Original Paper J Biomed Sci 2002;9:534–541

Received: April 2, 2002 Accepted: May 19, 2002

Gene Therapy for Mitochondrial Disease by Delivering Restriction Endonuclease SmaI into Mitochondria Masashi Tanaka a, b Harm-Jan Borgeld a, b Jin Zhang a, b Shin-ichi Muramatsu c Jian-Sheng Gong a, b Makoto Yoneda d Wakako Maruyama e Makoto Naoi b Tohru Ibi f Ko Sahashi f Masayo Shamoto e Noriyuki Fuku a, g Miyuki Kurata a Yoshiji Yamada a, b Kumi Nishizawa a Yukihiro Akao a, b Nobuko Ohishi b Shigeaki Miyabayashi h Hiraku Umemoto i Tatsuo Muramatsu i Koichi Furukawa j Akihiko Kikuchi k Imaharu Nakano c Keiya Ozawa l Kunio Yagi a, b a Department

of Gene Therapy, Gifu International Institute of Biotechnology, b Institute of Applied Biochemistry, Mitake, c Department of Neurology, Jichi Medical School, Tochigi, d Second Department of Internal Medicine, Fukui Medical University, Fukui, e Laboratory of Biochemistry and Metabolism, Department of Basic Gerontology, National Institute for Longevity Sciences, Obu, f Neurology Section, Department of Internal Medicine, Aichi Medical University, Aichi, g Japan Science and Technology Corporation, Tokyo, h Department of Pediatrics, Sendai National Hospital, Sendai, i Department of Applied Genetics and Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, j Department of Biochemistry, Graduate School of Medical Sciences, Nagoya University, k Department of Medical Mycology, Graduate School of Medical Sciences, Nagoya University, Nagoya, l Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan

Key Words Mitochondrial disease W Leigh’s disease W Neurogenic muscle weakness, ataxia and retinitis pigmentosa W Gene therapy W Restriction endonuclease

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 F0F1-ATPase. Our ultimate goal is to apply SmaI to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA) coexists with

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© 2002 National Science Council, ROC S. Karger AG, Basel 1021–7770/02/0096–0534$18.50/0 Accessible online at: www.karger.com/journals/jbs

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. Deliv-

Masashi Tanaka, MD, PhD Director, Department of Gene Therapy Gifu International Institute of Biotechnology Yagi Memorial Park, Mitake, Gifu 505-0116 (Japan) Tel. +81 574 68 0073, Fax +81 574 67 6627, E-Mail [email protected]

ery of restriction enzymes into mitochondria is a novel strategy for gene therapy of a special form of mitochondrial diseases. Copyright © 2002 National Science Council, ROC and S. Karger AG, Basel

Introduction

mtDNAs in the muscle. These results depended on mutant-free satellite cells, which could be stimulated to reenter the cell cycle and fuse with existing myofibers in response to signals for muscle growth or repair. However, Andrews et al. [3] tried bupivacaine-induced muscle regeneration for the treatment of ptosis in patients with mtDNA deletions, but they observed no functional recovery in treated patients. Because restriction endonucleases can recognize their target sequences highly specifically, we sought to utilize their specificity for gene therapy of a mitochondrial disease. Herein, we demonstrate that a restriction endonuclease targeting mitochondria specifically destroyed mutant mtDNA, thereby allowing repopulation by wild-type mtDNA.

