title: Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria.
abstract: The restriction endonuclease SmaI has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993T-->G mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F(0)F(1)-ATPase. Our ultimate goal is to apply SmaI 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 the SmaI gene fused to a mitochondrial targeting sequence in cybrids carrying the mutant mtDNA. Here, we demonstrate that mitochondria targeted by the SmaI 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-targeted EcoRI induced a decrease in cytochrome c 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.
authors.affiliation: Department of Gene Therapy, Gifu International Institute of Biotechnology, Mitake, Japan. mtanaka@giib.or.jp
authors.forename: Masashi
authors.initials: M
authors.lastname: Tanaka
authors.forename: Harm-Jan
authors.initials: HJ
authors.lastname: Borgeld
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Muramatsu
authors.forename: Jian-Sheng
authors.initials: JS
authors.lastname: Gong
authors.forename: Makoto
authors.initials: M
authors.lastname: Yoneda
authors.forename: Wakako
authors.initials: W
authors.lastname: Maruyama
authors.forename: Makoto
authors.initials: M
authors.lastname: Naoi
authors.forename: Tohru
authors.initials: T
authors.lastname: Ibi
authors.forename: Ko
authors.initials: K
authors.lastname: Sahashi
authors.forename: Masayo
authors.initials: M
authors.lastname: Shamoto
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Fuku
authors.forename: Miyuki
authors.initials: M
authors.lastname: Kurata
authors.forename: Yoshiji
authors.initials: Y
authors.lastname: Yamada
authors.forename: Kumi
authors.initials: K
authors.lastname: Nishizawa
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Akao
authors.forename: Nobuko
authors.initials: N
authors.lastname: Ohishi
authors.forename: Shigeaki
authors.initials: S
authors.lastname: Miyabayashi
authors.forename: Hiraku
authors.initials: H
authors.lastname: Umemoto
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Muramatsu
authors.forename: Koichi
authors.initials: K
authors.lastname: Furukawa
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kikuchi
authors.forename: Imaharu
authors.initials: I
authors.lastname: Nakano
authors.forename: Keiya
authors.initials: K
authors.lastname: Ozawa
authors.forename: Kunio
authors.initials: K
authors.lastname: Yagi
mesh_terms: D017209:Apoptosis / Q000235:genetics; D002460:Cell Line; D004272:DNA, Mitochondrial / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000008:administration & dosage / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000008:administration & dosage* / Q000235:genetics; D005347:Fibroblasts; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007888:Leigh Disease / Q000473:pathology; D028361:Mitochondrial Diseases / Q000235:genetics / Q000628:therapy*; D009154:Mutation; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics
chemical_list: D004272:DNA, Mitochondrial; D015246:Deoxyribonuclease EcoRI; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific