Allotopic Expression Research Articles

What limits the allotopic expression of nucleus-encoded mitochondrial genes? The case of the chimeric Cox3 and Atp6 genes

Allotopic expression is potentially a gene therapy for mtDNA-related diseases. Some OXPHOS proteins like ATP6 (subunit a of complex V) and COX3 (subunit III of complex IV) that are typically mtDNA-encoded, are naturally nucleus-encoded in the alga Chlamydomonas reinhardtii. The mitochondrial proteins whose genes have been relocated to the nucleus exhibit long mitochondrial targeting sequences ranging from 100 to 140 residues and a diminished overall mean hydrophobicity when compared with their mtDNA-encoded counterparts. We explored the allotopic expression of the human gene products COX3 and ATP6 that were re-designed for mitochondrial import by emulating the structural properties of the corresponding algal proteins. In vivo and in vitro data in homoplasmic human mutant cells carrying either a T8993G mutation in the mitochondrial atp6 gene or a 15 bp deletion in the mtDNA-encoded cox3 gene suggest that these human mitochondrial proteins re-designed for nuclear expression are targeted to the mitochondria, but fail to functionally integrate into their corresponding OXPHOS complexes.

Allotopic expression of mitochondrial-encoded genes in mammals: achieved goal, undemonstrated mechanism or impossible task?

Mitochondrial-DNA diseases have no effective treatments. Allotopic expression—synthesis of a wild-type version of the mutated protein in the nuclear-cytosolic compartment and its importation into mitochondria—has been proposed as a gene-therapy approach. Allotopic expression has been successfully demonstrated in yeast, but in mammalian mitochondria results are contradictory. The evidence available is based on partial phenotype rescue, not on the incorporation of a functional protein into mitochondria. Here, we show that reliance on partial rescue alone can lead to a false conclusion of successful allotopic expression. We recoded mitochondrial mt-Nd6 to the universal genetic code, and added the N-terminal mitochondrial-targeting sequence of cytochrome c oxidase VIII (C8) and the HA epitope (C8Nd6HA). The protein apparently co-localized with mitochondria, but a significant part of it seemed to be located outside mitochondria. Complex I activity and assembly was restored, suggesting successful allotopic expression. However, careful examination of transfected cells showed that the allotopically-expressed protein was not internalized in mitochondria and that the selected clones were in fact revertants for the mt-Nd6 mutation. These findings demonstrate the need for extreme caution in the interpretation of functional rescue experiments and for clear-cut controls to demonstrate true rescue of mitochondrial function by allotopic expression.

Leber Hereditary Optic Neuropathy Gene Therapy Clinical Trial Recruitment

To describe the patient profiles of the Leber hereditary optic neuropathy (LHON) Gene Therapy Clinical Trial, year 1. This study aims to identify and characterize affected patients and carriers with the G11778A mutation in mitochondrial DNA for planned gene therapy that will use "allotopic expression" by delivering a normal nuclear-encoded ND4 gene into the nuclei of retinal ganglion cells via an adeno-associated virus vector injected into the vitreous. Patients with LHON with visual loss as well as asymptomatic maternally related family members were molecularly screened for ND1, ND4, and ND6 mutations in mitochondrial DNA commonly associated with LHON. All patients and maternal relatives also underwent complete neuro-ophthalmic examination, automated visual field testing, pattern electroretinogram (PERG), and OCT3. Twenty-five subjects with LHON and 21 carriers positive for the G11778A mitochondrial DNA mutation were recruited. Three additional mutations in the ND4 gene, G11719A, G11947A, or G11914A, were detected. Mean retinal nerve fiber layer (RNFL) thickness was 78.3 μm up to 32 months after visual loss. It was 63.5 μm for all affected patients and 100.7 μm for carriers (P < .01). Mean PERG amplitude was lower in affected patients (40% of normal) than in carriers (94% of normal) (P < .01). Four carriers with PERG amplitudes less than 75% of normal had Early Treatment Diabetic Retinopathy Study acuity more than 20/25, mean defect more than -2 dB, and average RNFL thickness more than 80 μm. Potential candidates for future gene therapy may include affected patients, as late as 32 months after loss of vision, with mildly reduced RNFL thickness or carriers with low PERG amplitudes and normal RNFL thickness, if the PERG amplitude is a predictor of conversion to LHON in these carriers.

