Gene therapy for mitochondrial disease by delivering restriction endonucleaseSmaI into mitochondria |
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Authors: | Director Masashi Tanaka MD PhD Harm-Jan Borgeld Jin Zhang Shin-ichi Muramatsu Jian-Sheng Gong Makoto Yoneda Wakako Maruyama Makoto Naoi Tohru Ibi Ko Sahashi Masayo Shamoto Noriyuki Fuku Miyuki Kurata Yoshiji Yamada Kumi Nishizawa Yukihiro Akao Nobuko Ohishi Shigeaki Miyabayashi Hiraku Umemoto Tatsuo Muramatsu Koichi Furukawa Akihiko Kikuchi Imaharu Nakano Keiya Ozawa Kunio Yagi |
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Institution: | 1. Department of Gene Therapy, Gifu International Institute of Biotechnology, Yagi Memorial Park, 505-0116, Mitake, Gifu, Japan 2. Institute of Applied Biochemistry, Mitake 3. Department of Neurology, Jichi Medical School, Tochigi 4. Second Department of Internal Medicine, Fukui Medical University, Fukui 5. Laboratory of Biochemistry and Metabolism, Department of Basic Gerontology, National Institute for Longevity Sciences, Obu 6. Neurology Section, Department of Internal Medicine, Aichi Medical University, Aichi 7. Japan Science and Technology Corporation, Tokyo 8. Department of Pediatrics, Sendai National Hospital, Sendai 9. Department of Applied Genetics and Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 10. Department of Biochemistry,Graduate School of Medical Sciences, Nagoya University, Japan 11. Department of Medical Mycology, Graduate School of Medical Sciences, Nagoya University, Nagoya 12. Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan
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Abstract: | The restriction endonucleaseSmaI has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993TG mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F0F1-ATPase. Our ultimate goal is to applySmaI to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA) coexists with the wild-type mtDNA (heteroplasmy), and because only the mutant mtDNA, but not the wild-type mtDNA, is selectively restricted by the enzyme. For this purpose, we transiently expressed theSmaI gene fused to a mitochondrial targeting sequence in cybrids carrying the mutant mtDNA. Here, we demonstrate that mitochondria targeted by theSmaI enzyme showed specific elimination of the mutant mtDNA. This elimination was followed with repopulation by the wild-type mtDNA, resulting in restoration of both the normal intracellular ATP level and normal mitochondrial membrane potential. Furthermore, in vivo electroporation of the plasmids expressing mitochondrion-targetedEcoRI induced a decrease in cytochromec oxidase activity in hamster skeletal muscles while causing no degenerative changes in nuclei. Delivery of restriction enzymes into mitochondria is a novel strategy for gene therapy of a special form of mitochondrial diseases. |
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Keywords: | Mitochondrial disease Leigh's disease Neurogenic muscle weakness ataxia and retinitis pigmentosa Gene therapy Restriction endonuclease |
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