Review

Gene Therapy (2008) 15, 1017–1023; doi:10.1038/gt.2008.91; published online 22 May 2008

Progress and prospects: gene therapy for mitochondrial DNA disease

D S Kyriakouli1, P Boesch1, R W Taylor1 and R N Lightowlers1

1Mitochondrial Research Group, Medical School, Newcastle University, Newcastle upon Tyne, UK

Correspondence: Professor RN Lightowlers, Mitochondrial Research Group, Newcastle University, The Medical School, Framlington Place, Newcastle upon Tyne, Newcastle NE2 4HH, UK. E-mail: r.n.lightowlers@ncl.ac.uk

Received 13 February 2008; Revised 11 April 2008; Accepted 14 April 2008; Published online 22 May 2008.

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Abstract

Defects of the mitochondrial genome cause a wide variety of clinical disorders. Except for rare cases where surgery or transplant is indicated, there is no effective treatment for patients. Genetic-based therapies are consequently being considered. On account of the difficulties associated with mitochondrial (mt) transfection, alternative approaches whereby mitochondrial genes can be engineered and introduced into the nucleus (allotopic expression) are being attempted with some success, at least in cultured cells. Defects in the activities of multi-subunit complexes of the oxidative phosphorylation apparatus have been circumvented by the targeted expression of simple single subunit enzymes from other species (xenotopic expression). Although far from the clinic, these approaches show promise. Similarly, nuclear transfection with genes encoding restriction endonucleases or sequence-specific zinc finger-binding proteins destined for mitochondria has also proved successful in targeting mtDNA-borne pathogenic mutations. This is particularly important, as mutated mtDNA is often found in cells that also contain normal copies of the genome, a situation termed heteroplasmy. Shifting the levels of heteroplasmy towards the normal mtDNA has become the goal of a variety of invasive and non-invasive methods, which are also highlighted in this review.

Keywords:

mtDNA, heteroplasmy, mitochondria, mtDNA disease

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In brief

Progress

  • Strategies can be devised for gene therapy of mitochondrial DNA disorders.
  • Allotopic and xenotopic expression show great promise as treatment concepts for mtDNA disease.
  • The alternative oxidase may prove an attractive alternative for treating mtDNA disease.
  • The yeast soluble NADH oxidase can substitute for defective human complex I.
  • Allotopic expression and import of nucleic acids are being considered for rescuing pathogenic mt-tRNA mutations.
  • Rescue of mtDNA mutations may be possible through mitochondrial transfection.
  • Manipulating the mitochondrial genome may lead to rescue of an OXPHOS deficiency.
  • Germline therapy for mitochondrial DNA disease is being considered.

Prospects

  • Generation of numerous mice models heteroplasmic for pathogenic mtDNA mutations that can routinely be used to determine efficacy of any therapeutic. Although such mice do exist, there are currently very few. It is expected that within 5 years, there will be many models that can be used.
  • Determining the feasibility of using genes encoding single enzyme alternatives to OXPHOS complexes as possible interventions for mutations that affect protein-coding genes in mtDNA.
  • Production of antigenomic drugs that will allow targeting and removal of pathogenic mtDNA. Current work is promising and it is likely that within 3–5 years a pharmaceutical could be developed for oral or intravenous delivery that will be able to modulate levels of heteroplasmy.
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Introduction

The human mitochondrial genome (mtDNA) is found in many copies in all nucleated cells (Figure 1). This small (16569bp) genome is housed in the mitochondrial matrix and encodes 13 polypeptides, all of which are integral components of the complexes that couple oxidative phosphorylation (OXPHOS). In addition to these polypeptides, this remarkably compact molecule codes for the two ribosomal RNAs (16S and 12S mt-rRNA) and 22 tRNAs that are necessary and sufficient for intramitochondrial protein synthesis. As all 13 mitochondrially encoded polypeptides are essential members of the OXPHOS machinery that harnesses cellular respiration to ATP production, it is not surprising that mutations in this genome can cause a wide variety of disorders such as mitochondrial encephalopathy lactic acidosis with stroke-like episodes (MELAS), myoclonic epilepsy with ragged red fibres (MERRF) and leber's hereditary optic neuropathy (LHON).1 In addition, due to the ubiquity of mitochondria, the clinical presentation of mitochondrial DNA disorders can be multi-system, but often causes profound chronic progressive defects in muscle and nerve.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