Induction of Rapid and Highly Efficient Expression of the Human ND4 Complex I Subunit in the Mouse Visual System by Self-complementary Adeno-Associated Virus

To demonstrate the high efficiency and rapidity of allotopic expression of a normal human ND4 subunit of complex I in the vertebrate retina using a self-complementary adeno-associated virus (scAAV) vector for ocular gene delivery to treat acute visual loss in Leber hereditary optic neuropathy (LHON). The nuclear-encoded human ND4 subunit fused to the P1 isoform of subunit C of adenosine triphosphate synthase (ATPc) mitochondrial targeting sequence and FLAG epitope was packaged in scAAV2 capsids or single-stranded (ss) AAV2 capsids. These constructs were injected into the vitreous cavities of mice. The contralateral eyes were injected with scAAV-green fluorescent protein (GFP). One week later, pattern electroretinograms and gene expression of the human ND4 subunit and GFP were evaluated. Quantitative analysis of ND4FLAG-injected eyes was assessed relative to Thy1.2-labeled retinal ganglion cells (RGCs). Pattern electroretinogram amplitudes remained normal in eyes inoculated with scAAV-ND4FLAG, ssAAV-ND4FLAG, and GFP. Confocal microscopy revealed the typical perinuclear mitochondrial expression of scAAV-ND4FLAG in almost the entire retinal flat mount. In contrast, scAAV-GFP expression was cytoplasmic and nuclear. Relative to Thy1.2-positive RGCs, quantification of scAAV-ND4FLAG-positive RGCs was 91% and that of ssAAV-ND4FLAG-positive RGCs was 51%. Treatment of acute visual loss due to LHON may be possible with a normal human ND4 subunit gene of complex I, mutated in most cases of LHON, when delivered by an scAAV vector. Clinical Relevance Unlike most retinal degenerations that result in slowly progressive loss of vision over many years, LHON due to mutated mitochondrial DNA results in apoplectic, bilateral severe and usually irreversible visual loss. For rescue of acute visual loss in LHON, a highly efficient and rapid gene expression system is required.

The approaches for manipulating mitochondrial proteome

Over the past decade a large volume of research data has accumulated which has established a fundamental role for mitochondria in normal cellular functioning, as well as in various pathologies. Mitochondria play a pivotal role in metabolism and energy production, and are one of the key players involved in programmed cell death. On the other hand, mitochondrial dysfunction is implicated, directly or indirectly in numerous pathological conditions including inherited mitochondrial disorders, diabetes, cardiovascular and neurodegenerative diseases, and a variety of malignancies. The ability to modulate mitochondrial function by altering the diverse protein component of this organelle may be of great value for developing future therapeutic interventions. This review will discuss approaches used to introduce proteins into mitochondria. One group of methods utilizes strategies aimed at expressing proteins from genes in the nucleus. These include overexpression of nuclear-encoded mitochondrial proteins, allotopic expression, which is the re-coding and relocation of mitochondrial genes to the nucleus for expression and subsequent delivery of their gene products to mitochondria, and xenotopic expression, which is the nuclear expression of genes coding electron transport chain components from distant species, for delivery of their products to mammalian mitochondria. Additionally, antigenomic and progenomic strategies which focus on expression of mitochondrially targeted nuclear proteins involved in the maintenance of mtDNA will be discussed. The second group of methods considered will focus on attempts to use purified proteins for mitochondrial delivery. Special consideration has been given to the complexities involved in targeting exogenous proteins to mitochondria.

A single mutation in the first transmembrane domain of yeast COX2 enables its allotopic expression

During the course of evolution, a massive reduction of the mitochondrial genome content occurred that was associated with transfer of a large number of genes to the nucleus. To further characterize factors that control the mitochondrial gene transfer/retention process, we have investigated the barriers to transfer of yeast COX2, a mitochondrial gene coding for a subunit of cytochrome c oxidase complex. Nuclear-recoded Saccharomyces cerevisiae COX2 fused at the amino terminus to various alternative mitochondrial targeting sequences (MTS) fails to complement the growth defect of a yeast strain with an inactivated mitochondrial COX2 gene, even though it is expressed in cells. Through random mutagenesis of one such hybrid MTS-COX2, we identified a single mutation in the first Cox2 transmembrane domain (W56 --> R) that (i) results in the cellular expression of a Cox2 variant with a molecular mass indicative of MTS cleavage, which (ii) supports growth of a cox2 mutant on a nonfermentable carbon source, and that (iii) partially restores cytochrome c oxidase-specific respiration by the mutant mitochondria. COX2(W56R) can be allotopically expressed with an MTS derived from S. cerevisiae OXA1 or Neurospora crassa SU9, both coding for hydrophobic mitochondrial proteins, but not with an MTS derived from the hydrophilic protein Cox4. In contrast to some other previously transferred genes, allotopic COX2 expression is not enabled or enhanced by a 3'-UTR that localizes mRNA translation to the mitochondria, such as yeast ATP2(3)('-UTR). Application of in vitro evolution strategies to other mitochondrial genes might ultimately lead to yeast entirely lacking the mitochondrial genome, but still possessing functional respiratory capacity.

Converting NADH to NAD+ by nicotinamide nucleotide transhydrogenase as a novel strategy against mitochondrial pathologies during aging

Mitochondrial DNA defects are involved supposedly via free radicals in many pathologies including aging and cancer. But, interestingly, free radical production was not found increased in prematurely aging mice having higher mutation rate in mtDNA. Therefore, some other mechanisms like the increase of mitochondrial NADH/NAD(+) and ubiquinol/ubiquinone ratios, can be in action in respiratory chain defects. NADH/NAD(+) ratio can be normalized by the activation or overexpression of nicotinamide nucleotide transhydrogenase (NNT), a mitochondrial enzyme catalyzing the following very important reaction: NADH + NADP(+ )<--> NADPH + NAD(+). The products NAD(+) and NADPH are required in many critical biological processes, e.g., NAD(+) is used by histone deacetylase Sir2 which regulates longevity in different species. NADPH is used in a number of biosynthesis reactions (e.g., reduced glutathione synthesis), and processes like apoptosis. Increased ubiquinol/ubiquinone ratio interferes the function of dihydroorotate dehydrogenase, the only mitochondrial enzyme involved in ubiquinone mediated de novo pyrimidine synthesis. Uridine and its prodrug triacetyluridine are used to compensate pyrimidine deficiency but their bioavailability is limited. Therefore, the normalization of the ubiquinol/ubiquinone ratio can be accomplished by allotopic expression of alternative oxidase, a mitochondrial ubiquinol oxidase which converts ubiquinol to ubiquinone.

Optimized Allotopic Expression of the Human Mitochondrial ND4 Prevents Blindness in a Rat Model of Mitochondrial Dysfunction

Mitochondrial diseases due to mutations in mitochondrial DNA can no longer be ignored in most medical areas. With prevalence certainly higher than one in 6000, they probably represent the most common form of metabolic disorders. Despite progress in identification of their molecular mechanisms, little has been done with regard to therapy. We have recently optimized the allotopic expression for the mitochondrial genes ATP6, ND1, and ND4 and obtained a complete and long-lasting rescue of mitochondrial dysfunction in the human fibroblasts in which these genes were mutated. However, biosafety and benefit to mitochondrial function must be validated in animal models prior to clinical applications. To create an animal model of Leber Hereditary Optic Neuropathy (LHON), we introduced the human ND4 gene harboring the G11778A mutation, responsible of 60% of LHON cases, to rat eyes by in vivo electroporation. The treatment induced the degeneration of retinal ganglion cells (RGCs), which were 40% less abundant in treated eyes than in control eyes. This deleterious effect was also confirmed in primary cell culture, in which both RGC survival and neurite outgrowth were compromised. Importantly, RGC loss was clearly associated with a decline in visual performance. A subsequent electroporation with wild-type ND4 prevented both RGC loss and the impairment of visual function. Hence, these data provide the proof-of-principle that optimized allotopic expression can be an effective treatment for LHON, and they open the way to clinical studies on other devastating mitochondrial disorders.

Progress and prospects: gene therapy for mitochondrial DNA disease

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.

The optimized allotopic expression of ND1 or ND4 genes restores respiratory chain complex I activity in fibroblasts harboring mutations in these genes

Leber's Hereditary Optic Neuropathy (LHON) was the first maternally inherited mitochondrial disease identified and is now considered the most prevalent mitochondrial disorder. LHON patients harbor mutations in mitochondrial DNA (mtDNA). In about 90% of cases, the genes involved encode proteins of the respiratory chain complex I. Even though the molecular bases are known since 20 years almost all remains to be done regarding physiopathology and therapy. In this study, we report a severe decrease of complex I activity in cultured skin fibroblasts isolated from two LHON patients harboring mutations in ND4 or ND1 genes. Most importantly, we were able to restore sustainably (a) the ability to grow on galactose, (b) the ATP synthesis rate and (c) the complex I activity, initially impaired in these cells. Our strategy consisted of forcing mRNAs from nuclearly-encoded ND1 and ND4 genes to localize to the mitochondrial surface. The rescue of the respiratory chain defect observed was possible by discreet amounts of hybrid mRNAs and fusion proteins demonstrating the efficiency of their mitochondrial import. Hence, we confirmed here for two mitochondrial genes located in the organelle that the optimized allotopic expression approach represents a powerful tool that could ultimately be applied in human therapy for LHON.

Mitochondrial Diseases: Therapeutic Approaches

Therapy of mitochondrial encephalomyopathies (defined restrictively as defects of the mitochondrial respiratory chain) is woefully inadequate, despite great progress in our understanding of the molecular bases of these disorders. In this review, we consider sequentially several different therapeutic approaches. Palliative therapy is dictated by good medical practice and includes anticonvulsant medication, control of endocrine dysfunction, and surgical procedures. Removal of noxious metabolites is centered on combating lactic acidosis, but extends to other metabolites. Attempts to bypass blocks in the respiratory chain by administration of electron acceptors have not been successful, but this may be amenable to genetic engineering. Administration of metabolites and cofactors is the mainstay of real-life therapy and is especially important in disorders due to primary deficiencies of specific compounds, such as carnitine or coenzyme Q10. There is increasing interest in the administration of reactive oxygen species scavengers both in primary mitochondrial diseases and in neurodegenerative diseases directly or indirectly related to mitochondrial dysfunction. Aerobic exercise and physical therapy prevent or correct deconditioning and improve exercise tolerance in patients with mitochondrial myopathies due to mitochondrial DNA (mtDNA) mutations. Gene therapy is a challenge because of polyplasmy and heteroplasmy, but interesting experimental approaches are being pursued and include, for example, decreasing the ratio of mutant to wild-type mitochondrial genomes (gene shifting), converting mutated mtDNA genes into normal nuclear DNA genes (allotopic expression), importing cognate genes from other species, or correcting mtDNA mutations with specific restriction endonucleases. Germline therapy raises ethical problems but is being considered for prevention of maternal transmission of mtDNA mutations. Preventive therapy through genetic counseling and prenatal diagnosis is becoming increasingly important for nuclear DNA-related disorders. Progress in each of these approaches provides some glimmer of hope for the future, although much work remains to be done.

Allotopic mRNA Localization to the Mitochondrial Surface Rescues Respiratory Chain Defects in Fibroblasts Harboring Mitochondrial DNA Mutations Affecting Complex I or V Subunits

