Optimization of gene therapy approaches for hemophilia

• Project/Area Number: 15591022
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Hematology
• Research Institution: Jichi Medical School
• Principal Investigator: MIZUKAMI Hiroaki Jichi Medical School, Assistant Professor, 医学部, 講師 (20311938)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥3,000,000 (Direct Cost: ¥3,000,000); Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000); Fiscal Year 2003: ¥1,600,000 (Direct Cost: ¥1,600,000)
• Keywords: AAV Vectors / serotypes / hemophilia B / hemophilia A / coagulation factor IX / coagulation factor VIII / immune response / adipose tissue / 抗体

Research Abstract

The utility and tissue specificity ofg AAV serotypes were evaluated. For the muscle and liver, AAV1 and AAV8 showed the highest expression. Therapeutic levels of human coagulation factor IX was demonstrated for more than one year by muscle mediated gene transfer. In addition to these tissues, adipose tissue was examined as a target of gene transfer ; it is one of the most abundant tissue in the body, and are designed to secrete various proteins physiologically, making it suitable to supply transgene product into systemic circulation. Of note, the transduced tissue can be safely removed without any sequelae in case of unexpected events. However, efficient transduction of adipose tissue has not been feasible by conventional methods. In order to develop a practical method, we tested the enhancement effect of the excipients upon gene transfer. At first, AAV vectors encoding LacZ were administered into adipose tissue of Db/Db mice. Widespread beta-Gal expression was observed when a bio-comp atible surfactant was included in the vector solution. To validate the efficacy of this method, vectors encoding mouse erythropoietin (Epo) were utilized. Two weeks following vector injection, significant concentrations of plasma Epo were observed and the levels were almost comparable to that of muscle- or liver- mediated gene transfer. Plasma Epo concentrations kept similar levels during the observation period thereafter. Increased blood hemoglobin levels warrant biological activity of adipocyte-derived Epo. Efficient expression was also confirmed by both immunofluorescence staining of the adipose tissue using anti-Epo antibody and RT-PCR. In addition, following complete removal of transduced adipose tissue, increased plasma Epo concentrations returned normal, suggesting that other tissues were not transduced by this method. Although the precise mechanisms of this phenomenon are yet to be analyzed, our method leads to efficient gene transfer into adipocytes, adding a novel choice for the supplemental gene therapy approaches.