The human mitochondrial genome. This small (16569bp) genome is almost completely transcribed from both strands, initiating from one of two promoters (IH1, IH2) on the Heavy (H-) strand or the single promoter (IL) on the Light (L-) strand. All of these promoters and elements involved in replication initiation are found in the displacement (D-) loop, the only major non-coding region. The genome encodes 22 mt-tRNA (black diamonds), 2 mt-rRNA genes (fuscia) and 13 protein-coding genes (olive, ND1-6 encoding members of NADH:ubiquinone oxido-reductase; blue, Cytb encoding apocytochrome b of ubiquinol:cytochrome c oxido-reductase; orange, COI-III encoding members of cytochrome c oxidase: aquamarine, ATPase 6, 8 encoding two members of Fo-F1 ATP synthase). Important mutations that are referred to in this article are indicated. Single letter code is given for each mt-tRNA-encoding gene.

Full figure and legend (97K)

Cells are multiploid with respect to mtDNA, with most somatic cells containing at least 103 copies of the genome. Consequently, a pathogenic mutation may be present in just one, many, or all, depending on when the mutation occurred and how the mutated molecule was replicated and segregated during development. For most disorders, the pathogenic mutation is recessive, requiring a substantial percentage of the genome to carry the defect before an effect on OXPHOS can be measured. This ‘threshold’ varies between cell types and mutation sites but is normally between 60 and 95%.

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Strategies can be devised for gene therapy of mitochondrial DNA disorders

There is currently no effective treatment for the majority of these debilitating diseases. This has led researchers in the field to consider the prospect of gene therapy and related concepts. The approaches can roughly be divided into three groups: (1) rescue of a defect by expression of an engineered gene product from the nucleus (allotopic or xenotopic expression), (2) import of normal copies or relevant sections of mtDNA into mitochondria, and (3) manipulation of the mitochondrial genetic content. We briefly introduce these concepts below and indicate where significant progress has been made in the last 2 years.

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Allotopic and xenotopic expression show great promise as treatment concepts for mtDNA disease

In 1988, Nagley and colleagues2 were able to show that the respiratory defect in yeast carrying mutations in the mitochondrial MTATP8 gene could be rescued if a normal copy of the gene was engineered and introduced into the nucleus, resulting in cytosolic translation of the gene product, ATPase 8. Phenotypic rescue required the addition of a well-defined mitochondrial targeting and import sequence at its N-terminal. This ‘allotopically’ expressed mitochondrial gene product could be functionally integrated into the mitochondrial ATP synthase (complex V) and the deficiency could be rescued. Several years later, this finding was extended to cultured human cells. A different mitochondrial gene to encode a member of the ATP synthase, MTATP6, was also engineered for expression in the nucleus. Successful targeting of the ATPase 6 gene product to the mitochondrion was shown to partially suppress a respiratory-deficient phenotype caused by a pathogenic mutation in MTATP6. These experiments were met with optimism as a potential method for treating patients with mutations in mitochondrial protein-encoding genes such as those with neurogenic weakness, ataxia with retinitis pigmentosum (NARP) which are caused by a specific point mutation (m.8993T>G) in MTATP6. More recent and extensive analysis by Bokori-Brown and Holt conflict with these data.3 Similar allotopic experiments were performed to rescue a cell line carrying the identical mutation in MTATP6. The authors optimised the import of nuclear-encoded ATPase 6 into the human cell lines but after careful analysis of assembled complexes were unable to show integration of the imported protein into mature functional ATP synthase. Restoration of complex V activity in the original experiments was suggested to have been due to random clonal variations in ATP synthesis as a consequence of aneuploidy in the transfected population. Despite these discrepancies, the authors remained optimistic about the potential for allotopic expression as a treatment method.