The possibility of synthesizing mitochondrial DNA (mtDNA)-coded proteins in the cytosolic compartment, called allotopic expression, provides an attractive option for genetic treatment of human diseases caused by mutations of the corresponding genes. However, it is now appreciated that the high hydrophobicity of proteins encoded by the mitochondrial genome represents a strong limitation on their mitochondrial import when translated in the cytosol. Recently, we optimized the allotopic expression of a recoded ATP6 gene in human cells, by forcing its mRNA to localize to the mitochondrial surface. In this study, we show that this approach leads to a long-lasting and complete rescue of mitochondrial dysfunction of fibroblasts harboring the neurogenic muscle weakness, ataxia and retinitis Pigmentosa T8993G ATP6 mutation or the Leber hereditary optic neuropathy G11778A ND4 mutation. The recoded ATP6 gene was associated with the cis-acting elements of SOD2, while the ND4 gene was associated with the cis-acting elements of COX10. Both ATP6 and ND4 gene products were efficiently translocated into the mitochondria and functional within their respective respiratory chain complexes. Indeed, the abilities to grow in galactose and to produce adenosine triphosphate (ATP) in vitro were both completely restored in fibroblasts allotopically expressing either ATP6 or ND4. Notably, in fibroblasts harboring the ATP6 mutation, allotopic expression of ATP6 led to the recovery of complex V enzymatic activity. Therefore, mRNA sorting to the mitochondrial surface represents a powerful strategy that could ultimately be applied in human therapy and become available for an array of devastating disorders caused by mtDNA mutations.

Approaches to the treatment of mitochondrial diseases

Therapy for mitochondrial diseases is woefully inadequate. However, lack of a cure does not equate with lack of treatment. Palliative therapy is dictated by good medical practice and includes anticonvulsant medication, control of endocrine dysfunction, and surgical procedures. Removal of noxious metabolites is centered on combating lactic acidosis, but extends to other metabolites. Attempts to bypass blocks in the respiratory chain by administration of electron acceptors have not been successful, but this may be amenable to genetic engineering. Administration of metabolites and cofactors is the mainstay of real-life therapy and is especially important in disorders due to primary deficiencies of specific compounds, such as carnitine or coenzyme Q10 (CoQ10). There is increasing interest in the administration of reactive oxygen radicals (ROS) scavengers, both in primary mitochondrial diseases and in neurodegenerative diseases. Gene therapy is a challenge because of polyplasmy and heteroplasmy, but novel experimental approaches are being pursued. One important strategy is to decrease the ratio of mutant to wild-type mitochondrial genomes ("gene shifting") by different means: (1) converting mutated mitochondrial DNA (mtDNA) genes into normal nuclear DNA genes ("allotopic expression"); (2) importing cognate genes from other species ("xenotopic expression"); (3) correcting mtDNA mutations by importing specific restriction endonucleases; (4) selecting for respiratory function; and (5) inducing muscle regeneration. Germline therapy raises ethical problems but is being considered for prevention of maternal transmission of mtDNA mutations. Preventive therapy through genetic counseling and prenatal diagnosis is becoming increasingly important for nuclear DNA-related disorders.

Factors That Might Affect the Allotopic Replacement of a Damaged Mitochondrial DNA-Encoded Protein

The human mitochondrion contains a small circular genome that codes for 13 proteins, 22 tRNAs, and 2 rRNAs. The proteins are all inner membrane bound components of complexes involved in the electron transport system and ATP formation. Mutations to any of the 13 proteins affect cellular behavior because energy production could be decreased. Investigators have attempted to find methods to correct these mutated proteins. One way is to express the mitochondrial gene in the nucleus (called allotopic expression). The newly synthesized protein would have to be imported into mitochondria and assembled into complexes. This paper reviews some of the successful attempts to achieve allotopic expression and discusses some issues that might affect the ability to have the proteins properly inserted into the inner membrane.

AAV‐Mediated Gene Transfer Protects Against Mitochondrial Optic Neuropathy in Mice

