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Gene Therapy for Mitochondrial DNA Disease

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Gene Therapy for Mitochondrial DNA Disease Gene Therapy (2008) 15, 1017–1023 & 2008 Nature Publishing Group All rights reserved 0969-7128/08 $30.00 www.nature.com/gt REVIEW Progress and prospects: gene therapy for mitochondrial DNA disease DS Kyriakouli, P Boesch, RW Taylor and RN Lightowlers Mitochondrial Research Group, Medical School, Newcastle University, Newcastle upon Tyne, UK Defects of the mitochondrial genome cause a wide variety of Although far from the clinic, these approaches show promise. clinical disorders. Except for rare cases where surgery or Similarly, nuclear transfection with genes encoding restric- transplant is indicated, there is no effective treatment for tion endonucleases or sequence-specific zinc finger-binding patients. Genetic-based therapies are consequently being proteins destined for mitochondria has also proved success- considered. On account of the difficulties associated with ful in targeting mtDNA-borne pathogenic mutations. This is mitochondrial (mt) transfection, alternative approaches particularly important, as mutated mtDNA is often found in whereby mitochondrial genes can be engineered and cells that also contain normal copies of the genome, a introduced into the nucleus (allotopic expression) are being situation termed heteroplasmy. Shifting the levels of hetero- attempted with some success, at least in cultured cells. plasmy towards the normal mtDNA has become the goal of a Defects in the activities of multi-subunit complexes variety of invasive and non-invasive methods, which are also of the oxidative phosphorylation apparatus have been highlighted in this review. circumvented by the targeted expression of simple single Gene Therapy (2008) 15, 1017–1023; doi:10.1038/gt.2008.91; subunit enzymes from other species (xenotopic expression). published online 22 May 2008 Keywords: mtDNA; heteroplasmy; mitochondria; mtDNA disease In brief Progress Prospects Strategies can be devised for gene therapy of Generation of numerous mice models heteroplasmic mitochondrial DNA disorders. for pathogenic mtDNA mutations that can routinely Allotopic and xenotopic expression show great be used to determine efficacy of any therapeutic. promise as treatment concepts for mtDNA disease. Although such mice do exist, there are currently very The alternative oxidase may prove an attractive few. It is expected that within 5 years, there will be alternative for treating mtDNA disease. many models that can be used. The yeast soluble NADH oxidase can substitute for Determining the feasibility of using genes encoding defective human complex I. single enzyme alternatives to OXPHOS complexes as Allotopic expression and import of nucleic acids are possible interventions for mutations that affect being considered for rescuing pathogenic mt-tRNA protein-coding genes in mtDNA. mutations. Production of antigenomic drugs that will allow Rescue of mtDNA mutations may be possible targeting and removal of pathogenic mtDNA. Cur- through mitochondrial transfection. rent work is promising and it is likely that within 3–5 Manipulating the mitochondrial genome may lead to years a pharmaceutical could be developed for oral or rescue of an OXPHOS deficiency. intravenous delivery that will be able to modulate Germline therapy for mitochondrial DNA disease is levels of heteroplasmy. being considered. Introduction matrix and encodes 13 polypeptides, all of which are The human mitochondrial genome (mtDNA) is found in integral components of the complexes that couple many copies in all nucleated cells (Figure 1). This small oxidative phosphorylation (OXPHOS). In addition to (16 569 bp) genome is housed in the mitochondrial 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 Correspondence: Professor RN Lightowlers, Mitochondrial for intramitochondrial protein synthesis. As all 13 Research Group, Newcastle University, The Medical School, mitochondrially encoded polypeptides are essential Framlington Place, Newcastle upon Tyne, Newcastle NE2 4HH, UK. E-mail: [email protected] members of the OXPHOS machinery that harnesses Received 13 February 2008; revised 11 April 2008; accepted 14 April cellular respiration to ATP production, it is not surpris- 2008; published online 22 May 2008 ing that mutations in this genome can cause a wide Gene therapy for mitochondrial DNA disease DS Kyriakouli et al 1018 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. 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 func- Figure 1 The human mitochondrial genome. This small (16 569 bp) genome is almost completely transcribed from both strands, initiating tionally integrated into the mitochondrial ATP synthase from one of two promoters (IH1,IH2) on the Heavy (H-) strand or the (complex V) and the deficiency could be rescued. Several single promoter (IL) on the Light (L-) strand. All of these promoters years later, this finding was extended to cultured human and elements involved in replication initiation are found in the cells. A different mitochondrial gene to encode a member displacement (D-) loop, the only major non-coding region. The of the ATP synthase, MTATP6, was also engineered for genome encodes 22 mt-tRNA (black diamonds), 2 mt-rRNA genes expression in the nucleus. Successful targeting of the (fuscia) and 13 protein-coding genes (olive, ND1-6 encoding members of NADH:ubiquinone oxido-reductase; blue, Cytb encoding ATPase 6 gene product to the mitochondrion was shown apocytochrome b of ubiquinol:cytochrome c oxido-reductase; orange, to partially suppress a respiratory-deficient phenotype COI-III encoding members of cytochrome c oxidase: aquamarine, caused by a pathogenic mutation in MTATP6. These ATPase 6, 8 encoding two members of Fo-F1 ATP synthase). experiments were met with optimism as a potential Important mutations that are referred to in this article are indicated. method for treating patients with mutations in mito- Single letter code is given for each mt-tRNA-encoding gene. chondrial protein-encoding genes such as those with neurogenic weakness, ataxia with retinitis pigmentosum variety of disorders such as mitochondrial encephalo- (NARP) which are caused by a specific point mutation pathy lactic acidosis with stroke-like episodes (MELAS), (m.8993T4G) in MTATP6. More recent and extensive myoclonic epilepsy with ragged red fibres (MERRF) and analysis by Bokori-Brown and Holt conflict with these leber’s hereditary optic neuropathy (LHON).1 In addi- data.3 Similar allotopic experiments were performed to tion, due to the ubiquity of mitochondria, the clinical rescue a cell line carrying the identical mutation in presentation of mitochondrial DNA disorders can MTATP6. The authors optimised the import of nuclear- be multi-system, but often causes profound chronic encoded ATPase 6 into the human cell lines but after progressive defects in muscle and nerve. careful analysis of assembled complexes were unable to Cells are multiploid with respect to mtDNA, with show integration of the imported protein into mature most somatic cells containing at least 103 copies of the functional ATP synthase. Restoration of complex V genome. Consequently, a pathogenic mutation may be activity in the original experiments was suggested to present in just one, many, or all, depending on when the have been due to random clonal variations in ATP mutation occurred and how the mutated molecule was synthesis as a consequence of aneuploidy in the replicated and segregated during development. For most transfected population. Despite these discrepancies, the disorders, the pathogenic mutation is recessive, requiring authors remained optimistic about the potential for a substantial percentage of the genome to carry the defect allotopic expression as a treatment method. before an effect on OXPHOS can be measured. This In a twist to this approach, Corral-Debrinski et al.4 ‘threshold’ varies between cell types and mutation sites have attempted to optimise the allotopic expression of but is normally between 60 and 95%. such highly hydrophobic mitochondrial proteins by suggesting that targeting of the transcript to the outer mitochondrial membrane may facilitate co-translational Strategies can be devised for gene translocation of the gene product, thus preventing the therapy of mitochondrial DNA disorders accumulation of cytosolic aggregates. Further evidence was generated by using primary fibroblasts from patients There is currently no effective treatment for the majority with mutations in MTND4 (a mitochondrial gene of these debilitating diseases. This
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