Development of AAV (adeno-associated virus) vectors and their application to cancer therapy

2009 Fiscal Year Final Research Report

• Project/Area Number: 17016067
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Jichi Medical University
• Principal Investigator: OZAWA Keiya Jichi Medical University, 医学部, 教授 (30137707)
• Co-Investigator(Kenkyū-buntansha): MIZUKAMI Horoaki 自治医科大学, 医学部, 講師 (20311938) ; URABE Masashi 自治医科大学, 医学部, 講師 (40213516) ; MATSUSHITA Takashi 自治医科大学, 医学部, 助教 (20343444) ; OKADA Takashi 自治医科大学, 医学部, 講師 (00326828) ; TSUKAHARA Tomonri 自治医科大学, 医学部, 助教 (10362120)
• Project Period (FY): 2005 – 2009
• Keywords: 遺伝子 / ウイルス / 癌 / バイオテクノロジー / 遺伝子治療

Research Abstract

A) Development of AAV (adeno-associated virus)-mediated cancer gene therapy :
1) Establishment of AAV vector production system using baculovirus expression vectors : AAV vectors were efficiently produced in insect cells. We developed a column chromatographic method to isolate AAV vector particles from empty capsids, which can be adaptable to large-scale production of AAV vectors. Incorporation of the AAV p5 promoter into a transgene sequence increased the efficiency of AAV vector production.
2) Basic studies of the modification of AAV vectors : Chimeric type 5 AAV vectors with type 2 VP1 had a larger amount of VP 1 in their capsids and transduced target cells in a similar manner with parent type 5 AAV vectors.
3) Basic studies of AAV vector-mediated gene transfer and the regulation of transgene expression : We established standard methods of gene transfer into muscle, liver, adipose tissue and peritoneum. A histone deacetylase inhibitor enhanced AAV vector-mediated transgene expression in tumor cells.
4) Examination of strategies for cancer gene therapy using AAV vectors : We conducted gene therapy experiments for refractory cancers (e.g. hematogenous and/or lymphogenous metastasis, and peritoneal dissemination) and showed therapeutic efficacy in tumor-bearing animals.
B) Research on novel strategies for cancer gene therapy : For the treatment of refractory malignant lymphoma (B-cell non-Hodgkin lymphoma), we conducted experiments to develop a novel reinforced adoptive immuno-gene therapy using T-cells expressing a CAR (chimeric antigen receptor) targeting CD 19. We demonstrated that genetically engineered T-cells efficiently lyzed CD 19positive B-cell lymphoma cells in vitro.

Research Products

• [Journal Article] Generation of adeno-associated virus vector enabling functional expression of oxytocin receptor and fluorescence marker genes using the human eIF4G internal ribosome entry site element. (2009)
  • Author(s): Sato, K., Date, S., Aoyagi, Y., Kasahara, Y., Nawa, A., Mizukami, H., Hidema, S., Ozawa, K., Nishimori, K.
  • Journal Title: Biosci. Biotechnol. Biochem. 73(9) Pages: 2145-2148
  • Peer Reviewed
• [Journal Article] A convenient enzyme-linked immunosorbent assay for rapid screening of anti-adeno-associated virus neutralizing antibodies. (2009)
  • Author(s): Ito, T., Yamamoto, S., Hayashi, T., Kodera, M., Mizukami, H., Ozawa, K., Muramatsu, S.
  • Journal Title: Ann. Clin. Biochem. 46(Pt6) Pages: 508-510
  • Peer Reviewed
• [Journal Article] Scalable purification of adeno-associated virus serotype 1 (AAV1) and AAV8 vectors, using dual ion-exchange adsorptive membranes. (2009)
  • Author(s): Okada, T., Nonaka-Sarukawa, M., Uchibori, R., Kinoshita, K., Hayashita-Kinoh, H., Nitahara-Kasahara, Y., Takeda, S., Ozawa, K.
