Biosci Rep (2007) 27:139–150 DOI 10.1007/s10540-007-9042-3 ORIGINAL PAPER
Experimental Strategies Towards Treating Mitochondrial DNA Disorders Julie L. Gardner Æ Lyndsey Craven Æ Douglass M. Turnbull Æ Robert W. Taylor
Published online: 11 May 2007 Ó The Biochemical Society 2007
Abstract An extensive range of molecular defects have been identified in the human mitochondrial genome (mtDNA), causing a range of clinical phenotypes characterized by mitochondrial respiratory chain dysfunction. Sadly, given the complexities of mitochondrial genetics, there are no available cures for mtDNA disorders. In this review, we consider experimental, genetic-based strategies that have been or are being explored towards developing treatments, focussing on two specific areas which we are actively pursuing—assessing the benefit of exercise training for patients with mtDNA defects, and the prevention of mtDNA disease transmission.
Keywords Mitochondria mtDNA Mutation Heteroplasmy Treatment Exercise Satellite cell
Introduction Mitochondrial DNA (mtDNA) mutations—commonly large-scale, single deletions and point mutations—are now recognised as important causes of human disease [1, 2]. Many patients present with particular neurological syndromes [3] due to a specific mtDNA mutation, but there can be widespread clinical and genetic heterogeneity involving a number of organ systems. Such a widespread heterogeneity can be attributed to the phenomenon of heteroplasmy which describes the presence of two, discrete mtDNA genotypes—wild type and mutated molecules—on account of there being multiple copies of the mitochondrial genome in the same cell. In many patients, clinical J. L. Gardner L. Craven D. M. Turnbull R. W. Taylor (&) Mitochondrial Research Group, School of Neurology, Neurobiology and Psychiatry, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK e-mail:
[email protected] D. M. Turnbull R. W. Taylor Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, UK
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manifestation of disease only becomes evident when the number of mutated mtDNA molecules exceeds a certain threshold level. This threshold is commonly in the region of 60–90% mutated mtDNAs, can vary between organs depending upon their energy requirements, and reflects the incapacity of the remaining wild type mtDNA to compensate for the mutated mtDNA, leading to an impairment in oxidative phosphorylation and consequent cellular dysfunction. Whilst much has been learned in recent years about the clinical presentations, epidemiology and underlying molecular mechanisms associated with specific mtDNA mutations, available therapeutic interventions to help patients and their families remain somewhat limited. In this review, we consider the experimental and genetic-based strategies that are being considered to try and treat these devastating neurological disorders [4].
Current Treatments for Patients with Mitochondrial DNA Disease The pivotal role occupied by the respiratory chain in cellular metabolism poses acute difficulties in trying to overcome the respiratory defect and subsequent decline in the ability to supply cellular energy demands in the form of available ATP, a characteristic trait of all mitochondrial disorders. Biochemical strategies to increase the production of ATP have sought to bypass the block in electron transfer using artificial electron acceptors (e.g. menadione, vitamin C), minimise the free-radical induced damage that occurs as a result of a defective respiratory chain by administration of antioxidants (e.g. coenzyme Q10) or enhance residual enzyme activity (recently reviewed by DiMauro and colleagues [5] and also in this series [6]. Evaluating therapeutic intervention remains problematic on account of the phenotypic variability expressed in mitochondrial disorders, the small numbers of patients available for study make large-scale clinical trials extremely difficult to undertake and that in terms of clinical improvement, individual patients may respond in different ways. A consensus statement on the current best practice for the treatment of mitochondrial disorders has recently been published [7] and is considered elsewhere in this series, together with palliative interventions which may include ophthalmic splints or corrective surgery in patients with CPEO, gastrostomy in patients with severe swallowing problems leading to gastric stasis, intestinal pseudo-obstruction and severe constipation and cochlear implants in patients with sensorineural deafness [6].