In a twist to this approach, Corral-Debrinski et al.4 have attempted to optimise the allotopic expression of such highly hydrophobic mitochondrial proteins by suggesting that targeting of the transcript to the outer mitochondrial membrane may facilitate co-translational translocation of the gene product, thus preventing the accumulation of cytosolic aggregates. Further evidence was generated by using primary fibroblasts from patients with mutations in MTND4 (a mitochondrial gene encoding a member of complex I of the respiratory chain) known to cause Leber's hereditary optic neuropathy, thereby avoiding the complexities of aneuploid immortalised cell lines.5 Cis-acting elements in the 3′-untranslated regions from transcripts that had previously been shown to localise to mitochondria (SOD2 and COX10) were used in tandem with in-frame presequence coding regions to improve mitochondrial import of the allotopically expressed proteins. Transfections resulted in the rescue of the deficiency as evidenced by the restoration of OXPHOS and growth with galactose as a sole carbon source, a medium that requires efficient OXPHOS for cell proliferation. Although impressive, additional analysis by gel electrophoresis to show unequivocally that imported proteins are indeed integrated into fully assembled OXPHOS complexes is still required.

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The alternative oxidase may prove an attractive alternative for treating mtDNA disease

Allotopic expression is an encouraging strategy, but does require efficient mitochondrial import and correct integration of the allotropic protein into an assembled protein complex. Two entirely separate but equally exciting prospects have received attention in the last 2 years. Human mitochondria rely on a single cyanide-sensitive cytochrome c oxidase (COX) to reduce molecular oxygen. This multi-component complex is the terminal member of the mitochondrial electron transfer chain and contains three essential polypeptides encoded by mtDNA. Consequently, mtDNA mutations often cause defects in COX activity. Importantly, many other species encode cyanide-insensitive alternative oxidases (AOX). A very encouraging report recently showed that a single subunit AOX from the sea squirt Ciona intestinalis could be synthesised in the cytosol of cultured human cells and successfully targeted to the mitochondrial inner membrane.6 AOX caused no measurable cytotoxicity and transfectants showed impressive levels of cyanide-insensitive oxidation of mitochondrial metabolites. The authors also highlighted its important function as an anti-oxidant, preventing over-reduction of the ubiquinone pool, which is believed to be an important source of reactive oxygen species. To date, there has been no report of its use in rescuing a mitochondrial COX defect, but the potential for its use in treating human COX deficiencies is clear.

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The yeast-soluble NADH oxidase can substitute for defective complex I

A similar and equally elegant approach has also been used to by-pass defects in the initial part of the electron transfer chain. Human complex I, NADH:ubiquinone oxidoreductase is a profoundly complicated assembly of approximately 45 proteins, 7 of which are encoded by the mitochondrial genome. Numerous mitochondrial defects have been linked with mutations in these mitochondrial genes. In contrast, the yeast Saccharomyces cerevisiae does not possess a similar membrane-bound complex, but instead, uses just a single subunit NADH oxidase, termed as Ndi1. Back in 2001, Yagi and colleagues7 showed that mitochondrial targeting of Ndi1 in human cells defective in complex I activity corrected this defect. In the intervening years, the authors have built on this observation and showed that in addition to the potential for Ndi1 activity to treat patients with mtDNA disease associated with mtDNA-borne MTND mutations, this enzyme may prove useful for other complex I disorders. In particular, the authors have focussed on complex I deficiency in Parkinson's disease (PD). That the hallucinogen MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes a form of PD has been well described. This drug selectively inhibits complex I activity in humans, but has no effect on yeast Ndi1.7 Yagi and colleagues8 have now demonstrated that viral-mediated delivery and expression of Ndi1 in the substantia nigra protected against neurodegeneration after feeding rats with MPTP. These data tentatively support the use of yeast Ndi1 expression to compensate for any complex I deficiency in patients with PD.