Mitochondrial DNA (mtDNA) is the only extrachromosomal DNA in humans. It is a small (16,569-bp) genome [2] that encodes 13 essential peptides of enzymes for oxidative phosphorylation, 2 rRNAs and 22 tRNAs. Defects in this genome are now recognized as important causes of various diseases and may take the form of point mutations or rearrangements [8, 10, 21, 24]. There is no effective treatment for patients with mtDNA mutations at present. Holt et al. [11] reported a mitochondrial disease associated with mtDNA heteroplasmy (coexistence of mutant and wild-type mtDNA molecules in cells). This syndrome of neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) is caused by either the Mt8993T→G or Mt8993T→C mutation, resulting in a Leu156Arg or Leu156Pro replacement, respectively, in subunit a of mitochondrial ATP synthase. These mutations have also been detected in patients with Leigh’s disease (subacute necrotizing encephalomyelopathy) [19] as well as in patients with hypertrophic cardiomyopathy [16]. In the presence of heteroplasmy, there is a threshold whereby a certain level of mutant mtDNA is necessary before the disease becomes biochemically and clinically apparent. Based on the presence of heteroplasmy and the recessive nature of these mutations, it should be possible to treat patients either by selectively destroying mutant mtDNA or by inhibiting its replication, thereby only allowing propagation of wild-type mtDNA. To inhibit the replication of mutant mtDNA, Taylor et al. [22] utilized peptide nucleic acids complementary to mutant mtDNA. Although antigenomic peptide nucleic acids were demonstrated to specifically inhibit the replication of mutant but not wild-type mtDNA templates in vitro, the efficacy of this approach in vivo is unknown. Clark et al. [5] showed that the induction of necrosis by local injection of anesthetics into a myopathic patient’s skeletal muscle was followed by regeneration of muscle fibers from satellite cells (dormant myoblasts) without mutant mtDNA. Taivassalo et al. [20] reported that a short period of concentric exercise training induced remarkable increases in the ratio of wild-type to mutant

Construction of Plasmids to Deliver Restriction Endonucleases into Mitochondria The cDNA encoding the presequence of cytochrome c oxidase subunit IV (pCoxIV) from Saccharomyces cerevisiae was amplified from the plasmid pAK1, which was kindly provided by Prof. Toshiya Endo of the University of Nagoya, by use of the following primers: ecoRI-pCoxIV-U (5)-gga tcc GCA TAC AAA TAG ATA ACA A-3)) and apaI-xhoI-pCoxIV-L (5)-ggg ccc ctc gag GAG ATC TAG AGC TAC ACA AA-3)). The gene for SmaI [9] was amplified by PCR from total DNA isolated from Serratia marcescens by use of the primers apaI-xhoI-SmaI-U (5)-ggg ccc ctc gag CAA GCA GGG ATG ACC AAC TC-3)) and bamHI-SmaI-L (5)-gga tcc ATT GGG CCC GAG GCG GCG GTA GAA TAA AA-3)). The gene for EcoRI [15] was kindly provided by Prof. Ichizo Kobayashi of the University of Tokyo and was amplified by use of the primers apaI-xhoI-EcoRI-U (5)-ggg ccc ctc gag CAT CTA ATA AAA AAC AGT CA-3)) and bamHI-EcoRI-L (5)-gga tcc ATT GGG CCC GCT TAG ATG TAA GCT GTT CA-3)). The fused genes were amplified by PCR and then subcloned into either the plasmid pCR2.1-TOPO (Invitrogen, Carlsbad, Calif., USA) or pBluescript SK – (Stratagene, Heidelberg, Germany). The combined fusion genes were finally inserted into the EcoRI-

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Materials and Methods Introduction of mtDNA from Patient’s Fibroblasts into Human Ú0 Cells Skin fibroblasts were obtained from a patient with Leigh’s disease carrying the Mt8993T→G mutation. Enucleation of fibroblasts and fusion of enucleated cells (cytoplasts) with Ú0 human osteosarcoma cells (Ú0206) were carried out as described elsewhere [12, 13]. The resulting cybrids were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum supplemented with high glucose (4.5 g/ml), pyruvate (0.11 mg/ml) and uridine (0.1 mg/ml), in which even Ú0 cells without respiratory function can grow. Thus, we established two cybrid cell lines: NARP3-1 with a high mutant mtDNA ratio of approximately 98% and NARP3-2 with a relatively low mutant mtDNA ratio of approximately 60%. The human osteosarcoma cell line 143B, which carries wild-type mtDNA, was used as the control.

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Fig. 1. A schematic of the novel strategy for gene therapy of NARP disease caused by the Mt8993T→G mutation.

