水上 浩明

Mizukami, H., Okada, T., Ogasawara, Y., Matsushita, T., Urabe, M., Kume, A., and Ozawa, K.: Separate control of Rep and Cap expression utilizing mutant and wild-type loxP sequences and improved packaging system for adeno-associated virus vector production.

Kanazawa, T., Mizukami, H., Okada, T., Hanazono, Y., Kume, A., Nishino, A., Takeuchi, K., Kitamura, K., Ichimura, K., and Ozawa, K.: Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice.

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

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

Muramatsu S, Fujimoto K, Ikeguchi K, Shizuma N, Kawasaki K, Ono F, Shen Y, Wang L, Mizukami H, Kume A, Matsumura M, Nagatsu I, Urano F, Ichinose H, Nagatsu T, Terao K, Nakano I, Ozawa K:Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes

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 neuroreduction and promotes functional recovery in a rat model of Parkinson's disease

Kanazawa T, Mizukami H, Okada T, Hanazono Y, Kume A, Nishino H, Takeuchi K, Kitamura K, Ichimura K, Ozawa K:Suicide Gene Therapy using AAV-HSV<i>tk/</i>ganciclovir in Combination with Irradiation Results in Regeression of Human Head and Neck Cancer Xenografts in Nude Mice

Targeted integration of foreign DNA is ideal for gene therapy particularly when target cells such as hematopoietic cells actively divide and proliferate. Adeno-associated virus (AAV) has been shown to integrate its genome into a defined locus, AAVS1 (19q13.3-qter). The inverted terminal repeat (ITR) and Rep proteins are responsible for this site-specific integration, and a system has been developed that delivers a gene preferentially into AAVS1 by using these components of AAV. We examined whether this system could be: applied to gene transfer into K562 cells, Two rep, expression plasmids were tested, 1 driven by the cytomegalovirus (CMV) promoter (pCMVR78) and the other under the translational control of an internal ribosome entry site (pMGiR78) with mouse mammary tumor virus promoter. K562 cells were cotransfected with a rep plasmid and a plasmid containing a neo gene flanked by the ITRs. G418-resistant clones were isolated and analyzed by Southern blot analysis and fluorescence in situ hybridization (FISH). Southern blot analysis suggested AAVS1-specific integration of the neo gene in 6 (35%) of 17 clones when K562 cells were transfected with pMGiR78 by lipofection. FISH located the neo gene on chromosome 19 in 5 of these 6 clones (29%). Eight (32%) of 25 clones obtained by electroporation with pCMVR78 had the neo gene at AAVS1, according to Southern blot analysis, and 4 of these 8 clones (16%) were positive according to FISH analysis, These results suggest that site-specific integration of foreign DNA can be achieved at a significantly high rate in human hematopoietic cells using the AAV components. Int J Hematol. 2001;73:469-475. (C) 2001 The Japanese Society of Hematology.

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.