Mutations affecting mitochondria lead to a variety of diseases that have been hard to replicate in animals, because of the difficulty in manipulating mitochondrial DNA and because of embryonic lethality. We have used Adeno-associated virus (AAV) mediated RNA knockdown to produce two mouse models of Leber Hereditary Optic Neuropathy (LHON). Patients with LHON usually harbor mutations in subunits of Complex I of the respiratory chain and experience loss of vision beginning around age 20. In mice, blocking synthesis of subunits of this enzyme leads to the pathologic hallmarks of the disease—death of retinal ganglion cells, demyelination and loss of axons in the optic nerve. Using these mouse models, we tested two approaches for gene therapy of LHON. In the first, we delivered the gene for manganese superoxide dismutase (SOD2) to limit the accumulation of reactive oxygen species that result from Complex I defects. Delivery of SOD2 caused an 18% increase (p=0.001) in the survival of retinal ganglion cells and a proportional increase in the thickness of the optic nerve. In the second strategy, we re-coded the mitochondrial gene for the ND4 subunit of Complex I, attached a transport sequence to its amino terminus and expressed it as a nuclear gene--allotopic expression. Treatment of the optic nerve with AAV expressing this protein led to a 13% increase (p<0.01) in diameter of the optic nerve relative to eyes treated with a control virus. Thus both approaches prevented tissue injury resulting from defects to Complex I. These provide the first evidence in animals that gene therapy for mitochondrial disease may be possible.

Allotopic expression of a mitochondrial alternative oxidase confers cyanide resistance to human cell respiration

Human mitochondrial respiration is distinct from that of most plants, microorganisms and even some metazoans in that it reduces molecular oxygen only through the highly cyanide-sensitive enzyme cytochrome c oxidase. Here we show that expression of the cyanide-insensitive alternative oxidase (AOX), recently identified in the ascidian Ciona intestinalis, is well tolerated by cultured human cells and confers spectacular cyanide resistance to mitochondrial substrate oxidation. The expressed AOX seems to be confined to mitochondria. AOX involvement in electron flow is triggered by a highly reduced redox status of the respiratory chain (RC) and enhanced by pyruvate; otherwise, the enzyme remains essentially inactive. AOX expression promises to be a valuable tool to limit the deleterious consequences of RC deficiency in human cells and whole animals.

The 57th Annual Meeting of the American Academy of Neurology, Miami, Florida, April 9-16, 2005

The 57th Annual Meeting of the American Academy of Neurology, Miami, Florida, April 9-16, 2005

Targeting the NAD7 Subunit to Mitochondria Restores a Functional Complex I and a Wild Type Phenotype in the Nicotiana sylvestris CMS II Mutant Lacking nad7

The mitochondrial DNA of the Nicotiana sylvestris CMSII mutant carries a 72-kb deletion comprising the single copy nad7 gene that encodes the NAD7 subunit of the respiratory complex I (NADH-ubiquinone oxidoreductase). CMSII plants lack rotenone-sensitive complex I activity and are impaired in physiological and phenotypical traits. To check whether these changes directly result from the deletion of nad7, we constructed CMS transgenic plants (termed as CMSnad7) carrying an edited nad7 cDNA fused to the CAMV 35S promoter and to a mitochondrial targeting sequence. The nad7 sequence was transcribed and translated and the NAD7 protein directed to mitochondria in CMSnad7 transgenic plants, which recovered both wild type morphology and growth features. Blue-native/SDS gel electrophoresis and enzymatic assays showed that, whereas fully assembled complex I was absent from CMSII mitochondria, a functional complex was present in CMSnad7 mitochondria. Furthermore, a supercomplex involving complex I and complex III was present in CMSnad7 as in the wild type. Taken together, these data demonstrate that lack of complex I in CMSII was indeed the direct consequence of the absence of nad7. Hence, NAD7 is a key element for complex assembly in plants. These results also show that allotopic expression from the nucleus can fully complement the lack of a mitochondrial-encoded complex I gene.

Stable Transformation of CHO Cells and Human NARP Cybrids Confers Oligomycin Resistance (olir) Following Transfer of a Mitochondrial DNA–Encoded olirATPase6 Gene to the Nuclear Genome: A Model System for mtDNA Gene Therapy