  • Journal Title: Hum. Gene Ther. 20(9) Pages: 1013-1021
  • Peer Reviewed
• [Journal Article] Retroviral vector-producing mesenchymal stem cells for targeted suicide cancer gene therapy. (2009)
  • Author(s): Uchibori, R., Okada, T., Ito, T., Urabe, M., Mizukami, H., Kume, A., Ozawa, K.
  • Journal Title: J. Gene Med. 11 Pages: 373-381
  • Peer Reviewed
• [Journal Article] Systemic delivery of IL-10 by an AAV vector prevents vascular remodeling and end-organ damage in stroke-prone spontaneously hypertensive rat. (2009)
  • Author(s): Kume, A., Ookawara, S., Ikeda, U., Katayama, Y., Ozawa, K.
  • Journal Title: Gene Ther. 16(3) Pages: 383-91
  • Peer Reviewed
• [Journal Article] Overexpression of PTEN in ovarian cancer cells suppresses i. p. dissemination and extends survival in mice. (2008)
  • Author(s): Takei, Y., Saga, Y., Mizukami, H., Takayama, T., Ohwada, M., Ozawa, K., Suzuki, M.
  • Journal Title: Mol. Cancer. Ther. 7(3) Pages: 704-711
  • Peer Reviewed
• [Journal Article] Adeno-associated virus vector-mediated systemic interleukin-10 expression ameliorates hypertensive organ damage in Dahl salt-sensitive rats. (2008)
  • Author(s): Nonaka-Sarukawa, M., Okada, T., Ito, T., Yamamoto, K., Yoshioka, T., Nomoto, T., Hojo, Y., Shimpo, M., Urabe, M., Mizukami, H., Kume, A., Ikeda, U., Shimada, K., Ozawa, K.
  • Journal Title: J Gene Med. 10(4) Pages: 368-374
  • Peer Reviewed
• [Journal Article] Protection against aminoglycoside-induced ototoxicity by regulated AAV vector-mediated GDNF gene transfer into the cochlea. (2008)
  • Author(s): Liu, Y., Okada, T., Shimazaki, K., Sheykholeslami, K., Nomoto, T., Muramatsu, S., Mizukami, H., Kume, A., Xiao, S., Ichimura, K., Ozawa, K.
  • Journal Title: Mol. Ther. 16(3) Pages: 474-480
  • Peer Reviewed
• [Journal Article] Interleukin-10 expression mediated by an adeno-associated virus vector prevents monocrotaline-induced pulmonary arterial hypertension in rats. (2007)
  • Author(s): Ito, T., Okada, T., Miyashita, H., Nomoto, T., Nonaka-Sarukawa, M., Uchibori, R., Maeda, Y., Urabe, M., Mizukami, H., Kume, A., Takahashi, M., Ikeda, U., Shimada, K., Ozawa, K.
  • Journal Title: Circ. Res. 101 Pages: 734-741
  • Peer Reviewed
• [Journal Article] Adeno-associated virus-mediated prostacyclin synthase expression prevents pulmonary arterial hypertension in rats. (2007)
  • Author(s): Ito, T., Okada, T., Mimuro, J., Miyashita, H., Uchibori, R., Urabe, M., Mizukami, H., Kume, A., Takahashi, M., Ikeda, U., Sakata, Y., Shimada, K., Ozawa, K.
  • Journal Title: Hypertension 50 Pages: 531-536
  • Peer Reviewed
• [Journal Article] Transforming activity of purinergic receptor P2Y, G protein coupled, revealed by retroviral expression screening. (2007)
  • Author(s): Fujiwara, S., Yamashita, Y., Choi, Y.L., Watanabe, H., Kurashina, K., Soda, M., Enomoto, M., Hatanaka, H., Takada, S., Ozawa, K., Mano, H.
