Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria.
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.
Tanaka
Masashi
M
Department of Gene Therapy, Gifu International Institute of Biotechnology, Mitake, Japan. mtanaka@giib.or.jp
Borgeld
Harm-Jan
HJ
Zhang
Jin
J
Muramatsu
Shin-ichi
S
Gong
Jian-Sheng
JS
Yoneda
Makoto
M
Maruyama
Wakako
W
Naoi
Makoto
M
Ibi
Tohru
T
Sahashi
Ko
K
Shamoto
Masayo
M
Fuku
Noriyuki
N
Kurata
Miyuki
M
Yamada
Yoshiji
Y
Nishizawa
Kumi
K
Akao
Yukihiro
Y
Ohishi
Nobuko
N
Miyabayashi
Shigeaki
S
Umemoto
Hiraku
H
Muramatsu
Tatsuo
T
Furukawa
Koichi
K
Kikuchi
Akihiko
A
Nakano
Imaharu
I
Ozawa
Keiya
K
Yagi
Kunio
K
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
D004272:DNA, Mitochondrial; D015246:Deoxyribonuclease EcoRI; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific