療学会   日本癌学会   米国遺伝子治療学会   日本ウィルス学会   日本遺伝子治療学会   日本血液学会   日本内科学会   

Published Papers

  • Naoki Usui, Masahide Yoshida, Yuki Takayanagi, Naranbat Nasanbuyan, Ayumu Inutsuka, Hiroshi Kurosu, Hiroaki Mizukami, Yoshiyuki Mori, Makoto Kuro-O, Tatsushi Onaka
    Journal of neuroendocrinology e13026  2021/08 
    Fibroblast growth factor 21 (FGF21) modulates energy metabolism and neuroendocrine stress responses. FGF21 synthesis is increased after environmental or metabolic challenges. Detailed roles of FGF21 in the control of behavioural disturbances under stressful conditions remain to be clarified. Here, we examined the roles of FGF21 in the control of behavioural changes after social defeat stress in male rodents. Central administration of FGF21 increased the number of tyrosine hydroxylase-positive catecholaminergic cells expressing c-Fos protein, an activity marker of neurones, in the nucleus tractus solitarius and area postrema. Double in situ hybridisation showed that some catecholaminergic neurones in the dorsal medulla oblongata expressed β-Klotho, an essential co-receptor for FGF21, in male mice. Social defeat stress increased FGF21 concentrations in the plasma of male mice. FGF21-deficient male mice showed social avoidance in a social avoidance test with C57BL/6J mice (background strain of FGF21-deficient mice) and augmented immobility behaviour in a forced swimming test after social defeat stress. On the other hand, overexpression of FGF21 by adeno-associated virus vectors did not significantly change behaviours either in wild-type male mice or FGF21-deficient male mice. The present data are consistent with the view that endogenous FGF21, possibly during the developmental period, has an inhibitory action on stress-induced depression-like behaviour in male rodents.
  • Yoshihide Sehara, Yuka Hayashi, Kenji Ohba, Ryosuke Uchibori, Masashi Urabe, Ayumu Inutsuka, Kuniko Shimazaki, Kensuke Kawai, Hiroaki Mizukami
    Human Gene Therapy 1043-0342 2021/08
  • Mohammad Shahnaij, Mitsuhiro Iyori, Hiroaki Mizukami, Mayu Kajino, Iroha Yamagoshi, Intan Syafira, Yenni Yusuf, Ken Fujiwara, Daisuke S Yamamoto, Hirotomo Kato, Nobuhiko Ohno, Shigeto Yoshida
    Frontiers in immunology 12 612910 - 612910 2021 
    Hepatocyte infection by malaria sporozoites is a bottleneck in the life-cycle of Plasmodium spp. including P. falciparum, which causes the most lethal form of malaria. Therefore, developing an effective vaccine capable of inducing the strong humoral and cellular immune responses necessary to block the pre-erythrocytic stage has potential to overcome the spatiotemporal hindrances pertaining to parasite biology and hepatic microanatomy. We recently showed that when combined with a human adenovirus type 5 (AdHu5)-priming vaccine, adeno-associated virus serotype 1 (AAV1) is a potent booster malaria vaccine vector capable of inducing strong and long-lasting protective immune responses in a rodent malaria model. Here, we evaluated the protective efficacy of a hepatotropic virus, adeno-associated virus serotype 8 (AAV8), as a booster vector because it can deliver a transgene potently and rapidly to the liver, the organ malaria sporozoites initially infect and multiply in following sporozoite injection by the bite of an infected mosquito. We first generated an AAV8-vectored vaccine expressing P. falciparum circumsporozoite protein (PfCSP). Intravenous (i.v.) administration of AAV8-PfCSP to mice initially primed with AdHu5-PfCSP resulted in a hepatocyte transduction rate ~2.5 times above that seen with intramuscular (i.m.) administration. This immunization regimen provided a better protection rate (100% sterile protection) than that of the i.m. AdHu5-prime/i.m. AAV8-boost regimen (60%, p < 0.05), i.m. AdHu5-prime/i.v. AAV1-boost (78%), or i.m. AdHu5-prime/i.m. AAV1-boost (80%) against challenge with transgenic PfCSP-expressing P. berghei sporozoites. Compared with the i.m. AdHu5-prime/i.v. AAV1-boost regimen, three other regimens induced higher levels of PfCSP-specific humoral immune responses. Importantly, a single i.v. dose of AAV8-PfCSP recruited CD8+ T cells, especially resident memory CD8+ T cells, in the liver. These data suggest that boost with i.v. AAV8-PfCSP can improve humoral and cellular immune responses in BALB/c mice. Therefore, this regimen holds great promise as a next-generation platform for the development of an effective malaria vaccine.
  • Machi Horiai, Ayano Otsuka, Shizu Hidema, Yuichi Hiraoka, Ryotaro Hayashi, Shinji Miyazaki, Tamio Furuse, Hiroaki Mizukami, Ryoichi Teruyama, Masaru Tamura, Haruhiko Bito, Yuko Maejima, Kenju Shimomura, Katsuhiko Nishimori
    Scientific Reports 10 (1) 2020/12 
    AbstractAutism spectrum disorder (ASD) is a continuum of neurodevelopmental disorders and needs new therapeutic approaches. Recently, oxytocin (OXT) showed potential as the first anti-ASD drug. Many reports have described the efficacy of intranasal OXT therapy to improve the core symptoms of patients with ASD; however, the underlying neurobiological mechanism remains unknown. The OXT/oxytocin receptor (OXTR) system, through the lateral septum (LS), contributes to social behavior, which is disrupted in ASD. Therefore, we selectively express hM3Dq in OXTR-expressing (OXTR+) neurons in the LS to investigate this effect in ASD mouse models developed by environmental and genetic cues. In mice that received valproic acid (environmental cue), we demonstrated successful recovery of impaired social memory with three-chamber test after OXTR+ neuron activation in the LS. Application of a similar strategy to Nl3R451C knock-in mice (genetic cue) also caused successful recovery of impaired social memory in single field test. OXTR+ neurons in the LS, which are activated by social stimuli, are projected to the CA1 region of the hippocampus. This study identified a candidate mechanism for improving core symptoms of ASD by artificial activation of DREADDs, as a simulation of OXT administration to activate OXTR+ neurons in the LS.
  • Akira Arafune-Mishima, Hiroshi Abe, Toshiki Tani, Hiromi Mashiko, Satoshi Watanabe, Kazuhisa Sakai, Wataru Suzuki, Hiroaki Mizukami, Akiya Watakabe, Tetsuo Yamamori, Noritaka Ichinohe
    Neuroscience 446 145 - 156 2020/10 
    The pulvinar, the largest thalamic nucleus in the primate brain, has connections with a variety of cortical areas and is involved in many aspects of higher brain functions. Among cortico-pulvino-cortical systems, the connection between the middle temporal area (MT) and the pulvinar has been thought to contribute significantly to complex motion recognition. Recently, the common marmoset (Callithrix jacchus), has become a valuable model for a variety of neuroscience studies, including visual neuroscience and translational research of neurological and psychiatric disorders. However, information on projections from MT to the pulvinar in the marmoset brain is scant. We addressed this deficiency by injecting sensitive anterograde viral tracers into MT to examine the distribution of labeled terminations in the pulvinar. The injection sites were placed retinotopically according to visual field coordinates mapped by optical intrinsic imaging. All injections produced anterograde terminal labeling, which was densest in the medial nucleus of the inferior pulvinar (PIm), sparser in the central nucleus of the inferior pulvinar, and weakest in the lateral pulvinar. Within each subnucleus, terminations formed separate retinotopic fields. Most labeled terminals were small but these comingled with a few large terminals, distributed mainly in the dorsomedial part of the PIm. Our results further delineate the organization of projections from MT to the pulvinar in the marmoset as forming parallel complex networks, which may differentially contribute to motion processing. It is interesting that the densest projections from MT target the PIm, the subnucleus recently reported to preferentially receive direct retinal projections.
  • Ryota Watano, Tsukasa Ohmori, Shuji Hishikawa, Asuka Sakata, Hiroaki Mizukami
    Gene therapy 2020/02 [Refereed][Not invited]
     
    Adeno-associated virus (AAV) vectors can transduce hepatocytes efficiently in vivo in various animal species, including humans. Few reports, however, have examined the utility of pigs in gene therapy. Pigs are potentially useful in preclinical studies because of their anatomical and physiological similarity to humans. Here, we evaluated the utility of microminipigs for liver-targeted gene therapy. These pigs were intravenously inoculated with an AAV8 vector encoding the luciferase gene, and gene expression was assessed by an in vivo imaging system. Robust transgene expression was observed almost exclusively in the liver, even though the pig showed a low-titer of neutralizing antibody (NAb) against the AAV8 capsid. We assessed the action of NAbs against AAV, which interfere with AAV vector-mediated gene transfer by intravascular delivery. When a standard dose of vector was administered intravenously, transgene expression was observed in both NAb-negative and low-titer (14×)-positive subjects, whereas gene expression was not observed in animals with higher titers (56×). These results are compatible with our previous observations using nonhuman primates, indicating that pigs are useful in gene therapy experiments, and that the role of low-titer NAb in intravenous administration of the AAV vector shows similarities across species.
  • Kameda H, Murabe N, Odagaki K, Mizukami H, Ozawa K, Sakurai M
    The Journal of comparative neurology 527 (8) 1401 - 1415 0021-9967 2019/06 [Refereed][Not invited]
  • Ryosuke Uchibori, Takeshi Teruya, Hiroyuki Ido, Ken Ohmine, Yoshihide Sehara, Masashi Urabe, Hiroaki Mizukami, Junichi Mineno, Keiya Ozawa
    Molecular therapy oncolytics 12 16 - 25 2019/03 [Refereed][Not invited]
     
    Adoptive transfer of T cells expressing a chimeric antigen receptor (CAR) is a promising cell-based anticancer therapy. Although clinical studies of this approach show therapeutic efficacy, additional genetic modification is necessary to enhance the efficacy and safety of CAR-T cells. For example, production of an antitumor cytokine from CAR-T cells can potentially enhance their tumor-killing activity, but there are concerns that constitutive expression of anticancer molecules will cause systemic side effects. Therefore, it is important that exogenous gene expression is confined to the tumor locality. Here, we aimed to develop an inducible promoter driven by activation signals from a CAR. Transgene expression in T cells transduced with the CD19-targeted CAR and an inducible promoter, including inducible reporter genes (CAR-T/iReporter), was only induced strongly by co-culture with CD19-positive target cells. CAR-T/iReporter cells also showed redirected cytolysis toward CD19-positive, but not CD19-negative, tumor cells. Overall, our study indicated that the inducible promoter was selectively driven by activation signals from the CAR, and transduction with the inducible promoter did not affect original effector activities including interleukin-2 and interferon-γ production and the antitumor activity of CAR-redirected cytotoxic T lymphocytes. Moreover, this inducible promoter permits visualization and quantification of the activation status in CAR-T cells.
  • Yoshiba T, Saga Y, Urabe M, Uchibori R, Matsubara S, Fujiwara H, Mizukami H
    Oncology letters 17 (2) 2197 - 2206 1792-1074 2019/02 [Refereed][Not invited]
  • Kojima K, Nakajima T, Taga N, Miyauchi A, Kato M, Matsumoto A, Ikeda T, Nakamura K, Kubota T, Mizukami H, Ono S, Onuki Y, Sato T, Osaka H, Muramatsu SI, Yamagata T
    Brain : a journal of neurology 142 (2) 322 - 333 0006-8950 2019/02 [Refereed][Not invited]
  • Skibbe H, Reisert M, Nakae K, Watakabe A, Hata J, Mizukami H, Okano H, Yamamori T, Ishii S
    IEEE transactions on medical imaging 38 (1) 69 - 78 0278-0062 2019/01 [Refereed][Not invited]
  • Yusuf Y, Yoshii T, Iyori M, Yoshida K, Mizukami H, Fukumoto S, Yamamoto DS, Alam A, Emran TB, Amelia F, Islam A, Otsuka H, Takashima E, Tsuboi T, Yoshida S
    Frontiers in immunology 10 730 - 730 2019 [Refereed][Not invited]
     
    An ideal malaria vaccine platform should potently induce protective immune responses and block parasite transmission from mosquito to human, and it should maintain these effects for an extended period. Here, we have focused on vaccine development based on adeno-associated virus serotype 1 (AAV1), a viral vector widely studied in the field of clinical gene therapy that is able to induce long-term transgene expression without causing toxicity in vivo. Our results show the potential utility of AAV1 vectors as an extremely potent booster vaccine to induce durable immunity when combined with an adenovirus-priming vaccine in a rodent malaria model. We generated a series of recombinant AAV1s and human adenovirus type 5 (AdHu5) expressing either Plasmodium falciparum circumsporozoite protein (PfCSP) or P25 (Pfs25) protein. Heterologous two-dose immunization with an AdHu5-prime and AAV1-boost (AdHu5-AAV1) elicited robust and durable PfCSP- or Pfs25-specific functional antibodies over 280 days. Regarding protective efficacy, AdHu5-AAV1 PfCSP achieved high sterile protection (up to 80% protection rate) against challenge with transgenic Plasmodium berghei sporozoites expressing PfCSP. When examining transmission-blocking (TB) efficacy, we found that immunization with AdHu5-AAV1 Pfs25 maintained TB activity in vivo against transgenic P. berghei expressing Pfs25 for 287 days (99% reduction in oocyst intensity, 85% reduction in oocyst prevalence). Our data indicate that AAV1-based malaria vaccines can confer potent and durable protection as well as TB efficacy when administered following an AdHu5 priming vaccine, supporting the further evaluation of this regimen in clinical trials as a next-generation malaria vaccine platform.
  • Teppei Ebina, Yoshito Masamizu, Yasuhiro R. Tanaka, Akiya Watakabe, Reiko Hirakawa, Yuka Hirayama, Riichiro Hira, Shin-Ichiro Terada, Daisuke Koketsu, Kazuo Hikosaka, Hiroaki Mizukami, Atsushi Nambu, Erika Sasaki, Tetsuo Yamamori, Masanori Matsuzaki
    Nature Communications 9 (1) 1879  2041-1723 2018/12 [Refereed][Not invited]
     
    Two-photon imaging in behaving animals has revealed neuronal activities related to behavioral and cognitive function at single-cell resolution. However, marmosets have posed a challenge due to limited success in training on motor tasks. Here we report the development of protocols to train head-fixed common marmosets to perform upper-limb movement tasks and simultaneously perform two-photon imaging. After 2-5 months of training sessions, head-fixed marmosets can control a manipulandum to move a cursor to a target on a screen. We conduct two-photon calcium imaging of layer 2/3 neurons in the motor cortex during this motor task performance, and detect task-relevant activity from multiple neurons at cellular and subcellular resolutions. In a two-Target reaching task, some neurons show direction-selective activity over the training days. In a short-Term force-field adaptation task, some neurons change their activity when the force field is on. Two-photon calcium imaging in behaving marmosets may become a fundamental technique for determining the spatial organization of the cortical dynamics underlying action and cognition.
  • Yoshihide Sehara, Toshiki Inaba, Takao Urabe, Fumio Kurosaki, Masashi Urabe, Naoki Kaneko, Kuniko Shimazaki, Kensuke Kawai, Hiroaki Mizukami
    The European journal of neuroscience 48 (12) 3466 - 3476 0953-816X 2018/12 [Refereed][Not invited]
     
    Survivin, a member of the inhibitors of apoptosis protein gene family, inhibits the activity of caspase, leading to a halt of the apoptotic process. Our study focused on the neuroprotective effect of survivin after transient middle cerebral artery occlusion (MCAO) with intraparenchymal administration of an adeno-associated virus (AAV) vector. His-tagged survivin was cloned and packaged into the AAV-rh10 vector. Four-week-old Sprague-Dawley rats were injected with 4 × 109  vg of AAV-GFP or AAV-His-survivin into the right striatum and were treated 3 weeks later with transient MCAO for 90 min. Twenty-four hours after MCAO, functional and histological analyses of the rats were performed. The result showed that rats that had been treated with AAV-green fluorescent protein (GFP) and those that had been treated with AAV-His-survivin did not show a significant difference in neurological scores 24 hr after MCAO, however, infarction volume was significantly reduced in the AAV-His-survivin group compared to that in the AAV-GFP group. Although the neutrophil marker myeloperoxidase did not show a significant difference in the ischemic boundary zone, cells positive for active caspase-3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling were significantly decreased in the AAV-His-survivin group. In conclusion, survivin overexpression in the ischemic boundary zone induced by using an AAV vector has the potential for amelioration of ischemic damage via an antiapoptotic mechanism.
  • Fumio Kurosaki, Ryosuke Uchibori, Yoshihide Sehara, Yasushi Saga, Masashi Urabe, Hiroaki Mizukami, Koichi Hagiwara, Akihiro Kume
    Human gene therapy 29 (11) 1242 - 1251 1043-0342 2018/11 [Refereed][Not invited]
     
    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.
  • Murabe N, Mori T, Fukuda S, Isoo N, Ohno T, Mizukami H, Ozawa K, Yoshimura Y, Sakurai M
    Scientific reports 8 (1) 16536  2018/11 [Refereed][Not invited]
  • Ohmori T, Mizukami H, Katakai Y, Kawai S, Nakamura H, Inoue M, Shu T, Sugimoto H, Sakata Y
    International journal of hematology 108 (3) 239 - 245 0925-5710 2018/09 [Refereed][Not invited]
  • Yoshihide Sehara, Kuniko Shimazaki, Fumio Kurosaki, Naoki Kaneko, Ryosuke Uchibori, Masashi Urabe, Kensuke Kawai, Hiroaki Mizukami
    Neuroscience letters 682 27 - 31 0304-3940 2018/08 [Refereed][Not invited]
     
    Adeno-associated virus (AAV) is an ideal vector for gene transduction into the central nervous system because of its safety and efficiency. While it is currently widely used for clinical trials and is expected to become more widespread, the appropriate combination of viral serotypes and promoters have not been fully investigated. In this study, we compared the transduced gene expression of AAVrh10 to AAV5 in gerbil hippocampus using three different promoters, including cytomegalovirus (CMV), chicken β-actin promoter with the CMV immediate-early enhancer (CAG), and the Synapsin 1 (Syn1) promoter. Four-week-old male gerbils underwent stereotaxic injection with 1 × 1010 viral genome of AAV carrying green fluorescent protein (GFP). Quantification of the GFP-positive areas 3 weeks after injection showed that AAVrh10-CMV and AAVrh10-CAG were the most efficient (p < 0.001, compared with the control) and AAVrh10-Syn1 and AAV5-CMV were the next most efficient (p < 0.05, compared with the control). On the other hand, AAV5-Syn1 showed little expression, which was only observed at the injected site. In conclusion, we should note that some combinations of viral capsids and promoters can result in failure of gene delivery, while most of them will work appropriately in the transgene expression in the brain.
  • Akemi Shimada, Hisashi Ideno, Yoshinori Arai, Koichiro Komatsu, Satoshi Wada, Teruhito Yamashita, Norio Amizuka, Ernst Pöschl, Bent Brachvogel, Yoshiki Nakamura, Kazuhisa Nakashima, Hiroaki Mizukami, Yoichi Ezura, Akira Nifuji
    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 33 (8) 1532 - 1543 0884-0431 2018/08 [Refereed][Not invited]
     
    Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5-deficient (Anxa5-/- ) mice, the sizes of bone ridge outgrowths at the entheses of the tibias and femur were increased after age 7 weeks. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP-expressing cells were observed in the fibrocartilage layer in Anxa5-/- mice than in wild-type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5-/- mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5-/- mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail-suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5-/- and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of Anxa5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. Anxa5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in Anxa5-/- mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators. © 2018 American Society for Bone and Mineral Research.
  • Kondo T, Saito R, Otaka M, Yoshino-Saito K, Yamanaka A, Yamamori T, Watakabe A, Mizukami H, Schnitzer MJ, Tanaka KF, Ushiba J, Okano H
    Cell reports 24 (8) 2191 - 2195.e4 2018/08 [Refereed][Not invited]
     
    To understand brain circuits of cognitive behaviors under natural conditions, we developed techniques for imaging neuronal activities from large neuronal populations in the deep layer cortex of the naturally behaving common marmoset. Animals retrieved food pellets or climbed ladders as a miniature fluorescence microscope monitored hundreds of calcium indicator-expressing cortical neurons in the right primary motor cortex. This technique, which can be adapted to other brain regions, can deepen our understanding of brain circuits by facilitating longitudinal population analyses of neuronal representation associated with cognitive naturalistic behaviors and their pathophysiological processes.
  • Nakamura J, Watanabe S, Kimura H, Kobayashi M, Karasawa T, Kamata R, Usui-Kawanishi F, Sadatomo A, Mizukami H, Nagi-Miura N, Ohno N, Kasahara T, Minota S, Takahashi M
    Scientific reports 8 (1) 7601  2018/05 [Refereed][Not invited]
  • Naoto Sato, Yasushi Saga, Ryosuke Uchibori, Tomonori Tsukahara, Masashi Urabe, Akihiro Kume, Hiroyuki Fujiwara, Mitsuaki Suzuki, Keiya Ozawa, Hiroaki Mizukami
    International Journal of Oncology 52 (3) 687 - 696 1791-2423 2018/03 [Refereed][Not invited]
     
    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.
  • Abe H, Tani T, Mashiko H, Kitamura N, Hayami T, Watanabe S, Sakai K, Suzuki W, Mizukami H, Watakabe A, Yamamori T, Ichinohe N
    Frontiers in neuroanatomy 12 89 - 89 2018 [Refereed][Not invited]
     
    Neural activity in the middle temporal (MT) area is modulated by the direction and speed of motion of visual stimuli. The area is buried in a sulcus in the macaque, but exposed to the cortical surface in the marmoset, making the marmoset an ideal animal model for studying MT function. To better understand the details of the roles of this area in cognition, underlying anatomical connections need to be clarified. Because most anatomical tracing studies in marmosets have used retrograde tracers, the axonal projections remain uncharacterized. In order to examine axonal projections from MT, we utilized adeno-associated viral (AAV) tracers, which work as anterograde tracers by expressing either green or red fluorescent protein in infected neurons. AAV tracers were injected into three sites in MT based on retinotopy maps obtained via in vivo optical intrinsic signal imaging. Brains were sectioned and divided into three series, one for fluorescent image scanning and two for myelin and Nissl substance staining to identify specific brain areas. Overall projection patterns were similar across the injections. MT projected to occipital visual areas V1, V2, V3 (VLP) and V4 (VLA) and surrounding areas in the temporal cortex including MTC (V4T), MST, FST, FSTv (PGa/IPa) and TE3. There were also projections to the dorsal visual pathway, V3A (DA), V6 (DM) and V6A, the intraparietal areas AIP, LIP, MIP, frontal A4ab and the prefrontal cortex, A8aV and A8C. There was a visuotopic relationship with occipital visual areas. In a marmoset in which two tracer injections were made, the projection targets did not overlap in A8aV and AIP, suggesting topographic projections from different parts of MT. Most of these areas are known to send projections back to MT, suggesting that they are reciprocally connected with it.
  • Hiroshi Abe, Toshiki Tani, Hiromi Mashiko, Naohito Kitamura, Naohisa Miyakawa, Koki Mimura, Kazuhisa Sakai, Wataru Suzuki, Tohru Kurotani, Hiroaki Mizukami, Akiya Watakabe, Tetsuo Yamamori, Noritaka Ichinohe
    JOURNAL OF NEUROSCIENCE METHODS 286 102 - 113 0165-0270 2017/07 [Refereed][Not invited]
     