  • Journal Title: Leuk. Lymphoma. 48 Pages: 978-986
  • Peer Reviewed
• [Journal Article] Interferon-gamma and NF-kappaB mediate nitric oxide production by mesenchymal stromal cells. (2007)
  • Author(s): Oh, I., Ozaki, K., Sato, K., Meguro, A., Tatara, R., Hatanaka, K., Nagai, T., Muroi, K., Ozawa, K.
  • Journal Title: Biochem. Biophys. Res. Commun. 355 Pages: 956-962
  • Peer Reviewed
• [Journal Article] Prevention of diabetic retinopathy by intraocular soluble Flt-1 gene transfer in a spontaneously diabetic rat model. (2007)
  • Author(s): Ideno, J., Mizukami, H., Kakehashi, A., Saito, Y., Okada, T., Urabe, M., Kume, A., Kuroki, M., Kawakami, M., Ishibashi, S., Ozawa, K.
  • Journal Title: Int. J. Mol. Med. 19 Pages: 75-79
  • Peer Reviewed
• [Journal Article] Nitric oxide plays a critical role in suppression of T cell proliferation by mesenchymal stem cells. (2007)
  • Author(s): Sato, K., Ozaki, K., Oh, I., Meguro, A., Hatanaka, K., Nagai, T., Muroi, K., Ozawa, K.
  • Journal Title: Blood 109 Pages: 228-234
  • Peer Reviewed
• [Journal Article] Suppression of ovarian cancer by muscle-mediated expression of soluble VEGFR-1/Flt-1 using adeno-associated virus serotype 1-derived vector. (2006)
  • Author(s): Takei, Y., Mizukami, H., Saga, Y., Yoshimura, I., Hasumi, Y., Takayama, T., Kohno, T., Matsushita, T., Okada, T., Kume, A., Suzuki, M., Ozawa, K.
  • Journal Title: Int. J. Cancer 120 Pages: 278-284
  • Peer Reviewed
• [Journal Article] Adipose tissue as a novel target for in vivo gene transfer by adeno-associated viral vectors. (2006)
  • Author(s): Mizukami, H., Mimuro, J., Ogura, T., Okada, T., Urabe, M., Kume, A., Sakata, Y., Ozawa, K.
  • Journal Title: Hum. Gene Ther. 17 Pages: 921-928
  • Peer Reviewed
• [Journal Article] Phenotype correction of hemophilia A mice with adeno-associated virus vectors carrying the B domain-deleted canine factor VIII gene. (2006)
  • Author(s): Ishiwata, A., Mimuro, J., Kashiwakura, Y., Niimura, M., Takano, K., Ohmori, T., Madoiwa, S., Mizukami, H., Okada, T., Naka, H., Yoshioka, A., Ozawa, K., Sakata, Y.
  • Journal Title: Thromb. Res. 118 Pages: 627-635
  • Peer Reviewed
• [Journal Article] Utility of intraperitoneal administration as a route of AAV serotype 5 vector-mediated neonatal gene transfer. (2006)
  • Author(s): Ogura, T., Mizukami, H., Mimuro, J., Madoiwa, S., Okada, T., Matsushita, T., Urabe, M., Kume, A., Hamada, H., Yoshikawa, H., Sakata, Y., Ozawa, K.
  • Journal Title: Gene Med. 8 Pages: 990-997
  • Peer Reviewed
• [Journal Article] rAAV-mediated shRNA ameliorated 6 neuropathology in Huntington disease model mouse. Biochem. (2006)
  • Author(s): Machida, Y., Okada, T., Kurosawa, M., Oyama, F., Ozawa, K., Nukina, N.
  • Journal Title: Biophys. Res. Commun. 343 Pages: 190-197
  • Peer Reviewed
• [Journal Article] Removal of empty capsids from type 1 adeno-associated virus vector stocks by anion-exchange chromatography potentiates transgene expression. (2006)
  • Author(s): Urabe, M., Xin, K.Q., Obara, Y., Nakakura, T., Mizukami, H., Kume, A., Okuda, K., Ozawa, K.
