小澤 敬也

Tanaka, M., Borgeld, H. J., Zhang, J., Muramatsu, S., Gong, J. S., Yoneda, M., Maruyama, W., Naoi, M., Ibi, T., Sahashi, K., Shamoto, M., Fuku, N., Kurata, M., Yamada, Y., Nishizawa, K., Akao, Y., Ohishi, N., Miyabayashyi, S., Umemoto, H., Muramatus, T., Furukawa, K., Kikuchi, A., Nakano, I., Ozawa, K., and Yagi, K.: Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria

Xin, K. Q., Ooki, T., Mizukami, H., Hamajima, K., Okudela, K., Hashimoto, K., Kojima, Y., Jounai, N., Kumamoto, Y., Sasaki, S., Klinman, D., Ozawa, K., and Okuda, K.: Oral administration of recombinant adeno-associated virus elicits human immunodeficiency virus-specific immune responses

Hanazono, Y. Terao, K., Shibata, H., Nagashima, T., Ageyama, N., Asano, T., Ueda, Y., Katao, I., Kume, A., Hasegawa, M., and Ozawa, K.: Introduction of the green fluorescent protein gene into hematopoietic stem cells results in prolonged discrepancy of in vivo transduction levels between bone marrow progenitors and peripheral blood cells in nonhuman primates

Wang, L.-J., Lu, Y.-Y., Muramatsu, S., Ikeguchi, K., Fujimoto, K., Okada, T., Mizukami, H., Matsushita, T., Hanazono, Y., Kume, A., Nagatsu, Tl, Ozawa, K., and Nakano, I.: Neuroprotective effects of glial cell line-derived neurotrophic factor meddiated by an adeno-associated virus vector in a transgenic animal model of amyotrophic lateral sclerosis

Mishima, Y., Terui, Y., Mishima, Y., Katsuyama, M., Mori, M., Tomizuka, H., Takizawa, T., Miyazato, A., Ueda, M., Yamada, M., Hayasawa, H., Mizunuma, N., Ishizaka, Y., Ikeda, K., Kato, T., Ozawa, K., and Hatake, K.: New human myelodysplastic cell line, TER-3: G-CSF specific downregulation of Ca&lt;sup&gt;2+&lt;/sup&gt;/calmodulin-dependent protein kinase Ⅳ<br /> <br />

Wang, L., Muramatsu, S., Lu, Y., Ikeguchi, K., Fujimoto, K., Okada, T., Mizukami, H., Hanazono, Y., Kume, A., Urano, F., Ichinose, H., Nagatsu, T., Nakano, I., and Ozawa, K.: Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson&#039;s disease

Shimpo, M., Ikeda, U., Maeda, Y., Takahashi, M., Miyashita, H., Mizukami, H., Urabe, M., Kume, A., Takizawa, T., Shibuya, M., Ozawa, K., and Shimada, K.: AAV-mediated VEGF gene transfer into skeletal muscle stimulates angiogenesis and improves blood flow in a rat hindlimb ischemia model<br /> <br />

Muramatsu, S., Fujimoto, K., Ikeguchi, K., Shizuma, N., Kawasaki, K., Ono, F., Shen, Y., Wang, L., Mizukami, H., Kume, K., Matsumura, M., Nagatsu, N., Urano, F., Ichinose, H., Nagatsu, T., Terao, T., Nakano, I., and Ozawa, K.: Behavioral recovery in a primate model of Parkinson&#039;s disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes<br /> <br />

Kawada, T., Nakazawa, M., Nakauchi, S., Yamazaki, K., Shimamoto, R., Urabe, M., Nakata, J., Hemmi, C., Masui, F., Nakajima, T., Suzuki, J. I., Monahan, J., Sato, H., Masaki, T., Ozawa, K., and Toyo-Oka, T.; Rescue ofhereditary from of dilated cardiomyopathy by rAAV-mediated somatic gene therapy: Amelioration of morphological findings, carcolemmal permeability, cardiac performances, and the prognosis of TO-2 hamster

The murine sak gene encodes a putative serine-threonine kinase which is homologous to the members of the Plk/Polo family. Although Sak protein is presumed to be involved in cell growth mechanism, efforts have failed to demonstrate its kinase activity. Little has been, therefore, elucidated how Sak is regulated and how Sak contributes to cell proliferation. Tec is a cytoplasmic protein-tyrosine kinase (PTK) which becomes activated by the stimulation of cytokine receptors, lymphocyte surface antigens, heterotrimeric G protein-linked receptors, and integrins. To clarify the in vivo function of Tec, we have tried to isolate the second messengers of Tec by using the yeast two-hybrid screening. One of such Tec-binding proteins turned out to be Sak. In human kidney 293 cells, Sak became tyrosine-phosphorylated by Tec, and the serine-threonine kinase activity of Sak was detected only under the presence of Tec, suggesting Sak to be an effector molecule of Tec. In addition, Tec activity efficiently protects Sak from the "PEST" sequence-dependent proteolysis. Internal deletion of the PEST sequences led to the stabilization of Sak proteins, and expression of these mutants acted suppressive to cell growth. Our data collectively supports a novel role of Sak acting in the PTK-mediated signaling pathway.

Myelodysplastic syndrome (MDS) is a slowly progressing hematologic malignancy associated with a poor outcome. Despite the relatively high incidence of MDS in the elderly, differentiation of MDS from de novo acute myeloid leukemia (AML) still remains problematic. Identification of genes expressed in an MDS-specific manner would allow the molecular diagnosis of MDS. Toward this goal, AC133 surface marker-positive hematopoietic stem cell (HSC)-like fractions have been collected from a variety of leukemias in a large-scale and long-term genomics project, referred to as "Blast Bank," and transcriptome of these purified blasts from the patients with MDS were then compared with those from AML through the use of oligonucleotide microarrays. A number of genes were shown to be expressed in a disease-specific manner either to MDS or AML. Among the former found was the gene encoding the protein Delta-like (DIk) that is distantly related to the Delta-Notch family of signaling proteins. Because overexpression of DIk may play a role in the pathogenesis of MDS, the disease specificity of DIk expression was tested by a quantitative "realtime" polymerase chain reaction analysis. Examination of the Blast Bank samples from 22 patients with MDS, 31 with AML, and 8 with chronic myeloid leukemia confirmed the highly selective expression of the Dlk gene in the individuals with MDS. Dlk could be the first candidate molecule to differentiate MDS from AML. The proposal is made that microarray analysis with the Blast Bank samples is an efficient approach to extract transcriptome data of clinical relevance for a wide range of hematologic disorders. © 2001 by The American Society of Hematology.

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.

Mimuro, J., Muramatsu, H., Hakamada, Y., Mori, K., Kikuchi, J., Urabe, Ml, Madoiwa, S., Ozawa, K., and Sakata, Y.: Recombinant adeno-associated virus vector-transduced vascular endothelial cells express the thrombomodulin transgene under the regulation of enhanced plasminogen activator inhibitor-1 promoter

Ohmine, K., Ota, J., Ueda, M., Ueno, S., Yoshida, K., Yamashita, Y., Kirito, K., Imagawa, S., Nakamura, Y., Saito, K., Akutsu, M., Mitani, K., Kano, Y., Komatsu, N., Ozawa, k., and Mano H.: Characterization of stage progression in chronic myeloid leukemia by DNA microarray with purified hematopoietic stem cells

Mori, m., Hatake, K., Tanaka, M., Takatoku, M, Matsumoto, Y., Uchida, M., Kametaka, M., Nagai, T., Terui, Y., Tomizuka, H., Muroi, K., and Ozawa, K.: CAM-cytarabine, aclarubicin plus macrophage colony-stimulating factor in the treatment of acute myelogenous leukemia with trilineage dysplasia: usefulenss of in vitro apoptosis in leukemic cells

Okada, T., Mizukami, h., Urabe, M., Nomoto, T., Matsushita, T., Hanazono, Y., Jume, A., Tobita, K., and Ozawa, K.: Development and characterization of an antisense-mediated regulation system for adeno-associated virus vector production with introduction of Cre recombinase

Mimuro, J., Muramatsu, H., Hakamada, Y., Mori, K., Kikuchi, J., Urabe, Ml, Madoiwa, S., Ozawa, K., and Sakata, Y.: Recombinant adeno-associated virus vector-transduced vascular endothelial cells express the thrombomodulin transgene under the regulation of enhanced plasminogen activator inhibitor-1 promoter

Ohmine, K., Ota, J., Ueda, M., Ueno, S., Yoshida, K., Yamashita, Y., Kirito, K., Imagawa, S., Nakamura, Y., Saito, K., Akutsu, M., Mitani, K., Kano, Y., Komatsu, N., Ozawa, k., and Mano H.: Characterization of stage progression in chronic myeloid leukemia by DNA microarray with purified hematopoietic stem cells<br /> <br />