    Background: The brain of the common marmoset (Callithrix jacchus) is becoming a popular non-human primate model in neuroscience research. Because its brain fiber connectivity is still poorly understood, it is necessary to collect and present connection and trajectory data using tracers to establish a marmoset brain connectivity database. New method: To visualize projections and trajectories of axons, brain section images were reconstructed in 3D by registering them to the corresponding block-face brain images taken during brain sectioning. During preprocessing, autofluorescence of the tissue was reduced by applying independent component analysis to a set of fluorescent images taken using different filters. Results: The method was applied to a marmoset dataset after a tracer had been injected into an auditory belt area to fluorescently label axonal projections. Cortical and subcortical connections were clearly reconstructed in 3D. The registration error was estimated to be smaller than 200 pm. Evaluation tests on ICA-based autofluorescence reduction showed a significant improvement in signal and background separation. Comparison with existing methods: Regarding the 3D reconstruction error, the present study shows an accuracy comparable to previous studies using MRI and block-face images. Compared to serial section two-photon tomography, an advantage of the proposed method is that it can be combined with standard histological techniques. The images of differently processed brain sections can be integrated into the original ex vivo brain shape. Conclusions: The proposed method allows creating 3D axonal projection maps overlaid with brain area annotations based on the histological staining results of the same animal. (C) 2017 Elsevier B.V. All rights reserved.
  • Tsukasa Ohmori, Yasumitsu Nagao, Hiroaki Mizukami, Asuka Sakata, Shin-ichi Muramatsu, Keiya Ozawa, Shin-ichi Tominaga, Yutaka Hanazono, Satoshi Nishimura, Osamu Nureki, Yoichi Sakata
    SCIENTIFIC REPORTS 7 (1) 4159  2045-2322 2017/06 [Refereed][Not invited]
     
    Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B. Mutation-specific gene editing by simultaneous induction of homology-directed repair (HDR) sufficiently increased FIX levels to correct the disease phenotype. Insertion of F9 cDNA into the intron more efficiently restored haemostasis via both processes of non-homologous end-joining (NHEJ) and HDR following DSB. Notably, these therapies also cured neonate mice with haemophilia, which cannot be achieved with conventional gene therapy with AAV vector. Ongoing haemophilia therapy targeting the antithrombin gene with antisense oligonucleotide could be replaced by SaCas9/sgRNA-expressing AAV8 vector. Our results suggest that CRISPR/Cas9-mediated genome editing using an AAV8 vector provides a flexible approach to induce DSB at target genes in hepatocytes and could be a good strategy for haemophilia gene therapy.
  • F. Kurosaki, R. Uchibori, N. Mato, Y. Sehara, Y. Saga, M. Urabe, H. Mizukami, Y. Sugiyama, A. Kume
    GENE THERAPY 24 (5) 290 - 297 0969-7128 2017/05 [Refereed][Not invited]
     
    An efficient adeno-associated virus (AAV) vector was constructed for the treatment of respiratory diseases. AAV serotypes, promoters and routes of administration potentially influencing the efficiency of gene transfer to airway cells were examined in the present study. Among the nine AAV serotypes (AAV1-9) screened in vitro and four serotypes (AAV1, 2, 6, 9) evaluated in vivo, AAV6 showed the strongest transgene expression. As for promoters, the cytomegalovirus (CMV) early enhancer/chicken beta-actin (CAG) promoter resulted in more robust transduction than the CMV promoter. Regarding delivery routes, intratracheal administration resulted in strong transgene expression in the lung, whereas the intravenous and intranasal administration routes yielded negligible expression. The combination of the AAV6 capsid and CAG promoter resulted in sustained expression, and the intratracheally administered AAV6-CAG vector transduced bronchial cells and pericytes in the lung. These results suggest that AAV6-CAG vectors are more promising than the previously preferred AAV2 vectors for airway transduction, particularly when administered into the trachea. The present study offers an optimized strategy for AAV-mediated gene therapy for lung diseases, such as cystic fibrosis and pulmonary fibrosis.
  • Masafumi Takaji, Atsushi Takemoto, Chihiro Yokoyama, Akiya Watakabe, Hiroaki Mizukami, Keiya Ozawa, Hirotaka Onoe, Katsuki Nakamura, Tetsuo Yamamori
    SCIENTIFIC REPORTS 6 35809  2045-2322 2016/11 [Refereed][Not invited]
     
    The striatum plays important motor, associative and cognitive roles in brain functions. However, the rodent dorsolateral ( the primate putamen) and dorsomedial ( the primate caudate nucleus) striatum are not anatomically separated, making it difficult to distinguish their functions. By contrast, anatomical separation exists between the caudate nucleus and putamen in primates. Here, we successfully decreased dopamine D1 receptor ( D1R) or D2R mRNA expression levels selectively in the marmoset caudate using shRNA knockdown techniques, as determined using positron emission tomography imaging with specific D1R and D2R ligands and postmortem in situ hybridization analysis. We then conducted a voxel-based correlation analysis between binding potential values of PET imaging and visual discrimination learning task performance in these genetically modified marmosets to find a critical role for the caudate D2R but no apparent role for the caudate D1R. This latter finding challenges the current understanding of the mechanisms underlying D1R activation in the caudate.
  • Noriko Isoo, Takae Ohno, Mutsumi Isowaki, Satoshi Fukuda, Naoyuki Murabe, Hiroaki Mizukami, Keiya Ozawa, Masayoshi Mishina, Masaki Sakurai
    SCIENTIFIC REPORTS 6 34196  2045-2322 2016/09 [Refereed][Not invited]
     
    Neuronal plasticity is especially active in the young, during short windows of time termed critical periods, and loss of a critical period leads to functional limitations in the adults. The mechanism that governs the length of critical periods remains unknown. Here we show that levels of the NMDA receptor GluN2B subunit, which functions as a Ca2+ channel, declines in spinal cord synapses toward the end of the critical period for activity-dependent corticospinal synapse elimination. This period could be prolonged by blocking the decline of GluN2B, and after its termination the critical period could be reopened through upregulation of GluN2B. It is known that inhibitory neural activity increases with development in the CNS including the spinal cord. Suppression of the increasing inhibitory activity using low-dose strychnine also prolonged this critical period. During the strychnine-widened time window, Ca2+ influx through GluN2B channels returned to a level comparable to that seen during the critical period, though the level of GluN2B was slightly reduced. These findings indicate that loss of GluN2B subunits and the associated reduction in Ca2+ influx determines the end of the critical period in our in vitro CS system.
  • Peter Csikota, Anna Fodor, Diana Balazsfi, Otto Pinter, Hiroaki Mizukami, Stefan Weger, Regine Heilbronn, Mario Engelmann, Dora Zelena
    STRESS-THE INTERNATIONAL JOURNAL ON THE BIOLOGY OF STRESS 19 (4) 349 - 361 1025-3890 2016/07 [Refereed][Not invited]
     
    Vasopressin, a nonapeptide, signaling both as hormone in the blood and neuromodulator/neurotransmitter in the brain is considered to be causally involved in the pathological changes underlying anxiety and depression. In the present review we summarize experimental data obtained with Brattleboro rats as a model of congenital vasopressin-deficiency to test the hypothesis that central vasopressin signaling contributes to anxiety- and depression-like behavior. Male, female and lactating rats were studied. We focused on the paraventricular nucleus of the hypothalamus (PVN) and the septum, two brain areas in which vasopressin is proposed to control the endocrine and behavioral stress response, respectively. The presented data support the hypothesis that the behavioral changes seen in these rats are brought about by an altered vasopressin signaling at the brain level. Whereas vasopressin synthesized and released within the hypothalamus is primarily involved in endocrine regulation, vasopressin signaling in other brain areas may contribute to anxiety- and depression-like behavioral parameters. Further studies in this context might focus particularly on the interplay between extra-hypothalamic brain areas such as the septum and the medial amygdala.
  • Dongdong Wang, Yasushi Saga, Naoto Sato, Toshikazu Nakamura, Osamu Takikawa, Hiroaki Mizukami, Shigeki Matsubara, Hiroyuki Fujiwara
    INTERNATIONAL JOURNAL OF ONCOLOGY 48 (6) 2303 - 2309 1019-6439 2016/06 [Refereed][Not invited]
     
    Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme involved in tumor malignancy. However, the regulatory mechanism underlying its involvement remains largely uncharacterized. The present study aimed to investigate the hypothesis that NK4, an antagonist of hepatocyte growth factor (HGF), can regulate IDO and to characterize the signaling mechanism involved. Following successful transfection of the human ovarian cancer cell line SKOV-3 (which constitutively expresses IDO) with an NK4 expression vector, we observed that NK4 expression suppressed IDO expression; furthermore, NK4 expression did not suppress cancer cell growth in vitro [in the absence of natural killer (NK) cells], but did influence tumor growth in vivo. In addition, NK4 enhanced the sensitivity of cancer cells to NK cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. In an effort to clarify the mechanisms by which NK4 interacts with IDO, we performed investigations utilizing various biochemical inhibitors. The results of these investigations were as follows. First, c-Met (a receptor of HGF) tyrosine kinase inhibitor PHA-665752, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 both suppress IDO expression. Second, enhanced expression of PTEN (a known tumor suppressor) via negative regulation within a PI3K-AKT pathway, inhibits IDO expression. Conversely, neither the MEK1/2 inhibitor U0126 nor the STAT3 inhibitor WP1066 affects IDO expression. These results suggest that NK4 inhibits IDO expression via a c-Met-PI3K-AKT signaling pathway.
  • Shizu Hidema, Tomokazu Fukuda, Yuichi Hiraoka, Hiroaki Mizukami, Ryotaro Hayashi, Ayano Otsuka, Shingo Suzuki, Shinji Miyazaki, Katsuhiko Nishimori
    JOURNAL OF CELLULAR BIOCHEMISTRY 117 (5) 1099 - 1111 0730-2312 2016/05 [Refereed][Not invited]
     
    The neurohypophysial hormone oxytocin (OXT) and its receptor (OXTR) have critical roles in the regulation of pro-social behaviors, including social recognition, pair bonding, parental behavior, and stress-related responses. Supporting this hypothesis, a portion of patients suffering from autism spectrum disorder have mutations, such as single nucleotide polymorphisms, or epigenetic modifications in their OXTR gene. We previously reported that OXTR-deficient mice exhibit pervasive social deficits, indicating the critical role of OXTR in social behaviors. In the present study, we generated Oxtr cDNA(HA)-Ires-Cre knock-in mice, expressing both OXTR and Cre recombinase under the control of the endogenous Oxtr promoter. Knock-in cassette of Oxtr cDNA(HA)-Ires-Cre consisted of Oxtr cDNA tagged with the hemagglutinin epitope at the 3' end (Oxtr cDNA(HA)), internal ribosomal entry site (Ires), and Cre. Cre was expressed in the uterus, mammary gland, kidney, and brain of Oxtr cDNA(HA)-Ires-Cre knock-in mice. Furthermore, the distribution of Cre in the brain was similar to that observed in Oxtr-Venus fluorescent protein expressing mice (Oxtr-Venus), another animal model previously generated by our group. Social behavior of Oxtr cDNA(HA)-Ires-Cre knock-in mice was similar to that of wild-type animals. We demonstrated that this construct is expressed in OXTR-expressing neurons specifically after an infection with the recombinant adeno-associated virus carrying the flip-excision switch vector. Using this system, we showed the transport of the wheat-germ agglutinin tracing molecule from the OXTR-expressing neurons to the innervated neurons in knock-in mice. This study might contribute to the monosynaptic analysis of neuronal circuits and to the optogenetic analysis of neurons expressing OXTR. (C) 2015 Wiley Periodicals, Inc.
  • Yoshifumi Takahashi, Yasushi Saga, Takahiro Koyanagi, Yuji Takei, Shizuo Machida, Akiyo Taneichi, Hiroaki Mizukami, Yasufumi Sato, Shigeki Matsubara, Hiroyuki Fujiwara
    CANCER SCIENCE 107 (5) 629 - 637 1347-9032 2016/05 [Refereed][Not invited]
     
    Vasohibin-1 (VASH1) is a negative feedback regulator of angiogenesis, the first to be discovered, and was identified in vascular endothelial growth factor (VEGF)-stimulated vascular endothelial cells. Vasohibin-1 inhibits abnormal vascularization induced by various angiogenic factors including fibroblast growth factor and platelet-derived growth factor (PDGF), in addition to VEGF. By focusing on this characteristic of VASH1, we investigated the antitumor effects of VASH1 expression on ovarian cancer cells that produce different angiogenic factors. By using a high VEGF-producing ovarian cancer cell line, SHIN-3, and a high PDGF-producing ovarian cancer cell line, KOC-2S, the cells were transfected with either a VEGF antagonist, soluble VEGF receptor-1 (sVEGFR-1, or sFlt-1), or VASH1 genes to establish their respective cellular expression. The characteristics of these transfectants were compared with controls. We previously reported that the expression of sFlt-1 inhibited tumor vascularization and growth of high VEGF-producing ovarian cancer cells, reduced peritoneal dissemination and ascites development, and prolonged the survival time of the host. However, in the current study, the expression of sFlt-1 had no such effect on the high PDGF-producing ovarian cancer cells used here, whereas VASH1 expression inhibited tumor vascularization and growth, not only in high VEGF-producing cells, but also in high PDGF-producing cells, reduced their peritoneal dissemination and ascites, and prolonged the survival time of the host. These results suggest that VASH1 is an effective treatment for ovarian cancer cells that produce different angiogenic factors.
  • Motoi Kobayashi, Fumitake Usui, Tadayoshi Karasawa, Akira Kawashima, Hiroaki Kimura, Yoshiko Mizushina, Koumei Shirasuna, Hiroaki Mizukami, Tadashi Kasahara, Naoyuki Hasebe, Masafumi Takahashi
    SCIENTIFIC REPORTS 6 26489  2045-2322 2016/05 [Refereed][Not invited]
     
    NLRP3 inflammasomes recognize non-microbial danger signals and induce release of proinflammatory cytokine interleukin (IL)-1 beta, leading to sterile inflammation in cardiovascular disease. Because sterile inflammation is involved in doxorubicin (Dox)-induced cardiotoxicity, we investigated the role of NLRP3 inflammasomes in Dox-induced cardiotoxicity. Cardiac dysfunction and injury were induced by low-dose Dox (15 mg/kg) administration in NLRP3-deficient (NLRP3(-/-)) mice but not in wild-type (WT) and IL-1 beta(-/-) mice, indicating that NLRP3 deficiency enhanced the susceptibility to Dox-induced cardiotoxicity independent of IL-1 beta. Although the hearts of WT and NLRP3(-/-) mice showed no significant difference in inflammatory cell infiltration, macrophages were the predominant inflammatory cells in the hearts, and cardiac IL-10 production was decreased in Dox-treated NLRP3(-/-) mice. Bone marrow transplantation experiments showed that bone marrow-derived cells contributed to the exacerbation of Dox-induced cardiotoxicity in NLRP3(-/-) mice. In vitro experiments revealed that NLRP3 deficiency decreased IL-10 production in macrophages. Furthermore, adeno-associated virus-mediated IL-10 overexpression restored the exacerbation of cardiotoxicity in the NLRP3(-/-) mice. These results demonstrated that NLRP3 regulates macrophage IL-10 production and contributes to the pathophysiology of Dox-induced cardiotoxicity, which is independent of IL-1 beta. Our findings identify a novel role of NLRP3 and provided new insights into the mechanisms underlying Dox-induced cardiotoxicity.
  • Hiroaki Mizukami, Yoichi Sakata
    Gene Therapy and Cell Therapy Through the Liver: Current Aspects and Future Prospects 59 - 73 2016/01 [Refereed][Not invited]
     
    Adeno-associated virus (AAV) vector is widely used in gene transfer purposes. Not only the experimental gene transfer but also applications toward disease therapy are promising. Numerous attempts are ongoing for this purpose. One of the most prominent examples is the sustained clinical benefi t in hemophilia gene therapy targeting the liver. These successes have been brought chiefl y by the progress in vectorology, especially capsid development. At present, one of the biggest issues is the presence of neutralizing antibody (NAb) against AAV vector capsid. Challenges have been made to conquer this problem. Although there are still some hurdles for wide clinical application, use of this vector will soon be a common practice for the treatment of suitable disease conditions.
  • T. Kasahara, A. Takata, T. M. Kato, M. Kubota-Sakashita, T. Sawada, A. Kakita, H. Mizukami, D. Kaneda, K. Ozawa, T. Kato
    Molecular Psychiatry 21 (1) 39 - 48 1476-5578 2016/01 [Refereed][Not invited]
     
    Depression is a common debilitating human disease whose etiology has defied decades of research. A critical bottleneck is the difficulty in modeling depressive episodes in animals. Here, we show that a transgenic mouse with chronic forebrain expression of a dominant negative mutant of Polg1, a mitochondrial DNA (mtDNA) polymerase, exhibits lethargic behavioral changes, which are associated with emotional, vegetative and psychomotor disturbances, and response to antidepression drug treatment. The results suggested a symptomatic similarity between the lethargic behavioral change that was recurrently and spontaneously experienced by the mutant mice and major depressive episode as defined by DSM-5. A comprehensive screen of mutant brain revealed a hotspot for mtDNA deletions and mitochondrial dysfunction in the paraventricular thalamic nucleus (PVT) with similar defects observed in postmortem brains of patients with mitochondrial disease with mood symptoms. Remarkably, the genetic inhibition of PVT synaptic output by Cre-loxP-dependent expression of tetanus toxin triggered de novo depression-like episodes. These findings identify a novel preclinical mouse model and brain area for major depressive episodes with mitochondrial dysfunction as its cellular mechanism.
  • Hitoshi Maeda, Satoshi Fukuda, Hiroshi Kameda, Naoyuki Murabe, Noriko Isoo, Hiroaki Mizukami, Keiya Ozawa, Masaki Sakurai
    JOURNAL OF PHYSIOLOGY-LONDON 594 (1) 189 - 205 0022-3751 2016/01 [Refereed][Not invited]
     
    Key points Direct connections between corticospinal (CS) axons and motoneurons (MNs) appear to be present only in higher primates, where they are essential for discrete movement of the digits. Their presence in adult rodents was once claimed but is now questioned. We report that MNs innervating forearm muscles in infant rats receive monosynaptic input from CS axons, but MNs innervating proximal muscles do not, which is a pattern similar to that in primates. Our experiments were carefully designed to show monosynaptic connections. This entailed selective electrical and optogenetic stimulation of CS axons and recording from MNs identified by retrograde labelling from innervated muscles. Morphological evidence was also obtained for rigorous identification of CS axons and MNs. These connections would be transient and would regress later during development. These results shed light on the development and evolution of direct CS-MN connections, which serve as the basis for dexterity in humans. AbstractRecent evidence suggests there is no direct connection between corticospinal (CS) axons and spinal motoneurons (MNs) in adult rodents. We previously showed that CS synapses are present throughout the spinal cord for a time, but are eliminated from the ventral horn during development in rodents. This raises the possibility that CS axons transiently make direct connections with MNs located in the ventral horn of the spinal cord. This was tested in the present study. Using cervical cord slices prepared from rats on postnatal days (P) 7-9, CS axons were stimulated and whole cell recordings were made from MNs retrogradely labelled with fluorescent cholera toxin B subunit (CTB) injected into selected groups of muscles. To selectively activate CS axons, electrical stimulation was carefully limited to the CS tract. In addition we employed optogenetic stimulation after injecting an adeno-associated virus vector encoding channelrhodopsin-2 (ChR2) into the sensorimotor cortex on P0. We were then able to record monosynaptic excitatory postsynaptic currents from MNs innervating forearm muscles, but not from those innervating proximal muscles. We also showed close contacts between CTB-labelled MNs and CS axons labelled through introduction of fluorescent protein-conjugated synaptophysin or the ChR2 expression system. We confirmed that some of these contacts colocalized with postsynaptic density protein 95 in their partner dendrites. It is intriguing from both phylogenetic and ontogenetic viewpoints that direct and putatively transient CS-MN connections were found only on MNs innervating the forearm muscles in infant rats, as this is analogous to the connection pattern seen in adult primates. Key points Direct connections between corticospinal (CS) axons and motoneurons (MNs) appear to be present only in higher primates, where they are essential for discrete movement of the digits. Their presence in adult rodents was once claimed but is now questioned. We report that MNs innervating forearm muscles in infant rats receive monosynaptic input from CS axons, but MNs innervating proximal muscles do not, which is a pattern similar to that in primates. Our experiments were carefully designed to show monosynaptic connections. This entailed selective electrical and optogenetic stimulation of CS axons and recording from MNs identified by retrograde labelling from innervated muscles. Morphological evidence was also obtained for rigorous identification of CS axons and MNs. These connections would be transient and would regress later during development. These results shed light on the development and evolution of direct CS-MN connections, which serve as the basis for dexterity in humans.
  • Yoshifumi Takahashi, Yasushi Saga, Takahiro Koyanagi, Yuji Takei, Sizuo Machida, Akiyo Taneichi, Hiroaki Mizukami, Yasufumi Sato, Shigeki Matsubara, Hiroyuki Fujiwara
    INTERNATIONAL JOURNAL OF ONCOLOGY 47 (6) 2057 - 2063 1019-6439 2015/12 [Refereed][Not invited]
     
    Vasohibin-1 (VASH1) is expressed in vascular endothelial cells stimulated by several angiogenic growth factors and displays autocrine activity to regulate angiogenesis via a negative feedback mechanism. In this study, we investigated the effect of VASH1 on ovarian cancer progression using VASH1-expressing ovarian cancer cells in vitro and in vivo. The growth ability of ovarian cancer cells engineered to express the VASH1 gene remained unchanged in vitro. However, we showed that VASH1 secretion by tumor cells inhibited the growth of human umbilical vein endothelial cells. Further, animal experiments showed that VASH1 expression inhibited tumor angiogenesis and growth. In a murine model of peritoneal dissemination of ovarian cancer cells, VASH1 inhibited peritoneal dissemination and ascites, resulting in significantly prolonged survival in mice. This indicates that VASH1 exerts an antitumor effect on ovarian cancer by inhibiting angiogenesis in the tumor environment. These findings suggest that a novel therapy based on VASH1 could be a useful therapeutic strategy for ovarian cancer.
  • Osamu Sadakane, Yoshito Masamizu, Akiya Watakabe, Shin-Ichiro Terada, Masanari Ohtsuka, Masafumi Takaji, Hiroaki Mizukami, Keiya Ozawa, Hiroshi Kawasaki, Masanori Matsuzaki, Tetsuo Yamamori
    CELL REPORTS 13 (9) 1989 - 1999 2211-1247 2015/12 [Refereed][Not invited]
     
    Two-photon imaging with genetically encoded calcium indicators (GECIs) enables long-term observation of neuronal activity in vivo. However, there are very few studies of GECIs in primates. Here, we report a method for long-term imaging of a GECI, GCaMP6f, expressed from adeno-associated virus vectors in cortical neurons of the adult common marmoset (Callithrix jacchus), a small New World primate. We used a tetracycline-inducible expression system to robustly amplify neuronal GCaMP6f expression and up-and downregulate it for more than 100 days. We succeeded in monitoring spontaneous activity not only from hundreds of neurons three-dimensionally distributed in layers 2 and 3 but also from single dendrites and axons in layer 1. Furthermore, we detected selective activities from somata, dendrites, and axons in the somatosensory cortex responding to specific tactile stimuli. Our results provide a way to investigate the organization and plasticity of cortical microcircuits at subcellular resolution in non-human primates.
  • Yoshiyuki Kasahara, Yuko Tateishi, Yuichi Hiraoka, Ayano Otsuka, Hiroaki Mizukami, Keiya Ozawa, Keisuke Sato, Shizu Hidema, Katsuhiko Nishimori
    FRONTIERS IN ENDOCRINOLOGY 6 180  1664-2392 2015/11 [Refereed][Not invited]
     
    Recent papers have reported that oxytocin (Oxt) and the oxytocin receptor (Oxtr) may be involved in the regulation of food intake in mammals. We therefore suspected the Oxt/Oxtr system to be involved in energy homeostasis. In previous studies, we found a tendency toward obesity in Oxtr-deficient (Oxtr(-/-))mice, as well as impaired thermoregulation when these mice were exposed to cold conditions. In the present study, we observed the expression of Oxtr in the rostral medullary raphe (RMR), the brain region known to control thermogenesis in brown adipose tissue (BAT). Through immunohistochemistry, we detected neurons expressing Oxtr and c-Fos in the RMR of mice exposed to cold conditions. Up to 40% of Oxtr-positive neurons in RMR were classified as glutamatergic neurons, as shown by immunostaining using anti-VGLUT3 antibody. In addition, mice with exclusive expression of Oxtr in the RMR were generated by injecting an MV-Oxtr vector into the RMR region of Oxtr(-/-) mice. We confirmed the recovery of thermoregulatory ability in the manipulated mice during exposure to cold conditions. Moreover, mice with RMR-specific expression of Oxtr lost the typical morphological change in BAT observed in Oxtr(-/-) mice. Additionally, increased expression of the p-adrenergic receptor gene, Adrb3, was observed in BAT. These results are the first to show the critical role of RMR Oxtr expression in thermoregulation during cold conditions.
  • Koumei Shirasuna, Fumitake Usui, Tadayoshi Karasawa, Hiroaki Kimura, Akira Kawashima, Hiroaki Mizukami, Akihide Ohkuchi, Satoshi Nishimura, Junji Sagara, Tetsuo Noda, Keiya Ozawa, Shun'Ichiro Taniguchi, Masafumi Takahashi
    Nanotoxicology 9 (5) 554 - 567 1743-5404 2015/08 [Refereed][Not invited]
     
    Despite the increasing commercial use of nanoparticles, little is known about their effects on placental inflammation and pregnancy complications. In this study, nanosilica (NS) particles upregulated the inflammasome component nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) and induced placental inflammation and reactive oxygen species (ROS) generation, resulting in pregnancy complications. Furthermore, NS-induced pregnancy complications were markedly improved in Nlrp3< sup> -/-< /sup> mice but not in component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-deficient (Asc< sup> -/-< /sup> ) mice, indicating the independence of NLRP3 inflammasomes. Pregnancy complications in Nlrp3< sup> -/-< /sup> and Asc< sup> -/-< /sup> mice phenotypes were dependent on the balance between interleukin (IL)-1 and IL-10. NS-induced pregnancy complications were completely prevented by either inhibition of ROS generation or forced expression of IL-10. Our findings provide important information about NS-induced placental inflammation and pregnancy complications and the novel pathophysiological roles of NLRP3 and ASC in pregnancy.
  • Akiya Watakabe, Masanari Ohtsuka, Masaharu Kinoshita, Masafumi Takaji, Kaoru Isa, Hiroaki Mizukami, Keiya Ozawa, Tadashi Isa, Tetsuo Yamamori
    NEUROSCIENCE RESEARCH 93 144 - 157 0168-0102 2015/04 [Refereed][Not invited]
     
    Here we investigated the transduction characteristics of adeno-associated viral vector (AAV) serotypes 1, 2, 5, 8 and 9 in the marmoset cerebral cortex. Using three constructs that each has hrGFP under ubiquitous (CMV), or neuron-specific (CaMKII and Synapsin I (SynI)) promoters, we investigated (1) the extent of viral spread, (2) cell type tropism, and (3) neuronal transduction efficiency of each serotype. AAV2 was clearly distinct from other serotypes in small spreading and neuronal tropism. We did not observe significant differences in viral spread among other serotypes. Regarding the cell tropism, AAV1, 5, 8 and 9 exhibited mostly glial expression for CMV construct. However, when the CaMKII construct was tested, cortical neurons were efficiently transduced (>similar to 70% in layer 3) by all serotypes, suggesting that glial expression obscured neuronal expression for CMV construct. For both SynI and CaMKII constructs, we observed generally high-level expression in large pyramidal cells especially in layer 5, as well as in parvalbumin-positive interneurons. The expression from the CaMKII construct was more uniformly observed in excitatory cells compared with SynI construct. Injection of the same viral preparations in mouse and macaque cortex resulted in essentially the same result with some species-specific differences. (C) 2014 The Authors. Published by Elsevier Ireland Ltd.
  • Nishimori, K, Sato, K, Hidema, S, Yoshida, M, Mizukami, H
    Interdisciplinary Information Sciences 21 283 - 288 2015 [Refereed][Not invited]
  • Koumei Shirasuna, Fumitake Usui, Tadayoshi Karasawa, Hiroaki Kimura, Akira Kawashima, Hiroaki Mizukami, Akihide Ohkuchi, Satoshi Nishimura, Junji Sagara, Tetsuo Noda, Keiya Ozawa, Shun'ichiro Taniguchi, Masafumi Takahashi
    NANOTOXICOLOGY 9 (5) 554 - 567 1743-5390 2015 [Refereed][Not invited]
     
    Despite the increasing commercial use of nanoparticles, little is known about their effects on placental inflammation and pregnancy complications. In this study, nanosilica (NS) particles upregulated the inflammasome component nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) and induced placental inflammation and reactive oxygen species (ROS) generation, resulting in pregnancy complications. Furthermore, NS-induced pregnancy complications were markedly improved in N mice but not in component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-deficient (Asc) mice, indicating the independence of NLRP3 inflammasomes. Pregnancy complications in Nlrp3(-/-) and Asc(-/-) mice phenotypes were dependent on the balance between interleukin (IL)-1 a and IL-10. NS-induced pregnancy complications were completely prevented by either inhibition of ROS generation or forced expression of IL-10. Our findings provide important information about NS-induced placental inflammation and pregnancy complications and the novel pathophysiological roles of NLRP3 and ASC in pregnancy.
  • Tsutomu Kamiyama, Hiroshi Kameda, Naoyuki Murabe, Satoshi Fukuda, Noboru Yoshioka, Hiroaki Mizukami, Keiya Ozawa, Masaki Sakurai
    JOURNAL OF NEUROSCIENCE 35 (3) 1181 - 1191 0270-6474 2015/01 [Refereed][Not invited]
     
    The corticospinal (CS) tract is essential for voluntary movement, but what we know about the organization and development of the CS tract remains limited. To determine the total cortical area innervating the seventh cervical spinal cord segment (C7), which controls forelimb movement, we injected a retrograde tracer (fluorescent microspheres) into C7 such that it would spread widely within the unilateral gray matter (to >80%), but not to the CS tract. Subsequent detection of the tracer showed that, in both juvenile and adult mice, neurons distributed over an unexpectedly broad portion of the rostral two-thirds of the cerebral cortex converge to C7. This even included cortical areas controlling the hindlimbs (the fourth lumbar segment, L4). With aging, cell densities greatly declined, mainly due to axon branch elimination. Whole-cell recordings from spinal cord cells upon selective optogenetic stimulation of CS axons, and labeling of axons (DsRed) and presynaptic structures (synaptophysin) through cotransfection using exo utero electroporation, showed that over-growing CS axons make synaptic connections with spinal cells in juveniles. This suggests that neuronal circuits involved in the CS tract to C7 are largely reorganized during development. By contrast, the cortical areas innervating L4 are limited to the conventional hindlimb area, and the cell distribution and density do not change during development. These findings call for an update of the traditional notion of somatotopic CS projection and imply that there are substantial developmental differences in the cortical control of forelimb and hindlimb movements, at least in rodents.
  • Osamu Sadakane, Akiya Watakabe, Masanari Ohtsuka, Masafumi Takaji, Tetsuya Sasaki, Masatoshi Kasai, Tadashi Isa, Go Kato, Junichi Nabekura, Hiroaki Mizukami, Keiya Ozawa, Hiroshi Kawasaki, Tetsuo Yamamori
    eNeuro 2 (4) 2373-2822 2015 [Refereed][Not invited]
     
    Two-photon microscopy in combination with a technique involving the artificial expression of fluorescent protein has enabled the direct observation of dendritic spines in living brains. However, the application of this method to primate brains has been hindered by the lack of appropriate labeling techniques for visualizing dendritic spines. Here, we developed an adeno-associated virus vector-based fluorescent protein expression system for visualizing dendritic spines in vivo in the marmoset neocortex. For the clear visualization of each spine, the expression of reporter fluorescent protein should be both sparse and strong. To fulfill these requirements, we amplified fluorescent signals using the tetracycline transactivator (tTA)–tetracycline-responsive element system and by titrating down the amount of Thy1S promoter-driven tTA for sparse expression. By this method, we were able to visualize dendritic spines in the marmoset cortex by two-photon microscopy in vivo and analyze the turnover of spines in the prefrontal cortex. Our results demonstrated that short spines in the marmoset cortex tend to change more frequently than long spines. The comparison of in vivo samples with fixed samples showed that we did not detect all existing spines by our method. Although we found glial cell proliferation, the damage of tissues caused by window construction was relatively small, judging from the comparison of spine length between samples with or without window construction. Our new labeling technique for two-photon imaging to visualize in vivo dendritic spines of the marmoset neocortex can be applicable to examining circuit reorganization and synaptic plasticity in primates.
  • Wanpeng Cui, Hiroaki Mizukami, Michiko Yanagisawa, Tomomi Aida, Masatoshi Nomura, Yoshikazu Isomura, Ryoichi Takayanagi, Keiya Ozawa, Kohichi Tanaka, Hidenori Aizawa
    JOURNAL OF NEUROSCIENCE 34 (49) 16273 - 16285 0270-6474 2014/12 [Refereed][Not invited]
     
    The lateral habenula (LHb) regulates the activity of monoaminergic neurons in the brainstem. This area has recently attracted a surge of interest in psychiatry because studies have reported the pathological activation of the habenula in patients with major depression and in animal models. The LHb plays a significant role in the pathophysiology of depression; however, how habenular neurons are activated to cause various depression symptoms, such as reduced motivation and sleep disturbance, remain unclear. We hypothesized that dysfunctional astrocytes may cause LHb hyperactivity due to the defective uptake activity of extracellular glutamate, which induces depressivelike behaviors. We examined the activity of neurons in habenular pathways and performed behavioral and sleep analyses in mice with pharmacological and genetic inhibition of the activity of the glial glutamate transporter GLT-1 in the LHb. The habenula-specific inhibition of GLT-1 increased the neuronal firing rate and the level of c-Fos expression in the LHb. Mice with reduced GLT-1 activity in the habenula exhibited a depressive-like phenotype in the tail suspension and novelty-suppressed feeding tests. These animals also displayed increased susceptibility to chronic stress, displaying more frequent avoidant behavior without affecting locomotor activity in the openfield test. Intriguingly, the mice showed disinhibition of rapid eye movement sleep, which is a characteristic sleep pattern in patients with depression. These results provide evidence that disrupting glutamate clearance in habenular astrocytes increases neuronal excitability and depressive-like phenotypes in behaviors and sleep.
  • Jun Mimuro, Hiroaki Mizukami, Midori Shima, Tadashi Matsushita, Masashi Taki, Shinji Muto, Satoshi Higasa, Michio Sakai, Tsukasa Ohmori, Seiji Madoiwa, Keiya Ozawa, Yoichi Sakata
    JOURNAL OF MEDICAL VIROLOGY 86 (11) 1990 - 1997 0146-6615 2014/11 [Refereed][Not invited]
     
    Pre-existing antibodies against adeno-associated virus (AAV), caused by natural AAV infections, interfere with recombinant AAV vector-mediated gene transfer. We studied the prevalence of neutralizing antibodies against AAV serotypes 1, 2, 5, 8, and 9 in healthy subjects (n=85) and hemophilia patients (n=59) in a Japanese population. For healthy subjects, the prevalence of neutralizing antibodies against AAV serotypes 1, 2, 5, 8, and 9 was 36.5%, 35.3%, 37.6%, 32.9%, and 36.5%, respectively, while that in hemophilia patients was 39.7%, 28.8%, 35.6%, 32.9%, and 27.4%, respectively. There was no difference in the prevalence of neutralizing antibody against each AAV serotype between the healthy subjects and the hemophilia patients. The prevalence of neutralizing antibodies against all AAV serotypes increased with age in both healthy subjects and hemophilia patients. High titers of neutralizing antibodies against AAV2 (1:224) and AAV8 (1:224) were more evident in older individuals (42 years old). Approximately 50% of all screened individuals were seronegative for neutralizing antibodies against each AAV tested, while approximately 25% of individuals were seropositive for each AAV serotype tested. The prevalence of seronegativity for all AAV serotypes was 67.0% (healthy subjects, 68.6%; hemophilia patients, 65.0%) and 18.6% (healthy subjects, 20.5%; hemophilia patients, 15.7%) in young (<42 years old) and older subjects (42 years old), respectively. The findings from this study suggested that young subjects are more likely to be eligible for gene therapy based on AAV vectors delivered via an intravascular route because of the low prevalence of antibodies to AAV capsids. J. Med. Virol. 86:1990-1997, 2014. (c) 2013 Wiley Periodicals, Inc.
  • Eduard Ayuso, Veronique Blouin, Martin Lock, Susan McGorray, Xavier Leon, Mauricio R. Alvira, Alberto Auricchio, Stephanie Bucher, Abdelwahed Chtarto, K. Reed Clark, Christophe Darmon, Monica Doria, Will Fountain, Guangping Gao, Kai Gao, Mauro Giacca, Juergen Kleinschmidt, Barbara Leuchs, Catherine Melas, Hiroaki Mizukami, Marcus Mueller, Yvet Noordman, Olivier Bockstael, Keiya Ozawa, Catherine Pythoud, Marina Sumaroka, Richard Surosky, Liliane Tenenbaum, Inge van der Linden, Brigitte Weins, J. Fraser Wright, Xinhua Zhang, Lorena Zentilin, Fatima Bosch, Richard O. Snyder, Philippe Moullier
    HUMAN GENE THERAPY 25 (11) 977 - 987 1043-0342 2014/11 [Refereed][Not invited]
     
    Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50 x 10(11) pt/ml; CI, 4.26 x 10(11) to 6.75 x 10(11) pt/ml), vector genomes (mean, 5.75 x 10(11) vg/ml; CI, 3.05 x 10(11) to 1.09 x 10(12) vg/ml), and infectious units (mean, 1.26 x 10(9) IU/ml; CI, 6.46 x 10(8) to 2.51 x 10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.
  • Akiya Watakabe, Masafumi Takaji, Shigeki Kato, Kazuto Kobayashi, Hiroaki Mizukami, Keiya Ozawa, Sonoko Ohsawa, Ryosuke Matsui, Dai Watanabe, Tetsuo Yamamori
    FRONTIERS IN NEURAL CIRCUITS 8 110  1662-5110 2014/09 [Refereed][Not invited]
     
    Here we present a novel tracing technique to stain projection neurons in Golgi-like detail by double viral infection. We used retrograde lentiviral vectors and adeno-associated viral vectors (AAV) to drive "TET-ON/TET-OFF system" in neurons connecting two regions. Using this method, we successfully labeled the corticothalamic (CT) cells of the mouse somatosensory barrel field (S1BF) and motor cortex (M1) in their entirety. We also labeled contra- and ipsilaterally-projecting corticocortical (CC) cells of M1 by targeting contralateral M1 or ipsilateral Si for retrograde infection. The strength of this method is that we can observe the morphology of specific projection neuron subtypes en masse. We found that the group of CT cells extends their dendrites and intrinsic axons extensively below but not within the thalamorecipient layer in both Si BE and M1, suggesting that the primary target of this cell type is not layer 4. We also found that both ipsi- and contralateral targeting CC cells in M1 commonly exhibit widespread collateral extensions to contralateral M1 (layers 1-6), bilateral Si and S2 (layers 1, 5 and 6), perirhinal cortex (layers 1, 2/3, 5, and 6), striatum and claustrum. These findings not only strengthened the previous findings of single cell tracings but also extended them by enabling cross-area comparison of CT cells or comparison of CC cells of two different labeling.
  • Tsukasa Ohmori, Hiroaki Mizukami, Seiji Madoiwa, Keiya Ozawa, Yoichi Sakata
    [Rinsho ketsueki] The Japanese journal of clinical hematology 55 (8) 899 - 907 0485-1439 2014/08
  • 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 1349-7006 2014/01 [Refereed][Not invited]
     
    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 72h 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 48h. 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.
  • Yoshiyuki Kasahara, Keisuke Sato, Yuki Takayanagi, Hiroaki Mizukami, Keiya Ozawa, Shizu Hidema, Kyoung-Ha So, Teruo Kawada, Nao Inoue, Ikuo Ikeda, Sang-Gun Roh, Keiichi Itoi, Katsuhiko Nishimori
    ENDOCRINOLOGY 154 (11) 4305 - 4315 0013-7227 2013/11 [Refereed][Not invited]
     
    Oxytocin (OXT) and OXT receptor (OXTR) have been implicated in the regulation of energy homeostasis, but the detailed mechanism is still unclear. We recently showed late-onset obesity and impaired cold-induced thermogenesis in male OXTR knockout (Oxtr(-/-)) mice. Here we demonstrate that the OXTR in the hypothalamus has important functions in thermoregulation. Male Oxtr(-/-) mice failed to maintain their body temperatures during exposure to a cold environment. Oxtr(-/-) mice also showed decreased neuronal activation in the thermoregulatory hypothalamic region during cold exposure. Normal cold-induced thermogenesis was recovered in Oxtr (/) mice by restoring OXTR to the hypothalamus with an adeno-associated virus-Oxtr vector. In addition, brown adipose tissue (BAT) in Oxtr(-/-) mice contained larger lipid droplets in both 10- and 20-week-old compared with BAT from age-matched Oxtr(-/-) control mice. In BAT, the expression level of beta 3-adrenergic receptor at normal temperature was lower in Oxtr(-/-) mice than that in control mice. In contrast, alpha 2A-adrenergic receptor expression level was higher in BAT from Oxtr(-/-) mice in both normal and cold temperatures. Because beta 3- and alpha 2A-adrenergic receptors are known to have opposite effects on the thermoregulation, the imbalance of adrenergic receptors is suspected to affect this dysfunction in the thermoregulation. Our study is the first to demonstrate that the central OXT/OXTR system plays important roles in the regulation of body temperature homeostasis.
  • 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 0470-8105 2013/10 [Refereed][Not invited]
     
    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.
  • Uehara T, Kanazawa T, Mizukami H, Uchibori R, Tsukahara T, Urabe M, Kume A, Misawa K, Carey TE, Suzuki M, Ichimura K, Ozawa K
    Cancer science 1347-9032 2013/10 [Refereed][Not invited]
  • Yomayra F. Guzman, Natalie C. Tronson, Vladimir Jovasevic, Keisuke Sato, Anita L. Guedea, Hiroaki Mizukami, Katsuhiko Nishimori, Jelena Radulovic
    NATURE NEUROSCIENCE 16 (9) 1185 - U36 1097-6256 2013/09 [Refereed][Not invited]
     
    The nonapeptide oxytocin is considered beneficial to mental health due to its anxiolytic, prosocial and antistress effects, but evidence for anxiogenic actions of oxytocin in humans has recently emerged. Using region-specific manipulations of the mouse oxytocin receptor (Oxtr) gene (Oxtr), we identified the lateral septum as the brain region mediating fear-enhancing effects of Oxtr. These effects emerge after social defeat and require Oxtr specifically coupled to the extracellular signal-regulated protein kinase pathway.
  • 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 1347-9032 2013/08 [Refereed][Not invited]
     
    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 0006-291X 2013/08 [Refereed][Not invited]
     
    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.
  • Atsushi Yasumoto, Seiji Madoiwa, Yuji Kashiwakura, Akira Ishiwata, Tsukasa Ohmori, Hiroaki Mizukami, Keiya Ozawa, Yoichi Sakata, Jun Mimuro
    THROMBOSIS RESEARCH 131 (5) 444 - 449 0049-3848 2013/05 [Refereed][Not invited]
     
    Introduction: Factor VIII (FVIII) treatment for hemophilia A has difficulties in correcting bleeding diathesis in the presence of inhibitors. Materials and Methods: An adeno-associated virus type 8 (AAV8) vector containing the factor VII (FVII) gene or the activated factor VII (FVIIa) gene was used to investigate the therapeutic effect of FVII or FVIIa overexpression in FVIII-deficient mice with inhibitors. Results: Following repeated human FVIII injection, FVIII-deficient mice developed anti-human FVIII antibodies that cross-reacted with mouse FVIII. High transgene expression of murine FVII or murine FVIIa was achieved using the AAV8 vector and resulted in increased blood FVII activity greater than 800% of normal murine FVII levels in vector-injected FVIII-deficient mice. Thromboelastography analysis showed significant improvements in clotting time, clot formation time, a angle, and mean clot firmness in AAV8 vector-injected FVIII-deficient mice with inhibitors. Overexpression of FVIIa ameliorated the bleeding phenotype of FVIII-deficient mice with inhibitors and significantly increased the survival rate after tail clipping. In addition, overexpression of FVII increased the survival rate of FVIII-deficient mice with inhibitors after tail clipping though it was not as efficient as FVIIa overexpression. Conclusions: These data suggest that FVII overexpression is an alternative strategy for the treatment of hemophilia A with inhibitors. (C) 2013 Elsevier Ltd. All rights reserved.
  • Masaru Shimada, Shinya Abe, Toru Takahashi, Kazumasa Shiozaki, Mitsue Okuda, Hiroaki Mizukami, Dennis M. Klinman, Keiya Ozawa, Kenji Okuda
    PLOS ONE 8 (3) e57606  1932-6203 2013/03 [Refereed][Not invited]
     
    We previously reported on a monoclonal antibody (mAb) that targeted amyloid beta (A beta) protein. Repeated injection of that mAb reduced the accumulation of A beta protein in the brain of human A beta transgenic mice (Tg2576). In the present study, cDNA encoding the heavy and light chains of this mAb were subcloned into an adeno-associated virus type 1 (AAV) vector with a 2A/furin adapter. A single intramuscular injection of 3.0x10(10) viral genome of these AAV vectors into C57BL/6 mice generated serum anti-A beta Ab levels up to 0.3 mg/ml. Anti-A beta Ab levels in excess of 0.1 mg/ml were maintained for up to 64 weeks. The effect of AAV administration on A beta levels in vivo was examined. A significant decrease in A beta levels in the brain of Tg2576 mice treated at 5 months (prophylactic) or 10 months (therapeutic) of age was observed. These results support the use of AAV vector encoding anti-A beta Ab for the prevention and treatment of Alzheimer's disease.
  • Jun Mimuro, Hiroaki Mizukami, Shuji Hishikawa, Tomokazu Ikemoto, Akira Ishiwata, Asuka Sakata, Tsukasa Ohmori, Seiji Madoiwa, Fumiko Ono, Keiya Ozawa, Yoichi Sakata
    MOLECULAR THERAPY 21 (2) 318 - 323 1525-0016 2013/02 [Refereed][Not invited]
     
    Neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) are known to interfere with AAV vector-mediated gene transfer by intravascular delivery. Evading the inhibitory effects of antibodies against AAV vectors is necessary for efficient transfer of therapeutic genes clinically. For this purpose, we tested the efficacy of saline flushing in order to avoid contact of vectors with NAbs present in blood. Direct injection of the AAV8 vector carrying the factor IX (FIX) gene into the portal vein of macaques using saline flushing achieved transgene-derived FIX expression (4.7 +/- 2.10-10.1 +/- 5.45% of normal human FIX concentration) in the presence of NAbs. Expression was as efficient as that (5.43 +/- 2.59-12.68 +/- 4.83%) in macaques lacking NAbs. We next tested the efficacy of saline flushing using less invasive balloon catheter-guided injection. This approach also resulted in efficient expression of transgene-derived FIX (2.5 +/- 1.06-9.0 +/- 2.37%) in the presence of NAbs (14-56x dilutions). NAbs at this range of titers reduced the efficiency of transduction in the macaque liver by 100-fold when the same vector was injected into mesenteric veins without balloon catheters. Our results suggest that portal vein-directed vector delivery strategies with flushing to remove blood are efficacious for minimizing the inhibitory effect of anti-AAV antibodies.
  • Ryosuke Uchibori, Tomonori Tsukahara, Hiroyuki Mizuguchi, Yasushi Saga, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Keiya Ozawa
    CANCER RESEARCH 1 73 (1) 364 - 372 0008-5472 2013/01 [Refereed][Not invited]
     