BamHI site of the mammalian expression vector pMACSKkII (Miltenyi Biotec, Bergisch Gladbach, Germany), in which the inserted genes were expressed together with the mouse truncated MHC class I molecule, H-2Kk, for selection of transfected cells. The following fusion genes were constructed: pMACSKkII-pCoxIV-SmaI and pMACSKkII-pCoxIV-EcoRI. Transfection and Selection of Transfected Cells Cells were cultured in 10-cm dishes in 12 ml of the culture medium at 37 ° C under an atmosphere of 5% CO2 and 95% air. When cells had become 90–95% confluent, they were washed twice with PBS, and then used for transfection. The cells (approximately 6 ! 106) were transfected at 37 ° C for 24 or 48 h with 30 Ìg/dish of the plasmid dissolved in 3 ml of opti-MEM I reduced-serum medium with the use of 30 Ìl of Lipofectamine® 2000 reagent (Life Technologies, Rockville, Md., USA). After 24–48 h of incubation, the transfected cells were removed from the dish by trypsinization and then selected with magnetic microbeads (80 Ìl per 10-cm dish) coated with the monoclonal antibody against the mouse H-2Kk molecule by use of a magnetic MS column and a magnetic cell sorter (MiniMACS separation unit, Miltenyi Biotec). In typical experiments, the yield of selected cells was approximately 10% in a single cycle of transfection and selection. Selected cells were cultured for 2–3 days and then used for the next cycle of transfection and selection.

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Southern Blot Analysis Total DNA (2 Ìg) extracted from cells was digested with the restriction endonucleases. Restricted fragments were separated on a 1% agarose gel, transferred onto a Nylon membrane and hybridized with a fluorescein-dUTP-labeled human mtDNA probe. The membrane was washed, and the fragments were detected with the Gene Images Random-Prime Labelling and Detection System (Amersham Pharmacia Biotech, Buckinghamshire, England) according to the manufacturer’s instructions. Chemiluminescence of the fragments was analyzed with a bioimaging analyzer (Fujix LAS 1000, Fuji Photo Film, Tokyo, Japan). Measurement of ATP Content of Cells ATP was extracted from cells and measured using an ATP biofluorescence assay kit (HS II, Roche Diagnostics, Mannheim, Germany) with a luminometer (Lumat LB9501, Berthold, Bad Wildbad, Germany), according to the manufacturers’ instructions. Measurement of Mitochondrial Membrane Potential The mitochondrial membrane potential was measured by flow cytometry with a FACS-Calibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif., USA). The cultured cybrids growing on 60-mm culture dishes were incubated with the cationic lipophilic dye MitoTracker Orange CMTMRos at a concentration of 100 nM [18] (Molecular Probes, Eugene, Oreg., USA) for 30 min at 37 ° C in a humidified atmosphere of 5% CO2. The amount of fluorochrome incorporated into cells is dependent upon their

Tanaka et al.

mitochondrial transmembrane potential [1, 6]. After staining with the dye, cybrids were washed, harvested by trypsinization and then subjected to flow cytometry. In vivo Electroporation of the Plasmid Expressing Mitochondrion-Targeted EcoRI The expression plasmid pMACSKkII-pCoxIV-EcoRI (15 Ìg) together with the reporter plasmid pCAGGS-LacZ (15 Ìg) was injected into the tibialis anterior muscle of 3.5-week-old hamsters under general anesthesia. Electrical pulses (60 V, 0.02–0.04 A; electrode distance 6 mm; 6 times) were applied by use of a CUY645 hammerhead electrode (3 ! 5 mm) and a pulse generator (CUY21, BEX, Tokyo, Japan). The muscle tissue was excised 1 month after electroporation, and frozen sections (8 Ìm) were made. Tissue slices were stained for ß-galactosidase or cytochrome c oxidase.