Mori, m., Hatake, K., Tanaka, M., Takatoku, M, Matsumoto, Y., Uchida, M., Kametaka, M., Nagai, T., Terui, Y., Tomizuka, H., Muroi, K., and Ozawa, K.: CAM-cytarabine, aclarubicin plus macrophage colony-stimulating factor in the treatment of acute myelogenous leukemia with trilineage dysplasia: usefulenss of in vitro apoptosis in leukemic cells

Okada, T., Mizukami, h., Urabe, M., Nomoto, T., Matsushita, T., Hanazono, Y., Jume, A., Tobita, K., and Ozawa, K.: Development and characterization of an antisense-mediated regulation system for adeno-associated virus vector production with introduction of Cre recombinase

Hydrogen peroxide (H2O2) has previously been shown to inhibit the DNA binding activity of hypoxia inducible factor-1 (HIF-1), the accumulation of HIF-1α protein and erythropoietin (Epo) gene expression. Epo gene expression has been previously shown to be down-regulated through a GATA binding site at its promoter region. In this study, the effect of H2O2 on Epo gene expression under hypoxic conditions through a GATA transcription factor was investigated. Hypoxic induction was found to be inhibited upon the addition of H2O2, and this effect could be reversed through the addition of catalase. Hypoxic induction was found to be suppressed by co-transfection with a human GATA-2 cDNA expression plasmid. Transfection of Hep3B cells with a reporter gene bearing a mutation at the promoter GATA binding site was found to be only mildly affected by the addition of H2O2. Electrophoretic gel mobility shift assays (EMSAs), using the Epo promoter GATA site as a probe and the GATA-2 protein extracted from Hep3B cells, showed that addition of H2O2 enhanced the binding of GATA-2 while addition of catalase inhibited this binding. From these results, we conclude that H2O2 increases the binding activity of GATA-2 in a specific manner, thereby suppressing the activity of the Epo promoter and thus inhibiting Epo gene expression. © 2001 Wiley-Liss. Inc.

Two distinct signaling pathways regulate the survival of interleukin-3 (IL-3)-dependent hematopoietic progenitors. One originates from the membrane-proximal portion of the cytoplasmic domain of the IL-3 receptor (βc chain), which is shared by IL-3 and granulocyte-macrophage colony-stimulating factor and is involved in the regulation of Bcl-xL through activation of STAT5. The other pathway emanates from the distal region of the βc chain and overlaps with downstream signals from constitutively active Ras proteins. Although the latter pathway is indispensable for cell survival, its downstream targets remain largely undefined. Here we show that the expression of Bim, a member of the BH3-only subfamily of cell death activators, is downregulated by IL-3 signaling through either of two major Ras pathways: Raf/mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/mammalian target of rapamycin. Akt/phosphokinase B does not appear to play a significant role in this regulatory cascade. Bim downregulation has important implications for cell survival, since enforced expression of this death activator at levels equivalent to those induced by cytokine withdrawal led to apoptosis even in the presence of IL-3. We conclude that Bim is a pivotal molecule in cytokine regulation of hematopoietic cell survival.

Hematopoietic stem cells (HSCs) are desirable targets for gene therapy because of their self-renewal and multi-lineage differentiation abilities. Retroviral vectors are extensively used for HSC gene therapy. However, the initial human trials of HSC gene marking and therapy showed that the gene transfer efficiency into human HSCs with retroviral vectors was very low in contrast to the much higher efficiency observed in routine experiments. The more quiescent nature of human HSCs and the lower density of retroviral receptors on them hindered the efficient gene transfer with retroviral vectors. Since nonhuman primates have marked similarity to humans in all aspects including the HSC biology, their models are considered to be important to evaluate and improve gene transfer into human HSCs. Using these models, clinically relevant levels (around 10% or even more) of gene-modified cells in peripheral blood have recently been achieved after gene transfer into HSCs and their autologous transplantation. This has been made possible by improving ex vivo transduction conditions such as introduction of Flt-3 ligand and specific fibronectin fragment (CH-296) into ex vivo culture during transduction, and the use of retroviral vectors pseudotyped with the gibbon ape leukemia virus or feline endogenous retrovirus envelope. Other strategies including the use of lentiviral vectors and in vivo selective expansion of gene-modified cells with the drug resistance gene or selective amplifier gene (also designated the molecular growth switch) are now being tested to further increase the fraction of gene-modified cells using nonhuman primate models. In addition to the high gene transfer efficiency, high-level and long-term expression of transgenes in human HSCs and their progeny is also required for effective HSC gene therapy. For this purpose, other backbones of retroviral vectors such as the murine stem cell virus and cis-DNA elements, such as the β-globin locus control region and the chromatin insulator, also need to be tested in nonhuman primate models. Nonhuman primate studies will continue to provide an important framework for human HSC gene therapy. Well-designed nonhuman primate studies will also offer unique insights into the HSCs, immune system, and transplantation biology characteristic of large animals.