    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.
  • Katsusuke Hata, Hiroaki Mizukami, Osamu Sadakane, Akiya Watakabe, Masanari Ohtsuka, Masafumi Takaji, Masaharu Kinoshita, Tadashi Isa, Keiya Ozawa, Tetsuo Yamamori
    Journal of Neuroscience 33 (50) 19704 - 19714 0270-6474 2013 [Refereed][Not invited]
     
    Distinct anatomical regions of the neocortex subserve different sensory modalities and neuronal integration functions, but mechanisms for these regional specializations remain elusive. Involvement of epigenetic mechanisms for such specialization through the spatiotemporal regulation of gene expression is an intriguing possibility. Here we examined whether epigenetic mechanisms might play a role in the selective gene expression in the association areas (AAs) and the primary visual cortex (V1) in macaque neocortex. By analyzing the two types of area-selective gene promoters that we previously identified, we found a striking difference of DNA methylation between these promoters, i.e., hypermethylation in AA-selective gene promoters and hypomethylation in V1-selective ones. Methylation levels of promoters of each area-selective gene showed no areal difference, but a specific methyl-binding protein (MBD4) was enriched in the AAs, in correspondence with expression patterns of AA-selective genes. MBD4 expression was mainly observed in neurons. MBD4 specifically bound to and activated the AA-selective genes both in vitro and in vivo. Our results demonstrate that methylation in the promoters and specific methyl-binding proteins play an important role in the area-selective gene expression profiles in the primate neocortex. © 2013 the authors.
  • Naoto Sato, Yasushi Saga, Hiroaki Mizukami, Dongdong Wang, Suzuyo Takahashi, Hiroaki Nonaka, Hiroyuki Fujiwara, Yuji Takei, Shizuo Machida, Osamu Takikawa, Keiya Ozawa, Mitsuaki Suzuki
    ONCOLOGY REPORTS 28 (5) 1574 - 1578 1021-335X 2012/11 [Refereed][Not invited]
     
    This study examined the role of the immunosuppressive enzyme indoleamine-2,3-dioxygenase (IDO) in cervical cancer progression and the possible use of this enzyme for cervical cancer therapy. We analyzed IDO protein expression in 9 cervical cancer cell lines (SKG-I, -II, -IIIa, -IIIb, SiHa, CaSki, BOKU, HCS-2 and ME-180) stimulated with interferon-(gamma). IDO expression was observed in all cell lines except for SKG-IIIb. We transfected the human cervical cancer cell line CaSki that constitutively expresses IDO with a short hairpin RNA vector targeting IDO, and established an IDO-downregulated cell line to determine whether inhibition of IDO mediates cervical cancer progression. IDO downregulation suppressed tumor growth in vivo, without influencing cancer cell growth in vitro. Moreover, IDO downregulation enhanced the sensitivity of cervical cancer cells to natural killer (NK) cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. These findings indicate that downregulation of IDO controls cervical cancer progression by activating NK cells, suggesting IDO as a potential therapy for cervical cancer.
  • Tomokazu Fukuda, Masafumi Katayama, Takayuki Yoshizawa, Takahiro Eitsuka, Hiroaki Mizukami, Kiyotaka Nakagawa, Hisao Ito, Homika Komagata, Sanghoun Song, Sanggun Roh, Yumi Hoshino, Eimei Sato, Hirofumi Hanada, Katsuhiko Nishimori, Teruo Miyazawa, Takafumi Uchida
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 76 (7) 1372 - 1377 0916-8451 2012/07 [Refereed][Not invited]
     
    The pig is an important animal for both agricultural and medical purposes. However, the number of pig-derived cell lines is relatively limited when compared with mouse- and human-derived lines. We established in this study a retroviral conditional expression system for the Simian vacuolating virus 40 large T fragment (SV40T) which allowed us to efficiently establish pig embryonic fibroblast cell lines. The established cell lines showed high levels of cell proliferation and resistance to cellular senescence. A chromosome analysis showed that 84% of the cells had the normal karyotype. Transient expression of the Cre recombinase allowed us to excise the SV40T fragment from the genome. The development of this research tool will enable us to quickly establish new cell lines derived from various animals.
  • Naoto Sato, Yasushi Saga, Hiroaki Mizukami, Dongdong Wang, Hiroyuki Fujiwara, Yuji Takei, Shizuo Machida, Keiya Ozawa, Mitsuaki Suzuki
    ONCOLOGY REPORTS 27 (5) 1336 - 1340 1021-335X 2012/05 [Refereed][Not invited]
     
    The purpose of this study was to explore the possibility of targeted molecular therapy with anti-epidermal growth factor receptor (anti-EGFR) antibody (cetuximab) for the treatment of mucinous ovarian carcinoma. We analyzed EGFR protein expression and KRAS gene mutations in 5 mucinous ovarian carcinoma cell lines RMUG-L, RMUG-S, MN-1, OMC-1 and MCAS and evaluated the in vitro and in vivo effects of cetuximab on each. EGER expression was observed in all cell lines except for MN-1 cells, and a KRAS gene mutation at codon 12 was detected only in the MCAS cell line. Cetuximab inhibited RMUG-L and OMC-1 cell growth in vitro and completely blocked RMUG-L tumor growth in vivo. On the other hand, cetuximab did not affect MCAS cell growth in vitro and only partially reduced the MCAS tumor growth in vivo. These results suggest the possibility of targeted molecular therapy with cetuximab for mucinous ovarian carcinoma cells lacking a KRAS gene mutation.
  • M. Ogura, M. Urabe, T. Akimoto, A. Onishi, C. Ito, T. Ito, T. Tsukahara, H. Mizukami, A. Kume, S. Muto, E. Kusano, K. Ozawa
    GENE THERAPY 19 (5) 476 - 482 0969-7128 2012/05 [Refereed][Not invited]
     
    Varying degrees of metabolic abnormalities mediated by chronic inflammation are implicated in the chronic glomerular injuries associated with obesity. Interleukin (IL)-10, a pleiotropic cytokine, exerts anti-inflammatory effects in numerous biological settings. In the present study, we explored the biological benefits of adeno-associated virus (AAV) vector-mediated sustained IL-10 expression against the pathological renal characteristics observed in Zucker fatty rats (ZFRs). We injected an AAV vector, encoding rat IL-10 or enhanced green fluorescent protein (GFP) into male ZFRs at 5 weeks of age. Subsequently, the renal pathophysiological changes were analyzed. Persistent IL-10 expression significantly reduced the urinary protein excretion of ZFRs compared with GFP expression (47.1 +/- 11.6 mg per mg.creatinine versus 88.8 +/- 30.0 mg per mg.creatinine, P<0.01). The serum levels of IL-10 negatively correlated with the urinary protein in AAV-treated rats (r = -0.78, P<0.01). Renal hypertrophy, increased widths in the glomerular basement membrane, and the lack of uniformity and regularity of the foot process of the visceral glomerular epithelial cells of ZFRs were significantly blunted by IL-10 expression. IL-10 also abrogated the downregulation of glomerular nephrin observed in ZFRs treated with the GFP vector. Our findings provide insights into the potential benefit of the anti-inflammatory effects of IL-10 on the overall management of glomerulopathy induced by the metabolic disorders associated with obesity. Gene Therapy (2012) 19, 476-482; doi:10.1038/gt.2011.183; published online 24 November 2011
  • Dongdong Wang, Yasushi Saga, Hiroaki Mizukami, Naoto Sato, Hiroaki Nonaka, Hiroyuki Fujiwara, Yuji Takei, Shizuo Machida, Osamu Takikawa, Keiya Ozawa, Mitsuaki Suzuki
    INTERNATIONAL JOURNAL OF ONCOLOGY 40 (4) 929 - 934 1019-6439 2012/04 [Refereed][Not invited]
     
    This study examined the role of the immunosuppressive enzyme indoleamine-2,3-dioxygenase (IDO) in ovarian cancer progression, and the possible application of this enzyme as a target for ovarian cancer therapy. We transfected a short hairpin RNA vector targeting IDO into the human ovarian cancer cell line SKOV-3, that constitutively expresses IDO and established an IDO downregulated cell line (SKOV-3/shIDO) to determine whether inhibition of IDO mediates the progression of ovarian cancer. IDO downregulation suppressed tumor growth and peritoneal dissemination in vivo, without influencing cancer cell growth. Moreover, IDO downregulation enhanced the sensitivity of cancer cells to natural killer (NK) cells in vitro, and promoted NK cell accumulation in the tumor stroma in vivo. These findings indicate that downregulation of IDO controls ovarian cancer progression by activating NK cells, suggesting IDO targeting as a potential therapy for ovarian cancer.
  • Hiroya Yagi, Sho Sanechika, Hiroshi Ichinose, Chiho Sumi-Ichinose, Hiroaki Mizukami, Masashi Urabe, Keiya Ozawa, Akihiro Kume
    NEUROREPORT 23 (1) 30 - 34 0959-4965 2012/01 [Refereed][Not invited]
     
    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 1347-9032 2011/12 [Refereed][Not invited]
     
    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)
  • Katsuyuki Kaneda, Hironori Kasahara, Ryosuke Matsui, Tomoko Katoh, Hiroaki Mizukami, Keiya Ozawa, Dai Watanabe, Tadashi Isa
    PLOS ONE 6 (4) e18452  1932-6203 2011/04 [Refereed][Not invited]
     
    The superficial layer of the superior colliculus (sSC) receives visual inputs via two different pathways: from the retina and the primary visual cortex. However, the functional significance of each input for the operation of the sSC circuit remains to be identified. As a first step toward understanding the functional role of each of these inputs, we developed an optogenetic method to specifically suppress the synaptic transmission in the retino-tectal pathway. We introduced enhanced halorhodopsin (eNpHR), a yellow light-sensitive, membrane-targeting chloride pump, into mouse retinal ganglion cells (RGCs) by intravitreously injecting an adeno-associated virus serotype-2 vector carrying the CMV-eNpHR-EYFP construct. Several weeks after the injection, whole-cell recordings made from sSC neurons in slice preparations revealed that yellow laser illumination of the eNpHR-expressing retino-tectal axons, putatively synapsing onto the recorded cells, effectively inhibited EPSCs evoked by electrical stimulation of the optic nerve layer. We also showed that sSC spike activities elicited by visual stimulation were significantly reduced by laser illumination of the sSC in anesthetized mice. These results indicate that photo-activation of eNpHR expressed in RGC axons enables selective blockade of retino-tectal synaptic transmission. The method established here can most likely be applied to a variety of brain regions for studying the function of individual inputs to these regions.
  • Hiroya Yagi, Tsuyoshi Ogura, Hiroaki Mizukami, Masashi Urabe, Hiromi Hamada, Hiroyuki Yoshikawa, Keiya Ozawa, Akihiro Kume
    JOURNAL OF GENE MEDICINE 13 (2) 114 - 122 1099-498X 2011/02 [Refereed][Not invited]
     
    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.
  • Hiroaki Mizukami, Masashi Urabe, Akihiro Kume, Keiya Ozawa
    Open Diabetes Journal 4 (1) 119 - 122 1876-5246 2011 [Refereed][Not invited]
     
    Gene therapy is considered as one of the innovative treatment modalities for diabetic retinopathy (DR). Since genuine animal models of DR are limited, only a few studies have reported the efficacy of gene therapy. For preclinical study of DR, spontaneously diabetic Torii (SDT) rat is a valuable model. Fortunately, we could evaluate the efficacy of adeno-associated virus (AAV)-mediated gene therapy in SDT rats and proved that sFlt-1 expression prevented DR progression. Because of a limited number of large-animal models of DR, it is uncertain whether gene therapy experiments using dogs or monkeys allow reliable conclusions. On the other hand, owing to the recent progress in AAV-mediated gene therapy for retinal diseases in monkeys and humans, gene therapy for DR using AAV vectors may become a reality in the near future. © Mizukami et al.
  • Hiroaki Mizukami, Jun Mimuro, Akira Ishiwata, Fumiko Ono, Hiroya Yagi, Masashi Urabe, Akihiro Kume, Keiji Terao, Yasuhiro Yasutomi, Yoichi Sakata, Keiya Ozawa
    JOURNAL OF GENE MEDICINE 12 (12) 1061 - 1062 1099-498X 2010/12 [Refereed][Not invited]
  • Martin Lock, Susan McGorray, Alberto Auricchio, Eduard Ayuso, E. Jeffrey Beecham, Veronique Blouin-Tavel, Fatima Bosch, Mahuya Bose, Barry J. Byrne, Tina Caton, John A. Chiorini, Abdelwahed Chtarto, K. Reed Clark, Thomas Conlon, Christophe Darmon, Monica Doria, Anne Douar, Terence R. Flotte, Joyce D. Francis, Achille Francois, Mauro Giacca, Michael T. Korn, Irina Korytov, Xavier Leon, Barbara Leuchs, Gabriele Lux, Catherine Melas, Hiroaki Mizukami, Philippe Moullier, Marcus Mueller, Keiya Ozawa, Tina Philipsberg, Karine Poulard, Christina Raupp, Christel Riviere, Sigrid D. Roosendaal, R. Jude Samulski, Steven M. Soltys, Richard Surosky, Liliane Tenenbau, Darby L. Thomas, Bart van Montfort, Gabor Veres, J. Fraser Wright, Yili Xu, Olga Zelenaia, Lorena Zentilin, Richard O. Snyder
    HUMAN GENE THERAPY 21 (10) 1273 - 1285 1043-0342 2010/10 [Refereed][Not invited]
     
    A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 x 10(11) particles/ml; 95% confidence interval [CI], 7.89 x 10(11) to 1.05 x 10(12) particles/ml), vector genomes ({X}, 3.28 x 10(10) vector genomes/ml; 95% CI, 2.70 x 10(10) to 4.75 x 10(10) vector genomes/ml), transducing units ({X}, 5.09 x 10(8) transducing units/ml; 95% CI, 2.00 x 10(8) to 9.60 x 10(8) transducing units/ml), and infectious units ({X}, 4.37 x 10(9) TCID(50) IU/ml; 95% CI, 2.06 x 10(9) to 9.26 x 10(9) TCID(50) IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.
  • 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 1525-0016 2010/09 [Refereed][Not invited]
     
    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.
  • Akira Ishiwata, Jun Mimuro, Hiroaki Mizukami, Yuji Kashiwakura, Atsushi Yasumoto, Asuka Sakata, Tsukasa Ohmori, Seiji Madoiwa, Fumiko Ono, Midori Shima, Akira Yoshioka, Keiya Ozawa, Yoichi Sakata
    THROMBOSIS RESEARCH 125 (6) 533 - 537 0049-3848 2010/06 [Refereed][Not invited]
     
    Introduction: Gene therapy is expected to be the next generation therapy for hemophilia, and a good animal model is required for hemophilia gene therapy preclinical studies. Methods: Taking advantage of the human factor IX (FIX) specificity of monoclonal antibody 3A6, the epitope of which resides in the amino acid polypeptide segment including Ala 262 of human FIX, mutant macaque FIX with an amino acid substitution of Thr 262 to Ala (macaque FIX T262A) was generated and its reactivity to monoclonal antibody 3A6, biological activity and expression in vivo were studied. Results: Enzyme-linked immunosorbent assays (ELISAs) and Western blot analyses showed that monoclonal antibody 3A6 bound to human FIX and macaque FIX T262A but not to wild-type macaque FIX. Recombinant macaque FIX T262A exhibited a comparable coagulation activity to wild-type macaque FIX and human FIX. High expression of macaque FIX T262A was achieved in mice by injection of AAV8 vectors carrying the macaque FIX T262A gene and reached levels of up to 31.5 mu g/mL (1050% of the normal human FIX concentration). Macaque FIX T262A expressed in the liver of mice was as biologically active as that expressed in vitro. In addition, the macaque FIX T262A concentrations determined by a 3A6-based ELISA were not influenced by the presence of normal macaque plasma. Conclusions: The results of the present study suggest that macaque FIX T262A may be processed appropriately in vivo and that the macaque FIX T262A concentration in the macaque circulation can be quantified precisely by a monoclonal antibody 3A6-based ELISA. (C) 2010 Elsevier Ltd. All rights reserved.
  • Hiroaki Mizukami, Jun Mimuro, Akira Ishiwata, Fumiko Ono, Hiroya Yagi, Masashi Urabe, Akihiro Kume, Keiji Terao, Yasuhiro Yasutomi, Yoichi Sakata, Keiya Ozawa
    JOURNAL OF GENE MEDICINE 11 (12) 1177 - 1177 1099-498X 2009/12 [Refereed][Not invited]
  • Tetsuo Ito, Shigekazu Yamamoto, Tsukasa Hayashi, Mika Kodera, Hiroaki Mizukami, Keiya Ozawa, Shin-ichi Muramatsu
    ANNALS OF CLINICAL BIOCHEMISTRY 46 508 - 510 0004-5632 2009/11 [Refereed][Not invited]
     
    Background: Recombinant adeno-associated virus vectors based on serotype 2 (AAV-2) have become leading vehicles for gene therapy. Most humans in the general population have anti-AAV-2 antibodies as a result of naturally acquired infections. Pre-existing immunity to AAV-2 might affect the functional and safety consequences of AAV-2 vector-mediated gene transfer in clinical applications. Methods: An enzyme-linked immunosorbent assay (ELISA) method was developed using microwell plates coated with intact particles of recombinant AAV-2 vectors, and horseradish peroxidase-conjugated anti-human immunoglobulin G (HRP-IgG). Neutralizing antibody titres were analysed by assessing the ability of serum antibody to inhibit transduction into HEK293 cells of AAV vectors that express beta-galactosidase. Results: Anti-AAV-2 antibodies were detected by ELISA in two of 20 healthy subjects. The positivity criterion (optical density >0.5) in ELISA corresponded to the cut-off value (320-fold dilution of serum) in the AAV-2 neutralization assay. Influences of interfering substances were not observed. Conclusion: This ELISA method may be useful for rapid screening of anti-AAV-2 neutralizing antibodies in candidates for gene therapy.
  • Akira Ishiwata, Jun Mimuro, Hiroaki Mizukami, Yuji Kashiwakura, Katsuhiro Takano, Tsukasa Ohmori, Seiji Madoiwa, Keiya Ozawa, Yoichi Sakata
    JOURNAL OF GENE MEDICINE 11 (11) 1020 - 1029 1099-498X 2009/11 [Refereed][Not invited]
     
    Background Gene therapy for hemophilia A with adeno-associated virus (AAV) vectors involves difficulties in the efficient expression of factor VIII (FVIII) and in antibody formation against transgene-derived FVIII. Methods AAV8 vectors carrying the canine B domain deleted FVIII (cFVIII) gene under the control of the ubiquitous beta-actin promoter, the liver-specific human alpha 1 anti-trypsin promoter (HAAT) and the liver-specific hepatic control region (HCR) enhancer/human alpha 1 anti-trypsin promoter complex (HCRHAAT) were used for the expression of cFVIII in FVIII deficient (fviii(-/-)) mice. Results Addition of the hepatic control region enhancer element to the HAAT promoter successfully augmented HAAT promoter activity without loss of liver-specificity in vivo. Using this enhancer/promoter complex, a high cFVIII transgene expression was achieved, resulting in increased blood cFVIII activities to more than 100% of the normal canine FVIII levels in fviii(-/-) mice at a 1 : 10 lower dose of the AAV8 vector carrying the cFVIII gene driven by the HAAT promoter. Under short-term immunosuppression, neutralizing antibodies against cFVIII developed in only one out of six mice when the HAAT promoter was used for cFVIII expression, whereas all the mice developed neutralizing antibodies against cFVIII when the beta-actin promoter was used for cFVIII expression. No neutralizing antibodies against cFVIII developed in fviii(-/-) mice that received the AAV8 vector carrying the cFVIII gene driven by the HCRHAAT enhancer/promoter complex without immunosuppression. Conclusions These data suggest that AAV8 vector-mediated liver-restricted cFVIII gene expression is sufficient for immune hypo-responsiveness to transgene-derived cFVIII in fviii(-/-) mice. Copyright (C) 2009 John Wiley & Sons, Ltd.
  • Hiroaki Mizukami, Jun Mimuro, Akira Ishiwata, Fumiko Ono, Hiroya Yagi, Masashi Urabe, Akihiro Kume, Keiji Terao, Yasuhiro Yasutomi, Yoichi Sakata, Keiya Ozawa
    HUMAN GENE THERAPY 20 (11) 1481 - 1482 1043-0342 2009/11 [Refereed][Not invited]
  • Shinobu Kuratomi, Yoko Ohmori, Masayuki Ito, Kuniko Shimazaki, Shin-ichi Muramatsu, Hiroaki Mizukami, Hideki Uosaki, Jun K. Yamashita, Yuji Arai, Koichiro Kuwahara, Makoto Takano
    CARDIOVASCULAR RESEARCH 83 (4) 682 - 687 0008-6363 2009/09 [Refereed][Not invited]
     
    Hcn4, which encodes the hyperpolarization-activated, cyclic nucleotide-sensitive channel (I(h)), is a well-established marker of the cardiac sino-atrial node. We aimed to identify cis-elements in the genomic locus of the Hcn4 gene that regulate the transcription of Hcn4. We screened evolutionarily conserved non-coding sequences (CNSs) that are often involved in the regulation of gene expression. The VISTA Enhancer Browser identified 16 regions, termed CNS 1-16, within the Hcn4 locus. Using the luciferase reporter assay in primary neonatal rat cardiomyocytes, we found that CNS13 conferred a prominent enhancer activity (more than 30-fold) on the Hcn4 promoter. Subsequent mutation analysis revealed that the Hcn4 enhancer function was dependent on myocyte enhancer factor-2 (MEF2) and activator protein-1 (AP1) binding sequences located in CNS13. Electrophoretic mobility shift assay and chromatin immunoprecipitation confirmed that MEF2 and AP1 proteins bound CNS13. Furthermore, overexpression of a dominant negative MEF2 mutant inhibited the enhancer activity of CNS13, decreased Hcn4 mRNA expression and also decreased the amplitude of I(h) current in myocytes isolated from the inflow tract of embryonic heart. These results suggest that the novel enhancer CNS13 and MEF2 may play a critical role in the transcription of Hcn4 in the heart.
  • Keisuke Sato, Shiori Date, Yumi Aoyagi, Yoshiyuki Kasahara, Akihiko Nawa, Hiroaki Mizukami, Shizu Hidema, Keiya Ozawa, Katsuhiko Nishimori
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 73 (9) 2145 - 2146 0916-8451 2009/09 [Refereed][Not invited]
     
    We developed the AAV-Oxtr-IRES-Venus vector to rescue the oxytocin receptor (Oxtr) gene functionally at restricted regions in the brains of Oxtr knockout mice. First we chose human eIF4G gene-derived IRES to co-express Venus, a fluorescent marker gene, with Oxtr. With selected human eIF4G IRES, we constructed the AAV-Oxtr-IRES-Venus vector, and it caused expression of the Venus gene in the brain when 1 mu l of viral solution (9.4 x 10(7) vg) was injected into the medial amygdaloid nucleus. In primary neuronal cells transduced with this viral vector and followed by oxytocin administration, functional expression of OXTR was detected by Ca2+ imaging assay.
  • Ryosuke Uchibori, Takashi Okada, Takayuki Ito, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Keiya Ozawa
    JOURNAL OF GENE MEDICINE 11 (5) 373 - 381 1099-498X 2009/05 [Refereed][Not invited]
     
    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.
  • Hiroaki Mizukami, Jun Mimuro, Akira Ishiwata, Fumiko Ono, Hiroya Yagi, Masashi Urabe, Akihiro Kume, Keiji Terao, Yasuhiro Yasutomi, Yoichi Sakata, Keiya Ozawa
    MOLECULAR THERAPY 17 S314 - S315 1525-0016 2009/05 [Refereed][Not invited]
  • Hiroaki Mizukami, Jun Mimuro, Akira Ishiwata, Fumiko Ono, Hiroya Yagi, Masashi Urabe, Akihiro Kume, Terao Keiji, Yasuhiro Yasutomi, Yoichi Sakata, Keiya Ozawa
    HUMAN GENE THERAPY 19 (10) 1195 - 1196 1043-0342 2008/10 [Refereed][Not invited]
  • 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 0896-8411 2008/05 [Refereed][Not invited]
     