Point and deletion mutations and a general depletion of mammalian mitochondrial DNA (mtDNA) give rise to a wide variety of medical syndromes that are refractory to treatment, possibly including aging itself. While gene therapy directed at correcting such deficits in the mitochondrial genome may offer some therapeutic benefits, there are inherent problems associated with a direct approach. These problems are primarily due to the high mitochondrial genome copy number in each cell and the mitochondrial genome being "protected" inside the double-membrane mitochondrial organelle. In an alternative approach there is evidence that genes normally present in the mitochondrial genome can be incorporated into the nuclear genome. To extend such studies, we modified the Chinese Hamster Ovary (CHO) mtDNA-located ATPase6 gene (possessing a mutation which confers oligomycin resistance- oli(r)) by altering the mtDNA code to the universal code (U-code) to permit the correct translation of its mRNA in the cytoplasm. The U-code construct was inserted into the nuclear genome (nucDNA) of a wild type CHO cell. The expressed transgene products enabled the transformed CHO cell lines to grow in up to 1000 ng mL(-1) oligomycin, while untransformed sensitive CHO cells were eliminated in 1 ng mL(-1) oligomycin. This approach, termed allotopic expression, provides a model that may make possible the transfer of all 13 mtDNA mammalian protein-encoding genes to the nucDNA, for treatments of mtDNA disorders. The CHO mtATPase6 protein is 85% identical to both the mouse and human mtATPase6 protein; these proteins are highly conserved in the region of the oligomycin resistance mutation. They are also well conserved in the regions of the oligomycin resistance mutation of the mouse, and in the region of a mutation found in Leigh's syndrome (T8993G), also called NARP (neurogenic weakness, ataxia, retinitis pigmentosum). It is likely that the CHO oli(r) mtATPase6 Ucode construct could impart oligomycin-resistance in human and mouse cells, as well as function in place of the mutant ATPase subunit in a NARP cell line. Preliminary experiments on human cybrids homoplasmic for the NARP mutation (kindly supplied by D.C. Wallace), transformed with our construct, display an increased oligomycin resistance that supports these suppositions.

683. Cationic Mitochondriotropic Vesicles for DNA Delivery to Mitochondria

The number of diseases found to be associated with defects of the mitochondrial genome has grown significantly since 1988. Despite major advances in understanding mtDNA defects at the genetic and biochemical level, there is no satisfactory treatment available for a vast majority of patients. Objective limitations of conventional biochemical treatment for patients with defects of mtDNA warrant the exploration of gene therapeutic approaches. Two different strategies for mitochondrial gene therapy are imaginable1 The first involves expressing a wild-type copy of the defective gene in the nucleus, with cytoplasmic synthesis and subsequent targeting of the gene product to the mitochondria (“allotopic expression”). Besides the different codon usage in mitochondria, however, there are possibly four major difficulties in adapting this nuclear-cytosolic approach for mitochondrial gene therapy to mammalian cells2. First, the majority of mtDNA defects involve tRNAs and to date, no natural mechanism has been reported for the mitochondrial uptake of cytosolic tRNAs in mammalian cells. Second, it is generally agreed that the thirteen proteins encoded for by mtDNA are very hydrophobic peptides, which would not be readily imported by the mitochondrial protein import machinery. However, since the 13 mitochondrial coded proteins are not equally hydrophobic, the allotopic expression of at least some of the peptides appears as possible3. Third, it has been hypothesized that some of the proteins encoded by the mitochondrion may potentially be toxic if synthesized in the cytosol4. Fourth, according to a hypothesis termed co-location for redox regulation5, the co-location of mtDNA and its products may be essential for the rapid control of gene expression by the redox state in the mitochondrial matrix. Considering all problems associated with the nuclear-cytosolic approach the development of methods for the direct transfection of mitochondria6 as an alternative approach towards mitochondrial gene therapy seems highly warranted. We have developed a strategy for mitochondrial gene therapy which involves the transport of a DNA-mitochondrial leader sequence peptide conjugate to mitochondria using cationic mitochondriotropic vesicles, the liberation of this conjugate from the cationic vector upon contact with the mitochondrial outer membrane followed by DNA uptake via the mitochondrial protein import machinery. For the design of cationic mitochondriotropic vesicles we have utilized the self-assembly behavior of dequalinium, a cationic single-chain bola-amphiphile which is known to selectively accumulate in mitochondria. We found that such bola-amphiphiles are able to form liposome-like cationic vesicles (“bolasomes”), which we termed “DQAsomes” when prepared from dequalinium7,8. Data will be presented showing that DQAsomes fulfill all essential prerequisites for a mitochondria-specific DNA delivery system.