Gene Therapy Strategies for mtDNA Disorders For a number of reasons, the presence of multiple mtDNA copies within individual cells, the phenomenon of mtDNA heteroplasmy and the frustrating inability to manipulate the mitochondrial genome inside the organelle of living cells by DNA transfection techniques—progress towards developing generic and realistic treatments for patients with mitochondrial genetic defects has been frustratingly slow. The remainder of this review will briefly consider genetic-based strategies that have been or are being developed, before focussing on two specific areas which we are actively pursuing in collaboration with others—the role of exercise training as a treatment for patients with mtDNA defects, and the prevention of mtDNA disease transmission.
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Strategies to Complement the mtDNA Defect Allotopic expression, the term given to the expression of a gene in a different cellular compartment to its target location, was one of the first ways in which researchers approached mitochondrial gene therapy by exploiting the natural mechanisms of protein and nucleic acid import to introduce a normal copy of a gene to complement the defect. Although a number of potential obstacles needed to be overcome, the differences in codon recognition between mitochondria and the universal genetic code, concerns over whether the targeted protein would become integrated into functional respiratory chain complexes—the possibility of expressing a normal copy of a defective mitochondrial gene in the nucleus, synthesising the protein in the cytosol and conjugating a targeting sequence to facilitate import into mitochondria was attractive. Nagley and colleagues first established ‘‘proof of principle’’ using yeast as a model system by recoding the ATP8 gene, fusing it with the targeting signal of ATPase 9 from N. crassa and inserting the gene into the nucleus. The newly cytosolically-synthesised protein was correctly imported into mitochondria, assembled into a functioning ATPase complex and able to rescue a yeast mutant strain defective in the ATP8 gene [7]. More recently, this approach has recently been successfully applied to humans. By adding specific mitochondrial targeting presequences to the MTATP6 gene, Manfredi et al. were able to express wild type ATPase 6 protein allotopically from nucleus-transfected constructs in transmitochondrial cybrid cells homoplasmic for the m.8993T>G mutation and demonstrate partial rescue of the biochemical defect [8]. A similar strategy has been employed to express a modified MTND4 gene to complement the m.11778G>A LHON mutation, with transfected cells showing a restoration of ATP synthesis to normal levels [9]. Investigating the possibility of expressing mitochondrial genes from other species in human cells in order to correct the respiratory defect, Schon and colleagues used a nucleus-encoded MTATP6 gene from C. reinhardtii [10] to rescue the ATP synthesis defect in human cells harbouring the m.8993T>G mutation [11]. A similar approach has been adopted to treating complex I deficiency by utilising the NDI1 gene that codes for the single subunit NADH-quinone oxidoreductase dehydrogenase of S. cerevisiae (Ndi1) [12]. Using adeno-associated virus, the yeast gene could be introduced into a variety of cell lines, including C4T, a human cell line with a homoplasmic frameshift mutation in the MTND4 gene [13] in which the targeted Ndi1 gene product was incorporated into an active respiratory chain, thereby restoring functionality of complex I. Encouragingly, in vivo rodent studies of the NDI1 gene in brain and skeletal muscles have revealed sustained protein expression in mitochondria [14]. The import of small tRNA molecules to complement the genetic abnormality has also been explored. Mitochondrial tRNAs are not normally imported into human cells as all tRNAs required for mitochondrial translation are encoded by the mitochondrial genome, but other small RNA species have been reported to be mitochondriallytargeted and taken up. By studying the yeast cytosolic tRNALysCUU (tK1), Tarassov and colleagues have shown that tK1 and similar derivatives can be imported into isolated human mitochondria if the tRNA is aminoacylated and supplied with soluble factors including lysl-tRNA synthetases [15], and that yeast tRNAsLys derivatives can be expressed and imported into mitochondria within transmitochondrial cybrid cells and primary human fibroblasts carrying the m.8344A>G MTTK mutation that causes the MERRF phenotype. The imported tRNALys is correctly aminoacylated, able to participate in mitochondrial translation, and effects a partial rescue of mitochondrial