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Allotopic expression and import of nucleic acids are being considered for rescuing pathogenic mt-tRNA mutations

It has been a dramatic few years for reports of nucleic acid import into mammalian mitochondria, any of which may eventually prove beneficial for genetic therapies of mitochondrial disorders. In contrast to many eukaryotes, mammals encode all tRNAs required for mitochondrial protein synthesis on the mitochondrial genome (mt-tRNA). As many disorders of the mitochondrial genome are caused by mutations in mt-tRNA encoding genes, there has been great interest in rescuing these mutations by importing normal copies of the relevant tRNA from the cytosol.

Tarassov et al.9 showed that a respiratory defect in cell lines containing mutated mt-tRNALys could be partially rescued when the yeast homologue was expressed in the nucleus. These results suggested that a cryptic tRNA import system remains in human mitochondria. The exact tRNA import mechanism in human mitochondria is unclear, although the authors have begun to dissect the pathway in yeast mitochondria. Building on the theme of tRNA import as a potential mechanism for treating mtDNA disorders, Mukherjee et al.10 have exploited the highly prolific tRNA import process in protists. Initially, a large tRNA import complex (RIC) was isolated from the inner mitochondrial membrane of Leishmania tropica and reconstituted into phospholipid vesicles. The complex comprised 11 major polypeptides, three of which were mitochondrially encoded and five of the eight nuclear gene products were also members of the OXPHOS machinery. Remarkably, bathing cultured human cells in the purified RIC led to receptor-mediated import of the complex into the cytoplasm and integration into mitochondria via a mechanism that is currently unclear.11 Compelling evidence of cytosolic tRNA import comes from work with a cell line containing mtDNA with a large-scale deletion that removes various genes including MTTK, which encodes mt-tRNALys (Figure 1). Prior to incubation of cells with RIC, there was no evidence of any mitochondrial protein synthesis, which can normally be visualised after selective poisoning of cytosolic protein synthesis. Following RIC addition, protein translation was restored for gene products whose genes were not removed by the deletion. This restoration is likely to be due to imported cytosolic tRNALys functioning in mitochondrial translation.

It is remarkable that the large RIC complex can be transported across the plasma membrane and the outer mitochondrial membrane and integrated into the inner membrane. Work with RIC has now been extended to show that complexes preloaded with antisense RNA designed against various mt-mRNA can successfully transport these RNAs to the mitochondrial matrix of intact cells in culture. This leads not only to selective impairment of protein synthesis from the mt-mRNA target, but also degradation of this mt-mRNA.12 This is a very striking result as it is consistent with an RNA-mediated targeting and degradation pathway similar to that noted in the cytosol. The latter is mediated by the RISC complex and dicer for which there is no evidence of mitochondrial localisation. Clearly, this work from the laboratory of Adhya has quite profound consequences for the field of mammalian mitochondrial transfection and would benefit enormously from confirmatory experiments from independent laboratories.

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Rescue of mtDNA mutations may be possible through mitochondrial transfection

When a pathogenic nuclear mutation has been identified, one approach may be to re-introduce normal copies of the gene that might lead to rescue of the phenotype. There are major obstacles in using this strategy to overcome mitochondrial gene mutations. First, transfection is difficult, as there are effectively three membrane barriers. Second, even assuming that exogenous DNA can be imported, gene expression will be transient.

Stable integration may be mediated by homologous recombination. There is evidence that such recombination does occur in some somatic cells at low frequency, but it appears to be a rare event. Consequently, any imported DNA must contain the correct cis-acting elements to promote DNA replication, resolution and maintenance. To date, these elements are not fully characterised and even the detailed mechanism of mtDNA replication itself is controversial. Finally, assuming transfection and maintenance, there are still many hundreds or thousands of mtDNA molecules that will outweigh the transfected molecule. How can it be selected for and maintained in such a background?