Research Products

• [Journal Article] Overexpression of a hybrid gene consisting of the amino-terminal fragment of urokinase and carboxyl-terminal domain of bikunin suppresses invasion and migration of human ovarian cancer cells in vitro. (2004)
  • Author(s): Mochizuki S, et al.
  • Journal Title: Gene Ther and Mol Biol 8 Pages: 9-18
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Long-term correction of hyperphenylalaninemia by AAV-mediated gene transfer leads to behavioral recovery in phenylketonuria mice. (2004)
  • Author(s): Mochizuki S, et al.
  • Journal Title: Gene Ther 11 Pages: 1081-1086
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Adeno-associated virus vector-mediated interleukin-10 gene transfer inhibits atherosclerosis in apolipoprotein E-deficient mice. (2004)
  • Author(s): Yoshioka T, et al.
  • Journal Title: Gene Ther 11 Pages: 1772-1779
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Sustained transgene expression by human blood derived CD34(+) cells transduced with simian immuno-deficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID mice. (2004)
  • Author(s): Kikuchi J, et al.
  • Journal Title: J Gene Med 6 Pages: 1049-1060
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Specific detection of human coagulation factor IX in cynomolgus macaques. (2004)
  • Author(s): Mimuro J, et al.
  • Journal Title: J Thromb Haemost 2 Pages: 275-280
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Separate control of Rep and Cap expression utilizing mutant and wild-type loxP sequences and improved packaging system for adeno-associated virus vector production. (2004)
  • Author(s): Mizukami H, et al.
  • Journal Title: Mol Biotech 27 Pages: 7-14
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Topoisomerase inhibitors enhance the cytocidal effect of AAV-HSVtk/ganciclovir on head and neck cancer cells. (2004)
  • Author(s): Kanazawa T, et al.
  • Journal Title: Int J Oncol 25 Pages: 729-735
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Presynaptic localization of neprilysin contributes to efficient clearance of amyloid-beta peptide in mouse brain. (2004)
  • Author(s): Iwata N, et al.
  • Journal Title: J.Neurosci 24 Pages: 991-998
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Long-term correction of hyperphenylalaninemia by AAV-mediated gene transfer leads to behavioral recovery in phenylketonuria mice. (2004)
  • Author(s): Mochizuki, S., Mizukami, H., Ogura, T., Kure, S., Ichinohe, A., Kojima, K., Matsubara, Y., Kobayashi, E., Okada, T., Hoshika, A., Ozawa, K., Kume, A.
  • Journal Title: Gene Ther 11 Pages: 1081-1086
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Adeno-Associated Virus(AAV) vector-mediated liver- and muscle-directed transgene expression using various kinds of promoters and serotypes. (2004)
  • Author(s): Mochizuki, S., Mizukami, H., Kume, A., Muramatsu, S., Takeuchi, K., Matsushita, T., Okada, T., Kobayashi, E., Hoshika, A., Ozawa K.
  • Journal Title: Gene Ther and Mol Biol 8 Pages: 9-18
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Adeno-associated virus vector-mediated interleukin-10 gene transfer inhibits atherosclerosis in apolipoprotein E-deficient mice. (2004)
  • Author(s): Yoshioka, T., Okada, T., Maeda, Y., Ikeda, U., Shimpo, M., Nomoto, T., Takeuchi, K., Nonaka-Sarukawa, M., Ito, T., Takahashi, M., Matsushita, T., Mizukami, H., Hanazono, Y., Kume, A., Ookawara, S., Kawano, M., Ishibashi, S., Shimada, K., Ozawa, K.
  • Journal Title: Gene Ther 11 Pages: 1772-1779
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Sustained transgene expression by human cord blood derived CD34(+) cells transduced with simian immunodeficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID mice. (2004)
  • Author(s): Kikuchi, J., Mimuro, J., Ogata, K., Tabata, T., Ueda, Y., Ishiwata, A., Kimura, K., Takano, K., Madoiwa, S., Mizukami H., Hanazono, Y., Kume, A., Hasegawa, M., Ozawa, K., Sakata, Y.
  • Journal Title: J Gene Med 6 Pages: 1049-1060
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Specific detection of human coagulation factor IX in cynomolgus macaques. (2004)
  • Author(s): Mimuro, J., Mizukami, H., Ono, F., Madoiwa, S., Terao, K., Yoshioka, A., Ozawa, K., Sakata, Y.
  • Journal Title: J Thromb Haemost 2 Pages: 275-280
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Separate control of Rep and Cap expression utilizing mutant and wild-type loxP sequences and improved packaging system for adeno-associated virus vector production. (2004)
  • Author(s): Mizukami.H., Okada, T., Ogasawara, Y., Matsushita, T., Urabe, M., Kume, A., Ozawa, K.
  • Journal Title: Mol Biotechnol 27 Pages: 7-14
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Topoisomerase inhibitors enhance the cytocidal effect of AAV-HSVtk/ganciclovir on head and neck cancer cells. (2004)
  • Author(s): Kanazawa, T., Mizukami, H., Nishino, H., Okada, T., Hanazono, Y., Kume, A., Kitamura, K., Ichimura, K., Ozawa, K.
  • Journal Title: Int J Oncol 25 Pages: 729-735
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Presynaptic localization of neprilysin contributes to efficient clearance of amyloid-beta peptide in mouse brain. (2004)
  • Author(s): Iwata, N., Mizukami, H., Shirotani, K., Takaki, Y., Muramatsu, S., Lu, B., Gerard, N.P., Gerard, C., Ozawa, K., Saido, T.C.
  • Journal Title: J.Neurosci 24 Pages: 991-998
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Adeno-Associated Virus (AAV) vector-mediated liver- and muscle-directed transgene expression using various kinds of promoters and serotypes. (2004)
  • Author(s): Mochizuki S, et al.