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functions [16]. More recently, the RNA import complex from the kinetoplastid protozoa Leishmania has been shown to be taken up by human mitochondria via a caveolin-1dependent pathway, and facilitate the import of endogenous, cytosolic tRNAs into cells enabling the restoration of mitochondrial function [17]. Strategies to Manipulate mtDNA Heteroplasmy Several strategies have been employed to investigate shifting the critical balance of wild type and mutated mitochondrial genomes (threshold) which is so influential in determining the expression of the biochemical defect associated with mtDNA mutations. Given a small percentage of the wild type mtDNA is protective reflecting the recessive nature of many mutations, only minor adjustments to the balance of mutated to wild type genomes could theoretically result in a reversal of the associated biochemical defect. One such approach has trialled the use of sequence-specific nucleic acid derivatives—peptide nucleic acids (PNA)—to selectively inhibit the replication of (a subset of) mutated mtDNA in order to confer upon the wild type genome a distinct replicative advantage. This rationale forms the basis of the ‘‘antigenomic’’ approach and is theoretically applicable to all heteroplasmic mtDNA mutations including mtDNA deletions [18]. However, in spite of initial success in selectively inhibiting the replication single-stranded mtDNA templates with the m.8344A>G MERRF mutation in vitro [19] and targeting the molecule to cells and mitochondria [20, 21], many difficulties remain to be negotiated to fully realise the potential of this approach [4]. In a recent paper, a targeted-chimeric zinc finger methylase has been shown to bind and modify mutated mtDNA in cells carrying the m.8993T>G NARP mutation in a sequence-specific manner, and as such represents a new technology which may be applicable to treating heteroplasmic mtDNA mutations in the future [22]. Several other approaches to manipulate mtDNA heteroplasmy levels have met with success, including the targeting to mitochondria of a restriction endonuclease capable of differentiating between two mtDNA genotypes, which again could lead to the preferential elimination of the mutated genotype and propagation of the wild type genotype. Several groups have used various mitochondrially-targeted constructs to modulate mtDNA heteroplasmy in human cells carrying pathogenic mutations [23], hybrid cells carrying both mouse and rat mtDNA [24] in a mouse model which is heteroplasmic for the BALB and NZB mtDNA haplotypes [25]. Pharmacological approaches to shifting mtDNA heteroplasmy have also been investigated. Cell culture studies have shown that oligomycin, a specific inhibitor of mitochondrial ATP synthase, can significantly increase the fraction of wild-type molecules in cells that harbour the m.8933T>G NARP mutation under culture conditions (galactose as a carbon source) which specifically select for the wild-type molecule [26]. Based on the observation that this culture medium could also selectively kill cells homoplasmic for the ‘‘common’’ 4,977 bp mtDNA deletion, Schon and colleagues have demonstrated that a ketogenic medium can shift the heteroplasmy of cells containing a mixture of wild-type and partially deleted mtDNAs, possibly due to an intracellular selection for wild-type organelles [27].
Exercise Training Exercise training has been suggested as an approach to improve physical capacity and quality of life in patients with mtDNA mutations, either through improving the severe
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exercise intolerance in patients with high levels of mutation in muscle by endurance exercise protocols or through muscle satellite cell activation by resistance exercise protocols [28].