What progress has been made towards overcoming these barriers? It has been known for several years that isolated plant mitochondria can spontaneously import a low level of naked linearised DNA. This observation has now been extended to mammalian mitochondria, which also show a low efficiency of natural DNA import.13 Unlike previous anecdotal reports of DNA uptake, data showed that DNA could be imported and could act as a template for DNA synthesis. In addition, transcription was shown to occur only when the template carried a transcriptional promoter element and the primary transcript could be successfully processed and matured. This process of natural competence is not at all understood but has recently been used by Weissig and colleagues to load isolated mitochondria that were subsequently shown to be engulfed by cells when co-incubated.14 Uptake of isolated murine mitochondria had initially been shown by the authors to restore respiration on fusion with human A549 lung carcinoma cells devoid of mtDNA. This internalisation, similar to the method described over 25 years ago by Clark and Shay may prove to be a powerful technique for studying mitochondrial gene expression in humans but is unlikely to be immediately relevant to gene therapy for mtDNA disease.

Are there any other methods being investigated for transfecting mitochondria in whole cells? Several years ago, there was substantial interest in the use of DQasomes. These nonviral mitochondriotropic liposome derivatives could be loaded with DNA and had been shown to co-localise with mitochondria when added to cultured cells. Unfortunately, these earlier reports have not been greatly extended in the past 2 years. Another liposome-based carrier, MITO-Porter has also been shown to co-localise to mitochondria in intact cells.15 MITO-Porter loaded with GFP was shown in isolated mitochondria to be delivered to the intermembrane space and to co-localise with mitochondria in cultured cells. These data suggest that such liposomal preparations can fuse with the outer mitochondrial membrane, but will it prove to be able to access the inner membrane that is necessary for the cargo to access the mitochondrial matrix? Another approach to mitochondrial transfection is protofection. To our knowledge, however, details of this protein-mediated method for delivering complete mitochondrial genomes to mitochondria have not appeared as an original article in any peer-reviewed journal, so it is difficult to assess and to determine its potential for mediating gene therapy.

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Manipulating the mitochondrial genome may lead to rescue of an OXPHOS deficiency

Many patients that suffer from mtDNA disease are heteroplasmic, harbouring two subpopulations, one normal and one pathogenic. One approach to genetic therapy for these intractable disorders is to manipulate the relative amounts of these two subpopulations. This is particularly attractive, as most heteroplasmic pathogenic mutations are recessive, such that levels do not have to be altered greatly before biochemical defects can be rescued. Which methods are currently being investigated for manipulating levels of heteroplasmy (Figure 2)?

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Manipulating mtDNA heteroplasmy. An approach to treating patients who suffer from an mtDNA disorder where both pathogenic and wild type mtDNA co-exist (heteroplasmy) is to manipulate these levels with the aim of increasing the wild type at the expense of the pathogenic mtDNA, potentially reversing the biochemical defect. In this figure, heteroplasmic mitochondria containing both wild type (grey) and pathogenic (black) mtDNA are schematically represented. Transverse muscle sections illustrate how high levels of mutated mtDNA in heteroplasmic patients can result in mosaic muscle fibre staining for cytochrome c oxidase activity (left panel), a situation that can potentially be reversed by increasing the level of wild type mtDNA (right panel). Several different methods to modulate heteroplasmy are currently being explored. (A) Patients are being subjected to endurance or resistance training, increasing muscle bulk and promoting growth of new muscle. (B) Targeting restriction endonucleases to mitochondria has already been successfully employed in tissue culture to remove pathogenic mtDNA where the mutation generates a specific restriction site. This approach has also been used to modulate levels of heteroplasmy for polymorphic mtDNA in mice. (C) Cell membrane crossing oligomers (CMCOs) are being optimised to penetrate cells, target mitochondria and selectively bind to mutated mtDNA. These molecules are potentially capable of inhibiting mtDNA replication, although this has yet to be demonstrated. (D) Zinc-finger binding proteins that show sequence binding specificity have been targeted to mitochondria. By fusing the ZFB chimaera to a DNA methylase, it was shown that mtDNA carrying a specific mutation can be selectively methylated. It is postulated that ZFB proteins could be fused to DNases, leading to selective cleavage of mutated mtDNA.