  • Journal Title: Gene Ther and Mol Biol 8・1 Pages: 9-18
• [Journal Article] Long-term correction of hyperphenylalaninemia by AAV-mediated gene transfer leads to behavioral recovery in phenylketonuria mice. (2004)
  • Author(s): Mochizuki S, et al.
  • Journal Title: Gene Ther 11・13 Pages: 1081-6
• [Journal Article] Adeno-associated virus vector-mediated interleukin-10 gene transfer inhibits atherosclerosis in apolipoprotein E-deficient mice. (2004)
  • Author(s): Yoshioka T, et al.
  • Journal Title: Gene Ther 11・24 Pages: 1772-9
• [Journal Article] Sustained transgene expression by human cord blood derived CD34(+) cells transduced with simian immunodeficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID mice. (2004)
  • Author(s): Kikuchi J, et al.
  • Journal Title: J Gene Med 6・10 Pages: 1049-60
• [Journal Article] Specific detection of human coagulation factor IX in cynomolgus macaques. (2004)
  • Author(s): Mimuro J, et al.
  • Journal Title: J Thromb Haemost 2・2 Pages: 275-80
• [Journal Article] Separate control of Rep and Cap expression utilizing mutant and wild-type loxP sequences and improved packaging system for adeno-associated virus vector production. (2004)
  • Author(s): Mizukami H, et al.
  • Journal Title: Mol Biotech 27・1 Pages: 7-14
• [Journal Article] Persistent phenotypic correction of central diabetes insipidus using adeno-associated virus vector-expressing arginine-vasopressin in brattleboro rats. (2003)
  • Author(s): Ideno J, et al.
  • Journal Title: Mol Ther 8 Pages: 895-902
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Suppression of cell migration in ovarian cancer cells mediated by PTEN overexpression. (2003)
  • Author(s): Saga Y, et al.
  • Journal Title: Int J Oncol 23 Pages: 1109-1113
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Interleukin-10-mediated inhibition of angiogenesis and tumor growth in mice bearing VEGF-producing ovarian cancer. (2003)
  • Author(s): Kohno T, et al.
  • Journal Title: Cancer Res 63 Pages: 5091-5094
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice. (2003)
  • Author(s): Kanazawa T, et al.
  • Journal Title: Gene Ther 10 Pages: 51-58
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Persistent phenotypic correction of central diabetes insipidus using adeno-associated virus vector-expressing arginine-vasopressin in brattleboro rats. (2003)
  • Author(s): Ideno, J., Mizukami, H., Honda, K., Okada, T., Hanazono, Y., Kume, A., Saito, T., Ishibashi, S., Ozawa, K.
  • Journal Title: Mol Ther 8 Pages: 895-902
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Suppression of cell migration in ovarian cancer cells mediated by PTEN overexpression. (2003)
  • Author(s): Saga, Y., Mizukami, H., Takei, Y., Ozawa, K., Suzuki, M.
  • Journal Title: Int J Oncol 23 Pages: 1109-1113
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Interleukin-10-mediated inhibition of angiogenesis and tumor growth in mice bearing VEGF-producing ovarian cancer. (2003)
  • Author(s): Kohno, T., Mizukami, H., Suzuki, M., Saga, Y., Takei, Y., Shimpo, M., Matsushita, T., Okada, T., Hanazono, Y., Kume, A., Sato, I., Ozawa, K.
  • Journal Title: Cancer Res 63 Pages: 5091-5094
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice. (2003)
  • Author(s): Kanazawa, T., Mizukami, H., Okada, T., Hanazono, Y., Kume, A., Nishino, A., Takeuchi, K., Kitamura, K., Ichimura, K., Ozawa, K.
  • Journal Title: Gene Ther 10 Pages: 51-58
  • Description: 「研究成果報告書概要(欧文)」より
• [Patent(Industrial Property Rights)] Method of treating amino acid metabolic disorders using recombinant adeno-associated virus virions (2003)
  • Inventor(s): Keiya Ozawa, Hiroaki Mizukami, Akihiro Kume
  • Industrial Property Rights Holder: Keiya Ozawa, Hiroaki Mizukami, Akihiro Kume
  • Filing Date: 2003-04-16
  • Description: 「研究成果報告書概要(和文)」より
• [Patent(Industrial Property Rights)] Methods for treating and preventing vascular disease (2003)
  • Inventor(s): 小澤 敬也, 野本 達也, 岡田 尚巳, 水上 浩明
  • Industrial Property Rights Holder: 小澤 敬也, 野本 達也, 岡田 尚巳, 水上 浩明
  • Filing Date: 2003-10-20
  • Description: 「研究成果報告書概要(和文)」より
• [Patent(Industrial Property Rights)] Method of treating amino acid meta- bolic disorders using recombinant adeno-associated virus virions (2003)
  • Inventor(s): Keiya Ozawa, Hiroaki Mizukami, Akihiro Kume
  • Industrial Property Rights Holder: Keiya Ozawa, Hiroaki Mizukami, Akihiro Kume
  • Filing Date: 2003-04-16
• [Publications] Mizukami H, Okada T, et al.: "Separate control of Rep and Cap expression utilizing mutant and wild-type loxP sequence and improved packaging system for adeno-associated virus vector production."Mol Biotechnol. (in press).
• [Publications] Mimuro J, Mizukami H, et al.: "Specific detection of human coagulation factor IX in cynomolgus macaques."J.Thromb Haemost. (in press).
• [Publications] Takeda S, Takahashi M, et al.: "Successful Gene Transfer using Adeno-Associated Virus Vectors into the Kidney : Comparison among Adeno-Associated Virus Serotype 1 to 5 Vectors In Vitro and In Vivo."Nephron. (in press).
• [Publications] Iwata N, Mizukami H, et al.: "Presynaptic localization of neprilysin contributes to efficient clearance of amyloid-beta peptide in mouse brain."J.Neurosci. 24(4). 991-998 (2004)
• [Publications] Ideno J, Mizukami H, et al.: "Persistent phenotypic correction of central diabetes insipidus using adeno-associated virus vector-expressing arginine-vasopressin in brattleboro rats."Mol Ther. 8(6). 895-902 (2003)
• [Publications] Kohno T, Mizukarni H, et al.: "Interleukin-10-mediated inhibition of angiogenesis and tumor growth in mice bearing VEGI producing ovarian cancer."Cancer Res. 63(16). 5091-5094 (2003)