  • Journal Title: Mol. Ther. 13 Pages: 823-828
  • Peer Reviewed
• [Journal Article] Scalable generation of high-titer recombinant adeno-associated virus type 5 in insect cells. (2006)
  • Author(s): Urabe, M., Nakakura, T., Xin, K.Q., Obara, Y., Mizukami, H., Kume, A., Kotin, R.M., Ozawa, K.
  • Journal Title: J. Virol. 80 Pages: 1874-1885
  • Peer Reviewed
• [Journal Article] A histone deacetylase inhibitor enhances recombinant adeno-associated virus-mediated gene expression in tumor cells. (2006)
  • Author(s): Okada, T., Uchibori, R., Iwata-Okada, M., Takahashi, M., Nomoto, T., Nonaka-Sarukawa, M., Ito, T., Liu, Y., Mizukami, H., Kume, A., Kobayashi, E., Ozawa, K.
  • Journal Title: Mol. Ther. 13 Pages: 738-746
  • Peer Reviewed
• [Journal Article] Viral-mediated temporally controlled dopamine production in a rat model of Parkinson disease. (2006)
  • Author(s): Li, X.G., Okada, T., Kodera, M., Nara, Y., Takino, N., Muramatsu, C., Ikeguchi, K., Urano, F., Ichinose, H., Metzger, D., Chambon, P., Nakano, I., Ozawa, K., Muramatsu, S.
  • Journal Title: Mol. Ther. 13 Pages: 160-166
  • Peer Reviewed
• [Journal Article] Transforming activity of the lymphotoxin-beta receptor revealed by expression screening. (2005)
  • Author(s): Fujiwara, S., Yamashita, Y., Choin Y.L., Wada, T., Kaneda, R., Takada, S., Maruyama, Y., Ozawa, K., Mano, H.
  • Journal Title: Biochem. Biophys. Res. Commun. 338 Pages: 1256-1262
  • Peer Reviewed
• [Journal Article] Large-scale production of recombinant viruses by use of a large culture vessel with active gassing. (2005)
  • Author(s): Okada, T., Nomoto, T., Yoshioka, T., Nonaka-Sarukawa, M., Ito, T., Ogura, T., Iwata-Okada, M., Uchibori, R., Shimazaki, K., Mizukami, H., Kume, A., Ozawa, K.
  • Journal Title: Hum. Gene Ther. 16 Pages: 1212-1218
  • Peer Reviewed
• [Journal Article] Specific and efficient transduction of cochlear inner hair cells with recombinant adeno-associated virus type 3 vector. (2005)
  • Author(s): Liu, Y., Okada, T., Sheykholeslami, K., Shimazaki, K., Nomoto, T., Muramatsu, S., Kanazawa, T., Takeuchi, K., Ajalli, R., Mizukami, H., Kume, A., Ichimura, K., Ozawa, K.
  • Journal Title: Mol. Ther. 12 Pages: 725-733
  • Peer Reviewed
• [Journal Article] Repair of articular cartilage defect by autologous transplantation of basic fibroblast growth factor gene-transduced chondrocytes with adeno-associated virus vector. (2005)
  • Author(s): Yokoo, N., Saito, T., Uesugi, M., Kobayashi, N., Xin, K.Q., Okuda, K., Mizukami, H., Ozawa, K., Koshino, T.
  • Journal Title: Arthritis Rheum. 52 Pages: 164-170
  • Peer Reviewed
• [Journal Article] Efficient and stable Sendai virus-mediated gene transfer into primate embryonic stem cells with pluripotency preserved. (2005)
  • Author(s): Sasaki, K., Inoue, M., Shibata, H., Ueda, Y., Muramatsu, S., Okada, T., Hasegawa, M., Ozawa, K., Hanazono, Y.
  • Journal Title: Gene Ther. 12 Pages: 203-210
  • Peer Reviewed