Endurance Exercise A number of studies have previously demonstrated that exercise training was well tolerated by patients with mitochondrial myopathy, could improve exercise capacity and consequently induce physiological adaptations to reverse the effects of deconditioning and improve the underlying mitochondrial defect [29–31]. The effects of aerobic conditioning was investigated in a group of genetically-heterogenous patients with mitochondrial myopathy and following 14 weeks of endurance training, patients exhibited marked, physiological improvements on account of increased mitochondrial biogenesis due to an increase in the levels of wild type mtDNA [32]. However, mtDNA analysis revealed that mutation levels either stayed the same or, in some patients, increased, inferring preferential amplification of mutated DNA. This raised the question as to whether endurance training had potentially, long-term deleterious effects in spite of the obvious improvement in biochemical and physiological parameters Two recent studies have sought to assess the safety of endurance training in patients with heteroplasmic mtDNA mutations. The first focussed on a genetically-heterogeneous group of 20 patients (five with single, large-scale mtDNA deletions, one with a microdeletion, 13 with the m.3243A>G mutation and one with the m.8344A>G mutation) who underwent 12 weeks of endurance training, leading to an improvement in oxidative capacity but with no concomitant shift in mutated mtDNA levels in muscle [33]. A second study investigated the effect of endurance training in smaller, homogenous group of eight patients with single, large-scale mtDNA deletions. Following 14 weeks of exercise training, physiological assessment revealed improved oxygen utilisation, skeletal muscle oxygen extraction, peak capacity for work and submaximal exercise tolerance. As in the previous study, genetic analysis did not detect any change in the level of deleted mtDNA following exercise training [34]. Further training in four patients for an additional 14 weeks maintained these benefits, whereas detraining resulted in a loss of the physiological adaptations. These results are encouraging, however the authors suggest caution in recommending training to patients with other mtDNA mutations or suggesting exercise training as treatment as longerterm studies are still required to assess potential changes in mtDNA mutation as a result of exercise training. An interesting observation in the study by Taivassalo and colleagues [34] was the dramatic change seen in one of the patients who underwent de-training. This patient had 48% COX-deficient fibres at baseline and after 14 weeks of training, but this increased markedly to 79% COX-deficient fibres when she stopped training (Fig. 1). A knee injury acquired immediately post-training, unrelated to exercise, caused this patient to become significantly immobile and to limit physical activity to levels below that of her baseline condition. Despite this extreme form of deconditioning, no change was observed in the percentage of deleted mtDNA in all three biopsies (87% mutated mtDNA). Of note, however, was the finding of a 40% decrease in the copy number of total mtDNA in the COX-deficient fibres in the final biopsy which would support the hypothesis that the biochemical defect in COX activity relates to the amount of wild type molecules rather than the total percentage of mutated mtDNA (Fig. 2).
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A
C
B E Copy number per unit area
D
% deletion
100 95 90 85 80 baseline
14 wks
4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
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bas eline
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Biopsy
Fig. 1 Assessment of mitochondrial activities and mutation load following endurance strength training in a patient with a single, large-scale mtDNA deletion. Combined COX/SDH histochemistry showing the percentage of COX-deficient fibres in patient biopsies at (A) baseline, (B) following 14 weeks of training and (C) following a 14 week period of detraining. (D) Real-time PCR analysis shows no significant change in mtDNA deletion levels between the three biopsies, whilst there is a clear decrease in mtDNA copy number (E), although this does not reach statistical significance between baseline and 28 weeks (P = 0.08)
donor embryo pronuclei enucleation to remove pronuclei
Mutant mitochondria Normal mitochondria
Fig. 2 The concept of pronuclear transfer to prevent the transmission of mtDNA disease. In the first step, a donor embryo containing normal mitochondria is enucleated by removal of the pronuclei. The male and female pronuclei are subsequently removed from the fertilised embryo containing mutated mitochondria and transferred to the enucleated donor embryo. This results in an embryo containing nuclear DNA from the parents and healthy mitochondria from the donor
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Resistance Exercise Some patients with sporadic mtDNA mutations causing a predominantly myopathic phenotype harbour high levels of mutated mtDNA in mature muscle fibres, but low or undetectable levels in myoblast populations derived from the satellite cells [35, 36]. Satellite cells are undifferentiated, mononuclear myogenic precursor cells which proliferate in response to injury to repair or replace damaged muscle fibres. It has been proposed that the stimulation of muscle regeneration in vivo in these patients might restore a normal mitochondrial DNA genotype, and as such, several novel approaches to induce satellite cell incorporation (referred to as ‘‘gene shifting’’) have been explored. One study used the anaesthetic bupivacaine hydrochloride (0.75%) to induce limited muscle necrosis in a patient with a sporadic m.12320A>G MTTL2 mutation, in which the regenerating muscle fibres derived exclusively from the satellite cell population leading to an improvement in biochemical activity [37]. An independent study of another patient with a MTTL2 mutation revealed that the traumatic injury of the muscle biopsy procedure was in itself sufficient to promote satellite cell incorporation into existing muscle fibres [38], although resistance exercise training—either using concentric exercise (short contractions, which would induce muscle fibre hypertrophy) and eccentric exercise (lengthening contractions, which should lead to segmental necrosis) has proved to be a more convenient and applicable method to achieve this [39]. A clear decrease in the proportion of COX-deficient fibres was evidence following concentric training, whilst there was an increase in the percentage of fibres with lower levels of mtDNA mutation; an increase in muscle fibre diameter was also evident, especially in the COX-positive fibres. The authors did not expect a significant increase in the proportion of wild type mtDNA due to hypotrophy alone, and related the increase in wild-type mtDNA to the preferential amplification of satellite cell-derived mtDNA template. In an ongoing collaboration with Drs Taivassalo and Haller, we have investigated the effect of resistance exercise training in a homogenous group of patients with single, large-scale mtDNA deletions. Our preliminary data suggest that 14 weeks of resistance training leads to an increasing muscle fibre regeneration, measurable increases in muscle strength and a decrease in the percentage of COX-deficient fibres.