    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 1099-498X 2008/04 [Not refereed][Not invited]
     
    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.
  • 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 1099-498X 2008/04 [Refereed][Not invited]
     
    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.
  • Hiroaki Mizukami, Shin-Ichi Muramatsu, Fumiko Ono, Jun Mimuro, Yoichi Sakata, Masashi Urabe, Akihiro Kume, Keiji Terao, Imaharu Nakano, Keiya Ozawa
    JOURNAL OF GENE MEDICINE 10 (4) 454 - 454 1099-498X 2008/04 [Refereed][Not invited]
  • Hiroaki Mizukami, Akira Ishiwata, Fumiko Ono, Jun-Ichiro Takano, Koji Fujimoto, Jun Mimuro, Masashi Urabe, Akihiro Kume, Keiji Terao, Yoichi Sakata, Keiya Ozawa
    JOURNAL OF GENE MEDICINE 10 (4) 453 - 453 1099-498X 2008/04 [Refereed][Not invited]
  • 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 1525-0016 2008/03 [Not refereed][Not invited]
     
    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.
  • Yuji Takei, Yasushi Saga, Hiroaki Mizukami, Takeshi Takayama, Michitaka Ohwada, Keiya Ozawa, Mitsuaki Suzuki
    MOLECULAR CANCER THERAPEUTICS 7 (3) 704 - 711 1535-7163 2008/03 [Not refereed][Not invited]
     
    The main mode of progression of ovarian cancer is peritoneal dissemination, and its inhibition may lead to improved outcome. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) reportedly inhibits the proliferation, migration, and invasion of cancer cells. The purpose of this study is to explore the possibility of PTEN gene therapy for ovarian cancer. We transfected the ovarian cancer cell line SHIN-3 [vascular endothelial growth factor (VEGF) - hypersecretory cell line] with PTEN or luciferase (LUC) - expressing plasmid. After selection, PTEN-overexpressing cells (SHIN-3/PTEN) and control cells (SHIN-3/LUC) were obtained. SHIN-3/PTEN implanted s.c. into nude mice was examined for the change in tumor diameter and the number of new blood vessels. Mice with peritoneally disseminated tumors created by i.p. inoculation of the same cells were examined for changes in body weight and abdominal circumference and for survival time. The growth of s.c. SHIN-3/PTEN was significantly lower than that of control (P < 0.001). Compared with controls, mice with i.p. inoculated SHIN-3/PTEN showed significantly smaller increases in the body weight and abdominal circumference (P < 0.01) and a significantly longer survival time (P < 0.05). VEGF concentration in the supernatant of SHIN-3/PTEN was about half that of controls (P < 0.05). The number of new blood vessels in SHIN-3/PTEN was significantly smaller than that in controls (P < 0.001). Overexpression of PTEN suppressed tumor growth and peritoneal dissemination of VEGF-hypersecretory ovarian cancer cells and prolonged the survival time of the mice with peritoneal disseminated tumor. PTEN gene therapy could have therapeutic potential for ovarian cancer and exerts some of this effect by inhibiting angiogenesis.
  • 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 1525-0016 2008/03 [Refereed][Not invited]
     
    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.
  • Kazuhiro Suzumura, Tadamichi Hirano, Gakuhei Son, Yuji Iimuro, Hiroaki Mizukami, Keiya Ozawa, Jiro Fujimoto
    HEPATOLOGY INTERNATIONAL 2 (1) 80 - 88 1936-0533 2008/03 [Refereed][Not invited]
     
    Purpose Adeno-associated virus (AAV) vectors can achieve long-term gene expression and are now feasible for use in human gene therapy. We constructed hepatocyte growth factor (HGF) expressing AAV (AAV5-HGF) and examined its effect in two mouse hepatic fibrosis models. Methods A model of hepatic fibrosis was established by carbon tetrachloride (CCl4) administration in Balb/c mice. After the establishment of liver fibrosis, AAV5-HGF was injected once into the portal vein. Mice were killed 3, 6, 9, and 12 weeks after injection. Another model was established by bile duct ligation (BDL). Seven weeks after AAV5-HGF injection, mice underwent BDL, and were then killed 2 weeks after BDL. Results Mice that received AAV5-HGF achieved stable HGF expression both in the serum and liver for at least 12 weeks. In both models, significant improvement of the liver fibrosis was found in all mice receiving AAV5-HGF based on Azan-Mallory staining. Suppression of hepatic stellate cells (HSC) was confirmed by immunohistochemistry. Fibrogenic markers were significantly suppressed and collagenase activity increased in the livers of mice receiving AAV5-HGF. Conclusions A single injection of AAV vector containing HGF gene achieved long-term expression of HGF and resulted in resolution of mouse liver fibrosis. HGF gene therapy mediated by AAV is feasible for the treatment of liver fibrosis.
  • Yuji Takei, Yasushi Saga, Hiroaki Mizukami, Takeshi Takayama, Michitaka Ohwada, Keiya Ozawa, Mitsuaki Suzuki
    MOLECULAR CANCER THERAPEUTICS 7 (3) 704 - 711 1535-7163 2008/03 [Refereed][Not invited]
     
    The main mode of progression of ovarian cancer is peritoneal dissemination, and its inhibition may lead to improved outcome. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) reportedly inhibits the proliferation, migration, and invasion of cancer cells. The purpose of this study is to explore the possibility of PTEN gene therapy for ovarian cancer. We transfected the ovarian cancer cell line SHIN-3 [vascular endothelial growth factor (VEGF) - hypersecretory cell line] with PTEN or luciferase (LUC) - expressing plasmid. After selection, PTEN-overexpressing cells (SHIN-3/PTEN) and control cells (SHIN-3/LUC) were obtained. SHIN-3/PTEN implanted s.c. into nude mice was examined for the change in tumor diameter and the number of new blood vessels. Mice with peritoneally disseminated tumors created by i.p. inoculation of the same cells were examined for changes in body weight and abdominal circumference and for survival time. The growth of s.c. SHIN-3/PTEN was significantly lower than that of control (P < 0.001). Compared with controls, mice with i.p. inoculated SHIN-3/PTEN showed significantly smaller increases in the body weight and abdominal circumference (P < 0.01) and a significantly longer survival time (P < 0.05). VEGF concentration in the supernatant of SHIN-3/PTEN was about half that of controls (P < 0.05). The number of new blood vessels in SHIN-3/PTEN was significantly smaller than that in controls (P < 0.001). Overexpression of PTEN suppressed tumor growth and peritoneal dissemination of VEGF-hypersecretory ovarian cancer cells and prolonged the survival time of the mice with peritoneal disseminated tumor. PTEN gene therapy could have therapeutic potential for ovarian cancer and exerts some of this effect by inhibiting angiogenesis.
  • Hiroaki Mizukami, Akira Ishiwata, Fumiko Ono, Junichiro Takano, Koji Fujimoto, Jun Mimuro, Masashi Urabe, Akihiro Kume, Keiji Terao, Yoichi Sakata, Keiya Ozawa
    HUMAN GENE THERAPY 18 (10) 1044 - 1044 1043-0342 2007/10 [Refereed][Not invited]
  • 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 0009-7330 2007/09 [Not refereed][Not invited]
     
    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.
  • 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 0194-911X 2007/09 [Not refereed][Not invited]
     
    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 0009-7330 2007/09 [Refereed][Not invited]
     
    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.
  • Yuji Takei, Hiroaki Mizukami, Yasushi Saga, Ichiro Yoshimura, Yoko Hasumi, Takeshi Takayama, Takahiro Kohno, Takashi Matsushita, Takashi Okada, Akihiro Kume, Mitsuaki Suzuki, Keiya Ozawa
    INTERNATIONAL JOURNAL OF CANCER 120 (2) 278 - 284 0020-7136 2007/01 [Not refereed][Not invited]
     
    Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis in a variety of tumors. A soluble form of Flt-1 (sFlt-1), a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidences suggest the applicability of sFlt-1 in tumor suppression by means of anti-angiogenesis. We previously demonstrated the efficacy of sflt-1 gene expression in situ to suppress tumor growth and ascites in ovarian cancer. Here, we demonstrate the therapeutic applicability of muscle-mediated expression of sFlt-1 in tumor-bearing mice. Initially, tumor suppressive action was confirmed by inoculating sFlt-1-expressing ovarian cancer (SHIN-3) cells into mice, both subcutaneously and intraperitoneally. To validate the therapeutic efficacy in a more clinically relevant model, adeno-associated virus vectors encoding sflt-1 were introduced into mouse skeletal muscles and were subsequently inoculated with tumor cells. As a result, high serum sFlt-1 levels were constantly observed, and the growth of both subcutaneously- and intraperitoneally-inoculated tumors was significantly suppressed. No delay in wound healing or adverse events of neuromuscular damage were noted, body weight did not change, and laboratory data, such as those representing liver and renal functions, were not affected. These results indicate that sFlt-1 suppresses growth and peritoneal dissemination of ovarian cancer by the inhibition of angiogenesis, and thus suggest the usefulness of gene therapy for ovarian cancer. (c) 2006 Wiley-Liss, Inc.
  • Prevention of diabetic retinopathy by intraocular soluble flt-1 gene transfer in a spontaneously diabetic rat model.
    Ideno, J, Mizukami, H, Kakehashi, A, Saito, Y, Okada, T, Urabe, M, Kume, A, Kuroki, M, Kawakami, M, Ishibashi, S, Ozawa, K
    Int J Mol Med 19 75 - 9 2007 [Not refereed][Not invited]
  • Yuji Takei, Hiroaki Mizukami, Yasushi Saga, Ichiro Yoshimura, Yoko Hasumi, Takeshi Takayama, Takahiro Kohno, Takashi Matsushita, Takashi Okada, Akihiro Kume, Mitsuaki Suzuki, Keiya Ozawa
    INTERNATIONAL JOURNAL OF CANCER 120 (2) 278 - 284 0020-7136 2007/01 [Refereed][Not invited]
     
    Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis in a variety of tumors. A soluble form of Flt-1 (sFlt-1), a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidences suggest the applicability of sFlt-1 in tumor suppression by means of anti-angiogenesis. We previously demonstrated the efficacy of sflt-1 gene expression in situ to suppress tumor growth and ascites in ovarian cancer. Here, we demonstrate the therapeutic applicability of muscle-mediated expression of sFlt-1 in tumor-bearing mice. Initially, tumor suppressive action was confirmed by inoculating sFlt-1-expressing ovarian cancer (SHIN-3) cells into mice, both subcutaneously and intraperitoneally. To validate the therapeutic efficacy in a more clinically relevant model, adeno-associated virus vectors encoding sflt-1 were introduced into mouse skeletal muscles and were subsequently inoculated with tumor cells. As a result, high serum sFlt-1 levels were constantly observed, and the growth of both subcutaneously- and intraperitoneally-inoculated tumors was significantly suppressed. No delay in wound healing or adverse events of neuromuscular damage were noted, body weight did not change, and laboratory data, such as those representing liver and renal functions, were not affected. These results indicate that sFlt-1 suppresses growth and peritoneal dissemination of ovarian cancer by the inhibition of angiogenesis, and thus suggest the usefulness of gene therapy for ovarian cancer. (c) 2006 Wiley-Liss, Inc.
  • Junichi Ideno, Hiroaki Mizukami, Akihiro Kakehashi, Yuka Saito, Takashi Okada, Masashi Urabe, Akihiro Kume, Masatoshi Kuroki, Masanobu Kawakami, Shun Ishibashi, Keiya Ozawa
    INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 19 (1) 75 - 79 1107-3756 2007/01 [Refereed][Not invited]
     
    The number of patients suffering from diabetes mellitus is constantly rising worldwide, and diabetic retinopathy (DR) has become the most frequent cause of postnatal blindness. Vascular endothelial growth factor (VEGF) is known to play a central role during DR development. Thus, inhibiting the effects of VEGF may hamper the disease progression, and gene transfer of the soluble VEGF receptor sflt-1 is an attractive approach for this purpose. However, the lack of suitable animal models hindered the evaluation of this strategy. Recently, the spontaneously diabetic non-obese Torii (SDT) rat was established and is considered as one of the ideal models for human DR. In this study, we evaluated the efficacy of gene therapy in SDT rats by using adeno-associated viral vectors (AAV-sflt-1) injected into the subretinal space. Thirty weeks later, the progression of DR was assessed by fluorescein angiography using three parameters; the presence of an avascular area, extensive hyperfluorescein and arterial narrowing. These changes were significantly less evident in the 'treated' eyes than in the control. No adverse effects were observed throughout the study. These results indicate that local sflt-1 gene transfer inhibits DR progression in SDT rats and offers powerful therapeutic potential for the management of human DR.
  • Xiao Tian Wang, Paul Y. Liu, Jin Bo Tang, Hiroaki Mizukami, Ke-Qin Xin, Keiya Ozawa, Hiroshi Ushijima
    PLASTIC AND RECONSTRUCTIVE SURGERY 119 (1) 227 - 234 0032-1052 2007/01 [Refereed][Not invited]
     
    Background: Transfer of exogenous growth factor genes to injured tendons offers a promising method for strengthening tendon repairs. Adeno-associated virus vectors have advantages of being both nonpathogenic and nontoxic. The authors explored the efficiency of transduction of intrasynovial tenocytes with different serotypes of adeno-associated virus (AAV) and the persistency of its expression of a growth factor transgene. Methods: Tenocytes were obtained from cultures of rat intrasynovial tendons and distributed to 82 wells in eight culture plates and to 30 culture dishes. The tenocytes in the wells were treated with AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, and AAV8 vectors containing the lacZ gene, and plasmid vectors (pCMV beta-lacZ). The tenocytes were stained with in situ P-galactosidase 5 days later. The basic fibroblast growth factor (bFGF) gene was cloned to the AAV2 vector to construct the AAV2-bFGF vector, which transduced tenocytes in culture dishes. Expression of the transgene was measured over 3 weeks and analyzed statistically. Results: AAV2 effectively delivered exogenous genes to proliferating intrasynovial tenocytes. In contrast, other tested adeno-associated viruses transduced tenocytes minimally or not at all. The efficiency of gene transfer by AAV2, indicated by the percentage of cells with positive beta-galactosidase staining, was significantly greater than that by a plasmid vector (p = 0.001). Expression of the bFGF gene in tenocytes transduced with the AAV2-bFGF was significantly higher than that in the control over the 3-week period (p < 0.01). Conclusions: Gene transfer to tenocytes by AAV2 is more efficient than that by a plasmid vector. However, other adeno-associated virus serotypes cannot effectively transduce tenocytes. The bFGF gene can be delivered to intrasynovial tenocytes by the AAV2 vector effectively, and the gene transfer significantly increases expression of bFGF gene over 3 weeks.
  • Ke-Qin Xin, Hiroaki Mizukami, Masashi Urabe, Yoshihiko Toda, Kaori Shinoda, Atsushi Yoshida, Kenji Oomura, Yoshitsugu Kojima, Motohide Ichino, Dennis Klinman, Keiya Ozawa, Kenji Okuda
    JOURNAL OF VIROLOGY 80 (24) 11899 - 11910 0022-538X 2006/12 [Refereed][Not invited]
     
    The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity study of AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 gene induced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cell-mediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells) and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of AAV-based vaccine and gene therapy.
  • Hiroaki Mizukami, Jun Mimuro, Akira Ishiwata, Fumiko Ono, Takashi Matsushita, Takashi Okada, Masashi Urabe, Akihiro Kume, Keiji Terao, Yoichi Sakata, Keiya Ozawa
    JOURNAL OF GENE MEDICINE 8 (12) 1460 - 1460 1099-498X 2006/12 [Refereed][Not invited]
  • Hiroaki Mizukami, Jun Mimuro, Tsuyoshi Ogura, Takashi Okada, Masashi Urabe, Akihiro Kume, Yoichi Sakata, Keiya Ozawa
    HUMAN GENE THERAPY 17 (9) 921 - 928 1043-0342 2006/09 [Refereed][Not invited]
     
    Traditionally, skeletal muscle and liver are the preferred target organs for gene transfer to supply a transgene product into the systemic circulation. In this respect, adipose tissue presents a number of attractive features. However, adipose tissue transduction in vivo has not been feasible by conventional methods. To solve this issue, we tested the utility of excipients in adeno-associated virus (AAV) vector-mediated gene transfer and found that Pluronics are suitable for this purpose. In a histological analysis of adipose tissue in db/db mice, Pluronic F88 showed the greatest augmentative effect on beta-galactosidase expression in combination with the AAV1 vector. When the vector encoding mouse erythropoietin (Epo) was used in the same manner, increased plasma Epo concentrations were observed (230 +/- 80 versus 58 +/- 14 mU/ml). Moreover, the plasma Epo concentration returned to the normal level after the surgical removal of transduced adipose tissue. No damage was observed in the transduced tissue. Our results indicate that the proposed method is safe and efficient for gene transfer into adipose tissues, thus providing an alternative for supplemental gene therapy.
  • Tsuyoshi Ogura, Hiroaki Mizukami, Jun Mimuro, Seiji Madoiwa, Takashi Okada, Takashi Matsushita, Masashi Urabe, Akihiro Kume, Hirorni Hamada, Hiroyuki Yoshikawa, Yoichi Sakata, Keiya Ozawa
    JOURNAL OF GENE MEDICINE 8 (8) 990 - 997 1099-498X 2006/08 [Refereed][Not invited]
     
    Background Gene transfer into a fetus or neonate can be a fundamental approach for treating genetic diseases, particularly disorders that have irreversible manifestations in adulthood. Although the potential utility of this technique has been suggested, the advantages of neonatal gene transfer, have not been widely investigated. Here, we tested the usefulness of neonatal gene transfer using adeno-associated virus (AAV) vectors by comparing the, administration routes and vector doses. Methods To determine the optimal administration route neonates were, subjected to intravenous (iv) or intraperitoneal (ip) injections of AAV5-based vectors encoding the human coagulation factor IX (hfIX) gene, and the dose response was examined. To determine the distribution of transgene expression, vectors encoding lacZ or luciferase (luc) genes were used and assessed by X-gal staining and in vivo imaging, respectively. After the observation period, the vector distribution across tissues was quantified. Results The factor IX concentration was higher in ip-injected mice than in iv-injected mice. All transgenes administered by ip injection were more efficiently expressed in neonates than in adults. The expression was confined to the peritoneal tissue. Interestingly, a sex-related difference was observed in transgene expression in adults, whereas this difference was not apparent in neonates. Conclusions AAV vector administration to neonates using the ip route was clearly advantageous in obtaining robust transgene expression. Vector genomes and transgene expression were observed mainly in the peritoneal tissue. These findings indicate the advantages of neonatal gene therapy and would help in designing strategies for gene therapy using AAV vectors. Copyright (c) 2006 John Wiley & Sons, Ltd.
  • Hiroaki Mizukami, Jun Mimuro, Akira Ishiwata, Fumiko Ono, Takashi Matsushita, Takashi Okada, Masashi Urabe, Akihiro Kume, Keiji Terao, Yoichi Sakata, Keiya Ozawa
    MOLECULAR THERAPY 13 S3 - S3 1525-0016 2006/05 [Refereed][Not invited]
  • T Okada, R Uchibori, M Iwata-Okada, M Takahashi, T Nomoto, M Nonaka-Sarukawa, T Ito, Y Liu, H Mizukami, A Kume, E Kobayashi, K Ozawa
    MOLECULAR THERAPY 13 (4) 738 - 746 1525-0016 2006/04 [Refereed][Not invited]
     
    The transduction of cancer cells using recombinant adeno-associated virus (rAAV) occurs with low efficiency, which limits its utility in cancer gene therapy. We have previously sought to enhance rAAV-mediated transduction of cancer cells by applying DNA-damaging stresses. In this study, we examined the effects of the histone deacetylase inhibitor FR901228 on tumor transduction mediated by rAAV types 2 and 5. FR901228 treatment significantly improved the expression of the transgene in four cancer cell lines. The cell surface levels of alpha v integrin, FGF-R1, and PDGF-R were modestly enhanced by the presence of FR901228. These results suggest that the superior transduction induced by the HDAC inhibitor was due to an enhancement of transgene expression rather than increased viral entry. Furthermore, we characterized the association of the acetylated histone H3 in the episomal AAV vector genome by using the chromatin immunoprecipitation assay. The results suggest that the superior transduction may be related to the proposed histone-associated chromatin form of the rAAV concatemer in transduced cells. In the analysis with subcutaneous tumor models, strong enhancement of the transgene expression as well as therapeutic effect was confirmed in vivo. The use of this HDAC inhibitor may enhance the utility of rAAV-mediated transduction strategies for cancer gene therapy.
  • M Urabe, KQ Xin, Y Obara, T Nakakura, H Mizukami, A Kume, K Okuda, K Ozawa
    MOLECULAR THERAPY 13 (4) 823 - 828 1525-0016 2006/04 [Refereed][Not invited]
     
    Production of recombinant adeno-associated virus (rAAV) results in substantial quantities of empty capsids or virus-like particles (VLPs), virus protein shells without the vector genome. The contaminating VLPs would interfere with transduction by competing for cell-surface receptors and, when administered in vivo, contribute to antigen load, which may elicit a stronger immune response. Density-gradient ultracentrifugation provides a means to separate VLPs from rAAV particles, but is not feasible for large-scale preparations of vectors. Since the compositions of the VLP and vector differ by the single-stranded DNA genome, we hypothesized that the isoelectric point of the vector may differ from that of the VLP. In an attempt to separate type I rAAV particles from VLPs by ionexchange chromatography, we tested a number of buffer systems and found that trimethylammonium sulfate, or [(CH3)(4)N](2)SO4, effectively separated rAAV1 particles from VLPs. The rAAV1-GFP chromatographically separated from VLPs induced stronger GFP expression in HEK293 cells than rAAV1-GFP contaminated with VLPs. The transduction of mouse muscles with rAAV1-SEAP (secreted form of alkaline phosphatase) isolated from VLPs also showed higher serum SEAP levels than rAAV1 SEAP with VLPs. These results suggest that chromatographic separation of rAAV1 from empty capsids increased the efficacy of rAAV1.
  • M Urabe, T Nakakura, KQ Xin, Y Obara, H Mizukami, A Kume, RM Kotin, K Ozawa
    JOURNAL OF VIROLOGY 80 (4) 1874 - 1885 0022-538X 2006/02 [Refereed][Not invited]
     