Results

We first established the cybrid cell line NARP3-1 by cytoplasmic transfer of mitochondria from fibroblasts of a patient with Leigh’s disease into the Ú0206 osteosarcoma cell line lacking mtDNA. The NARP3-1 cybrids had a high percentage of mutant mtDNA (approximately 98%). The ATP content of the NARP3-1 cybrids was 70% of that of the 143B cells carrying the wild-type mtDNA. The mitochondrial membrane potential of the NARP3-1 cybrids was markedly lower (by 69%) than that of the 143B cells. The low mitochondrial membrane potential is ascribable to the fact that the ATP supplied from glycolysis cannot be utilized to generate the mitochondrial membrane potential because of the blockade of the proton channel by the Leu156Arg replacement in subunit a of F0F1-ATPase. To deliver SmaI into mitochondria, we fused the SmaI gene to the presequence that encodes the signal peptide of cytochrome c oxidase subunit IV (pCoxIV) and then inserted this fused gene into a mammalian expression vector (pMACSKkII), as illustrated in figure 1. When we transfected the NARP3-1 cybrids with the plasmid containing the fused gene (pMACSKkII-pCoxIV-SmaI), the mutant mtDNA was completely eliminated, and only a trace amount of wild-type mtDNA was detected (fig. 2, day 2). Because the amount of total DNA applied to each lane in figure 2 was the same, the Southern blot signal in each sample reflects the mtDNA content in individual cells. At this stage, the transfected NARP3-1 cybrids were similar to the Ú0206 cells; namely, they had a low mitochondrial membrane potential (fig. 3) due to a deficiency of the wild-type mtDNA. When NARP3-2 cybrids, which had a lower percentage of mutant mtDNA (approximately 60%), were transfected, the membrane potential in-

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Fig. 2. Elimination of mutant mtDNA and repopulation by wild-

type mtDNA after import of SmaI into mitochondria. NARP3-1 cybrids were transfected with the pMACSKkII-pCoxIV-SmaI plasmid, and transfected cells were then selected with the MACSelect system. Five cycles of transfection and selection were repeated. Total DNA was extracted from the cybrids before, 2 days after and 23 days after import of SmaI into the mitochondria. DNA samples (2 Ìg) were digested either with AflII alone or with both AflII and SmaI and then subjected to Southern blot analysis. The wild-type mtDNA (16,569 bp) was linearized by the restriction enzyme AflII, whereas the mutant mtDNA with Mt8993T→G mutation was further restricted by SmaI into two fragments (7,586 and 8,983 bp).

Fig. 3. Normalization of the mitochondrial membrane potential after elimination of mutant mtDNA and repopulation by wild-type mtDNA. The mitochondrial membrane potential was measured by flow cytometry with MitoTracker Orange CMTMRos. The mean fluorescence intensity of the cybrids (104 cells) was determined. The mitochondrial membrane potential of transfected NARP3-1 cybrids is expressed as a percentage of that of control 143B cells (fluorescence intensity of 110–138 in arbitrary units). An average of 3 different determinations is shown together with the standard deviation.

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Fig. 4. Normalization of ATP content after elimination of mutant

mtDNA. NARP3-1 cybrids were cultured for up to 3 weeks after 5 cycles of transfection and selection. ATP content per milligram of total cellular protein was measured before transfection, on day 10 and on day 20 after completion of selection. The ATP content of the transfected cybrids is expressed as a percentage of that of control 143B cells (84 nmol/mg protein on average). An average of 3 different determinations is shown together with the standard deviation.

Fig. 5. Depletion of total mtDNA by mitochondrion-targeted EcoRI.

NARP3-2 cybrids were twice transfected with pMACSKkII/pCoxIVEcoRI (on day 1 for the first cycle and on day 3 for the second cycle) and twice selected with the MACSelect system (on day 2 for the first cycle and on day 4 for the second cycle). Total DNA (2 Ìg) extracted from the cybrids was digested with restriction enzyme AflII, separated by agarose gel electrophoresis and then analyzed by Southern blotting for mtDNA. The minus (–) and plus (+) signs denote untransfected cybrids which passed through the magnetic column and selected cybrids that were bound to the magnetic column by microbeads coated with the anti-H-2Kk monoclonal antibody, respectively. After the first transfection, the amount of mtDNA in cybrids bound to the column (+ lane in the first selection) was slightly less than that in cybrids in the pass-through fraction (– lane in the first selection). After the second transfection, both cybrids in the pass-through fraction (– lane) and those bound to the magnetic column (+ lane) contained only trace amounts of mtDNA (*).