Germline Therapy—Preventing mtDNA Disease Transmission Maternal transmission of the mitochondrial genome means that a heteroplasmic woman carrying an mtDNA mutation is at significant risk of passing the defect to her children. The outcome for specific pregnancies remains difficult to predict, however, as the proportion of mutant mtDNA passed from mother to child can vary dramatically with each pregnancy [40]. This is largely due to the mitochondrial genetic bottleneck that occurs during development, which makes accurate genetic counselling extremely challenging and leaves considerable uncertainty for families affected by mtDNA disease. Prenatal Genetic Diagnosis Prenatal diagnosis, including amniocentesis and chorionic villus sampling, is widely used to detect autosomal and chromosomal abnormalities in fetal tissues. It has also been used to detect certain mtDNA disorders [41–43], but its use is limited as
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different mtDNA mutations may not all behave in the same way. There is also concern whether the mutation load detected in the prenatal sample will reflect other fetal tissues, as with heteroplasmic mtDNA disorders there is often remarkable tissue specific differences in the level of heteroplasmy. The present evidence suggests that this should not be a problem and it is hoped that with increasing knowledge of mtDNA mutations, improved prenatal diagnosis may become available for more families with mtDNA disorders. Preimplantation Genetic Diagnosis Preimplantation genetic diagnosis (PGD) is a technique that allows the diagnosis of genetic disease using the polar body from an unfertilised oocyte or one to two single cells from an early embryo. This approach provides genetic screening before a pregnancy is established and allows only unaffected embryos to be transferred to the uterus for implantation. Since the introduction of PGD [44], it has been used to detect an expanding number of genetic disorders. However, although it has been considered as an option for women at risk of transmitting an mtDNA mutation, there is only one report of PGD being successfully completed for a mitochondrial DNA disease [45]. Experiments performed by our group and others to determine the mtDNA copy number in individual human blastomeres have shown that each individual cell contains on average around 100,000 copies of mtDNA. Although there is variability in copy number between cells from different embryos, the high copy number suggests that PGD should be feasible for mtDNA disease and eliminates the problems associated with single cell analysis, such as amplification failure. PGD for mitochondrial disease aims to detect the proportion of mutant mtDNA in the embryo. This can be done by analysing mtDNA in the first polar body [46] or one to two blastomeres from a preimplantation embryo. Using a heteroplasmic mouse model, it has been shown that the level of heteroplasmy is virtually identical between the ooplasm and polar body of a mature oocyte, and also between the blastomeres of each 2-, 4- and 6–8 cell embryo [47]. This suggests that the level of heteroplasmy detected in the polar body or individual blastomere is representative of the level in the embryo. However, problems in interpreting this data have been highlighted, as only embryos with undetectable or very low mutant loads would be suitable for transfer. The situation would not be as clear for embryos containing intermediate levels of mutant mtDNA [48]. Cytoplasmic Transfer This technique involves the transfer of normal mitochondria (in cytoplasts) to an oocyte containing mutant mtDNA, thus reducing the effect of any mtDNA defect [49]. Cytoplasmic transfer between human oocytes has been used to try and improve the success rate of assisted reproduction treatments [50], although concerns have been raised as to the safety and effectiveness of the procedure [51, 52]. Despite this, some of the children born were heteroplasmic with low levels of mtDNA from the donor oocyte [53]. Although this demonstrates that changes in heteroplasmy can occur, it is likely that this technique will have limited use in patients with mtDNA disease. Experiments in mice suggest that the amount of mtDNA that can be transferred is relatively small [48] and so the relative proportion of mutated to wild-type mtDNA is unlikely to change significantly.