Full figure and legend (71K)

Exercise training, either by endurance training to promote mitochondrial biogenesis or by resistance exercise protocols to promote muscle satellite cell activation has long been proposed as a means of treating patients with high levels of heteroplasmic mtDNA mutations in muscle. Endurance training improves exercise capacity, inducing physiological adaptations, which reverse the effects of muscle deconditioning and lead to a resolution of the underlying mitochondrial biochemical defect. Nevertheless, concerns have been raised as to whether endurance training has potentially long-term deleterious effects, given that the mutated mtDNA load in some patients was noted to increase following training. To assess the safety of endurance training, Taivassalo et al.16 studied the effects of 14 weeks of exercise in a group of patients with single, large-scale mtDNA deletions. Markers of physiological improvement (oxygen utilisation, skeletal muscle oxygen extraction, peak capacity for work and submaximal exercise tolerance) were evident, although genetic analysis did not reveal any change in the level of deleted mtDNA following exercise training. Further training for an additional 14 weeks maintained these benefits, whereas detraining resulted in a loss of the physiological adaptations. Follow-up studies to assess whether long-term training might induce changes in mtDNA mutation load are currently in progress.

Some patients with sporadic mtDNA mutations harbour high levels of mutated mtDNA in mature muscle fibres, but low or undetectable levels in muscle satellite cells. The stimulation of muscle regeneration, and concomitant incorporation of satellite cell mtDNA into mature muscle (gene shifting), is therefore an attractive approach to manipulate mtDNA heteroplasmy levels in vivo. Indeed, anaesthetic injection, traumatic biopsy injury and resistance exercise (both eccentric and concentric) training protocols have all been shown to be of benefit in inducing muscle regeneration in individual patients. Ongoing studies in patients with single, large-scale mtDNA deletions suggest that 12 weeks of resistance training induces an increase in satellite cell number and muscle regeneration, leading to improvements in strength and oxidative capacity as reflected by a decrease in the percentage of muscle fibres deficient in mitochondrial COX activity.17

An elegant approach to removing pathogenic mtDNA is by targeting restriction endonucleases to mitochondria of heteroplasmic cells. First pioneered by Carlos Moraes in rodent and Masashi Tanaka in human cell lines, specific mtDNA subpopulations in a heteroplasmic background can be degraded by endonucleases expressed from introduced genes engineered to generate enzymes with mitochondrial targeting sequences. Heteroplasmy was shown to shift substantially towards the genome lacking the restriction site. This was potentially of direct relevance to disease, as the heteroplasmic m.8993T>C mutation was shown to be markedly reduced on targeting of the restriction site by endonuclease SmaI. It could be envisaged that the engineered gene encoding the targeted endonuclease could be delivered to patients, but the approach would appear to be limited to the very few human pathogenic mtDNA mutations that result in a restriction site unique to the pathogenic mtDNA. Moraes and colleagues have, however, shown that the generated restriction site need not be unique.18 Heteroplasmic mice carrying two distinct genomes were subjected to adenoviral-mediated delivery of a gene encoding the targeted endonuclease ScaI. The two genomes each carried multiple ScaI sites, five in one genome and three in the second genome. Heteroplasmy was shown to shift in the liver and skeletal muscle, although high-level expression did lead to mtDNA depletion. These data, however, are promising and suggest that if expression levels are controlled it may be possible to treat patients who have pathogenic mtDNA mutations that create additional restriction sites.