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creased without the transient decrease found in the NARP3-1 cybrids after transfection (data not shown). To examine whether there was repopulation by wildtype mtDNA after destruction of the mutant mtDNA, we continued to culture the posttransfection NARP3-1 cybrids for 3 weeks. As also shown in figure 2, the wild-type mtDNA had propagated markedly by day 23. This repopulation by wild-type mtDNA was associated with normalization of both the mitochondrial membrane potential (fig. 3) and the cellular ATP content (fig. 4). When control plasmid pMACSKkII without the insert was used for transfection, no significant changes in the ratio of mutant to wild-type mtDNA, ATP level or membrane potential were observed. To examine the effect of another restriction endonuclease on both nuclear DNA and mtDNA, we delivered EcoRI into mitochondria. After the first cycle of transfection and selection, the selected cybrids (fig. 5, +), which were retained by the magnetic column bearing microbeads coated with the monoclonal antibody against the H-2Kk molecule on their cellular surface, contained a decreased amount of mtDNA compared with unselected cybrids (fig. 5, –), which were not retained by the column. After the second cycle of transfection and selection, both the retained (+) and nonretained (–) cybrids were depleted of mtDNA (fig. 5). This indicates that most of the NARP3-2 cybrids were converted to the Ú0 state after the second transfection. Interestingly, we detected no intermediary mtDNA fragments restricted with EcoRI after the first cycle of transfection and selection, but we only observed a decreased amount of mtDNA (fig. 5, +). This finding indicates that the destruction of restricted mtDNA is an allor-none process. Although some of the restricted mtDNA fragments may have been repaired by putative mitochondrial ligases, they were eventually digested by intramitochondrial exonucleases. The restricted mtDNA seemed to disappear swiftly from the cells as a part of physiological processes that maintain an intact mtDNA pool against various types of oxidative damage to the mitochondrial genome. We detected no signs of apoptosis, such as the appearance of apoptotic bodies or the formation of DNA ladders. These results indicate that restriction enzymes imported into the mitochondria are safely segregated within the mitochondrial matrix compartment without attacking nuclear DNA, at least under the cell culture conditions used here. To confirm whether the transient expression of mitochondrion-targeted restriction endonucleases can be safe-

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Fig. 6. Decreased cytochrome c oxidase activity after in vivo electroporation of the plasmid expressing the mitochondrion-targeted EcoRI. Plasmids pMACSKkII-pCoxIV-EcoRI and pCAGGS-LacZ were used to cotransfect the tibialis anterior muscle of 3.5-week-old hamsters by in vivo electroporation. The muscle tissue was excised 1 month after electroporation, and frozen sections (8 Ìm) were made. Tissue slices were stained for ß-galactosidase (a) or cytochrome c oxidase (b) activity. Asterisks denote ß-galactosidase-positive muscle fibers with decreased cytochrome c oxidase activity.

ly applied for in vivo therapy, we introduced two types of plasmids, one expressing ß-galactosidase and the other expressing mitochondrially targeted EcoRI, into hamster skeletal muscles by in vivo electroporation. One month after a single injection and in vivo electroporation, most of the muscle fibers with ß-galactosidase activity (fig. 6a), representing the LacZ gene coexpressed with pCoxIVEcoRI, exhibited decreased cytochrome c oxidase activity (fig. 6b). In vivo electroporation of plasmid DNA for mitochondrion-targeted SmaI or EcoRI into hamster skeletal muscle induced no degenerative changes in nuclei.

In the present study, we succeeded in eliminating mutant mtDNA with the Mt8993T→G mutation from cultured cybrids using a novel strategy employing the mitochondrion-targeted restriction enzyme SmaI (fig. 1). Because the Mt8993T→G mutation affects only the pro-

ton-translocating activity of F0F1-ATPase [4, 7, 23], the NARP3-1 cybrids exhibited no defects in the respiratory chain, which mainly contributes to the mitochondrial membrane potential. In the case of NARP3-1 cybrids, the mitochondrial membrane potential was markedly decreased (fig. 3) in association with acute elimination of mutant mtDNA by transfection (fig. 2). After repopulation by wild-type mtDNA, both the mitochondrial membrane potential (fig. 3) and the cellular ATP content (fig. 4) were normalized to levels comparable to those of wild-type cells. In order to eliminate mutant mtDNA, we needed 5 cycles of transfection and selection for SmaI (fig. 2), whereas we needed only 1 or 2 cycles for EcoRI (fig. 5). After the first transfection with mitochondrion-targeted EcoRI, the mtDNA content of selected cells decreased only slightly. After the second transfection and selection, mtDNA was depleted in both the pass-through fraction and the selected fraction. The mtDNA in the first transfected cells would have been digested by EcoRI during the culture before the second transfection and selection. Eco-