    We established a method for production of recombinant adeno-associated virus type 5 (rAAV5) in insect cells by use of baculovirus expression vectors. One baculovirus harbors a transgene between the inverted terminal repeat sequences of type 5, and the second expresses Rep78 and Rep52. Interestingly, the replacement of type 5 Rep52 with type 1 Rep52 generated four times more rAAV5 particles. We replaced the N-terminal portion of type 5 VP1 with the equivalent portion of type 2 to generate infectious AAV5 particles. The rAAV5 with the modified VP1 required alpha 2-3 sialic acid for transduction, as revealed by a competition experiment with an analog of alpha 2-3 sialic acid. rAAV5-GFP/Neo with a 4.4-kb vector genome produced in HEK293 cells or Sf9 cells transduced COS cells with similar efficiencies. Surprisingly, Sf9-produced humanized Renilla green fluorescent protein (hGFP) vector with a 2.4-kb vector genome induced stronger GFP expression than the 293-produced one. Transduction of murine skeletal muscles with Sf9-generated rAAV5 with a 3.4-kb vector genome carrying a human secreted alkaline phosphatase (SEAP) expression cassette induced levels of SEAP more than 30 times higher than those for 293-produced vector 1 week after injection. Analysis of virion DNA revealed that in addition to a 2.4- or 3.4-kb single-stranded vector genome, Sf9-rAAV5 had more-abundant forms of approximately 4.7 kb, which appeared to correspond to the monomer duplex form of hGFP vector or truncated monomer duplex SEAP vector DNA. These results indicated that rAAV5 can be generated in insect cells, although the difference in incorporated virion DNA may induce different expression patterns of the transgene.
  • Xin, KQ, Mizukami, H, Urabe, M, Toda, Y, Shinoda, K, Yoshida, A, Oomura, K, Kojima, Y, Ichino, M, Klinman, D, Ozawa K, Okuda, K
    J Virol 80 11899 - 910 2006 [Not refereed][Not invited]
  • Ogura, T, Mizukami, H, Mimuro, J, Madoiwa, S, Okada, T, Matsushita, T, Urabe, M, Kume, A, Hamada, H, Yoshikawa, H, Sakata, Y, Ozawa, K
    J Gene Med 8 990 - 7 2006 [Not refereed][Not invited]
  • Mizukami, H, Mimuro, J, Ogura, T, Okada, T, Urabe, M, Kume, A, Sakata, Y, Ozawa, K
    Hum Gene Ther 17 921 - 8 2006 [Not refereed][Not invited]
  • Akira Ishiwata, Jun Mimuro, Yuji Kashiwakura, Masanori Niimura, Katsuhiro Takano, Tsukasa Ohmori, Seiji Madoiwa, Hiroaki Mizukami, Takashi Okada, Hiroyuki Naka, Akira Yoshioka, Keiya Ozawa, Yoichi Sakata
    THROMBOSIS RESEARCH 118 (5) 627 - 635 0049-3848 2006 [Refereed][Not invited]
     
    Adeno-associated virus (AAV) vectors carrying the B domain-deleted canine FVIII (BDD cFVIII) gene utilizing the beta-actin minimum promoter (167b) pseudotyped with serotype 1 (AAV1-beta-actin-cFVIII) and serotype 8 (AAV8-beta-actin-cFVIII) were developed to express cFVIII in hemophilia A mice. FVIII clotting activities measured by the APTT method increased in hemophilia A mice with intramuscular injection of AAV1-beta-actin-cFVIII in a dose-dependent manner. Therapeutic FVIII levels (2.9 +/- 1.0%) in hemophilia A mice with the AAV1-beta-actin-cFVIII dose of 1x10(12) gc/body were achieved, suggesting partial correction of the phenotype with AAV1-beta-actin-cFVIII vectors. FVIII clotting activity levels in hemophilia A mice with intravenous injection of AAV8-beta-actin-cFVIII also were increased dose-dependently, achieving therapeutic FVIII levels (5-90%) in hemophilia A mice with the AAV8-beta-actin-cFVIII doses of 1-3 x 10(11) gc/body and supernormal FVIII levels (180-670%) in hemophilia A mice with the AAV8-beta-actin-cFVIII dose of 1 x 10(12) gc/body. Transduction of the liver with AAV8-beta-actin-cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding the FVIII production and antibody formation. These data suggested that both AM and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy. (c) 2005 Elsevier Ltd. All rights reserved.
  • A Hiraide, N Yokoo, KQ Xin, K Okuda, H Mizukami, K Ozawa, T Saito
    HUMAN GENE THERAPY 16 (12) 1413 - 1421 1043-0342 2005/12 [Refereed][Not invited]
     
    The objective of this study was to establish the potency of adeno-associated virus (AAV) as a viral vector to transport the basic fibroblast growth factor (bFGF) gene into synovial tissue, and to evaluate the consequent repair of articular cartilage defects. In the in vitro study, LacZ- and bFGF-encoding genes were transduced into rabbit synoviocytes by recombinant adeno-associated virus (AAV) vector, and the cells were cultured for 2 weeks. The percentage of successfully transduced LacZ-positive cells was assessed by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining, and the concentration of bFGF in the culture supernatant was confirmed by bFGF-specific enzyme-linked immunosorbent assay. In the in vivo study, 12- to 14-week-old Japanese white rabbits (all female) were used. AAV-bFGF was administered into an artificially created full-thickness defect (5 mm in diameter and 3 nun deep) in the patellar groove of the distal femur. Cartilage repair was subsequently monitored at 4, 8, and 12 weeks, by macroscopic and histological examination, and results were graded on the basis of semiquantitative scores. lacZ gene expression in synoviocytes reached more than 93% within the first 2 weeks, and the mean bFGF concentration in the culture supernatant of the bFGF gene-transduced group was significantly increased (p < 0.01). Semiquantitative macroscopic and histological assessment indicated that the average score was significantly better in the bFGF-transduced group throughout the observation period, suggesting better cartilage repair. These results demonstrate that gene transfer into synoviocytes, using the AAV vector, was a potent method of gene transduction. Moreover, after intraarticular administration of AAV-bFGF, constant expression of bFGF in the knee joints resulted in substantial cartilage regeneration that, with further long-term study, could possibly merit consideration for clinical application.
  • YH Liu, T Okada, K Sheykholeslami, K Shimazaki, T Nomoto, SI Muramatsu, T Kanazawa, K Takeuchi, R Ajalli, H Mizukami, A Kume, K Ichimura, K Ozawa
    MOLECULAR THERAPY 12 (4) 725 - 733 1525-0016 2005/10 [Refereed][Not invited]
     
    Recombinant adeno-associated virus (AAV) vectors are of interest for cochlear gene therapy because of their ability to mediate the efficient transfer and long-term stable expression of therapeutic genes in a wide variety of postmitotic tissues with minimal vector-related cytotoxicity. In the present study, seven AAV serotypes (AAV1-5, 7, 8) were used to construct vectors. The expression of EGFP by the chicken P-actin promoter associated with the cytomegalovirus immediate-early enhancer in cochlear cells showed that each of these serotypes successfully targets distinct cochlear cell types. In contrast to the other serotypes, the AAV3 vector specifically transduced cochlear inner hair cells with high efficiency in vivo, while the AAV1, 2, 5, 7, and 8 vectors also transduced these and other cell types, including spiral ganglion and spiral ligament cells. There was no loss of cochlear function with respect to evoked auditory brain-stem responses over the range of frequencies tested after the injection of AAV vectors. These findings are of value for further molecular studies of cochlear inner hair cells and for gene replacement strategies to correct recessive genetic hearing loss due to monogenic mutations in these cells.
  • T Okada, T Nomoto, T Yoshioka, M Nonaka-Sarukawa, T Ito, T Ogura, M Iwata-Okada, R Uchibori, K Shimazaki, H Mizukami, A Kume, K Ozawa
    HUMAN GENE THERAPY 16 (10) 1212 - 1218 1043-0342 2005/10 [Refereed][Not invited]
     
    Adenovirus and adeno-associated virus (AAV) vectors are increasingly used for gene transduction experiments. However, to produce a sufficient amount of these vectors for in vivo experiments requires large-capacity tissue culture facilities, which may not be practical in limited laboratory space. We describe here a large-scale method to produce adenovirus and AAV vectors with an active gassing system that uses large culture vessels to process labor-and cost-effective infection or transfection in a closed system. Development of this system was based on the infection or transfection of 293 cells on a large scale, using a large culture vessel with a surface area of 6320 cm(2). A minipump was connected to the gas inlet of the large vessel, which was placed inside the incubator, so that the incubator atmosphere was circulated through the vessel. When active gassing was employed, the productivity of the adenovirus and AAV vectors significantly increased. This vector production system was achieved by improved CO2 and air exchange and maintenance of pH in the culture medium. Viral production with active gassing is particularly promising, as it can be used with existing incubators and the large culture vessel can readily be converted for use with the active gassing system.
  • M Maruyama, M Higuchi, Y Takaki, Y Matsuba, H Tanji, M Nemoto, N Tomita, T Matsui, N Iwata, H Mizukami, SI Muramatsu, K Ozawa, TC Saido, H Arai, H Sasaki
    ANNALS OF NEUROLOGY 57 (6) 832 - 842 0364-5134 2005/06 [Refereed][Not invited]
     
    Amyloid β peptide (Aβ) has been implicated in Alzheimer's disease (AD) as an initiator of the pathological cascades. Several lines of compelling evidence have supported major roles of Aβ-degrading enzyme neprilysin in the pathogenesis of sporadic AD. Here, we have shown a substantial reduction of cerebrospinal fluid (CSF) neprilysin activity (CSF-NEP) in patients with AD-converted mild cognitive impairment and early AD as compared with age-matched control subjects. The altered CSF-NEP likely reflects changes in neuronal neprilysin, since transfer of neprilysin from brain tissue into CSF was demonstrated by injecting neprilysin-carrying viral vector into the brains of neprilysin-deficient mice. Interestingly, CSF-NEP showed an elevation with the progression of AD. Along with a close association of CSF-NEP with CSF tau proteins, this finding suggests that presynaptically located neprilysin can be released into CSF as a consequence of synaptic disruption. The impact of neuronal damages on CSF-NEP was further demonstrated by a prominent increase of CSF-NEP in rats exhibiting kainate-induced neurodegeneration. Our results unequivocally indicate significance of CSF-NEP as a biochemical indicator to pursue a pathological process that involves decreased neprilysin activity and Aβ-induced synaptic toxicity, and the support the potential benefits of neprilysin up-regulation in ameliorating neuropathology in prodromal and early AD.
  • Y Takei, H Mizukami, Y Saga, H Kobayashi, M Suzuki, T Matsushita, K Ozawa, M Suzuki
    INTERNATIONAL JOURNAL OF CANCER 113 (1) 54 - 58 0020-7136 2005/01 [Not refereed][Not invited]
     
    A Kunitz-type protease inhibitor, bikunin, is known to suppress the invasion and metastasis of cancer cells. HI8, a carboxyl-terminal domain of bikunin, is an active site of this glycoprotein. To increase its affinity for cancer cells, we constructed a chimeric gene, ATF-HI8, and investigated the anti-invasive and anti-migratory activity of ATF-HI8 on ovarian cancer cells. ATF-HI8-expressing plasmid and ATF-expressing plasmid were introduced into the highly invasive and metastatic ovarian cancer cell line HRA. The properties of the established cell line (HRA/ATF-HI8) were compared to those of the HRA/ATF and the HRA/luciferase (HRA/LUC, control) cell lines in terms of cell proliferation, invasion and migration. As a result, (i) there were no differences in cell proliferation between HRA/ATF-HI8 and HRA/LUC; (ii) the invasion and migration of HRA/ATF-HI8 cells were significantly inhibited compared to those of HRA/LUC cells; (iii) the migration, but not the invasion, of HRA/ATF cells was significantly inhibited compared to that of HRA/LUC. These results indicate that the overexpression of ATF-HI8 inhibits the invasion and migration of ovarian cancer cells without affecting cell proliferation and suggest that HI8 is involved in the anti-invasive and the anti-migratory activities, and the addition of ATF brought about the increase in the anti-migratory activity of HI8. The above findings suggest the applicability of therapeutic strategies targeting the inhibition of peritoneal invasion and dissemination of ovarian cancer by the use of the chimeric gene ATF-HI8. (C) 2004 Wiley-Liss, Inc.
  • Y Takei, H Mizukami, Y Saga, H Kobayashi, M Suzuki, T Matsushita, K Ozawa, M Suzuki
    INTERNATIONAL JOURNAL OF CANCER 113 (1) 54 - 58 0020-7136 2005/01 [Refereed][Not invited]
     
    A Kunitz-type protease inhibitor, bikunin, is known to suppress the invasion and metastasis of cancer cells. HI8, a carboxyl-terminal domain of bikunin, is an active site of this glycoprotein. To increase its affinity for cancer cells, we constructed a chimeric gene, ATF-HI8, and investigated the anti-invasive and anti-migratory activity of ATF-HI8 on ovarian cancer cells. ATF-HI8-expressing plasmid and ATF-expressing plasmid were introduced into the highly invasive and metastatic ovarian cancer cell line HRA. The properties of the established cell line (HRA/ATF-HI8) were compared to those of the HRA/ATF and the HRA/luciferase (HRA/LUC, control) cell lines in terms of cell proliferation, invasion and migration. As a result, (i) there were no differences in cell proliferation between HRA/ATF-HI8 and HRA/LUC; (ii) the invasion and migration of HRA/ATF-HI8 cells were significantly inhibited compared to those of HRA/LUC cells; (iii) the migration, but not the invasion, of HRA/ATF cells was significantly inhibited compared to that of HRA/LUC. These results indicate that the overexpression of ATF-HI8 inhibits the invasion and migration of ovarian cancer cells without affecting cell proliferation and suggest that HI8 is involved in the anti-invasive and the anti-migratory activities, and the addition of ATF brought about the increase in the anti-migratory activity of HI8. The above findings suggest the applicability of therapeutic strategies targeting the inhibition of peritoneal invasion and dissemination of ovarian cancer by the use of the chimeric gene ATF-HI8. (C) 2004 Wiley-Liss, Inc.
  • N Yokoo, T Saito, M Uesugi, N Kobayashi, KQ Xin, K Okuda, H Mizukami, K Ozawa, T Koshino
    ARTHRITIS AND RHEUMATISM 52 (1) 164 - 170 0004-3591 2005/01 [Refereed][Not invited]
     
    Objective. To examine the effects of basic fibroblast growth factor (bFGF) gene-transduced chondrocytes on the repair of articular cartilage defects. Methods. LacZ gene or bFGF gene was transduced into primary isolated rabbit chondrocytes with the use of a recombinant adeno-associated virus (AAV) vector. These gene-transduced chondrocytes were embedded in collagen gel and transplanted into a full-thickness defect in the articular cartilage of the patellar groove of a rabbit. The efficiency of gene transduction was assessed according to the percentage of LacZ-positive cells among the total number of living cells. The concentration of bFGF in the culture supernatant was measured by enzyme-linked immunosorbent assay to confirm the production by bFGF gene-transduced chondrocytes. At 4, 8, and 12 weeks after transplantation, cartilage repair was evaluated histologically and graded semiquantitatively using a histologic scoring system ranging from 0 (complete regeneration) to 14 (no regeneration) points. Results. LacZ gene expression by chondrocytes was maintained until 8 weeks in >85% of the in vitro population. LacZ-positive cells were found at the transplant sites for at least 4 weeks after surgery. The mean concentration of bFGF was significantly increased in bFGF gene-transduced cells compared with control cells (P < 0.01). Semiquantitative histologic scoring indicated that the total score was significantly lower in the bFGF-transduced group than in the control group throughout the observation period. Conclusion. These results demonstrated that gene transfer to chondrocytes by an ex vivo method was established with the AAV vector, and transplantation of bFGF gene-transduced chondrocytes had a clear beneficial effect on the repair of rabbit articular cartilage defects.
  • J Kikuchi, J Mimuro, K Ogata, T Tabata, Y Ueda, A Ishiwata, K Kimura, K Takano, S Madoiwa, H Mizukami, Y Hanazono, A Kume, M Hasegawa, K Ozawa, Y Sakata
    JOURNAL OF GENE MEDICINE 6 (10) 1049 - 1060 1099-498X 2004/10 [Refereed][Not invited]
     
    Background Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34(+) cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII). Methods CD34(+) cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34(+) cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34(+) cells and expression of transgenes were studied. Results We could efficiently transduce CD34(+) cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 +/- 0.1%) at MOI of 5 x 10(3) vector genome/cell. After transducing CD34(+) cells with SIVhFVIII, hFVIII was produced (274.3 +/- 20.1 ng) from 106 CD34(+) cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34(+) cells (5-10 x 10(5)) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34(+) cells and production of hFVIII (minimum 1.2 +/- 0.9 ng/mL, maximum 3.6 +/- 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells. Conclusions Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients. Copyright (C) 2004 John Wiley Sons, Ltd.
  • T Kanazawa, H Mizukami, H Nishino, T Okada, Y Hanazono, A Kume, K Kitamura, K Ichimura, K Ozawa
    INTERNATIONAL JOURNAL OF ONCOLOGY 25 (3) 729 - 735 1019-6439 2004/09 [Refereed][Not invited]
     
    Adeno-associated virus (AAV) is a non-pathogenic virus with a single-strand DNA genome. AAV vectors have several unique properties suited for gene therapy applications. However, an obstacle to their application is a low efficiency of transgene expression, mainly due to a limited second-strand synthesis. Previously, we reported that gamma-rays enhanced the transduction efficiency and cytocidal effect of AAV vector harboring the herpes simplex virus-thymidine kinase (AAVtk) and ganciclovir (GCV) system. In the present study, we investigated whether topoisomerase inhibitors (etoposide and camptothecin) enhance the AAV vector-mediated transgene expression and the killing effect by AAVtk/GCV system. The enhancement of transgene expression was observed in a concentration -dependent manner on human laryngeal carcinoma cells (HEp-2 cells) and HeLa cells. Southern analysis confirmed that etoposide enhanced the double-strand synthesis of the AAV vector genome in HEp-2 cells and HeLa cells. The cells were efficiently killed by AAVtk/GCV system, as expected. More importantly, both etoposide and camptothecin augmented the cytocidal effect of the AAVtk/GCV system. These findings suggest that the combination of AAV-mediated suicide gene therapy and treatment with topoisomerase inhibitors may have synergistic therapeutic effects in the treatment of cancers.
  • 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 0969-7128 2004/09 [Refereed][Not invited]
     
    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.
  • T Matsushita, T Okada, T Inaba, H Mizukami, K Ozawa, P Cojosi
    JOURNAL OF GENERAL VIROLOGY 85 2209 - 2214 0022-1317 2004/08 [Refereed][Not invited]
     
    Although the adenoviral Ell, E2A, E4 and VA RNA regions are required for efficient adeno-associated virus (AAV) vector production, the role that the individual Ell genes (E1A, E1B19K, E1B55K and protein IX) play in AAV vector production has not been clearly determined. Ell mutants were analysed for their ability to mediate AAV vector production in HeLa or KB cells, when cotransfected with plasmids encoding all other packaging functions. Disruption of E1A and E1B19K genes resulted in vector yield reduction by up to 10- and 1 00-fold, respectively, relative to the wild-type Ell. Interruption of the E1B55K and protein IX genes had a modest effect on vector production. Interestingly, expression of anti-apoptotic E1B19K cellular homologues such as BcI-2 or Bcl-X-L fully complemented E1B19K mutants for AAV vector production. These findings may be valuable for the future development of packaging cell lines for AAV vector production.
  • 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 0969-7128 2004/07 [Not refereed][Not invited]
     
    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.
  • K Ishibashi, T Midorikawa, H Kikuchi, Y Fujiwara, H Izuno, T Hatakeyama, M Saito, K Miyakawa, H Mizukami, K Maezawa, H Nemoto, Y Sanada
    HEPATO-GASTROENTEROLOGY 51 (58) 1165 - 1167 0172-6390 2004/07 [Refereed][Not invited]
     
    We report a 67-year-old man with highly symptomatic polycystic liver disease. Fenestration was selected to treat symptoms because the cysts were scattered diffusely and the normal liver volume was limited. Although this patient was relieved from symptoms of liver cysts consequently, several severe postoperative complications including disseminated intravascular coagulation, respiratory failure, liver failure, and biliary leakage occurred resulting in a 6-month postoperative hospital stay. Although various treatments for symptomatic adult polycystic liver disease have been advocated, a definitive treatment remains controversial, especially in diffuse adult polycystic liver disease. Fenestration is one of the alternative treatments for the patients whose cysts are difficult to resect. However high morbidity rate should be carefully assessed, if extensive fenestration is needed to treat diffuse adult polycystic liver disease. Further consideration of appropriate treatments for diffuse adult polycystic liver disease is needed.
  • H Mizukami, T Okada, Y Ogasawara, T Matsushita, M Urabe, A Kume, K Ozawa
    MOLECULAR BIOTECHNOLOGY 27 (1) 7 - 14 1073-6085 2004/05 [Refereed][Not invited]
     
    Adeno-associated virus (AAV) vectors are a practical choice for gene transfer, and demand for them is increasing. To cope with the necessity in the near future, we have developed a number of approaches to establish packaging cell lines for the production of AAV vectors. In our previous study, a-highly regulated expression of large Rep proteins was obtained by using,the Cke-loxP. switching system. Therefore, in the present study, to regulate Cap expression as well, we developed an inducible expression system for both Rep and Cap proteins by using an additional set of mutant loxP sequences. The mutants possess two base alterations in the spacer region of loxP and recombine specifically with the same counterpart in the presence of Cre. By using two separate plasmids, one with mutant and the other with wild-type loxP sequences, the expression of two different proteins can be induced simultaneously by Cre recombinase. When the LacZ-encoding plasmid vector was used as a packaging model, a significant packaging titer of 2.1 x 10(10) genome copies per 10-cm dish was obtained. These results indicate the importance of controlling Cap expression, in addition to Rep, to achieve an optimum production rate for AAV vectors.
  • J Mimuro, H Mizukami, F Ono, S Madoiwa, K Terao, A Yoshioka, K Ozawa, Y Sakata
    JOURNAL OF THROMBOSIS AND HAEMOSTASIS 2 (2) 275 - 280 1538-7933 2004/02 [Refereed][Not invited]
     
    After screening for species-specific antihuman factor (F)IX monoclonal antibodies, we found that antibody 3A6 did not bind to cynomolgus FIX. The 3A6 epitope was found to include Ala262 of human FIX. The 3A6 antibody was used as a catching antibody in an enzyme immunoassay (EIA) for specific detection of human FIX in cynomolgus macaque plasma. No significant increase of substrate hydrolysis was observed when EIA buffer containing cynomolgus macaque plasma was subjected to the 3A6-based EIA. Addition of up to 30% cynomolgus macaque plasma or canine plasma to the assay did not alter detection of human FIX. Three cynomolgus macaques were injected with human FIX (10 U kg(-1); i.v.) and the circulating human FIX was quantified in the macaque plasma. The FIX level in the circulation increased to 470 +/- 37.6 ng mL(-1) at 1 h after the injection and gradually decreased to 1.79 +/- 1.1 ng mL(-1) by day 5, which is approximately 0.06% of the normal human plasma FIX concentration. These data suggest that the cynomolgus macaque can be used as a primate model for studying hemophilia B gene therapy by transduction of macaque organs with vectors to express human FIX in vivo and detection of human FIX using the 3A6 monoclonal antibody.
  • Separate control of Rep and Cap expression utilizing mutant and wild-type loxP sequences and improved packaging system for adeno-associated virus vector production.
    Mizukami, H, Okada, T, Ogasawara, Y, Matsushita, T, Urabe, M, Kume, A, Ozawa, K
    Mol Biotechnol 27 7 - 14 2004 [Not refereed][Not invited]
  • N Iwata, H Mizukami, K Shirotani, Y Takaki, S Muramatsu, B Lu, NP Gerard, C Gerard, K Ozawa, TC Saido
    JOURNAL OF NEUROSCIENCE 24 (4) 991 - 998 0270-6474 2004/01 [Refereed][Not invited]
     
    A local increase in amyloid-beta peptide (Abeta) is closely associated with synaptic dysfunction in the brain in Alzheimer's disease. Here, we report on the catabolic mechanism of Abeta at the presynaptic sites. Neprilysin, an Abeta-degrading enzyme, expressed by recombinant adeno-associated viral vector-mediated gene transfer, was axonally transported to presynaptic sites through afferent projections of neuronal circuits. This gene transfer abolished the increase in Abeta levels in the hippocampal formations of neprilysin-deficient mice and also reduced the increase in young mutant amyloid precursor protein transgenic mice. In the latter case, Abeta levels in the hippocampal formation contralateral to the vector-injected side were also significantly reduced as a result of transport of neprilysin from the ipsilateral side, and in both sides soluble Abeta was degraded more efficiently than insoluble Abeta. Furthermore, amyloid deposition in aged mutant amyloid precursor protein transgenic mice was remarkably decelerated. Thus, presynaptic neprilysin has been demonstrated to degrade Abeta efficiently and to retard development of amyloid pathology.
  • Takeda S, Takahashi M, Mizukami H, Kobayashi E, Takeuchi K, Hakamata Y, Kaneko T, Yamamoto H, Ito C, Ozawa K, Ishibashi K, Matsuzaki T, Takata K, Asano Y, Kusano E
    Nephron. Experimental nephrology 4 96 e119 - 26 1660-8151 2004 [Refereed][Not invited]
  • Shin-Ichi Takeda, Masafumi Takahashi, Hiroaki Mizukami, Eiji Kobayashi, Koichi Takeuchi, Yoji Hakamata, Takashi Kaneko, Hisashi Yamamoto, Chiharu Ito, Keiya Ozawa, Kenichi Ishibashi, Toshiyuki Matsuzaki, Kuniaki Takata, Yasushi Asano, Eiji Kusano
    Nephron - Experimental Nephrology 96 (4) e119 - e126 1660-2129 2004 [Refereed][Not invited]
     