A concept that received wide coverage several years ago was the possibility of antigenomic therapy. This required the design and use of a drug that could be imported into mitochondria, bind selectively to the mutated mtDNA and inhibit it from replicating, allowing the normal copy to propagate with time, and thus rescue the defective OXPHOS phenotype. But what could this antigenomic molecule be? At first, there was great interest in the use of mitochondrially targeted peptide nucleic acids (PNAs). These molecules still retained the ability to Watson:Crick base pair and showed remarkable binding selectivity even at physiological salt concentrations and temperatures. Initial experiments were encouraging but eventually it was determined that the targeted PNAs although co-localised to mitochondria in whole cells did not get across the inner membrane, and so could not access mtDNA. Modified molecules termed as ‘cell membrane crossing oligomers’ (CMCO) have now been designed, with greater polarity than traditional PNAs. It has now proved possible to target and import CMCO:PNA hybrid molecules into mitochondria in whole cells. Although these new molecules have great promise as potential drugs for treating heteroplasmic patients, many important experiments need to be performed to determine whether they can inhibit mtDNA replication in whole cells and be delivered to defective mitochondria in animals.

The possibility of selective targeting of mtDNA has also been explored by Minczuk et al.19 These authors have attempted to use the short zinc-finger peptides that show sequence-specific DNA binding capabilities. The authors generated chimaeric ZFPs linked to a DNA methylase carrying an additional mitochondrial presequence. Following import, chimaeras were shown to bind mtDNA and to leave a signature methylation pattern at various sites on the mitochondrial genome, a genome that is not normally methylated. In addition, by using the NARP m.8993T>G cell line, popular for many of these studies, the authors showed methylation patterns that differed from the wild-type mtDNA control. By linking these domains to a nuclease it may be possible to target a mutated subpopulation in a heteroplasmic patient. This is an exciting and impressive achievement, but its long-term usage will be limited by the requirement for transfection and successful expression in defective cells and tissues.

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Germline therapy for mitochondrial DNA disease is being considered

Patients with mtDNA disease often wish to have children. However, the knowledge that mtDNA disease is a genetic disorder and can be transmitted down the maternal line can preclude this decision. Would it be possible to generate a zygote from the parents' gametes using standard in vitro fertilisation techniques and then replace the defective mitochondrial genetic complement? A single-cell zygote containing normal copies of mtDNA would need to be enucleated to produce a cytoplast. An egg from the patient would then be fertilised in vitro with the sperm from the partner. Finally, an embryo would have to be reconstructed from pronuclei carefully removed from the patients fertilised oocyte and fused to the cytoplast.20 Studies in mice have indeed suggested that it may be possible to prevent transmission of mutated mtDNA by pronuclear transfer. Following reconstruction of such a murine embryo, fused zygotes were transferred to the oviducts of pseudopregnant females and offspring were shown to be normal.21 Could such an approach be possible in humans? Following the decision in 2005 by the UK Human Fertilisation and Embryology Authority to approve research into the feasibility of this approach in humans, experiments have been initiated using abnormally fertilised embryos. Although results of these studies have yet to be published, this approach may bring hope to patients with mtDNA disorders who wish to have children and for whom oocyte donation is either not possible or not desired.

Future prospects for genetic therapy of mtDNA disease

Considering the recent output, it is clear that scientists are now claiming a variety of methods for transfecting mitochondria. Although impressive, it is difficult to see how this work will be of direct relevance to treatment unless the transfected material can be autonomously maintained. Allotopic expression and the use of simple single-subunit enzymes that can potentially replace the activity of OXPHOS complexes are both areas of great potential. The drawback is that they will require the delivery of foreign genes to affected cells, which is a similar problem to many conventional gene therapy approaches. Manipulating heteroplasmy remains a particularly attractive idea, especially if the intervention is non-invasive or is mediated by a pharmaceutical agent. One area that has been resurrected recently and shows great promise is the use of CMCO-based molecules that can enter cells and mitochondria naturally and initial experiments suggest these molecules can influence heteroplasmy and mitochondrial gene expression. Before any of these therapeutic approaches can be used in clinical trials, it will be necessary to show their efficacy in animal models. Unfortunately, it has proven difficult to establish mice that are heteroplasmic for pathogenic mutations. To move forward, it is important that more models become available.

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References

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