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Discussion

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RI cuts human mtDNA at 3 sites, whereas SmaI cuts the mutant mtDNA at only 1 site. Differences in the number of recognition sites may influence the efficiency of elimination of mtDNA between the two restriction enzymes. Alternatively, the activity and stability in cultured human cells may differ between these enzymes. The effect of the EcoRI plasmid delivered by in vivo electroporation was milder in hamster skeletal muscle (fig. 6) than that in NARP3-1 cybrids. After 1 month, we detected slightly decreased cytochrome c oxidase activity in transfected hamster muscle fibers. We examined cytochrome c oxidase activity histochemically, because its three subunits (CO1, CO2 and CO3) are encoded by mtDNA. In our previous analysis, we did not detect decreased activity of cytochrome c oxidase 2 weeks after electroporation of muscle fibers. The slow decrease in cytochrome c oxidase activity is probably ascribable to the slow decay of the previously synthesized enzyme complex in nondividing muscle cells. The immediate appearance of mitochondrial dysfunction in cultured cybrids after expression of mitochondrion-targeted endonuclease can be explained by the rapid turnover of mitochondrial enzymes associated with cellular proliferation. We confirmed in both cultured cybrids and hamster skeletal muscles that exogenous endonucleases imported into the mitochondria exerted no toxic effects on nuclear DNA. This phenomenon may be relevant to the fact that endogenous mitochondrial exonucleases or proteases either in the matrix space or in the intermembrane space do not attack nuclear DNA or cellular proteins except under certain pathological conditions. Because extranuclear genomes, such as mtDNA and chloroplast DNA, have limited sizes, we can select a restriction enzyme that restricts a mutant genome. This technique can be also be applied in selecting a novel mutant that is resistant to the restriction enzyme. The present results indicate that the use of a mitochondrion-targeted restriction enzyme which specifically recognizes a mutant mtDNA provides a novel strategy for gene therapy of mitochondrial diseases. The advantage of this strategy is that the transient expression of the restriction enzyme is sufficient for the elimination of the mutant mtDNA. Once the mutant mtDNA is eliminated, the wild-type mtDNA becomes the major population in the cell, resulting in complete conversion of the mitochondrial genome from the mutant to the wild-type form. At present, adeno-associated virus (AAV) vectors seem to be the safest and most efficient method for gene therapy. Delivery of genes encoding dopamine-synthesizing enzymes by use of AAV vectors has been successfully

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applied in a primate model of Parkinson’s disease [14, 17]. AAV vectors are expected to be useful for eliminating mutant mtDNA from the central nervous system as well as from cardiac and skeletal muscles. To supplement a defective subunit 6 of F0F1-ATPase by gene therapy, the expression must be persistent. In contrast, expression of mitochondrion-targeted restriction enzymes to eliminate mutant mtDNA from tissue can be transient. After elimination of the mutant mtDNA, the wild-type subunit 6 will be supplied from the wild-type mtDNA. Therefore, it would be desirable to control the expression of mitochondrion-targeted SmaI by an appropriate on/off system to ensure the safety of gene therapy.

Acknowledgements This paper was presented at the 10th International Congress on Cardiovascular Pharmacotherapy WHF/ISCP Joint International Symposium on Cardiomyopathy in the 21st Century held on March 29, 2001, in Kyoto, Japan, and at the International Workshop on Mitochondria held on August 17, 2001, in Kaohsiung, Taiwan. This work was supported in part by grants from the programs Grants-inAid for Research Projects on Intractable Diseases (to M.T.) of the Ministry of Health, Welfare and Labor of Japan and Grants-in-Aid for Fundamental Scientific Research (to M.T.) and Grants-in-Aid for Scientific Research on Priority Areas (to M.T.) of the Ministry of Education, Science and Culture, Japan.

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