    Background/Aim: Gene transfer into the kidney has great potential as a novel therapeutic approach. However, an efficient method of gene transfer into the kidney has not been established. We explored the transduction efficiency of renal cells in vitro and in vivo using adeno-associated virus (AAV) serotype 1-5 vectors encoding the β-galactosidase gene. Methods: In the in vitro study, rat kidney epithelial cell line NRK52E cells were transfected with AAV serotype derived vectors. In the in vivo study, AAV serotype derived vectors were selectively injected into the kidney using a catheter-based gene delivery system in rats and mice mimicking the clinical procedure. The efficiency of gene expression was histologically evaluated on the basis of the β-galactosidase expression. Results: AAV serotype 1, 2, and 5 vectors transduced in rat kidney epithelial cell line NRK52E cells in vitro, whereas AAV serotype 3 or 4 vectors showed no transduction. In addition, the kidney-specific injection of AAV serotype 2 vectors successfully transduced in tubular epithelial cells, but not in glomerular, blood vessel, or interstitial cells in vivo, whereas the rest of the serotypes showed no transduction. Conclusion: Since kidney-specific gene delivery via the renal artery by catheterization is highly feasible in humans, these findings provide useful information for promising strategies in renal gene therapy. Copyright © 2004 S. Karger AG, Basel.
  • J Ideno, H Mizukami, K Honda, T Okada, Y Hanazono, A Kume, T Saito, S Ishibashi, K Ozawa
    MOLECULAR THERAPY 8 (6) 895 - 902 1525-0016 2003/12 [Refereed][Not invited]
     
    Adeno-associated virus (AAV) vector is suitable for gene transfer to the central nervous system. However, the efficacy of gene therapy for neuroendocrine disease is still unknown. In this study, we injected AAV vector encoding arginine-vasopressin (AVP) stereotaxically into the bilateral hypothalamus of Brattleboro rats. Brattleboro rats show a central diabetes insipidus (CDI) phenotype and growth retardation due to a complete deficiency of AVP. Following injection, both urine volume and urine osmolality normalized, and these therapeutic effects persisted for more than 50 weeks. In addition to phenotypic correction, secretion of transgene-derived AVP was enhanced after 24 h water deprivation or hypertonic saline injection, and water diuresis was demonstrated after acute water loading. Also, reduced body weight and low plasma insulin-like growth factor 1 levels of Brattleboro rats were restored after AVP gene transduction, suggesting the importance of AVP in growth. These findings indicate that hypothalamic neurons of Brattleboro rats can produce and release mature AVP following AAV-mediated gene transduction, resulting in long-term phenotypic correction of CDI. Moreover, the fact that transgene-derived AVP was secreted adequately in response to stimuli, even if it was expressed constitutively, suggests advantages of gene therapy for neuroendocrine diseases and offers a basis to investigate AVP function.
  • A Itoh, T Okada, H Mizuguchi, T Hayakawa, H Mizukami, A Kume, M Takatoku, N Komatsu, Y Hanazono, K Ozawa
    JOURNAL OF GENE MEDICINE 5 (11) 929 - 940 1099-498X 2003/11 [Refereed][Not invited]
     
    Background Although adenoviral vectors primarily derived from the adenovirus serotype 5 (Ad5) are widely used for many gene transfer applications, they cannot efficiently infect hematopoietic cells, since these cells do not express the coxsackie-adenoviral receptor (CAR). Methods We have developed a soluble fusion protein that bridges adenoviral fibers and the c-Kit receptor to alter Ad5 tropism to immature hematopoietic cells. The CAR-SCF fusion protein consists of the extracellular domains of CAR and stem cell factor (SCF). The human megakaryoblastic leukemia cell lines UT-7 and M07e, human chronic myelogenous leukemia cell line K-562, and erythroleukemia cell line TF-1 were used to assess CAR-SCF-assisted Ad5-mediated gene transfer. Hematopoietic cell lines were infected with an Ad5 vector (Ad5-eGFP) or a fiber-mutant Ad5/F35 (Ad5/F35-eGFP) expressing the enhanced green fluorescent protein gene in the presence or absence of CAR-SCF. Results Twenty-four hours after infection, more than 80% of M07e cells infected in the presence of CAR-SCF were eGFP-positive, compared with very few eGFP-positive cells following AdS-eGFP infection in the absence of CAR-SCF. The enhancement of Ad5-eGFP infection by CAR-SCF was greater than that caused by Ad5/F35-eGFP (50%). The ability of CAR-SCF to enhance Ad5-eGFP infectivity was highly dependent on cellular c-Kit expression levels. Furthermore, CAR-SCF also enhanced Ad5-mediated gene transfer into human primary CD34(+) cells. Conclusions The CAR-SCF fusion protein assists Ad5-mediated transduction to c-Kit(+) CAR(-) hematopoietic cells. The use of this fusion protein would enhance a utility of Ad5-mediated hematopoietic cell transduction strategies. Copyright (C) 2003 John Wiley Sons Ltd.
  • Y Saga, H Mizukami, Y Takei, K Ozawa, M Suzuki
    INTERNATIONAL JOURNAL OF ONCOLOGY 23 (4) 1109 - 1113 1019-6439 2003/10 [Refereed][Not invited]
     
    Peritoneal dissemination is the major progression pathway of ovarian cancer, and its control is important for improvement of the prognosis. PTEN is a tumor suppressor gene, and is known to inhibit cancer cell growth and migration. To investigate the possibility of gene therapy using PTEN for ovarian cancer, we introduced PTEN cDNA into an ovarian cancer cell line HRA carrying wild-type PTEN, and examined the effects in vitro and in vivo. Using PTEN cDNA cloned from a human liver cDNA library, a PTEN expression vector was constructed. This vector was introduced into HRA cells by the standard calcium phosphate precipitation method, and an HRA cell line overexpressing PTEN (HRA/PTEN) was established. On the cell migration test by in vitro scratch wound healing assay, the number of migrating cells was 6.3 +/- 0.9 cells/mm(2) in HRA/PTEN, which was significantly smaller than that in the control (39.7 +/- 3.2 cells/mm(2)) (p<0.01). No significant differences were observed in the in vitro cell growth or in vivo tumor growth between HRA/PTEN and the control. The findings described above, show that enhanced expression of PTEN inhibits ovarian cancer cell migration, suggesting that gene therapy approaches using PTEN for control of peritoneal dissemination of ovarian cancer are possible.
  • Y Saga, M Suzuki, H Mizukami, T Kohno, Y Takei, M Fukushima, K Ozawa
    INTERNATIONAL JOURNAL OF CANCER 106 (3) 324 - 326 0020-7136 2003/09 [Refereed][Not invited]
     
    The prognosis of cancers of various organs overexpressing thymidylate synthase (TS) has been reported to be poor. It has been suggested that the poor prognosis is partly due to a low sensitivity of TS-overexpressing tumors to TS-targeting 5-fluorouracil (5-FU). To investigate the relationship between TS expression and sensitivity to 5-FU, we used the TS-overexpressing cervical cancer cell line SKG-II/TS and SKG-I/TS that had been established by TS gene transfer. The 50% growth inhibitory concentration (IC50) of 5-FU for SKG-II/TS was 24 +/- 6.0 muM, which was 6 times as high as that for the control (4.0 +/- 1.1 muM), showing significantly decreased sensitivity to 5-FU (p < 0.01). The IC50 of 5-FU for SKG-I/TS was 90 +/- 15 muM, which was over 2 times as high as that for the control (40 +/- 0.6 muM), showing significantly decreased sensitivity to 5-FU (p < 0.05). Thus, TS-overexpressing tumors have decreased sensitivity to 5-FU, which may be one of the factors that determine the prognosis of these tumors. (C) 2003 Wiley-Liss, Inc.
  • T Kohno, H Mizukami, M Suzuki, Y Saga, Y Takei, M Shimpo, T Matsushita, T Okada, Y Hanazono, A Kume, Sato, I, K Ozawa, K Ozawa
    CANCER RESEARCH 63 (16) 5091 - 5094 0008-5472 2003/08 [Refereed][Not invited]
     
    Interleukin-10 (IL-10) is an immunosuppressive cytokine produced by T lymphocytes and drawing attention as an inhibitor of tumor angiogenesis. In this study, we investigated antiangiogenic and tumor suppressive effects of IL-10 in ovarian cancer cells. mIL-10-expressing phismid was transferred into two ovarian cancer cell lines, SHIN-3 [vascular endothelial growth factor (VEGF) producing] and KOC-2S (non-VEGF producing). After selection, mIL-10-expressing cells were obtained as SHIN-3/mIL-10 and KOC-2S/mIL-10. No significant differences were observed in in vitro growth properties between mIL-10-expressing cells and control (luciferase expressing) cells in either KOC-2S or SHIN-3. The angiogenic activities of mIL-10-expressing cells were measured by dorsal air sac assay, which detected the number of newly formed blood vessels within a chamber in vivo. In addition, tumor formation was evaluated by s.c. tumor transplantation, and survival was monitored after i.p. injection of ovarian cancer cells into BALB/c nude mice. Both in vivo angiogenic activity and tumor growth were significantly inhibited in SHIN-3/mIL-10 cells compared with the control. Moreover, peritoneal dissemination was inhibited, and the survival period was significantly prolonged (mean survival days >90 versus 36). In contrast, in the case of KOC-2S cells, no significant differences were observed in any of the parameters tested. These results indicate that IL-10 has suppressive effects on angiogenesis, tumor growth, and peritoneal dissemination of VEGF-producing ovarian cancer cells. Although the mechanisms of the antiangiogenic effect of IL-10 are still unclear, the potential usefulness of IL-10-mediated gene therapy of ovarian cancer was suggested.
  • M Kametaka, A Kum, T Okada, H Mizukami, Y Hanazono, K Ozawa
    CANCER SCIENCE 94 (7) 639 - 643 1347-9032 2003/07 [Refereed][Not invited]
     
    Allogeneic bone marrow transplantation and donor lymphocyte infusion are powerful treatments for chemotherapy-resistant leukemia. Tumor eradication is attributed to a graft-versus-leukemia reaction by the donor-derived cytotoxic T lymphocytes (CTLs), but the same cell population may cause severe graft-versus-host disease. One strategy to suppress harmful CTL activity is to incorporate a suicide gene into the donor lymphocytes prior to infusion, and to destroy these cells when they aggressively attack nonmalignant host tissues. In this study, we investigated the feasibility of using a Fas-estrogen receptor fusion protein (MfasER) to control T cell-mediated cytotoxicity, based on our previous finding that the chimera transmits a Fas-mediated death signal through activation by estrogen binding. A murine CTL line CTLL-2 was transfected with a vector encoding MfasER, and the growth, viability and cytotoxic activity of the transfected cells (CTLL/MfasER) were analyzed. The expression of apoptosis-related proteins such as Fas ligand and perforin was also investigated. In the absence of estrogen, CTLL/MfasER showed similar growth to parental CTLL-2, and the killing activity was preserved. Addition of 10(-7) M estrogen induced a rapid apoptosis of CTLL/MfasER, and the cytotoxicity was severely impaired. A decrease of Fas ligand and perforin in the estrogen-treated CTLL/MfasER was seen in an immunoblot analysis. These functional and biochemical analyses showed that the estrogen-inducible apoptosis in MfasER-expressing CTLs rapidly terminated their target cell killing. The feasibility of using the MfasER-estrogen system to control graft-versus-host disease was demonstrated.
  • KQ Xin, T Ooki, N Jounai, H Mizukami, K Hamajima, Y Kojima, K Ohba, Y Toda, SI Hirai, DM Klinman, K Ozawa, K Okuda
    JOURNAL OF GENE MEDICINE 5 (5) 438 - 445 1099-498X 2003/05 [Refereed][Not invited]
     
    Background DNA vaccines have been used to induce both humoral and cellular immune responses against infectious microorganisms. This study explores whether DNA vaccine immunogenicity can be improved by introducing inverted terminal repeats (ITRs) from adeno-associated virus (AAV) into the regulatory region of the DNA plasmid. Methods CMV promoter-driven HIV Env expressing plasmid (pCMV-HIV) and the pCMV-HIV plasmid introduced ITRs (pITR/CMV-HIV) were transfected in HEK293 cells with LipofectAmine. The HIV Env expression was quantified with Western blot. Fifty mug of pCMV-HIV or pITR/CMV-HIV plasmid with RIBI adjuvant were immunized to BALB/c mice on days 0, 14 and 28 by intramuscular route, and HIV-specific serum IgG titer was detected 2, 6, 10, 14 and 18 weeks after the first immunization. HIV-specific tetramer assay and HIV-specific IFN-gamma ELIspot assay were performed 1 week after the last immunization. The immune mice were intravenously challenged with a vaccinia virus expressing the HIV env gene 1 week after the last immunization. Results Significantly higher level of HIV Env expression was achieved by pITR/CMV-HIV plasmid. BALB/c mice immunized with pITR/CMV-HIV plasmid generated significantly higher HIV-specific antibody, higher cellular immune responses and lower viral loading than animals immunized with pCMV-HIV plasmid. Conclusions AAV ITRs enhance CMV-dependent up-regulation of transgene expression and immunogenicity of DNA vaccine. Copyright (C) 2002 John Wiley Sons, Ltd.
  • T Nomoto, T Okada, K Shimazaki, H Mizukami, T Matsushita, Y Hanazono, A Kume, K Katsura, Y Katayama, K Ozawa
    NEUROSCIENCE LETTERS 340 (2) 153 - 157 0304-3940 2003/04 [Refereed][Not invited]
     
    Genetic modification of the gerbil hippocampal neuronal cells in vivo helps us understand the mechanisms of neuronal function under various circumstances such as ischemic insult. In this study, we examined the distinct distribution of the recombinant adeno-associated virus type 2 (rAAV2) and rAAV5 vectors for gene delivery to primary cultured cells and the gerbil hippocampus. Mixed cortical cultures containing both neurons and astrocytes from E17 rat embryos were infected with rAAVs containing the Cytomegalovirus virus (CMV) promoter. rAAV2 was preferably transduced to neurons, whereas rAAV5 was inclined to be transduced to astrocytes in vitro. rAAV2 and rAAV5 vectors, each with the CMV or Rous sarcoma virus (RSV) promoter, were injected into the gerbil hippocampus using a stereotaxic apparatus. Five days after injection, transgene expression was analyzed with X-gal staining. In the gerbil hippocampus, rAAV5 with the CMV promoter achieved, a higher overall transgene expression than rAAV2 with the CMV promoter. The transgene expression of rAAV2 with the RSV promoter was found in the pyramidal and granular cells, while the transgene expression of rAAV5 with the RSV promoter was preferentially found in the granular cells. These findings would be valuable in optimizing rAAV-mediated gene transfer to the gerbil hippocampus. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.
  • A Kume, M Koremoto, RF Xu, T Okada, H Mizukami, Y Hanazono, M Hasegawa, K Ozawa
    JOURNAL OF GENE MEDICINE 5 (3) 175 - 181 1099-498X 2003/03 [Refereed][Not invited]
     
    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.
  • YY Lu, LJ Wang, S Muramatsu, K Ikeguchi, K Fujimoto, T Okada, H Mizukami, T Matsushita, Y Hanazono, A Kume, T Nagatsu, K Ozawa, Nakano, I
    NEUROSCIENCE RESEARCH 45 (1) 33 - 40 0168-0102 2003/01 [Refereed][Not invited]
     
    Adeno-associated virus (AAV) vector has been developed as an attractive gene delivery system with proven safety. Glial cell line-derived neurotrophic factor (GDNF) is proposed to be a promising therapeutic agent for amyotrophic lateral sclerosis (ALS) and other motor neuron diseases. The purpose of this report was to investigate transgenic GDNF expression at different time points post AAV mediated GDNF intramuscular delivery. An AAV vector was constructed to encode a recombinant fusion of GDNF tagged with a FLAG sequence at the C-terminal (AAV-GDNF) to distinguish it from its endogenous counterpart. A single intramuscular injection of AAV-GDNF led to substantial expression of transgenic GDNF which remained for at least 10 months in transduced gastrocnemius muscle. This transgenic GDNF was distributed in a large number of myofibers, mainly in the vicinity of the sarcolemma and predominantly concentrated at the sites of neuromuscular junctions (NMJs). Furthermore, transgenic GDNF, but not P-galactosidase expressed as a control, was detected in the motoneurons that projected axons to the injected muscles, thus, indicating retrograde axonal transportation of the transgenic GDNF. This study provides a basis for a strategy of intramuscular AAV-GDNF delivery to protect motoneurons as a possible means of ALS treatment. (C) 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Muramatsu S, Wang L, Ikeguchi K, Fujimoto K, Nakano I, Okada T, Mizukami H, Hanazono Y, Kume A, Nakano I, Ozawa K
    International review of neurobiology 55 205 - 222 0074-7742 2003 [Refereed][Not invited]
  • A Kume, Y Hanazono, H Mizukami, T Okada, K Ozawa
    INTERNATIONAL JOURNAL OF HEMATOLOGY 76 (4) 299 - 304 0925-5710 2002/11 [Refereed][Not invited]
     
    Although gene transfer into hematopoietic stem cells holds a considerable therapeutic potential, clinical trials targeting this cell compartment have achieved limited success. Poor transduction efficiency with gene transfer vectors used in human studies has hindered delivering therapeutic genes to clinically relevant numbers of target cells. One way to overcome the low-efficiency problem is by selecting or expanding the number of genetically modified cells to a suprathreshold level to achieve clinical efficacy. This approach may be further classified into 2 categories: one is to transfer a drug resistance gene and eliminate unmodified cells with cytotoxic drugs, and the other is to confer a direct growth advantage on target cells. This review aims at an overview of recent advances involving these strategies, with some details of "selective amplifier genes," a novel system that we have developed for specific expansion of genetically modified hematopoietic cells.
  • N Kobayashi, T Koshino, M Uesugi, N Yokoo, KQ Xin, K Okuda, H Mizukami, K Ozawa, T Saito
    JOURNAL OF RHEUMATOLOGY 29 (10) 2176 - 2180 0315-162X 2002/10 [Refereed][Not invited]
     
    Objective. To evaluate both the potential for transferring genes to periosteal cells using an adeno-associated virus (AAV) vector and the potential for gene expression after transplantation of those cells to a cartilage defect in vivo. Methods. Periosteum was obtained from the tibia of 6-week-old rabbits and enzymatically digested. The isolated periosteum derived cells were cultured and the subconfluence cells were infected with a recombinant AAV expressing the LacZ gene (AAV-LacZ). Collagen gel containing the LacZ transferred, periosteum derived cells was transplanted into a full thickness articular cartilage defect in 10 rabbits. Results. Infected cells still growing on the plate continued to express LacZ at least 12 weeks after AAV infection, with the highest percentage of LacZ positive cells reaching 74.4%. The LacZ positive cells were recognized at the transplant sites in 8 out of 10 knees. Conclusion. Gene expression in periosteum derived cells was sustained in vitro for at least 12 weeks using the AAV vector, and for 2 weeks ex vivo after transplantation into a cartilage defect.
  • Okada T, Nomoto T, Shimazaki K, Lijun W, Lu Y, Matsushita T, Mizukami H, Urabe M, Hanazono Y, Kume A, Muramatsu S, Nakano I, Ozawa K
    Methods (San Diego, Calif.) 2 28 237 - 247 1046-2023 2002/10 [Refereed][Not invited]
  • KQ Xin, T Ooki, H Mizukami, K Hamajima, K Okudela, K Hashimoto, Y Kojima, N Jounai, Y Kumamoto, S Sasaki, D Klinman, K Ozawa, K Okuda
    HUMAN GENE THERAPY 13 (13) 1571 - 1581 1043-0342 2002/09 [Refereed][Not invited]
     
    Oral vaccines can induce both systemic and mucosal immunity. Mucosal immunity, especially regional cell-mediated immunity, plays an important role in protecting individuals from infectious diseases such as acquired immunodeficiency syndrome. In this study, a recombinant adeno-associated virus vector expressing human immunodeficiency virus type 1 env gene (AAV-HIV) was orally administered to BALB/c mice. Systemic and regional immunity was induced in the mice. Furthermore, the immunization significantly reduced viral load after an intrarectal challenge with a recombinant vaccinia virus expressing HIV env gene. Moreover, we also show that dendritic cells might contribute to the AAV-HIV vector- induced immune responses.
  • A Kume, H Mizukami, T Okada, Y Hanazono, K Sugamura, K Ozawa, F Takaku
    PROCEEDINGS OF THE JAPAN ACADEMY SERIES B-PHYSICAL AND BIOLOGICAL SCIENCES 78 (7) 211 - 216 0386-2208 2002/09 [Refereed][Not invited]
     
    The cytokine receptor common gamma chain (gammac) plays a pivotal role in transmitting multiple interleukin signals. Its gene mutations cause profound lymphoid maldevelopment, a disorder known as X-linked severe combined immunodeficiency (X-SCID). Without successful bone marrow transplantation, affected patients would die in infancy or early childhood due to severe and recurrent infections. Therefore, development of gene therapy strategies has been awaited for those without suitable marrow donors. We constructed a retrovirus vector carrying the gammac and the enhanced green fluorescent protein (EGFP) genes for a preclinical study with a murine X-SCID model. After transduction of X-SCID bone marrow and infusion into myeloablated X-SCID recipients, robust lymphoid reconstitution was observed in all the examined lymphocyte subsets such as CD4(+)-T, CD8(+)-T, B and NK cells. A long-term EGFP expression was demonstrated in both lymphoid and myeloid cells, suggesting that this type of vector is useful in evaluating regimens of stem cell-directed gene therapy.
  • LJ Wang, YY Lu, S Muramatsu, K Ikeguchi, K Fujimoto, T Okada, H Mizukami, T Matsushita, Y Hanazono, A Kume, T Nagatsu, K Ozawa, Nakano, I
    JOURNAL OF NEUROSCIENCE 22 (16) 6920 - 6928 0270-6474 2002/08 [Refereed][Not invited]
     
    Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive lethal disease that involves selective annihilation of motoneurons. Glial cell line-derived neurotrophic factor (GDNF) is proposed to be a promising therapeutic agent for ALS and other motor neuron diseases. Because adeno-associated virus (AAV) has been developed as an attractive gene delivery system with proven safety, we explored the therapeutic efficacy of intramuscular delivery of the GDNF gene mediated by an AAV vector (AAV-GDNF) in the G93A mouse model of ALS. We show here that AAV-GDNF leads to substantial and long-lasting expression of transgenic GDNF in a large number of myofibers with its accumulation at the sites of neuromuscular junctions. Detection of GDNF labeled with FLAG in the anterior horn neurons, but not beta-galactosidase expressed as a control, indicates that most of the transgenic GDNF observed there is retrogradely transported GDNF protein from the transduced muscles. This transgenic GDNF prevents motoneurons from their degeneration, preserves their axons innervating the muscle, and inhibits the treated-muscle atrophy. Furthermore, four-limb injection of AAV-GDNF postpones the disease onset, delays the progression of the motor dysfunction, and prolongs the life span in the treated ALS mice. Our finding thus indicates that AAV-mediated GDNF delivery to the muscle is a promising means of gene therapy for ALS.
  • A Kume, M Koremoto, H Mizukami, T Okada, Y Hanazono, K Sugamura, K Ozawa
    BONE MARROW TRANSPLANTATION 30 (2) 113 - 118 0268-3369 2002/07 [Refereed][Not invited]
     