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Pronuclear Transfer Nuclear transfer is a technique that could be used to prevent the transmission of mtDNA disease. The strategy involves the transfer of nuclear DNA from an oocyte with mutant mtDNA to an enucleated oocyte from a female with normal mtDNA [54] (Fig. 2). This would result in healthy offspring with all the characteristics of the parents (from the nuclear genes) but without the mtDNA mutation. There are several different approaches that could be considered, including nuclear transfer between unfertilised oocytes, but the strategy we believe will be most successful is pronuclear transfer between single cell zygotes. The technique was first described by Mcgrath and Solter [55] and has been developed in mice with considerable success following the birth of live offspring [56–59]. This approach involves transfer of a karyoplast containing the male and female pronuclei, distinct structures that become visible in the oocyte following fertilisation and eventually fuse to form the nucleus in the embryo. As the karyoplast contains the pronuclei surrounded by a small amount of cytoplasm, it is inevitable that mitochondria of karyoplast origin will be transferred to the recipient zygote. This could potentially present a problem for the prevention of mtDNA disease, as mutant mitochondria will be transferred to the enucleated zygote. Following pronuclear transfer between single cell mouse zygotes, one study reported that reconstructed zygotes contained on average 19% mtDNA of karyoplast origin, although most offspring generated from these reconstructed embryos were found to contain a lower percentage and produced exclusively homoplasmic first generation offspring [56, 57]. The study also reported that the average amount of karyoplast derived mtDNA present in adult tissue varied, with heteroplasmy levels in a founder female ranging from 6% (lung) to 69% (heart). Her progeny were all heteroplasmic, but some second and third generation offspring were homoplasmic. They detected heteroplasmy up to the fifth generation, but with lessened tissue and litter variability, indicating a low but stable and persistent transmission of both mtDNA species down the maternal line. Similar results were also reported by our group following pronuclear transfer in mice [59]. Our data revealed that blastocysts cultured from reconstructed zygotes contained 16.3 ± 8.4% mtDNA of karyoplast origin, which from a clinical point of view means an 83.7% reduction in the mutant load. Our study also demonstrated the ability to generate mice with low levels of karyoplast derived mtDNA. The level of heteroplasmy varied in tissues from 10% (muscle) to 37% (liver). A recent study directly addressed the issue of pronuclear transfer to prevent the transmission of mtDNA disease using transmitochondrial mice (mito-mice). These mice express respiration defects and mitochondrial diseases due to accumulation of mtDNA carrying a large-scale mtDNA rearrangement [60]. The study carried out pronuclear transfer from mito-mice zygotes with an average of 35% mutant mtDNA to enucleated normal zygotes [58]. The resultant progeny had an average of 11% mutant mtDNA in tail and did not express the mitochondrial disease phenotype throughout their lives. This demonstrates that pronuclear transfer is an effective method to prevent the transmission of mtDNA disease in mice. Following approval for a research licence from the Human Fertilisation and Embryology Authority in the UK, studies are in progress to determine whether pronuclear transfer is a feasible option for the prevention of transmission of mtDNA disease in human embryos. These studies will use abnormally fertilised human embryos and will determine the feasibility of the technique, the percentage of mtDNA of karyoplast origin and the presence of either cytogenetic or epigenetic abnormalities.
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Whilst this approach is very much a possibility for the future in terms of a treatment, developing techniques to improve the chances of a mother with mtDNA disease having a healthy child remains a priority for families, clinicians and scientists alike. Acknowledgements Work in our laboratory is supported by the Muscular Dystrophy Campaign, Wellcome Trust, Department of Health and the Newcastle upon Tyne Hospitals NHS Foundation Trust.
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