Peritoneal dissemination is the most frequent progression pathway of ovarian cancer and is therefore a key step to improve the prognosis. NK4, a large part of the α-chain of hepatocyte growth factor, is known to inhibit cancer cell migration. To characterize the function of NK4 and investigate its potential role in gene therapy of ovarian cancer, we introduced NK4 cDNA to an ovarian cancer cell line HRA and investigated its effects both in vitro and in vivo. HRA cells were transfected with either NK4 or luciferase-expression plasmids. After selection, NK4-expressing HRA cells (HRA/NK4) and the control cells (HRA/LUC) were obtained. NK4 was detected in the culture supernatant of HRA/NK4 by Western analysis. Migration capabilities of the cells were evaluated in vitro by scratch wound healing assay. The number of migrated cells was significantly smaller in the HRA/NK4 cultures than that in the control cultures (HRA or HRA/LUC). Also, the culture supernatant of HRA/NK4 significantly suppressed migration of control cells. This suppressive effect was observed when NK4-expressing cells were mixed with control cells at the ratio of 25% or more. In the in vivo experiments, HRA transfectants were injected intraperitoneally. The number of intraperitoneal tumors of HRA/NK4 was much smaller than that of control. In mice injected with HRA/NK4, ascites formation was suppressed and the survival was significantly prolonged. These findings suggest that NK4-mediated gene therapy can improve the prognosis of ovarian cancer by suppressing peritoneal dissemination.

A recently reported system for recombinant adeno-associated virus (rAAV) production does not require infection of a helper virus and depends on the transfection with a huge amount of three plasmids: AAV-vector, AAV-helper, and adenovirus-helper plasmids. Toward simplifying rAAV production, as a first step, we tested the use of the rAAV itself instead of the AAV-vector plasmid as a source of rAAV DNA and determined the optimal timing of infection and dose of the input rAAV. When 293 cells were infected just after transfection with 100 particles/cell of rAAV, irrespective of the purity, CsCl-purified or crude, up to 2000 particles/cell of rAAV were produced (9- to 20-fold self-amplification), a yield comparable to that obtained by an adenovirus-free transfection. These results indicate that infection of rAAV can greatly reduce the amount of plasmid DNA for a large-scale transfection. This strategy will also be useful when applied to packaging cell lines inducibly expressing Rep and Cap proteins. (C) 2000 Academic Press.

A variety of growth factor receptors induce the tyrosine phosphorylation of a nonreceptor protein-tyrosine kinase Tec as well as that of a Tec-binding protein of 62 kDa. Given the similarity in properties between this 62-kDa protein and p62(Dok-1), the possibility that these two proteins are identical was investigated. Overexpression of a constitutively active form of Tec in a pro-B cell line induced the hyperphosphorylation of endogenous Dok-1. Tec also associated with Dok-1 in a phosphorylation-dependent manner in 293 cells. Tec mediated marked phosphorylation of Dok-1 both in vivo and in vitro, and this effect required both the Tec homology and Src homology 2 domains of Tec in addition to its kinase activity. Expression of Dok-1 in 293 cells induced inhibition of Ras activity, suggesting that Dok-1 is a negative regulator of Ras. In the immature B cell line Ramos, cross-linking of the B cell antigen receptor (BCR) resulted in tyrosine phosphorylation of Dok-1, and this effect was markedly inhibited by expression of dominant negative mutants of Tec. Furthermore, overexpression of Dok-1 inhibited activation of the c-fos pro-rooter induced by stimulation of the BCR. These results suggest that Tec is an important mediator of signaling from the BCR to Dok-1.

Parkinson's disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxy-dopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.