    The cytokine receptor common gamma chain (gammac) plays a pivotal role in multiple interleukin signaling, and gammac gene mutations cause an X-linked form of SCID (X-SCID). Recently, gammac gene transfer into the autologous X-SCID BM achieved appreciable lymphocyte reconstitution, contrasting with the limited success in previous gene therapy trials targeting hematopoietic stem cells. To understand the mechanisms underlying this success, we examined the repopulating potential of the wild-type (WT) BM cells using an X-SCID mouse model. Limited numbers of WT cells were infused into non-ablated WT and X-SCID hosts. Whereas no appreciable engraftment was observed in WT recipients, donor-derived lymphocytes repopulated well in X-SCID, reaching 37% (10(6) cells given) and 53% (10(7) cells given) of the normal control value 5 months post BMT. A lineage analysis showed a predominance of the donor-derived lymphocytes (CD4(+) T, CD8(+) T, B and NK cells) in X-SCID while the donor-derived granulocytes and monocytes engrafted poorly. These results showed a selective advantage of WT cells in X-SCID, and that the advantage was restricted to lymphocytes. In human gene therapy for X-SCID, an analogous growth advantage would greatly enhance the repopulation of lymphocytes derived from a very small number of gammac gene-supplemented precursors.
  • Y Saga, H Mizukami, M Suzuki, T Kohno, M Urabe, K Ozawa, Sato, I
    CLINICAL CANCER RESEARCH 8 (5) 1248 - 1252 1078-0432 2002/05 [Refereed][Not invited]
     
    Purpose: PTEN is a tumor suppressor gene that was identified on chromosome 40q23. In addition to its original function as a tumor suppressor, this gene product was recently reported to enhance the sensitivity of cancer cells to anticancer agents. It is for the purpose of this study to investigate its function and the mechanisms by which PTEN enhances the sensitivity of ovarian cancer to antitumor agents. Experimental Design: PTEN cDNA was introduced into the ovarian cancer cell line SHIN-3 and a high-expression cell line (SHIN-3/PTEN) was established. This cell line and a control were further analyzed. Results: SHIN-3 cells did not carry any mutations in its genome after sequencing. In vitro examination of sensitivity to anticancer agents showed that the 50% growth -inhibitory concentration value for irinotecan metabolite (SN-38) in SHIN-3/PTEN was 800 nM, a 6.6-fold higher sensitivity compared with that of the control (5300 nM). There were no differences in sensitivity to cisplatin, paclitaxel, or gemcitabine between SHIN-3/PTEN and the controls. The percentage of apoptotic cells in SHIN-3/PTEN was 16.6 +/- 0.7% 24 h after addition of SN-38, a significant increase over controls (8.6 +/- 0.9%; P < 0.01). Lower topoisomerase I activity was observed in SHIN-3/PTEN, compared with controls. Conclusions: These results indicate that high PTEN expression enhances the sensitivity, of ovarian cancer cells to irinotecan and the induction of apoptosis and the suppression of topoisomerase I activity in cancer cells are suggested as possible mechanisms attributable to high PTEN expression.
  • Y Hasumi, H Mizukami, M Urabe, T Kohno, K Takeuchi, A Kume, M Momoeda, H Yoshikawa, T Tsuruo, M Shibuya, Y Taketani, K Ozawa
    CANCER RESEARCH 62 (7) 2019 - 2023 0008-5472 2002/04 [Refereed][Not invited]
     
    Vascular endothelial growth factor (VEGF), a bifunctional protein enhancing vascular permeability and stimulating endothelial growth, is thought to be responsible for fluid accumulation and angiogenesis in ascites tumors. To investigate the effects of stable expression of the soluble form of Fit-1 VEGF receptor (sFit-1), a known endogenous inhibitor of VEGF, on the malignant ascites tumors, we cotransduced RMG-1 human ovarian cancer cells with adeno-associated virus vectors carrying the sFlt-1 cDNA and Neo gene or Neo gene alone and isolated both the sFlt-1-expressing clone and the Neo-expressing clone. In vitro growth characteristics were essentially the same. As expected, conditioned medium collected from the sFlt-1-expressing cells significantly inhibited the human umbilical vein endothelial cell proliferation in the presence of recombinant VEGF. Expression of sFlt-1 significantly suppressed RMG-1 cell-induced angiogenesis in vivo in the mouse dorsal air sac assay model. We then inoculated sFlt-1- or Neo alone-expressing cells i.p. into female BALB/c nude mice. The average volume of ascites fluid, number of leaked RBCs, and number of cancer cells were significantly lower in mice injected with sFlt-1-expressing cells than in the controls. Survival time was significantly prolonged in mice injected with sFlt-1-expressing cells. These results suggest that inhibition of VEGF activity by sFlt-1 expression may provide a means to control carcinomatous ascites and angiogenesis of malignant ascites tumors.
  • M Shimpo, U Ikeda, Y Maeda, M Takahashi, H Miyashita, H Mizukami, M Urabe, A Kume, T Takizawa, M Shibuya, K Ozawa, K Shimada
    CARDIOVASCULAR RESEARCH 53 (4) 993 - 1001 0008-6363 2002/03 [Refereed][Not invited]
     
    Objectives: Clinical trials on therapeutic angiogenesis using vascular endothelial growth factor (VEGF) are ongoing, however the benefits of these therapies are still controversial. To establish a more efficient gene transfer method for ischemic diseases, we investigated the therapeutic potential of adeno-associated virus (AAV)-mediated VEGF gene transfer. Methods: We produced VEGF(165)-express in AAV vectors (AAV-VEGF). HEK-293 cells were transduced with AAV-VEGF in vitro and VEGF expression and secretion were examined. We used a rat ischemic hindlimb model and AAV-VEGF was administered intramuscularly into the ischemic limb. Gene expression was evaluated by RT-PCR and ELISA. Six weeks after gene transfer, we measured the blood flow of limb vessels and the skin temperature of limbs. Histochemical examination was performed to illustrate capillary growth. Results: Western blotting and ELISA revealed VEGF protein expression and secretion from AAV-VEGF-transduced HEK-293 cells. VEGF mRNA and protein expression was consistently observed in the injected muscle at least 10 weeks after the injection, while no VEGF mRNA could be detected at remote organs. The mean blood flow in AAV-VEGF-transduced ischemic limbs was significantly higher than in AAV-LacZ-transduced limbs. Capillary density was significantly higher in AAV-VEGF-injected tissues than in AAV-LacZ-injected tissues. Conclusions: This study demonstrates that (1) AAV-mediated VEGF gene transfer into rat skeletal muscles is efficient and stable without ectopic expression, and (2) AAV-mediated VEGF gene transfer stimulates angiogenesis and thereby improves blood flow in a rat hindlimb ischemia model. These findings suggest that AAV-mediated VEGF gene transfer may be useful for treatment of ischemic diseases. (C) 2002 Elsevier Science BY All rights reserved.
  • L Wang, S Muramatsu, Y Lu, K Ikeguchi, K Fujimoto, T Okada, H Mizukami, Y Hanazono, A Kume, F Urano, H Ichinose, T Nagatsu, Nakano, I, K Ozawa
    GENE THERAPY 9 (6) 381 - 389 0969-7128 2002/03 [Refereed][Not invited]
     
    Glial cell line-derived neurotrophic factor (GDNF) is a strong candidate agent in the neuroprotective treatment of Parkinson's disease (PD). We investigated whether adeno-associated viral (AA V) vector-mediated delivery of a GDNF gene in a delayed manner could prevent progressive degeneration of dopaminergic (DA) neurons, while preserving a functional nigrostriatal pathway. Four weeks after a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), rats received injection of AA V vectors expressing GDNF tagged with FLAG peptide (AAV-GDNFflag) or beta-galactosidase (AAV-LacZ), into the lesioned striatum. Immunostaining for FLAG demonstrated retrograde transport of GDNFflag to the substantia nigra (SN). The density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive or cholera toxin subunit B (CTB, neuronal tracer)-labeled neurons in the SN were significantly greater in the AAV-GDNFflag group than in the AA V-LacZ group. Dopamine levels and those of its metabolites in the striatum were remarkably higher in the AAV-GDNFflag group compared with the control group. Consistent with anatomical and biochemical changes, significant behavioral recovery was observed from 4-20 weeks following AAV-GDNFflag injection. These data indicate that a delayed delivery of GDNF gene using AA V vector is efficacious even 4 weeks after the onset of progressive degeneration in a rat model of PD.
  • SI Muramatsu, KI Fujimoto, K Ikeguchi, N Shizuma, K Kawasaki, F Ono, Y Shen, LJ Wang, H Mizukami, A Kume, M Matsumura, Nagatsu, I, F Urano, H Ichinose, T Nagatsu, K Terao, Nakano, I, K Ozawa
    HUMAN GENE THERAPY 13 (3) 345 - 354 1043-0342 2002/02 [Refereed][Not invited]
     
    One potential strategy for gene therapy of Parkinson's disease (PD) is the local production of dopamine (DA) in the striatum induced by restoring DA-synthesizing enzymes. In addition to tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), GTP cyclohydrolase I (GCH) is necessary for efficient DA production. Using adeno-associated virus (AAV) vectors, we previously demonstrated that expression of these three enzymes in the striatum resulted in long-term behavioral recovery in rat models of PD. We here extend the preclinical exploration to primate models of PD. Mixtures of three separate AAV vectors expressing TH, AADC, and GCH, respectively, were stereotaxically injected into the unilateral putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys. Coexpression of the enzymes in the unilateral putamen resulted in remarkable improvement in manual dexterity on the contralateral to the AAV-TH/-AADC/-GCH-injected side. Behavioral recovery persisted during the observation period (four monkeys: 48 days, 65 days, 50 days, and >10 months, each). TH-immunoreactive (TH-IR), AADC-IR, and GCH-IR cells were present in a large region of the putamen. Microdialysis demonstrated that concentrations of DA in the AAV-TH/-AADC/-GCH-injected putamen were increased compared with the control side. Our results show that AAV vectors efficiently introduce DA-synthesizing enzyme genes into the striatum of primates with restoration of motor functions. This triple transduction method may offer a potential therapeutic strategy for PD.
  • Okada T, Shimazaki K, Nomoto T, Matsushita T, Mizukami H, Urabe M, Hanazono Y, Kume A, Tobita K, Ozawa K, Kawai N
    Methods in enzymology 346 378 - 393 0076-6879 2002 [Refereed][Not invited]
  • Y Saga, M Suzuki, H Mizukami, M Urabe, M Fukushima, K Ozawa, Sato, I
    ONCOLOGY 63 (2) 185 - 191 0030-2414 2002 [Refereed][Not invited]
     
    It has been shown that there is an inverse relationship between the level of thymidylate synthase (TS) and therapeutic outcomes in patients with malignancies including cervical cancer. To clarify the mechanism of the poor prognosis of cervical cancer with high TS expression, we introduced TS cDNA to the human uterine cervical cancer cell line SKG-II and evaluated the effect of TS expression on its radiosensitivity. After selection, stable transformants of SKG-II cells expressing high level of TS (SKG-II/TS) and control cells (SKG-II/luciferase) were obtained. The level of TS measured by the FdUMP-TS binding assay was significantly higher (p < 0.05) in SKG-II/TS than in control (2.0 +/- 0.1 and 1.3 +/- 0.1 pmol/mg, respectively). No difference was observed in in vitro cell growth or in vivo tumor growth. On evaluation of in vitro radiosensitivity, the 50% growth inhibitory dose (ID50) was 3.1 +/- 0.1 Gy in SKG-II/TS and was significantly higher (p < 0.01) than that in control (2.2 +/- 0.1 Gy). From these results, it is suggested that one of the reasons of poor outcome of cervical cancer to radiation is high TS expression.
  • KQ Xin, K Hamajima, H Mizukami, N Jounai, Y Kojima, K Hashimoto, Y Kumamoto, K Mazui, K Ozawa, K Okuda
    XIV INTERNATIONAL AIDS CONFERENCE: BASIC SCIENCES 233 - 236 2002 [Refereed][Not invited]
     
    The ability of an adeno-associated virus vector expressing the env gene from HIV-1 (AAV-HIV) to induce a protective mucosal immune response when administered orally was studied. A single oral administration of AAV-HIV stimulated strong HIV-specific mucosal, humoral immunity and generated MHC class I restricted CTL in BALB/c mice. This response significantly reduced the viral load after intra-rectal challenge with a recombinant vaccinia virus expressing the HIV env gene. These results demonstrate that oral administration of an AAV-based HIV vaccine elicits a strong immune response that protects against mucosal infection.
  • K Ozawa, S Muramatsu, K Fujimoto, K Ikeguchi, Y Shen, LJ Wang, T Okada, H Mizukami, Y Hanazono, A Kume, H Ichinose, T Nagatsu, K Terao, Nakano, I
    MAPPING THE PROGRESS OF ALZHEIMER'S AND PARKINSON'S DISEASE 51 459 - 462 0099-9962 2002 [Refereed][Not invited]
  • M Muramatsu, Y Hanazono, Y Ogasawara, T Okada, H Mizukami, A Kume, H Mizoguchi, K Ozawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 285 (4) 891 - 896 0006-291X 2001/07 [Refereed][Not invited]
     
    Since hematopoietic stem cells (HSCs) differentiate readily ex vivo resulting in the loss of self-renewal and engraftment abilities, the transient block of differentiation is essential to maintain those abilities during their ex vivo expansion culture. To this end, we developed a method of reversible integration of the dominant negative retinoic acid receptor (DN-RAR) gene, a differentiation-blocking gene, into cells utilizing the Cre/loxP-dependent gene recombination system. The murine immature hematopoietic 32D cells differentiate into mature neutrophils upon G-CSF treatment. However, 32D cells transduced with a retroviral vector expressing the DN-RAR gene put between two loxP sites continued to proliferate without showing differentiation even in the presence of G-CSF. After the cells were fully amplified, the cells were transduced with the Cre recombinase gene. The cells then restored the ability to differentiate into mature neutrophils upon G-CSF treatment. PCR analysis showed that the DN-RAR gene was efficiently removed from the genome by introduction of the Cre gene. This system may eventually be applicable to the ex vivo expansion of HSCs. (C) 2001 Academic Press.
  • T Kanazawa, M Urabe, H Mizukami, T Okada, A Kume, H Nishino, J Monahan, K Kitamura, K Ichimura, K Ozawa
    CANCER GENE THERAPY 8 (2) 99 - 106 0929-1903 2001/02 [Refereed][Not invited]
     
    Adeno-associated virus (AAV) vector has several unique properties suited for gene therapy applications. However, relatively low efficiency of transgene expression, which is mainly due to a limited second-strand synthesis from the single-stranded AAV genome, can be a problem in some applications that require potent gene expression such as antitumor applications. Recently, gamma -ray irradiation has been reported to enhance the second-strand synthesis of the AAV genome, and consequently transgene expression. We demonstrate here that an AAV vector harboring the herpes simplex virus type-1 thymidine kinase (HSVtk) is able to kill cancer cells more efficiently when used in combination with gamma -ray irradiation. A human maxillary sinus cancer cell line, NKO-1, was efficiently killed in combination with HSVtk transduction and ganciclovir (GCV), as expected. More importantly, gamma -ray irradiation of practical dosages augmented the cytocidal effect of the HSVtk/GCV system. Southern analysis indicated that gamma -rays enhanced the double-strand synthesis of the rAAV genome in NKO-1 cells. These findings suggest that the combination of rAAVtk/GCV suicide gene therapy with radiotherapy has synergistic effects in the treatment of cancers and may lead to a reduction of the potential toxicity of both rAAVtk/GCV and gamma -ray irradiation.
  • A Handa, S Muramatsu, JM Qiu, H Mizukami, KE Brown
    JOURNAL OF GENERAL VIROLOGY 81 (Pt 8) 2077 - 2084 0022-1317 2000/08 [Refereed][Not invited]
     
    Although adeno-associated virus (AAV)-2 has a broad tissue-host range and can transduce a wide variety of tissue types, some cells, such as erythro-megakaryoblastoid cells, are non-permissive and appear to lack the AAV-2 receptor. However, limited studies have been reported with the related dependovirus AAV-3, We have previously cloned this virus, characterized its genome and produced an infectious clone. In this study, the gene for green fluorescent protein (GFP) was inserted into AAV-2- and AAV-3-based plasmids and recombinant viruses were produced. These viruses were then used to transduce haematopoietic cells and the transduction efficiencies were compared. In contrast to recombinant (r) AAV-2, rAAV-3 successfully transduced erythroid and megakaryoblastoid cells, although rAAV-2 was superior in transduction of lymphocyte-derived cell lines. Recently, it was reported that heparan sulphate can act as a receptor of AAV-2, The infectivity of rAAV-2 and rAAV-3 was tested with mutant cell lines of Chinese hamster ovary cells that were defective for heparin or heparan sulphate expression on the cell surface. There was no correlation between the ability of rAAV-2 or rAAV-3 to infect cells and the cell surface expression of heparan sulphate and, although heparin blocked both rAAV-2 and rAAV-3 transduction, the ID50 of rAAV-3 was higher than that of rAAV-2. In addition, virus-binding overlay assays indicated that AAV-2 and AAV-3 bound different membrane proteins. These results suggest not only that there are different cellular receptors for AAV-2 and AAV-3, but that rAAV-3 vectors may be preferred for transduction of some haematopoietic cell types.
  • Nishizono-Maher A, Sakamaki H, Mizukami H, Kuraki T, Minakawa K, Masuda Y
    BMJ (Clinical research ed.) 306 (6881) 830 - 831 0959-8138 1993/03 [Refereed][Not invited]
  • A. Nishizono-Maher, H. Sakamaki, H. Mizukami, T. Kuraki, K. Minakawa, Y. Masuda
    British Medical Journal 306 (6881) 830 - 831 0959-8146 1993 [Refereed][Not invited]
  • T OHTSUKI, Y KATSURA, H MIZUKAMI, Y MATSUURA, F KIMURA, M OHNISHI, N NAGATA, K MOTOYOSHI
    AMERICAN JOURNAL OF HEMATOLOGY 41 (1) 50 - 56 0361-8609 1992/09 [Refereed][Not invited]
     
    A 65-year-old man with marked leukocytosis was admitted for diagnosis and treatment. His peripheral blood leukocyte count was 37,500/mu-l and the leukocytes consisted of mature neutrophil-like cells. A high neutrophil alkaline phosphatase score and a normal bone marrow cell karyotype suggested that the patient had chronic neutrophilic leukemia rather than chronic myeloid leukemia. Several neutrophil functions, such as superoxide production, nitroblue tetrazolium reduction activity, and phagocytosis, were elevated. These data and the morphological features (toxic granules and Dohle bodies) indicated that the patient's neutrophils were in an activated stage.
  • H MIZUKAMI, N SATO
    EXPERIMENTAL HEMATOLOGY 20 (4) 482 - 485 0301-472X 1992/05 [Refereed][Not invited]
     
    Granulocyte colony-stimulating factor (G-CSF) is known to act on the neutrophilic granulocytes from chronic myelogenous leukemia (CML) patients to induce neutrophil alkaline phosphatase (NAP) activity. Gamma-interferon (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been reported to suppress NAP induction with G-CSF. We confirmed that this inhibitory effect of GM-CSF is accompanied by the decrease of the NAP mRNA level. Moreover, we found that the simultaneous addition of retinoic acid completely neutralized this inhibitory effect of GM-CSF. Recovery of the NAP activity brought about by the retinoic acid was also accompanied by the increase of NAP mRNA level. These results indicate that retinoic acid neutralizes the inhibitory effect of GM-CSF on the induction of NAP activity through the change of the NAP mRNA level.
  • F KIMURA, Y TAKEMURA, T OHTSUKI, H MIZUKAMI, S TAKAGI, K YAMAMOTO, N NAGATA, K MOTOYOSHI
    INTERNATIONAL JOURNAL OF HEMATOLOGY 55 (2) 147 - 155 0925-5710 1992/04 [Refereed][Not invited]
     
    To investigate the physiologic role of macrophage colony-stimulating factor (M-CSF) in hematological recovery from bone marrow hypoplasia, we used an enzyme-linked immunosorbent assay to measure serial changes of the serum M-CSF level during 25 intensification chemotherapy courses given to seven patients with acute non-lymphocytic leukemia who were in complete remission. Three M-CSF peaks were observed during therapy: the first peak was during or just after chemotherapy, the second peak was around the leukocyte nadir, and the third peak coincided with a rapid increase in the monocyte count. We could find no significant correlation between the height of the second peak and the time from the initiation of therapy to hematological recovery. On the other hand, there was a significant positive correlation between the height of the second peak and the interval from the last day of chemotherapy to the peak (r = 0.62, p = 0.001), and there was a significant negative correlation between the peak height and the time from the peak until hematological recovery (defined as a neutrophil count of over 500/mu-l (r = -0.63, p = 0.001) and a leukocyte count of over 1,000/mu-l (r = 0.55, p = 0.008). However, we found only a weak correlation between the peak height and monocyte recovery. These data suggest that increased M-CSF levels lead to the stimulation of granulocyte progenitors, and that we can predict the time of neutrophil recovery by monitoring the serum M-CSF level and finding its peak.
  • T OHTSUKI, Y MATSUURA, S SUZU, H MIZUKAMI, M OHNISHI, F KIMURA, N NAGATA, K MOTOYOSHI
    ACTA HAEMATOLOGICA 87 (3) 156 - 159 0001-5792 1992 [Refereed][Not invited]
     
    A 71-year-old woman with leukocytosis was admitted for treatment of malignant lymphoma. During the clinical course, neutrophilia of unknown origin occurred in parallel with the progression of the malignant lymphoma. The supernatant of lymphoma tissue culture contained a high titer of granulocyte colony-stimulating factor (G-CSF), and lymphoma cells were positive when immunohistochemically stained by anti-G-CSF antibody. Western blot analysis and mouse colony assay of the supernatant also confirmed that the lymphoma produced G-CSF.
  • N SATO, H MIZUKAMI, K TANI, S ASANO
    EUROPEAN JOURNAL OF HAEMATOLOGY 46 (2) 107 - 111 0902-4441 1991/02 [Refereed][Not invited]
     
    We examined steady-state levels of mRNA for alkaline phosphatase in neutrophils (NAP) treated with granulocyte colony-stimulating factor (G-CSF). The amount of mRNA for NAP was shown to increase after 6 hours of culture with G-CSF when no increase in NAP activity was yet observed, and the transcript was the greatest after 20-24 h of culture with G-CSF. Treatment of neutrophils with both G-CSF and retinoic acid augmented the amount of mRNA for NAP over the amount obtained by G-CSF alone, which was most marked at 24 h. These results show that both G-CSF-mediated NAP induction and its potentiation by retinoic acid are due to the increased levels of mRNA for NAP.

Books etc

  • 『糖尿病学ー基礎と臨床』
    ()
    西村書店 2007 9784890133550

MISC

Industrial Property Rights

  • 特願2019-19247:組換えアデノ随伴ウイルスを含むマラリアワクチン  2019年/02/06
    吉田 栄人, 伊從 光洋, 吉井 達也, 水上 浩明
  • 特願2017-004198:肝臓ゲノム上の凝固関連因子遺伝子を破壊するためのAAVベクター  2017年/01/17
    大森司, 長尾恭光, 水上浩明, 坂田飛鳥, 坂田洋一, 西村智, 小澤敬也, 村松慎一, 富永眞一, 花園豊

Research Grants & Projects

  • 1. 遺伝子導入に関連する分子基盤とin vivoにおける組織特異性の解明 2. AAV各血清型に由来するベクターの特性の解明 3. 導入遺伝子産物に対する免疫反応の解析とその制御 4. ベクターに対する免疫反応が遺伝子導入に与える影響の解析 5. 血友病・悪性腫瘍・神経疾患などに対する新規治療法の開発
    厚生労働科学研究費補助金
  • Gene transfer
    Health and Labour Sciences Research Grants


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